Epigenetic Mechanisms of Cell Programming and Reprogramming (Reports of China's Basic Research) 9811974187, 9789811974182

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Reports of China’s Basic Research

Gang Pei Editor

Epigenetic Mechanisms of Cell Programming and Reprogramming

Reports of China’s Basic Research Editor-in-Chief Wei Yang, National Natural Science Foundation of China, Beijing, China Zhejiang University, Hangzhou, Zhejiang, China

The National Natural Science Foundation of China (NSFC) was established on February 14, 1986. Upon its establishment, NSFC was an institution directly under the jurisdiction of the State Council, tasked with the administration of the National Natural Science Fund from the Central Government. In 2018, it became managed by the Ministry of Science and Technology (MOST) but kept its due independence in operation. Since its establishment, NSFC has comprehensively introduced and implemented a rigorous and objective merit-review system to fulfill its mission of supporting basic research, fostering talented researchers, developing international cooperation and promoting socioeconomic development. Featuring science, basics, and advances, the series of Reports of China’s Basic Research is organized by the NSFC to present the overall level and pattern of China’s basic research, share innovative achievements, and illustrate excellent breakthroughs in key fields. It covers various disciplines including but not limited to, computer science, materials science, life sciences, engineering, environmental sciences, mathematics, and physics. The series will show the core contents of the final reports of the Major Programs and the Major Research Plans funded by NSFC, and will closely follow the frontiers of basic research developments in China.

Gang Pei Editor

Epigenetic Mechanisms of Cell Programming and Reprogramming

Editor Gang Pei Tongji University Shanghai, China

Supported by the NSFC Major Research Plan “Epigenetic Mechanisms of Cell Programming and Reprogramming” ISSN 2731-8907 ISSN 2731-8915 (electronic) Reports of China’s Basic Research ISBN 978-981-19-7418-2 ISBN 978-981-19-7419-9 (eBook) https://doi.org/10.1007/978-981-19-7419-9 Jointly published with Zhejiang University Press The print edition is not for sale in China Mainland. Customers from China Mainland please order the print book from: Zhejiang University Press. © Zhejiang University Press 2023 This work is subject to copyright. All rights are solely and exclusively licensed by the Publisher, whether the whole or part of the material is concerned, specifically the rights of reprinting, reuse of illustrations, recitation, broadcasting, reproduction on microfilms or in any other physical way, and transmission or information storage and retrieval, electronic adaptation, computer software, or by similar or dissimilar methodology now known or hereafter developed. The use of general descriptive names, registered names, trademarks, service marks, etc. in this publication does not imply, even in the absence of a specific statement, that such names are exempt from the relevant protective laws and regulations and therefore free for general use. The publishers, the authors, and the editors are safe to assume that the advice and information in this book are believed to be true and accurate at the date of publication. Neither the publishers nor the authors or the editors give a warranty, expressed or implied, with respect to the material contained herein or for any errors or omissions that may have been made. The publishers remain neutral with regard to jurisdictional claims in published maps and institutional affiliations. This Springer imprint is published by the registered company Springer Nature Singapore Pte Ltd. The registered company address is: 152 Beach Road, #21-01/04 Gateway East, Singapore 189721, Singapore

Reports of China’s Basic Research

Editorial Board Editor-in-Chief Wei Yang Associate Editors Wen Gao Ruiping Gao Editors Yu Han Changrui Wang Yonghe Zheng Zhongwen Zheng Feng Feng Yanze Zhou Tiyu Gao Weitong Zhu Qingguo Meng Yongjun Chen Shengming Du Qidong Wang Ming Li Yuwen Qin Ziyou Gao Erdan Dong Zhiyong Han Xinquan Yang Shengli Ren

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Preface to the Series

As Lao Tzu said, “A huge tree grows from a tiny seedling; a nine-storied tower rises from a heap of earth.” Basic research is the fundamental approach to fostering innovation-driven development, and its level becomes an important yardstick for measuring the overall scientific and national strength of a country. Since the beginning of the twenty-first century, China’s overall strength in basic research has been consistently increasing. With respect to input and output, China’s input in basic research increased by 14.8 times from 5.22 billion yuan in 2001 to 82.29 billion yuan in 2016, with an average annual increase of 20.2%. In the same period, the number of China’s scientific papers included in the Science Citation Index (SCI) increased from lower than 40,000 to 324,000; China rose from the 6th to the 2nd place in global ranking in terms of the number of published papers. In regard to the quality of output, in 2016, China ranked No. 2 in the world in terms of citations in 9 disciplines, among which the materials science ranked No. 1; as of October 2017, China ranked No. 3 in the world in the numbers of both Highly Cited Papers (top 1%) and Hot Papers (top 0.1%), with the latter accounting for 25.1% of the global total. In talent cultivation, in 2006, China had 175 scientists (136 of whom from the Chinese mainland) included in Thomson Reuters’ list of Highly Cited Researchers, ranking 4th globally and 1st in Asia. Meanwhile, we should also be keenly aware that China’s basic research is still facing great challenges. First, funding for basic research in China is still far less than that in developed countries—only about 5% of the R&D funds in China are used for basic research, a much lower percentage than 15%–20% in developed countries. Second, competence for original innovation in China is insufficient. Major original scientific achievements that have global impact are still rare. Most of the scientific research projects are just a follow-up or imitation of existing research, rather than groundbreaking research. Third, the development of disciplines is not balanced, and China’s research level in some disciplines is noticeably lower than the international level—China’s Field-Weighted Citation Impact (FWCI) in disciplines just reached 0.94 in 2016, lower than the world average of 1.0.

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The Chinese government attaches great importance to basic research. In the 13th Five-Year Plan (2016–2020), China has established scientific and technological innovation as a priority in all-round innovation and has made strategic arrangements to strengthen basic research. General Secretary XI Jinping put forward a grand blueprint of making China into a world-leading power in science and technology in his speech delivered at the National Conference on Scientific and Technological Innovation in 2016, and emphasized that “we should aim for the frontiers of science and technology, strengthen basic research and make major breakthroughs in pioneering basic research and groundbreaking and original innovations” at the 19th CPC National Congress on October 18, 2017. With more than 30 years of unremitting exploration, the National Natural Science Foundation of China (NSFC), one of the main channels for supporting basic research in China, has gradually shaped a funding pattern covering research, talent, tools and convergence, and has taken action to vigorously promote basic frontier research and the growth of scientific research talent, reinforce the building of innovative research teams, deepen regional cooperation and exchanges, and push forward multidisciplinary convergence. As of 2016, nearly 70% of China’s published scientific papers were funded by the NSFC, accounting for 1/9 of the total number of published papers all over the world. Facing the new strategic target of building China into a strong country in science and technology, the NSFC will conscientiously reinforce forward-looking planning and enhance the efficiency of evaluation, so as to achieve the strategic goal of making China progressively share the same level with major innovative countries in research total volume, contribution and groundbreaking researchers by 2050. The series of Advances in China’s Basic Research and the series of Reports of China’s Basic Research proposed and planned by the NSFC emerge against such a background. Featuring science, basics and advances, the two series are aimed at sharing innovative achievements, diffusing performances of basic research and leading breakthroughs in key fields. They closely follow the frontiers of basic research developments in China and publish excellent innovation achievements funded by the NSFC. The series of Advances in China’s Basic Research mainly presents the important original achievements of the programs funded by the NSFC and demonstrates the breakthroughs and forward guidance in key research fields; the series of Reports of China’s Basic Research shows the core contents of the final reports of Major Programs and Major Research Plans funded by the NSFC to make a systematic summarization and give a strategic outlook on the achievements in the funding priorities of the NSFC. We hope not only to comprehensively and systematically introduce backgrounds, scientific significance, discipline layouts, frontier breakthroughs of the programs and a strategic outlook for the subsequent research, but also to summarize innovative ideas, enhance multidisciplinary convergence, foster the continuous develop of research in concerned fields and promote original discoveries. As Hsun Tzu remarked, “When earth piles up into a mountain, wind and rain will originate thereof. When waters accumulate into a deep pool, dragons will come to live in it.” The series of Advances in China’s Basic Research and Reports of China’s Basic Research are expected to become the “historical records” of China’s basic research. They will provide researchers with abundant scientific research material

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and vitality of innovation and will certainly play an active role in making China’s basic research prosper and building China’s strength in science and technology.

Academician of the Chinese Academy of Sciences Beijing, China

Preface

Epigenetics, an emerging discipline since the late 1980s, refers to “the study of changes in gene function that are heritable but do not entail a change in the DNA sequence.” How is the selective expression of the same genome determined in different types of cells within multicellular organisms? How do cells induce and memorize the gene expression in response to changes in the internal and external environment of the organism? The answers to these questions can be found with the epigenetic research. Epigenetic research started from the epigenetic phenomena observed in plants and animals, followed by the discoveries of a series of epigenetic modifications, and then the identification of multiple epigenetic regulators. Gradually, it became to focus on the regulation of gene expression by chromatin structure. The scientific community came to understand that epigenetic factors and transcription factors jointly determine the spatiotemporal expression of genes. In 2006, Japanese scientist Shinya Yamanaka achieved the somatic cell reprogramming with transcription factors. Chinese scientists quickly realized the importance of epigenetic mechanisms in the processes of cell programming and reprogramming. Initiated by Pei Gang, Meng Anming, Chen Runsheng, Shang Yongfeng, Cao Xiaofeng, Sun Fanglin, Xi Zhen and other scientists, departments of Life Sciences, Chemical Sciences and Information Sciences of the National Natural Science Foundation of China (NSFC) jointly supported the Major Research Plan Program (MRP) of “Epigenetic Mechanisms of Cell Programming and Reprogramming” in 2008. It funded 156 projects, including the fostering projects, key projects and integrated projects, with an investment of RMB 190 million within 8 years. This MRP has actively encouraged forward-looking, original and systematic research. By the end of 2016, it completed all proposed scientific objectives and made a series of major scientific achievements that attracted worldwide attention. For example, our scientists have, for the first time in the world, analyzed the 30-nm chromatin structure and pointed out that the tetra-nucleosome is an important structural and regulatory unit in chromatin; the “seesaw model” of cell reprogramming has been suggested and the system for inducing somatic cell reprogramming with small chemical molecules has been significantly optimized; the technologies of haploid embryonic stem cells and “semi-cloned” mice have been successfully xi

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established to generate “artificial spermatids”; the transdifferentiation from fibroblasts to hepatocyte-like cells has been first achieved in the world, which has been gradually developed toward the application of the bioartificial liver. Series of breakthroughs have also been made in the epigenome research. DNA and histone methylomes in mammalian early embryos have been first established, and the mechanisms to regulate activities of many important epigenetic modifying enzymes, such as DNA methylases and demethylases, have been analyzed. This MRP has cultivated numerous world-class excellent scientists, improved the level of research on epigenetics and cell fate determination in China and achieved the leapfrog progress from “follow-up” to “world-leading position.” The successful completion of this MRP has provided new directions for further explorations of the roles of epigenetic regulation in cell fate determination. Current research still focuses on the discovery and functional verification of key epigenetic factors; however, the understanding on the chromatin dynamic changes in cell reprogramming, changes in cell epigenomes and their regulation on transcriptomes, and mechanisms for determining cell fate transitions remains elusive. Our scientists should continue to give full play to the interdisciplinary advantages, deepen the research on epigenetic transcriptomes, single-cell transcriptomes and epigenomes, epigenome editing, four-dimensional genome and the mechanisms for intergenerational inheritance of epigenetics, crack the genetic codes stored in the form of chromatin and comprehensively analyze the role of epigenetics in cell fate determination, to make unremitting efforts for the overall improvement and sustainable development of the innovation ability of China’s epigenetic research. Gang Pei Academian of the Chinese Academy of Sciences, Shanghai, China

Contributors

Yongfeng Shang Hangzhou Normal University, Hangzhou, Zhejiang, China Fanglin Sun Tongji University, Shanghai, China Zuoyan Zhu Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China Runsheng Chen Institute of Biophysics, Chinese Academy of Sciences, Beijing, China Xiaofeng Cao Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Zhen Xi Nankai University, Tianjin, China

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Contents

1 Project Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1 Introduction . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.1 Arrangement and Comprehensive Integration of This MRP . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.1.2 Interdisciplinary Cooperation . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2 Research Overview . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.1 Overall Scientific Objectives . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.2.2 Key Scientific Issues . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 1.3 Significant Progress . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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2 Research in China and International Research . . . . . . . . . . . . . . . . . . . . . 2.1 Introduction of Epigenetic Research Plans . . . . . . . . . . . . . . . . . . . . . . 2.2 Status Quo of the Epigenetic Research . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3 Development Trends of the Epigenetic Research . . . . . . . . . . . . . . . . . 2.3.1 Mechanisms of Epigenetic Regulation of Gene Expression . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.2 Discovery, Identification, and Function Studies of New Epigenetic Modifications, Modifying Enzymes, and Reader Proteins . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.3 Mechanisms of Establishment and Maintenance of Epigenetic Information . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.4 Epigenetic Regulation on Reproduction and Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.5 Molecular Mechanisms of Epigenetic Regulation on Cell Programming and Reprogramming . . . . . . . . . . . . . . . . . . . . . . 2.3.6 Higher-Order Chromatin Structures and Subnuclear Structures . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.7 Origins and Evolution of Epigenetic Regulatory Networks . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 2.3.8 Cancer, Neurodegenerative Diseases, and Other Major Diseases Related to Epigenetic Regulation . . . . . . . . . . . . . . . .

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3 Major Research Achievements . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1 New Epigenetic Regulators and Chromatin Remodeling Factors as Well as Their Biological Functions and Mechanisms . . . . . . . . . . . 3.1.1 New Mechanisms of Interactive Regulation Between DNA Methylation and Histone Methylation Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.2 New Regulation Mechanisms of DNA Methylase and Demethylase Activities . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.3 New Histone Modification Codes and the Interpretation Mechanisms . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.4 Higher-Order Structure of 30-Nm Chromatin Fibers and Their Dynamic Regulation Mechanisms . . . . . . . . . . . . . . 3.1.5 Mechanisms of Centromeric Chromatin Establishment and Nucleosome Assembly . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.1.6 Roles of Histone Variants in Chromatin Assembly . . . . . . . . . 3.1.7 New Interaction Patterns Between Epigenetic Regulation and DNA-Damage Repair . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2 Discovery of Epigenetic Regulation Mechanisms for Stem Cell Self-renewal and Somatic Cell Reprogramming, and Breakthroughs in Animal Cloning and Reproductive Technologies . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.1 Establishment of ESCs and Semi-cloning Technologies to Make “Artificial Spermatids” . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.2 First Utilization of the Polar Body Genome Transfer for Preventing the Transmission of Inherited Mitochondrial Diseases . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.3 The Seesaw Model of Lineage Specifiers Inducing Somatic Cells to iPSC . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.4 Safety Evaluation of Traditional Methods of iPSC Induction by Yamanaka Factors . . . . . . . . . . . . . . . . . . . . . . . . . 3.2.5 Exploration of the Regulation Mechanisms of Somatic Cell Reprogramming and Discovery of New Regulators . . . . 3.2.6 Discovery of New Signals for Maintaining Stem Cell Self-renewal and Developmental Pluripotency . . . . . . . . . . . . . 3.3 Epigenetic Mechanisms Related to Cell Differentiation and Transdifferentiation, Ontogeny, and Diseases . . . . . . . . . . . . . . . . 3.3.1 Discovery of the Key Factors to Promote the Transdifferentiation of Somatic Cells to Hepatocytes . . . . 3.3.2 Discovery of the Epigenetic Mechanisms and Cell Signaling Pathways Inducing the Differentiation and Transdifferentiation from Embryonic Stem Cells or Somatic Cells to Neurons . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.3.3 Discovery of Important Disease-related Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . .

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3.4 Establishment of Epigenetic Maps to Reveal the Characteristics and Rules of Epigenetic Modifications During the Embryonic Development . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.1 Discovery of Distribution and Change Rules of Genome-Wide Histone Modifications in Pre-implantation Embryos . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.2 Establishment of Single-Cell Transcriptome Sequencing and Analysis Tools . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 3.4.3 Establishment of Genome-Wide Methylation Profiles to Reveal the Inheritance and Evolution Rules of Epigenetic Modifications . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4 Outlook . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 4.1 Directions to Be Strengthened in China’s Epigenetic Research . . . . . 4.2 Strategic Needs of the Epigenetic Research in China . . . . . . . . . . . . . . 4.3 Conceptions and Suggestions for Further Research . . . . . . . . . . . . . . .

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References . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 51 Index . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . 55

Chapter 1

Project Overview Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen, Xiaofeng Cao, and Zhen Xi

1.1 Introduction The Major Research Plan of “Epigenetic Mechanisms of Cell Programming and Reprogramming” (hereinafter referred to as “this MRP”) was launched by the National Natural Science Foundation of China (NSFC) during the 11th Five-Year Plan period, which was started in October 2008 and concluded at the end of 2016. It has funded 156 projects with a total investment of RMB 190 million, including 68 fostering projects, 23 key projects, and 59 integrated projects, covering the fields supported by NSFC departments of Life Sciences, Chemical Sciences, and Information Sciences. Epigenetics, an emerging discipline since the late 1980s, studies the molecular mechanisms of changes in cell phenotypes expressed by heritable genes without any G. Pei (B) Tongji University, Shanghai, 200000, China e-mail: [email protected] Y. Shang Hangzhou Normal University, Hangzhou, Zhejiang, China F. Sun Tongji University, Shanghai, China Z. Zhu Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China R. Chen Institute of Biophysics, Chinese Academy of Sciences, Beijing, China X. Cao Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Z. Xi Nankai University, Tianjin, China © Zhejiang University Press 2023 G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming, Reports of China’s Basic Research, https://doi.org/10.1007/978-981-19-7419-9_1

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alterations of the DNA sequence. The epigenetic regulation mechanism, as a common way of gene expression regulation in life phenomena, is one of the important mechanisms to regulate growth, development, aging, and disease. Especially, epigenetic regulation plays a decisive role in stem cell maintenance, self-renewal and differentiation, individual aging and dysplasia, development of complex conditions such as cancer, diabetes, mental illness, and nervous system diseases. The orderly responses of individual life to environmental factors (including those of nutrition, physical chemistry, and psychology) largely depend on the effective operation of epigenetic regulation networks. It also plays an important role in plant development, resistance, and formation of heterosis.

1.1.1 Arrangement and Comprehensive Integration of This MRP Since it was organized and implemented, this MRP has always followed NSFC’s general principle of “limited goals to enable stable supports, and integrated themes to realize leapfrog development.” With focuses on the key scientific issues of the overall scientific objectives, it has funded 68 fostering projects, 23 key projects, and 59 integrated projects. Epigenetics gradually rose in the late 1980s. After 2000, research in this field has been widely valued and become hotspots in life sciences. Studies on cell programming and reprogramming cover the basic scientific questions of epigenetics. In 2006, scientists of the USA and Japan reported the establishment of induced pluripotent stem cell, symbolizing a new development stage of the research on reprogramming of somatic cells. Before this MRP was launched, great progress had been made in epigenetic research worldwide, but scientists’ understanding of epigenetic mechanisms was still the tip of the iceberg. Many key problems remained unsolved. For instance, how do DNA methyltransferases (DNMTs) selectively act on target genes? How can we clone and identify DNA demethylases? How can we decode the composition and recognition of the “histone code”? How do the higher-order structures of chromatin interact with epigenetic information? How does noncoding RNA participate in the epigenetic regulation? What are the molecular mechanisms of the epigenetic plasticity and cell reprogramming? What are the relationships between epigenetic regulation and environment, disease, aging, etc.? What are the features of the composition, origin, and evolution of epigenetic regulatory networks? At the beginning of this MRP, the epigenetic research was an emerging field where there was little gap between Chinese scientists and their international counterparts in research level and opportunities outweighed challenges. The Advisory Expert Group enhanced the top-down design and utilized the advantages of our young scientists engaged in epigenetic research, to focus on the international frontier and carry out innovative research.

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Centering on the scientific objectives and the key scientific issues, this MRP initially established 5 research directions: (1) Molecular mechanisms of establishment and maintenance of epigenetic information; (2) Epigenetic mechanisms of directional differentiation of stem cells; (3) Epigenetic mechanisms of somatic cell reprogramming; (4) Epigenetic mechanisms of tissue and organ development and regeneration; (5) Origins and evolution of epigenetic networks. Four years after the launch of this MRP, the Advisory Expert Group, based on a comprehensive investigation on the progress of funded projects, aimed at the cuttingedge scientific questions and development trends in epigenetics, and condensed the research directions into three integrated themes, to further fulfill the strategy of “focusing on goals to make key breakthroughs” of this MRP: (1) Molecular mechanisms and biological significance of DNA methylation and demethylation; (2) Epigenetic mechanisms of cell reprogramming; (3) Higher-order structures and dynamic changes of nuclear chromatin and noncoding RNAs during the cell reprogramming. As a result of the joint efforts of the Advisory Expert Group, the Project Review Group, and project scientists, this MRP has fully completed the planned scientific objectives and achieved numerous breakthroughs with great international influences in the three integrated themes, which has promoted the overall improvement and leapfrog development of epigenetic research in China.

1.1.2 Interdisciplinary Cooperation During the implementation of this MRP, the Advisory Expert Group took into consideration both the current situation of epigenetic research in China and the international frontier and development trends in this field. It vigorously promoted multidisciplinary convergence among cell biology, biochemistry, developmental biology, structural biology, bioinformatics, and clinical medicine, and, through various forms of project support, promptly applied the latest ideas and technologies of related disciplines to epigenetic research. It thus has made outstanding contributions to the comprehensive leapfrog development of epigenetic research in China. Especially, the following measures should be noted. (1) Cooperation from such disciplines as cell biology, biochemistry, genetics, developmental biology, genesiology, and evolution was promoted to catapult China into world’s top ranks of somatic cell reprogramming, nuclear transfer, and semi-cloned technology. Specially, the number of the papers on haploid stem cell research published in high-impact journals has exceeded the sum of those

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published by other countries in the world, suggesting that China is in a leading position in this area. (2) The convergence between epigenetics and physics was actively encouraged. With the introduction of the research means of structural biology, the analysis on the spatial structures of DNA, RNA, histone modifiers, and higher-order structures of chromatin was enhanced. It has effectively promoted the discovery and identification of key molecules and the elucidation of their mechanisms, making China a leader in this area. (3) The convergence with mathematics and information science was emphasized. The means of computational biology were introduced for the massive data analysis in the epigenetic research, which has effectively promoted the discovery of key nodes in the interactions of the epigenetic signal networks and the elucidation of systems biology mechanisms. Since its implementation, this MRP has followed NSFC’s general principle of “limited goals to enable stable supports, and integrated themes to realize leapfrog development.” With focuses on the scientific frontiers of related epigenetic areas such as somatic cell reprogramming, ontogeny, cell differentiation, and diseases, the Advisory Expert Group and the Management Group enhanced the top-down design, refined scientific objectives, and carried out innovative research, which have made major contributions to the comprehensive upgrade of the original innovation in the basic research of epigenetics in China.

1.2 Research Overview 1.2.1 Overall Scientific Objectives The scientific objectives of this MRP were to, with the application of interdisciplinary research methods, understand the rules and characteristics for formation, maintenance, and functions of the epigenetic information during cell programming and reprogramming, elucidate the mechanisms of the epigenetic regulation in cell growth, development, and environmental adaptation, and to reveal the mechanisms of the composition, evolution, and operation of epigenetic networks.

1.2.2 Key Scientific Issues This MRP aimed to explore the following core scientific themes: (1) Molecular mechanisms of establishment and maintenance of epigenetic information; (2) Epigenetic mechanisms of directional differentiation of stem cells;

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(3) Epigenetic mechanisms of somatic cell reprogramming; (4) Epigenetic mechanisms of tissue and organ development and regeneration; (5) Origins and evolution of epigenetic networks.

1.3 Significant Progress During its implementation, this MRP has achieved breakthroughs with great international influences in the core scientific themes and realized the leapfrog development of China’s epigenetic research from “follow-up” to “world-leading position.” The representative achievements are as follows. (1) New epigenetic regulators and chromatin remodeling factors have been discovered, and their biological functions and mechanisms have been revealed. (2) The technologies of haploid embryonic stem cells and semi-cloned mice have been successfully established, and “artificial spermatids” have been made with the manipulation of imprinted genes. New regulation mechanisms and methods of somatic cell reprogramming have been found, and the “seesaw model” of cell reprogramming has been suggested. (3) Several regulation mechanisms of cell differentiation and trans-differentiation have been revealed. Especially, the key factors to promote the transdifferentiation of somatic cells to hepatocytes have been discovered, which has provided a solid foundation for the clinical application of the bioartificial liver. Multiple disease-related epigenetic modifications have been found. (4) For the first time in the world, the epigenetic map has been established with high-throughput data collection to reveal the characteristics of epigenetic modifications during the early embryonic development of different species and the rules of genetic evolution, which has enriched our understanding of the origins and evolution of epigenetic networks. (5) The 30-nm chromatin structure has been first analyzed in the world, and it has been indicated that the tetra-nucleosome is an important unit of structure and regulation in chromatin. (6) The polar body genome transfer has been first used to prevent the transmission of inherited mitochondrial diseases. After the completion of this MRP, the development trends in the fields are compared in Table 1.1.

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Table 1.1 Comparison of development trends before and after the completion of this MRP Core scientific themes

Status of China’s research at the start of this MRP

Status of international research at the end of this MRP

Strengths and gaps of China’s research at the end of this MRP

Molecular mechanisms of establishment and maintenance of epigenetic information

Despite China’s gaps with the international advanced levels, a group of young scientists returned to China and were gearing up

Internationally, rapid development has been made in this area, but China has taken the lead in some areas

The research on the mechanisms of DNA methylation and demethylation, discovery of new DNA methylation modifications and their mechanisms, and higher-order structures of chromatin has kept ahead internationally. The research on other areas is equivalent to the international research level

Epigenetic mechanisms of somatic cell reprogramming

Internationally, Europe and America enjoyed the absolute leading positions in the research on the mechanisms of somatic cell reprogramming, while China just started

The international research level of the mechanisms of somatic cell reprogramming has been similar to that in China, but lagged behind it in terms of the research on the haploid stem cells

China has ranked top in the research on the haploid stem cells. It has been similar in the international research level of the mechanisms of somatic cell reprogramming, but the number of its research teams in this area is still small

Epigenetic mechanisms of cell differentiation and transdifferentiation, tissue and organ development and regeneration

There were big gaps between China and international leaders in terms of cell differentiation and transdifferentiation as well as organ development and regeneration

Internationally, there have been many studies on the transdifferentiation of other types of cells, such as nerve cells, myocardial cells, and blood cells

China has become a leader in the research on the transdifferentiation of hepatocytes, and an average player in other areas such as the transdifferentiation of nerve cells

Origins and evolution of epigenetic networks

The research foundation of this area in China was relatively poor

The international research advantages over China is gradually becoming smaller

China has rose to the top of the research on the reprogramming of the epigenetic modifications during the early embryonic development, leaving the rest of the world behind

Chapter 2

Research in China and International Research Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen, Xiaofeng Cao, and Zhen Xi

The classic definition of epigenetics is “the study of changes in gene function that are heritable but do not entail a change in DNA sequence.” The epigenetic phenomenon was first observed in Drosophila melanogaster in the 1930s. Since the 1970s, a series of epigenetic markers were found. Subsequently, a large number of epigenetic regulators were identified, leading to gradual understanding of their biological significance. With an analysis of the biological mechanisms of several classical epigenetic phenomena, the focus of epigenetic research evolved to the regulation of chromatin structure on gene expression. At the beginning of the twenty-first century, scientists learned that the epigenetic regulation mechanism, as a common way of gene expression regulation in life phenomena, is one of the important mechanisms of regulating growth, development, aging, and disease. Especially, epigenetic regulation plays a decisive role in stem cell maintenance, self-renewal and differentiation, G. Pei (B) Tongji University, Shanghai, 200000, China e-mail: [email protected] Y. Shang Hangzhou Normal University, Hangzhou, Zhejiang, China F. Sun Tongji University, Shanghai, China Z. Zhu Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China R. Chen Institute of Biophysics, Chinese Academy of Sciences, Beijing, China X. Cao Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Z. Xi Nankai University, Tianjin, China © Zhejiang University Press 2023 G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming, Reports of China’s Basic Research, https://doi.org/10.1007/978-981-19-7419-9_2

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individual aging and dysplasia, development of complex conditions such as cancer, diabetes, mental illness, and nervous system diseases. The orderly responses of individual life to environmental factors (including those of nutrition, physical chemistry, and psychology) largely depend on the effective operation of epigenetic regulation networks. A growing number of epigenetic regulators have become new drug targets for the treatment of cancer, neurodegenerative diseases, and other major diseases. Epigenetic regulation also plays an important role in plant development, resistance, and formation of heterosis. The main epigenetic regulation mechanisms include DNA methylation modifications, histone modifications, histone variants, noncoding RNAs, nucleosome positioning, and higher-order structures of chromatin. Research on cell programming and reprogramming covers the basic scientific questions of epigenetics and studies classic changes in heritable cell traits without alterations in the DNA sequence. In 2006, Japanese scientist Shinya Yamanaka first reported the reprogramming of somatic cells with transcription factors. This achievement promoted the research of somatic reprogramming into a new stage of development, focusing on three main directions: (i) finding new factors and technologies to improve reprogramming efficiency and biosafety; (ii) exploring the regulation mechanisms of programming and reprogramming; (iii) developing new treatments for specific diseases with cell programming and reprogramming technologies. To study the mechanisms of programming and reprogramming and apply them to clinical and regenerative medicine to produce both economic and social benefits, we should explore the epigenetic regulation mechanisms in depth. Before this MRP was launched, great progress had been made in epigenetic research worldwide, but scientists’ understanding of epigenetic mechanisms was still the tip of the iceberg. Many key problems remained unsolved, including selectivity of DNA methyltransferases (DNMTs) acting on target genes, cloning and identification of DNA demethylases, the composition and recognition mechanisms of various histone modifications, interaction of higher-order structures of chromatin with epigenetic information, participation of noncoding RNA in the epigenetic regulation, molecular mechanisms of the epigenetic plasticity and cell reprogramming, relationships between epigenetic regulation and environment, disease, aging, and the features of the composition, origins, and evolution of epigenetic regulatory networks. Since epigenetic phenomena were discovered at the beginning of the twentieth century, the research themed by epigenetics had almost covered all aspects of growth and development of organisms, individual health, and environmental response. As time goes, the research methods and the understanding are constantly improving, and the research focus of epigenetics is also gradually changing. From the discovery of classic epigenetic phenomena, to identification of numerous epigenetic modifiers and regulators, to comprehensive epigenomics, the research on the epigenetic regulation mechanisms remains a hot area of life sciences, and also one of the most active and path-breaking fields.

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2.1 Introduction of Epigenetic Research Plans In April 2003, the Human Genome Project was declared complete. The National Human Genome Research Institute (NHGRI) of the USA launched the Encyclopedia of DNA Elements (ENCODE) project in September of the same year, which aimed to analyze and identify all functional elements encoded in the human genome. The project is now in the fourth phase. Its first phase of pilot and the second phase of technology development were carried out simultaneously (2003–2007). There were 8 research teams participating in the pilot phase, 12 teams in the phase of technology development, and some teams involved in the comparison, calculation, and analysis of sequencing data. The total investment of the two phases were about 55 million dollars. In 2007, ENCODE published 29 papers in Nature and Genome Research to report its achievements in the phases. In the third phase of production (2007–2017), the project scaled and globalized the research, and established the data integration center and data analysis center, with an investment of about 130 million dollars. In September 2012, the findings of this phase were reported in 30 papers in Nature, Genome Biology, and Genome Research. Starting in February 2017, its Phase IV funded the first batch of 19 research programs and 2 data centers, with a total investment of 33 million dollars. The National Institutes of Health (NIH) of the USA launched the Roadmap Epigenomics Project in 2007. It had two goals: (i) to develop comprehensive reference epigenome maps, i.e., the structure and organization of epigenome; (ii) to develop new technologies for comprehensive epigenomic analyses, i.e., to deeply understand the functions and significance of epigenome and to discover new epigenome components. Five initiatives of the project were implemented: (i) 10 million dollars per year for building the Reference Epigenome Mapping Center; (ii) 1.5 million dollars per year for each grantee to develop and run the Epigenomics Data Analysis and Coordination Center which would support the Reference Epigenome Mapping Center and be responsible for delivering standardized data to the National Center for Biotechnology (NCBI); (iii) funding the development of new technologies that would revolutionize epigenetic research approaches; (iv) funding the discovery of novel epigenetic marks in mammalian cells; (v) supporting research on epigenomic changes of human health and disease. Based on the Roadmap Epigenomics Project, the NIH also invested 190 million dollars to the research under International Human Epigenome Consortium (IHEC) founded in Paris, France in 2010. In 2015, the NIH announced the launch of the 4D Nucleome Program with the goal of studying the genome conformation and nuclear organization in an interdisciplinary manner and developing improved research approaches with new cutting-edge technologies. The program planned to support the following six aspects: (i) establishment of the Nuclear Organization and Function Interdisciplinary Consortium (NOFIC); (ii) development of new technologies for studying higher-order structures of chromatin and their interactions; (iii) funding studies on subcellular structures; (iv) development of high-throughput, high-resolution, and high-content microscopy

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imaging technologies; (v) establishment of the 4D Nucleome Network Organizational Hub to promote cooperation and source sharing; (vi) establishment of the Data Coordination and Integration Center. It funded the first batch of 29 five-year research projects, with a total investment of 25 million dollars. To conclude, the international epigenetic research programs, represented by those of the USA, focus on the research of big data omics with more descriptive results and the development of new technologies.

2.2 Status Quo of the Epigenetic Research The early epigenetic research focused on the discovery of classic epigenetic phenomena, identification of the relationship between chromatin modifications and gene expression regulation, identification and purification of epigenetic regulators, and studies of the physiological functions of epigenetic regulators with traditional methods of genetics and biochemistry. In the 1960s, there were quite prospective predictions of the functions of various chromatin modifications. Thanks to the development of technologies in genetics and molecular biology since the 1990s, the methods for studying chromatin biology were becoming increasingly mature and diverse. Meanwhile, the role of epigenetic regulators directly regulating gene expression was finally confirmed in eukaryotic cells. In 1996, C. David Allis of Rockefeller University and Stuart Schreiber of Harvard University of the USA respectively cloned and identified a histone acetyltransferase and a deacetylase, and their findings were published in Cell and Science of the same year. The progress led people to focus on the epigenetic modifiers and regulators of various types. The period from the 1990s to the early twenty-first century saw the blowout development of the research on epigenetic modifiers and regulators. It was during the studying of their mechanisms that the epigenetic theories and methods were continuously improved. Recently, with the advances in genomics and high-throughput sequencing technologies, epigenetics, which is at the forefront of the life sciences, has entered a new stage of development. This MRP started in 2008 when the epigenetic research was in full swing internationally, especially the identification of new epigenetic modifiers and regulators. Since the technical means were increasingly mature, the international competition in this field was fierce, with many breakthroughs achieved. Take the research on histone methylation as an example. In 1999, Michael Stallcup of the University of Southern California, the USA, cloned the first histone arginine methyltransferase. In 2000, Thomas Jenuwein of the Research Institute of Molecular Pathology (IMP) in Vienna, Austria, cloned the first histone lysine methyltransferase. In 2001, the teams of Thomas Jenuwein and Tony Kouzarides of Cambridge University, the UK, independently reported the first reader protein binding with histone methylation modification in Nature at the same time. In 2002, various research teams in the USA and Europe analyzed the crystal structures of histone modification reader proteins and catalytic domains of histone methyltransferases. Meanwhile, in the keen world competition in the research area of histone demethylases, Yang Shi of Harvard

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University of the USA discovered the first histone demethylase in 2004; he and Yi Zhang of the University of North Carolina of the USA independently found a new class of histone demethylases (containing the JMJ domain). Subsequently, the studies on histone methylation gradually turned to explore the physiological functions of known factors. In 2006, Shinya Yamanaka of Kyoto University in Japan achieved the reprogramming of somatic cells with transcription factors, which had set off an upsurge internationally and promoted the stem cell research into a new stage. With years of accumulation of the research in genetics and developmental biology, Japan had many achievements in the field of epigenetics and stem cells. In 2009, Anjana Rao of Harvard University at that time and Nathaniel Heintz of the Rockefeller University independently reported in Science of the same issue the discovery of the phenomenon that methylated DNA could be oxidized in mammalian cells. Then, the research on the mechanism of oxidative DNA demethylation started. These achievements, which were attributed to the long-term accumulation and hard work of epigenetic researchers worldwide, led to a world research pattern dominated by the USA, Europe, and Japan. Epigenetic research institutes and centers were springing up, and the main force of research was a large number of institutes and universities, including Johns Hopkins University, Harvard University, the National Cancer Institute, Cold Spring Harbor Laboratory, University of Southern California, University of Virginia, Massachusetts Institute of Technology, the Rockefeller University, New York University, University of California (San Francisco), University of California (Berkeley), University of California (Los Angeles), University of California (San Diego), Stanford University, University of Massachusetts, University of Washington (Seattle), Pennsylvania State University, Washington University in St. Louis, Ohio State University, etc. of the USA; Cambridge University, Oxford University, University of Edinburgh, and John Innes Centre of the UK; Max Planck Institutes of Germany, Curie Institute of France, and Institute of Physical and Chemical Research (RIKEN) of Japan. In these institutions and universities, various important scientists in the field of epigenetics emerged, for example, Shiv Grewal (heterochromatin) from National Cancer Institute, Yi Zhang (histone-modifying enzymes) of Harvard University, Steven Jacobson (DNA methylation in plants) of University of California (Los Angeles), Job Dekker (higher-order structures of chromatin) of University of Massachusetts, Bing Ren (higher-order structures of chromatin) of the University of California (San Diego), Wolf Reik (DNA methylation in mammals) of Cambridge University, Edith Heard (X chromosome inactivation) of Curie Institute, and Hiroyuki Sasaki (genomic imprinting) of Kyushu University of Japan. Before this MRP, China just started the research in epigenetics and kept strengthening its research teams. Nevertheless, some of its research work was recognized by international peers, and relevant results were published in Cell, Nature, and other international journals. The representative work included the study on a G proteincoupled receptor (GPCR) of the adrenaline receptor and epigenetic regulation by Pei Gang’s team of Shanghai Institutes for Biological Sciences of Chinese Academy of Sciences, with the results published in Cell in 2005; the regulation of a histone and epigenetic proteins on higher-order chromatin structures by Sun Fanglin’s team of

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Fig. 2.1 Number of papers on epigenetic research published by major countries and collected in the Web of Science Core Collection during 2009–2016

Tsinghua University, with the results published in Genes & Development in 2006; the regulation mechanism of DNA demethylation on gene silencing by Gong Zhizhong’s team of China Agricultural University, with the results published in Plant Cell in 2006 and EMBO Reports in 2007. On the whole, compared with the international counterparts, the epigenetic research in China started later with smaller team size. In terms of the number of papers, there was a total of 12,542 articles and reviews in epigenetics from 2000 to 2008 that were collected in the Web of Science Core Collection. Half of them were published by researchers from the USA (46.4%), while fewer than 5% were from China, ranking the 7th internationally. Since this MRP was launched, the epigenetic research in China has saw rapid progress. From 2009 to 2016, 6,728 articles and reviews were published, accounting for 13.1% of the related papers in the world (Fig. 2.1) and exceeding the UK in 2014. From 2014 to 2016, the number of the papers on epigenetics published in academic journals by the researchers in China was second only to the USA (Fig. 2.2). With support from this MRP, our scientists have made several major achievements and published a series of original papers in the world’s top journals, which has won unanimous praise at home and abroad and has produced great international influences. Eight years after the launch of this MRP, in the field of epigenetics, China has surpassed Japan in terms of paper quality and quantity and talent building, and has become another epigenetic research center on a par with the USA and Europe.

2.3 Development Trends of the Epigenetic Research The current development trends of the epigenetic research could be summarized as follows.

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Fig. 2.2 Number of papers on epigenetic research published by major countries and collected in the Web of Science Core Collection during 2004–2016

2.3.1 Mechanisms of Epigenetic Regulation of Gene Expression This area focuses on the interactions and regulation between epigenetic modifiers and regulators, and the regulation modes (excluding enzymatic reactions) of epigenetic modifiers on gene expression. It also pays attention to the mechanisms that cells respond to changes in intracellular and extracellular environment via epigenetic regulation. At present, the catalytic processes and molecular mechanisms of most epigenetic modifying enzymes are basically clear. Further studies on the regulation mechanisms of epigenetic factors help to understand why gene expression is highly spatiotemporally specific and why its roles present complex dynamic changes.

2.3.2 Discovery, Identification, and Function Studies of New Epigenetic Modifications, Modifying Enzymes, and Reader Proteins This area focuses on the interdisciplinary studies of biochemistry and structural biology to analyze the functions and mechanisms of epigenetic modifying enzymes and modification reader proteins.

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2.3.3 Mechanisms of Establishment and Maintenance of Epigenetic Information This area focuses on the interdisciplinary research with developmental biology and systems biology to study the carriers and mechanisms of the transgenerational inheritance of epigenetic information in different model organisms. It emphasizes distinguishing the contribution of different elements to the establishment and maintenance of epigenetic information and their regulation mechanisms, and studying the molecular mechanisms of the establishment and maintenance of spatiotemporally specific epigenetic information in cells.

2.3.4 Epigenetic Regulation on Reproduction and Development This area focuses on the mechanisms of establishing and maintaining epigenetic modifications and their dynamic changes during these processes, as well as the molecular mechanisms of epigenetic factors integrating signal transduction pathways and regulating cell differentiation.

2.3.5 Molecular Mechanisms of Epigenetic Regulation on Cell Programming and Reprogramming This area focuses on the interdisciplinary research with genetics and bioinformatics to study, from the perspective of systems biology, the dynamic changes and regulation mechanisms of chromatin modifications and higher-order structures during cell fate changes, as well as the mechanisms of the heterogeneity of cell changes during these processes. It also emphasizes improving the efficiency of cell reprogramming in vitro with regulations on epigenetic factors.

2.3.6 Higher-Order Chromatin Structures and Subnuclear Structures This area focuses on the interdisciplinary research with physics and computational biology, and especially, the development of new technologies to study the composition and dynamic changes of higher-order chromatin structures. It also emphasizes the research of general principles for the composition of higher-order chromatin structures.

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2.3.7 Origins and Evolution of Epigenetic Regulatory Networks This area focuses on the interdisciplinary research with bioinformatics to establish the epigenetic maps during early embryonic development of different species and study the rules of the inheritance and evolution of epigenetic information.

2.3.8 Cancer, Neurodegenerative Diseases, and Other Major Diseases Related to Epigenetic Regulation This area focuses on the regulatory mechanisms of epigenetic factors in disease occurrence, development, and deterioration. It also emphasizes the interdisciplinary research with chemical biology to develop potential targeted drugs of small-molecule compounds on epigenetic factors.

Chapter 3

Major Research Achievements Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen, Xiaofeng Cao, and Zhen Xi

The successful implementation of this MRP has promoted the overall improvement and leapfrog development of the epigenetic research in China. A series of breakthroughs with great international influences have been achieved in discovering new epigenetic regulators and chromatin remodeling factors and revealing their biological functions and mechanisms; analyzing the regulation mechanisms for stem cell self-renewal and somatic cell reprogramming and developing new cloning methods; revealing the rules for epigenetic regulation of cell differentiation and transdifferentiation, ontogeny, and disease occurrence and development; and understanding the composition and operation of epigenetic networks at the whole genome level.

G. Pei (B) Tongji University, Shanghai, 200000, China e-mail: [email protected] Y. Shang Hangzhou Normal University, Hangzhou, Zhejiang, China F. Sun Tongji University, Shanghai, China Z. Zhu Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China R. Chen Institute of Biophysics, Chinese Academy of Sciences, Beijing, China X. Cao Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Z. Xi Nankai University, Tianjin, China © Zhejiang University Press 2023 G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming, Reports of China’s Basic Research, https://doi.org/10.1007/978-981-19-7419-9_3

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3.1 New Epigenetic Regulators and Chromatin Remodeling Factors as Well as Their Biological Functions and Mechanisms Epigenetic regulation is a common way of gene expression regulation in life phenomena. Its important means include DNA methylation modification, histone modification, nucleosome assembly, establishment and recognition of the histone code, as well as formation, maintenance, and transformation of higher-order chromatin structures. Therefore, this MRP has supported the research directions of discovering and identifying the new molecules for DNA methylation modification and histone modifications, exploring the mechanisms for nucleosome assembly and the composition, recognition, and functions of the histone code, studying the regulation mechanisms and functions of higher-order chromatin structures and the transformation between heterochromatin and euchromatin, and clarifying the roles of important signal transduction pathways in the establishment and maintenance of epigenetic information. It has also actively encouraged structural biology research and achieved a series of breakthroughs.

3.1.1 New Mechanisms of Interactive Regulation Between DNA Methylation and Histone Methylation Modifications DNA methylation can cause changes in chromatin structure, DNA conformation, DNA stability, and interactions between DNA and proteins, to control gene expression. The enzymes involved in DNA methylation are mainly DNMT1, DNMT3A, and DNMT3B. Previous studies have established a connection between DNA methylation and histone modifications and revealed a histone-guided mechanism for the establishment of DNA methylation. The ATRX-DNMT3-DNMT3L (ADD) domain of DNMT3A recognizes unmethylated histone H3 (H3K4me0). The histone H3 tail stimulates the enzymatic activity of DNMT3A in vitro, whereas the molecular mechanism remains elusive. With the support of this MRP, our scientists have determined the crystal structures of DNMT3A-DNMT3L (autoinhibitory form of DNMT3A) and DNMT3ADNMT3L-H3 (active form) complexes at 3.82 and 2.90 Å resolution, respectively. Structural and biochemical analyses indicated that the ADD domain of DNMT3A interacted with and inhibited enzymatic activity of the catalytic domain (CD) through blocking its DNA-binding affinity. Histone H3 (but not H3K4me3) could disrupt ADD-CD interaction, induce a large movement of the ADD domain, and thus release the autoinhibition of DNMT3A. The finding has added another layer of regulation of DNA methylation to ensure that DNMT3A is mainly activated at proper targeting loci when unmethylated H3K4 is present, and has strongly supported a negative correlation between H3K4me3 and DNA methylation across the mammalian genome (Guo

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et al. 2015). This study has provided a new insight into an unexpected autoinhibition and histone H3-induced activation of the de novo DNA methyltransferase after its initial genomic positioning, and thus has been recommended by the professional academic review website F1000. Another study has found the interactive mechanism of methylated H3K9 and methylated DNA (Liu et al. 2013), and put forward a new model for maintaining DNA methylation: UHRF1 could bind to DNA replication forks more effectively through the cooperative binding of hemi-methylated DNA and methylated histone, and thus recruited DNMT1 for the maintenance of DNA methylation. In the mouse model, the study further demonstrated that UHRF1-mediated cross-talk between histone modification and DNA methylation via recognizing H3K9 methylation. It also indicated that H3K9 methylation plays an auxiliary rather than decisive role in DNA methylation in mammalian cells. This work has shown that the interaction between histone modification and DNA methylation in different species differs in the degree and the mechanism (Zhao et al. 2016a). Meanwhile, our scientists have discovered the negative regulation of de novo DNA methylation by UHRF1/2, and proposed that UHRF1/2 promote DNA methylation maintenance and inhibit de novo DNA methylation, to support the hypothesis of cellular DNA methylation homeostasis (Jia et al. 2016). Additionally, another study has found that hemi-methylated DNA (formed after DNA replication) binds itself to epigenetic regulator UHRF1 and opens its closed conformation to facilitate its recognition of histone modification, which ensures UHRF1 to precisely locate itself in specific regions of the genome and play a role in maintaining DNA methylation (Fang et al. 2016).

3.1.2 New Regulation Mechanisms of DNA Methylase and Demethylase Activities In recent years, light-controlled proteins have become a powerful tool for studying the spatiotemporal regulation of cellular signal transduction. Compared with ultravioletactivated probes, two-photon-activatable probes can significantly reduce cytotoxity and thus have a broad application prospect. Our scientists utilized protein chemical synthesis as a core technique and developed light-controlled protein probes targeting immune cells. With these probes, they studied the directional migration of immune cells under precise spatiotemporal stimulation and the activation mechanism. Through the development of two-photon-controlled chemokine probes, they and their collaborators explored the mechanism of directional migration of immune cells in living tissues and designed and synthesized the first two-photon-activatable chemokine probe hCCL5** . With its high spatiotemporal resolution, they showed at the single-cell level that “T cells perceive the directional cue without relying on PI3K activities, which are nonetheless required for persistent migration” (Chen et al. 2015b). Additionally, utilizing the good tissue penetration of the two-photon technique, they and their collaborators realized the directed migration of immune

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cells in living tissues (ears and lymph nodes of mice). This probe is an ideal molecular tool for understanding and potentially manipulating cell positioning and related cell biology. Based on the developed light-controlled protein antigens, they revealed the early signal transduction dynamics of B cell activation. HEL-K96NPE, the first light-controlled protein antigen for B cell, was developed with light-controlled amino acid screening and protein chemical synthesis. They found that for hen egg lysozyme (HEL) and its antibody HyHEL-10 (or B cells from MD4 transgenic mice expressing HyHEL-10), masking a single site (Lys96 of HEL) was sufficient to block the interaction as high as 20 pm with a large interaction surface (1800 Å2 ). Combined with the high-speed high-resolution live cell imaging, HEL-K96NPE was used to monitor the early formation of B cell synapse and the periodic responses of calcium influx in a real-time manner. This probe is a promising tool for in-depth studies on the early signal transduction dynamics of B cell activation. 5-methylcytosine (5mC), often called “the 5th base,” is a methylated cytosine base in mammalian genome. TET protein is an oxidase in mammalian cells, which can function in DNA demethylation. The mammalian TET plays a vital role in critical life processes, such as epigenetic reprogramming of fertilized eggs, pluripotent stem cell differentiation, and medullary hematopoiesis, and its inactivation is also closely related to multiple diseases, especially hematological tumors. The research on the DNA demethylation centered on TET is one of the most active areas in epigenetics. Previous studies have shown that during the demethylation, TET oxidizes 5mC into 5-hydroxymethylcytosine (5hmC, the 6th base) and continues to catalyze it into 5-formylcytosine (5fC, the 7th base) and 5-carboxylcytosine (5caC, the 8th base). 5hmC is relatively stable in cells, and its content is much higher than that of 5fC and 5caC. There remained no reasonable biological explanation for this phenomenon. Our scientists have uncovered the mystery with research methods of structural biology, biochemistry, computational biology, and other disciplines. Structural analyses indicated that the orientation of 5mC in the TET catalytic cavity made it prone to be captured by the catalytic domain and oxidized into 5hmC. Due to the existence of oxygen, 5hmC and 5fC were restrained within the catalytic cavity and were less prone to further oxidation, resulting in the decrease of TET activity to these two bases. With such a difference in catalytic efficiency, it was easy for TET to oxidize 5mC into 5hmC. After the generation of 5hmC, however, it was not easy for TET to further oxidize it into 5fC or 5caC. As a result, cellular 5hmC was relatively stable and significantly more prevalent than 5fC/5caC. The study has demonstrated the mechanism of substrate preference of TET and provided an explanation for the stable existence of 5hmc in the genome at the molecular level. In specific gene domains, TETs might be activated by specific regulators to produce TETs with high activity to iteratively oxidize 5hmC into 5fC and 5caC. This discovery has solved a conundrum in epigenetics and also provided a new idea and method for revealing the molecular mechanisms of the stepwise catalysis of other proteins (Hu et al. 2015). Our scientists have found that the zinc finger protein SALL4A preferentially binds itself to 5hmC-modified DNA. The Sall4a gene is important during the early embryonic development, and its mutation leads to the autosomal dominant condition of Duane radial ray syndrome. The Sall4a knockout mouse embryos stop developing

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during the peri-implantation and die soon. The study found that in the mouse embryonic stem cells, the majority of SALL4A localized at enhancers and its chromatin association largely depended on TET1. Further analyses on the cytosine modification at the Sall4a-binding sites in the genome indicated that these sites were depleted of stable 5hmC, but were enriched for the further oxidized products of 5fC and 5caC, suggesting a possible role of Sall4a in facilitating further oxidation of 5hmC. As expected, the researchers found that knockout of Sall4a resulted in elevated 5hmC at the original Sall4a-binding sites, and reduced the stable binding of TET2 which is not conducive to the further oxidation of 5hmC (Xiong et al. 2016a). This study has enriched the understanding of DNA oxidation and demethylation regulated by TET family proteins and proposed the concept of the cooperative stepwise oxidation of 5mC: SALL4A does not function as a general 5hmC-binding protein like the role of MeCP2 for 5mC; instead, it is a cell type and genomic region-specific regulator of 5hmC; it recruits TET2 at specific sites and facilitates further oxidation of 5hmC into 5fC and 5caC to fine-tune gene expression regulation. This discovery has promoted our understanding of the dynamics of DNA methylation and its functional roles in embryonic stem cells and cell reprogramming. It has been introduced and recommended on F1000. Previous studies have shown that USP7 binds itself to DNMT1 and regulates DNMT1 stability through acetylation and ubiquitination. However, the molecular mechanism for USP7-mediated stabilization of DNMT1 remains largely unknown. To answer this question, our scientists determined the crystal structure of human DNMT1 in complex with USP7 at 2.9 Å resolution. Structural and biochemical analyses revealed that the interaction between the two proteins was primarily mediated by the KG linker of DNMT1 and a previously uncharacterized acidic pocket that acted as a substrate-binding site near the C-terminus of USP7. Mutations of these acidic residues disrupted the interaction between DNMT1 and USP7, leading to increased turnover of DNMT1. Acetylation of lysine residues of the KG linker impaired the DNMT1-USP7 interaction and promoted proteasomal degradation of DNMT1. Treatment with histone deacetylase (HDAC) inhibitors resulted in increased acetylated DNMT1 and decreased total DNMT1 protein. This negative correlation was observed in differentiated neuronal cells and pancreatic cancer cells. These findings have revealed that USP7-mediated stabilization of DNMT1 is regulated by acetylation and provided a structural basis for the design of inhibitors by targeting the DNMT1-USP7 interaction surface (Cheng et al. 2015).

3.1.3 New Histone Modification Codes and the Interpretation Mechanisms Histone modifications, one of basic mechanisms of epigenetic regulation, are considered to constitute a type of “histone codes.” They regulate the interpretation of genetic information at the chromatin level and play an important role in the processes such

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as gene expression and cell fate determination. In recent years, many new histone modifications have been found, one of which is histone lysine acylation, such as acetylation (ac), propionylation (pr), butyrylation (bu), and crotonylation (cr). There have been various studies on the histone acetylation, whereas the histone crotonylation is a type of newly discovered modification codes conserved from yeast to human. It is closely associated with active transcription and gene activation, and regulates the biological processes such as gene expression and gamete maturation. Since the histone crotonylation was reported by Zhao Yingming’s team of the University of Chicago in 2011 (Tan et al. 2011), the research on its generation, elimination, and identification mechanisms has become a hotspot. The opportunity and challenge that follows is to discover the specific recognition reader of crotonylation as a direct interpreter of the “histone code.” With the support of this MRP, our scientists have discovered new histone crotonylation readers. They published three high-quality papers (Xiong et al. 2016b; Li et al. 2016c; Zhao et al. 2016a) and reported two such readers—YEATS and DPF domains. According to the structural and functional analyses of the YEATS domains of the epigenetic regulators AF9 and YEATS2 and the DPF domains of MOZ, both domains have been found to be histone crotonylation readers with preference. These studies have elucidated the molecular and cellular mechanisms that these domains promote gene transcription by specifically reading the histone crotonylation codes. Notably, the important work of YEATS mentioned above has been specially emphasized in three heavyweight reviews. They believe that this discovery, as a breakthrough in the area of histone modification regulation, has opened a new direction of metabolic and epigenetic research and deepened our biological understanding of crotonylation modification. The histone methylation is another important type of codes. Our scientists have discovered and demonstrated that KIAA1718 (KDM7A) is a dual-specificity histone demethylase for both H3K9 and H3K27 (Huang et al. 2010). The studies on the histone demethylase ceKDM7A from Caenorhabditis elegans found that it binds itself to H3K4me3 to demethylate H3K9me2 and H3K27me2, and demonstrated the mechanism that the methylation associated with transcription repression (H3K9me2 and H3K27me2) and the methylation associated with transcription activation (H3K4me3) distribute in a mutually exclusive manner (Lin et al. 2010; Yang et al. 2010). Six co-crystal structures of ceKDM7A with peptides containing different histone methylation modifications have been completed to explain its specificity and substrate recognition mechanisms from the perspective of structure. Our researchers have found that PHF8 is a histone H3K9 demethylase locating in nucleolar region and regulating rRNA transcription (Zhu et al. 2010). The related findings were published in Cell Research and recommended as a featured article on the cover. It was commented by experts in the same issue and won “Sanofy-Cell Research Outstanding Paper Award of 2010.” Additionally, the 20 different histone lysine demethylases that have been identified so far can only eliminate the methylation modifications at the 4 lysine sites of H3K4, H3K9, H3K27, and H3K36. There are many important lysine methylation sites in histone, including H3K79 and H4K20, and their demethylases are waiting

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to be discovered. Also, the arginine demethylases have not been found yet. With the support of this MRP, our scientists have established a system for the genomewide screening of novel histone demethylases and their methylation regulation, and utilized it to discover the new demethylase KDM9A/9B of H4K20. This family is a new histone lysine demethylase that has no conservatism with LSD and JmjC families. KDM9A/9B could regulate the methylation of H4K20 at its binding domain, activate gene transcription by removing H4K20me1 methylation, and activate the transcription of repetitive sequences by removing H4K20me3 methylation. Related research is being carried out.

3.1.4 Higher-Order Structure of 30-Nm Chromatin Fibers and Their Dynamic Regulation Mechanisms Using the in vitro self-developed chromatin assembly technology and cryogenic electron microscopy, our scientists have, for the first time in the world, analyzed the high-resolution cryogenic electron microscopy structures (11 Å) of 30-nm chromatin fibers. The structures show a left-handed double-helical twist of the repeating tetranucleosomal structural units (Fig. 3.1). With the single-molecule magnetic tweezers, the study has explored in depth the dynamic processes of establishing and regulating the 30-nm chromatin fiber structure (Song et al. 2014). The study was specially introduced by Science editors with the title of “Double helix, Double.” In the same issue, it was evaluated in “The as Perspective” by Professor Andrew Travers of Cambridge University in the UK, as the origin of DNA double helix structure model, with the article titled “The 30-nm Fiber Redux.” The findings have been collected in the latest editions of the world-famous biochemistry textbooks Fundamentals of Biochemistry: Life at the Molecular Level and Lehninger Principles of Biochemistry. This paper has been recommended by F1000 as “Exceptional.” The 3D electron microscopic structure of the 30-nm chromatin fibers has been collected in “A Tale of Chromatin and Transcription in 100 Structures” (Cramer 2014) by Dr. Patrick Cramer, Director at the Max Planck Institute for Biophysical Chemistry in Germany. Our scientists have made breakthroughs in solving the major scientific question of the higher-order structure of 30-nm chromatin fibers, making China a global leader in the research area of chromatin structure. The following studies have established and improved the MNase-Seq technique for mapping the genome-wide chromatin structure—gMNaseSeq (a method for analyzing the chromatin structure in nucleus). Through modifications of MNase, the spatial resolution of the MNase-Seq technique was improved. They further demonstrated the dynamic regulation of the 3D structure of 30-nm chromatin fibers. For the first time, they have reported that the “tetranucleosomes-on-astring” appears as an important intermediate structure during the folding of 30-nm chromatin fibers. The histone chaperone FACT negatively regulates the structure of tetranucleosomal unit during the gene transcription (Li et al. 2016a).

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Fig. 3.1 Unraveling the 3D organization of 30-nm chromatin fibers by cryo-electron microscopy (Song et al. 2014)

3.1.5 Mechanisms of Centromeric Chromatin Establishment and Nucleosome Assembly With the support of this MRP, our scientists have systematically studied the mechanisms of recognition and targeting of the histone variant CENP-A during the centromeric chromatin establishment as well as the assembly into nucleosomes. The study analyzed the crystal structure of a centromeric CENP-A-H4 heterodimer in complex with the histone chaperone HJURP, revealed the specific recognition mechanism between CENP-A and HJURP, and discovered that the important residue Ser68 of CENP-A played a critical role in HJURP recognition (Hu et al. 2011). The following study has found that the phosphorylation of CENP-A at Ser68 regulates its specific recognition by HJURP, and orchestrates its dynamic assembly during the cell cycle (Yu et al. 2015). These findings have provided strong evidence for understanding the centromeric nucleosome assembly. According to the comment on F1000, “the results have revealed that HJURP can specifically recognize the serine at site 68 of CENP-A, which will become a focus of related work in the future.” Dr. Patrick Cramer also collected this work in “A Tale of Chromatin and Transcription in 100 Structures.”

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3.1.6 Roles of Histone Variants in Chromatin Assembly The histone variants of H3.1, H3.3, and centromere-specific CenH3 in the histone H3 family remain conserved in both plants and animals (from Drosophila melanogaster to humans). H3.3 and H3.1 of Arabidopsis differ in only 4 amino acids—amino acids 31 and 41 in the N-terminal tail and amino acids 87 and 90 in the core domain. Through the delicate dynamic cell biology analysis of Arabidopsis histone H3.3, H3.1, and their mutants in nucleolar rDNA, our scientists have proposed and verified the model in which amino acids 87 and 90 in the core domain of H3.3 guide nucleosome assembly, whereas amino acids 31 and 41 in the N-terminal tail guide nucleosome disassembly (Shi et al. 2011). To study the molecular mechanisms of the recognition and assembly of the histone variant H3.3, our scientists have analyzed the crystal structure of a DAXX- H3.3H4 complex and revealed the molecular mechanism for the recognition of H3.3 by the complex of the H 3.3-specific chaperone DAXX and HIRA, which has laid the foundation for outlining the storage pathway of H3.3 and understanding its specific recognition and assembly mechanisms. Additionally, the study has discovered that H3.3 and H2A.Z function together to dynamically regulate the chromatin structures over the enhancer and promoter regions, to maintain the self-renewal of stem cells and promote their neural differentiation (Chen et al. 2013).

3.1.7 New Interaction Patterns Between Epigenetic Regulation and DNA-Damage Repair Homologous recombination (HR) is essential for maintaining genome integrity and variability. Before HR, chromatin needs to release DNA, and its structure needs to be reconstructed after HR. The study has shown that depletion of either the nucleosome assembly protein 1 (NAP1) group of H2A/H2B-type histone chaperones or NAP1related protein (NRP) group proteins in Arabidopsis cause a reduction in HR in plants. The hyperrecombinogenic phenotype caused by the depletion of CAF-1, the H3/H4-type histone chaperones, relied on NRP, but the telomere shortening phenotype did not, suggesting that the dependence was specific in HR. The analysis of DNA lesions, H3K56 acetylation, and expression of DNA repair genes argued for a role of NAP1/NRP participating in HR via nucleosome disassembly/reassembly. For the first time, the study has established a crucial function for NAP1 family of H2A/H2B-type histone chaperones in somatic HR in eukaryotes (Gao et al. 2012). Histone demethylase KDM5B, a member of the JmjC domain-containing histone demethylases, becomes enriched in DNA-damage sites due to its poly-ribosylation and recognition of histone variants. It changes the structures and states of the chromatin in the damaged regions with its demethylase activity and recruits Ku70 and BRCA1, the essential components of nonhomologous end-joining and homologous

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recombination, respectively, to regulate double-strand DNA-damage repair to maintain the genome stability. The results help to analyze the epigenetic roles in the maintenance of genetic fidelity and to understand the genomic instability related diseases, such as cancer (Li et al. 2014a).

3.2 Discovery of Epigenetic Regulation Mechanisms for Stem Cell Self-renewal and Somatic Cell Reprogramming, and Breakthroughs in Animal Cloning and Reproductive Technologies Somatic cell reprogramming remains one of the hotspots in life sciences. It can be realized through somatic cell nuclear transfer. In 2006, Japanese scientist Shinya Yamanaka reported to induce somatic cells into pluripotent stem cells, similar to embryonic stem cells, by introducing four transcription factors, Oct3/4, Sox2, cMyc, and Klf4. The establishment and discovery of the induced pluripotent stem cells (iPSCs) has not only created a new method for somatic cell reprogramming, but also a milestone in the history of life sciences. Somatic cell reprogramming plays an important role in regenerative medicine and the development and utilization of drugs, as well as in the treatment of various genetic and functional diseases of human beings. After this MRP was launched, the Advisory Expert Group took stock and believed that somatic cell reprogramming would become a hotspot in the future frontier research, and provided strong support for the research in this area. As a result, China has made great achievements in the research of animal cloning, reproductive technology, and somatic reprogramming mechanisms in a short time and become a world leader in this research area.

3.2.1 Establishment of ESCs and Semi-cloning Technologies to Make “Artificial Spermatids” The haploid stem cell is a new type of artificial cells. With the support of this MRP, our scientists have made a series of important progress in the establishment and application of mammalian haploid stem cells. They have, for the first time, established the rat and mouse androgenetic haploid embryonic stem cell lines that can replace sperms to complete the reproductive process and the monkey parthenogenetic haploid embryonic stem cell lines; developed the genetic screening and modification technologies based on the haploid embryonic stem cells (ESCs); applied the haploid stem cells as a new technology and tool to the research of reproductive and developmental biology which produced the offspring of two female mice; and for the first time created a new type of cells—mammalian interspecific hybrid allodiploid stem cells. Their achievements have enriched the theories and systems of the reproductive and

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developmental research, expanded the research scope, and presented the important application prospects of haploid stem cells for reproductive biology, developmental biology, genetics, and evolutionary biology. The studies have been published in Nature, Cell, Cell Stem Cell, and other journals. The number of the papers published by China in high-impact journals has exceeded the sum of those published by other countries in the world, suggesting that China is in a leading position in the haploid stem cell technologies and relevant research fields. (1) Establishment of mammalian haploid stem cell lines to study their features and functions With the nuclear transfer technique, our scientists have established the androgenetic haploid embryonic stem (ahES) cell lines by transferring a sperm into an enucleated oocyte to generate androgenetic haploid embryos. The cells possessed the ability to differentiate into three layers of epiblasts and germ cells. After injecting into the cytoplasm of M II phase oocytes, they could replace a sperm to fertilize an egg. The embryo could further develop into a healthy and fertile organism—a “semi-cloned” mouse. This is called semi-cloned. In the study, a neomycin-resistant gene neor, controlled by the PGK promoter, was electroporated into the ahES cell line AHGFP-4, and the transgenic macrohaploid stem cell lines were established after the G418 drug selection. Three transgenic cell lines were selected for the intracytoplasmic ahES-cell injection (ICAI). They found that all the three lines could get due fetuses, and 7 and 1 healthy alive transgenic animals were obtained from 2 cell lines, respectively. The study has provided a new model for studying the basic questions such as reproduction and genomic imprinting, a new method for obtaining transgenic animals, and a new idea for the development of assisted reproductive technology. The findings were published in Nature in 2012 (Li et al. 2012a), and selected as one of the Top 10 Scientific Advances in China of the same year. Another study has established the haploid epiblast stem cells (hEpiSCs) to provide a new tool for the genetic screening of haploid stem cells and the research on the developmental mechanisms such as diploid maintenance (Shuai et al. 2015). (2) Reproductive and developmental studies with haploid stem cells The mammalian interspecific hybrid allodiploid embryonic stem cells (AdESCs) have been generated with haploid ESCs. Our scientists have reported the generation of mouse–rat hybrid AdESCs by fusing androgenetic and parthenogenetic haploid ESCs of the two species, which avoids the reproductive isolation barrier that the fusion of the sperm and egg of mice with rats’ cannot develop (Li et al. 2016b). The AdESCs have the ability to differentiate into all three germ layers as well as early stage germ cells while maintaining a stable allodiploid genome. The gene expression analyses have revealed the unique high-parent or low-parent expression patterns and biological traits in these AdESCs. The analyses on the two types could lead to effective explorations of the molecular regulation mechanisms of the trait differences between species. Additionally, rather than the “random inactivation” model common in mammals, the X chromosomes in the interspecific hybrid cells follow the model of mouse X chromosome-specific inactivation. With this feature,

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the study has systematically identified the mouse X inactivation-escaping genes. This has been the first case of artificially created interspecific hybrid embryonic stem cells in the form of stable diploid, which provides a new model and tool for the research of evolutionary biology, developmental biology, and genetics. The results were published in Cell in 2016. Our scientists have bred healthy fertile mice by haploid fusing cells and demonstrated that the interactions between parental genomes before the blastocyst stage are not necessary for embryo development, which provides a new idea for developmental biology and the development of assisted reproductive technology (Li et al. 2015). Precise imprinting modification on the haploid embryonic stem cells can realize the “homosexual” reproduction (Li et al. 2016c). Imprinted genes are those with different expression patterns on paternal and maternal chromosomes. The haploid stem cells are suitable imprinted gene research platforms because they have genetic materials only from paternal or maternal sources and the advantages of ESCs in gene manipulation. With the parthenogenetic ESCs as the platform, the study had the two paternal methylated imprinting control regions knocked out. By repairing the imprinted gene expression pattern of the region, and injecting the knockout parthenogenetic haploid ESCs into oocytes, the parthenogenetic embryos with two mothers were generated, and parthenogenetic mice could be produced in relatively high efficiency. The study has provided a new way for mammalian parthenogenesis, a new idea for animal reproduction and breeding research, and also a new research method for the exploration of imprinting abnormalities and treatment methods. (3) Genetic screening and modifications with the haploid stem cell technology The “fertilization” capacity of the haploid cells gradually loses with the passage of cells. Especially, after gene editing, it is difficult to obtain healthy semi-cloned mice by injecting them into the oocytes. With the support of this MRP, our scientists have shown that the spermatid-like haploid cells (artificial spermatids) are produced by deleting H19-DMR and IG-DMR regulating two paternally imprinted genes, and they are proved to be able to efficiently support the generation of genetically modified semi-cloned mice. Importantly, these artificial spermatids carrying a CRISPR-Cas9 library can further produce various mice with different mutations, which provides technology supports for organism-wide genetic screening and modifications in mice (Zhong et al. 2015). It has been further demonstrated that the haploid cells from the oocytes, after deleting H19-DMR and IG-DMR, also have the capacity of the artificial sperm, which has realized the effective parthenogenetic development in mammals. Additionally, the parthenogenetic haploid ESCs from human oocytes have been generated (Zhong et al. 2016). In haploid stem cells, CRISPR-Cas9 can be used for efficient treatment of genetic diseases. Generally, the direct injection of the CRISPR-Cas9 system into zygotes with the genetic defect of cataracts for gene editing could cure such defect in mice. However, this method had two problems: a low rate of the newborn mice being cured (about 30%) and a small amount of off-target effects (Wu et al. 2013). To solve the problems, our scientists produced spermatogonial stem cells (SSCs) carrying the homozygous mutant gene from the testis of the cataract mice and established a series

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of cell lines from single SSC expansion after CRISPR-Cas9-mediated correction of the genetic defect. After further analyses, they then transferred the SSCs from the cell lines carrying the corrected gene without off-target mutations and maintaining normal epigenetic traits into recipient testes to produce healthy mice. Their findings have provided a new idea for human gene therapy (Wu et al. 2015). When the paper was published, it was released globally in Cell and got 7 comments or special reports in Cell Stem Cell, Nature, Nature Review Genetics, Addgene Blog, Sci Bull, MIT Technology Review, and on F1000. It is believed that this study has demonstrated that “the CRISPR-Cas9 technology can be used for treating human genetic diseases,” starting a craze of gene therapies. Both papers have been ranked in the world’s top 1% in the Percentiles for Papers Published by Field, 2006–2016 of Essential Science Indicators (ESI).

3.2.2 First Utilization of the Polar Body Genome Transfer for Preventing the Transmission of Inherited Mitochondrial Diseases Our scientists have used polar bodies as donor genomes and placed them in the cytoplasm of healthy donor oocytes, to realize the genome transfer—namely mitochondrial replacement (Wang et al. 2014b). This study has developed the cell organelle replacement technology in addition to the stem cell therapy, providing a new strategy and path for treating refractory diseases. The study was published in Cell and has been introduced in the articles in Nature and Nature Review Genetics, titled “Assisted Reproductive Technologies to Prevent Human Mitochondrial Disease Transmission” and “Clinical Genetics: Mitochondrial Replacement Techniques under the Spotlight” respectively, stating that the invention “is important to prove the feasibility of polar body transfer and significantly improve the efficiency of mitochondrial transfer therapy.” Dr. Herman, President of American College of Medical Genetics and Genomics (ACMG), has praised the study, believing that it has provided interesting hypothesis, new discoveries, and advanced intervention means for mitochondrial disease intervention. The Human Fertilization and Embryology Authority (HFEA) of the UK released a 45-page Review of the Safety and Efficacy of Polar Body Transfer to Avoid Mitochondrial Disease as a reference for the British public and parliament to discuss the amendment of the law to allow “mitochondrial DNA replacement.” According to the review, the polar body transfer (PBT) techniques invented by Chinese scientists to prevent mitochondrial diseases might offer five advantages over maternal spindle transfer (MST) and pronuclear transfer (PNT) techniques because they: may reduce mtDNA carryover; reduce the risk, when compared to MST, of leaving chromosomes behind (as these are all packaged within the polar body); avoid the need to use cytoskeletal inhibitors to allow removal of the spindle or pronuclei from the patient’s oocyte or zygote; involve the use of more conventional micromanipulation procedures, which will reduce the chance of damaging the patient’s

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karyoplast or the donor’s oocyte/embryo and therefore lead to greater efficiency; raise the possibility of carrying out both PB1T and MST, or PB2T and PNT, to double the chances of success for each patient cycle. In the past, China’s research and development of the mitochondrial replacement techniques, an area of high biological technology with international competition, was nearly blank. The invention of the polar body transfer for mitochondrial replacement has made us a global leader in this research area and contributed to technological innovation. This research has won the second prize of National Science and Technology Award of China and been selected as one of the Top 10 Scientific Advances in China 2014.

3.2.3 The Seesaw Model of Lineage Specifiers Inducing Somatic Cells to iPSC The traditional induced pluripotent stem cell (iPSC) technology is to establish pluripotency by introducing pluripotency-related genes into somatic cells. Our scientists have found that lineage specifiers can substitute for pluripotency-associated factors to induce somatic cells into iPSCs. Based on the new discovery, a “seesaw” model has been proposed to explain the theory of cell fate determination in a new prospective. Pluripotency is maintained as a consequence of the balance of different lineage-specifying forces. There might be a universal principle to induce reprogramming by balancing such forces. For the first time, our scientists have reported that all six members of the GATA transcription factor family, as lineage specifiers, could substitute for OCT4, a Yamanaka factor. Additionally, they have found that all members of the GATA family could inhibit the expression of the ectodermal-lineage genes, which is consistent with the “seesaw” model. They then have determined that the pluripotency-related gene Sall4 serves as a bridge linking the GATA transcription factor to the pluripotency circuit (Shu et al. 2015). The dynamic “seesaw” model has been found in the reprogramming of somatic cells by small-molecular compounds into chemically-induced pluripotent stem cells (CiPSCs). Different from traditional transcription factor-induced reprogramming, at the early stage of reprogramming, small molecules start the XEN-related gene expression to tilt the “seesaw” and make the cells into XEN-like states; at the later stage of reprogramming, the expression of Sox2 is induced by switching to the mice ESC culture conditions to restore the tilted “seesaw” to the balance position, so as to make cell transitions to pluripotency (Zhao et al. 2015b). These results have indicated that the seesaw model is a universal model to change cell fate (Fig. 3.2).

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Fig. 3.2 Seesaw model for induction of pluripotency in mouse somatic cells with lineage specifiers (Shu et al. 2013)

3.2.4 Safety Evaluation of Traditional Methods of iPSC Induction by Yamanaka Factors Previous studies on somatic cell nuclear transfer (SCNT) have shown that somatic cells could be reprogrammed and mice generated by SCNT can be successfully re-cloned to more than 25 generations through serial nuclear transfer technology. However, for the iPSC induction, another classic method of the somatic cell reprogramming, the number of generations of all-iPSC mice that can be serially produced using this inducible iPSC system has not been determined. Additionally, since the introduction of the iPSC technology, scientists have been making comprehensive evaluations on the safety and feasibility of its clinical application, and assessing whether there are gene mutations and whether it has an impact on the development potential. This affects not only the cell quality but also the safety of future applications. For the first time in the world, our scientists have used a Tet-on system to establish the OSKM iPSCs for up to six generations. They found that the viability of the iPSC mice decreased with increasing generations, mainly because mutations accumulated throughout the sequential reprogramming process. This finding has further suggested that iPSC induced by traditional methods may have certain risks. The results were published in Nature Communications in 2015 and cited by many papers in Cell Stem Cell and other journals.

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3.2.5 Exploration of the Regulation Mechanisms of Somatic Cell Reprogramming and Discovery of New Regulators Despite the wide use of the method of Yamanaka factor-induced iPSC, we know little about the dynamic binding models of Yamanaka factors in the reprogrammed cells and the mechanisms of their bindings to regulate transcriptome changes and determine cell transitions. With the support of this MRP, our scientists have carried out systematic research on the core pluripotency factor Oct4 with the secondary reprogramming system and high-throughput sequencing technologies. They have demonstrated that at different stages of the somatic cell reprogramming, Oct4 binds itself to the genome in a hierarchical fashion with primed epigenetic modifications; the Oct4 binding plays an important role in the hierarchical activation of the pluripotency circuitry during the reprogramming (Chen et al. 2016a). Additionally, pluripotency factors, such as Oct4, have been found to bind themselves to ESC-specific promoters and make ubiquitously expressed genes express stem-cell-specific transcripts (Feng et al. 2016). Our scientists have discovered that N(6)-methyladenosine (m6A) RNA methylation is regulated by microRNAs (miRNAs) and promotes reprogramming to pluripotency. More than 100 types of modifications have been identified in eukaryotic RNAs so far, among which m6A RNA methylation is one of the most prevalent modifications of messenger RNAs (mRNAs) in higher-level organisms. The m6A modification participates in the regulation of splicing, transportation, stability, and translation efficiency of mRNAs, and it is implicated in obesity, cancer, and other abnormal physiological functions and human diseases. Our scientists have reported the m6A modification profiles in the mRNA transcriptomes of mouse ESCs, iPSCs, neural stem cells (NSCs), and testicular sertoli cells (SCs), and identified the difference in m6A distribution between pluripotent and differentiated cell types. Bioinformatic analyses indicated that the m6A-enriched signature sequences preferred to complementarily pair with microRNA sequences. Multi-level experiments of cell and molecular biology demonstrated that microRNAs regulate m6A modification in the corresponding sites in mRNAs via a sequence pairing mechanism. Increased m6A modifications enhanced the expressions of Oct4 and other key pluripotent regulatory genes, to promote the reprogramming of mouse fibroblasts to pluripotent stem cells (Chen et al. 2015a). The results have revealed a new mechanism of microRNA regulating mRNA methylation via sequence pairing and discovered the important role of m6A modification, which promotes the reprogramming of somatic cells to pluripotent stem cells. The study has made groundbreaking achievements in analyzing the site selection mechanism for m6A modification, expanding new functions of microRNAs, and discovering new regulators of the cell reprogramming. It has been published as a cover article in Cell Stem Cell, and selected as the feature report of the issue, “Stem Cell Highlights from Cell Press,” and one of Abcam Monthly Excellent Epigenetics Papers. Our scientists have discovered that E3 ligase RNF20-mediated H2B ubiquitination regulates chromatin relaxation at the early stage of cell reprogramming, affecting the

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recruitment of gene transcription factors, leading to the initial expressions of pluripotent genes at the early stage of reprogramming. After the depletion of RNF20, the establishment of the pluripotency gene network at the early stage of cell reprogramming failed. Additionally, in iPSC, RNF20 regulated the DNA-damage responses during the replication through H2B ubiquitination. After the depletion of RNF20, the DNA double-strand breaks in iPSC generated during the replication could not be repaired by homologous recombination, resulting in unstable genomes and finally apoptosis. A germ cell-specific knockout of RNF20 resulted in abnormal meiosis and male infertility (Xu et al. 2016).

3.2.6 Discovery of New Signals for Maintaining Stem Cell Self-renewal and Developmental Pluripotency Human embryonic stem cells (hESCs) can self-renew and differentiate into all cell types in vitro and thus have a broad application prospect in organ regeneration and cell replacement therapy. However, the molecular mechanisms of maintaining hESC self-renewal and developmental pluripotency remain poorly understood, preventing safe and effective clinical applications of hESC-differentiated cells. As a result, it is particularly important to delve into the mechanisms how hESCs maintain their own features. Through a genome-wide transcription factor siRNA screen of transcription factors for hESC self-renewal, our scientists have identified a series of genes that play an important role in the maintenance of hESC identity. PHB is found to have a unique role in maintaining the right histone methylation modifications in hESCs, which helps the hESC self-renewal and the reprogramming of human somatic cells. Further analyses indicated that PHB interacted with histone H3.3 chaperone HIRA complexes and stabilized the protein levels of HIRA complex components. Additionally, in hESCs, PHB and HIRA jointly regulated the genome-wide H3.3 enrichment at chromatin, especially the enrichment of H3.3 involved in regulation at the promoters of isocitrate dehydrogenase (IDH) genes and their expressions, thus to control the production of a-ketoglutarate (a-KG), a key metabolite regulating ESC fate, which led to the establishment of correct level of histone methylation and the maintenance of hESC self-renewal and their epigenetic features. Based on the findings, our scientists have proposed an epigenetic-metabolic circuit for the maintenance of hESC identity (Zhu et al., 2017). Histone demethylases play a key role in the establishment and maintenance of developmental pluripotency, but not all of them have been identified, and their functions during the processes remain unclear. Our scientists have revealed that Jmjd1c/Kdm3c, H3K9 demethylases, can inhibit the activation of MAPK/Erk signaling pathway and EMT by regulating the expression of miR-200 and miR290/295 family, and then promote ESC self-renewal and the maintenance of developmental pluripotency. During the somatic cell reprogramming, interference or

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knockout of Jmjd1c expression significantly reduced the reprogramming efficiency, while overexpression of miR-200 family could partially recover the reprogramming efficiency of Jmjd1c-null MEF. This has provided a new perspective for the establishment and maintenance of developmental pluripotency, and laid a theoretical foundation for the application of pluripotent cells in cell therapy and regenerative medicine. BMP signaling, through downstream Smad proteins, upregulates the expression of phosphatase DUSP9 at the transcriptional level to make ERK protein dephosphorylated and deactivated, to sustain low ERK activity, which helps cells to maintain the state of self-renewal and nondifferentiation. Therefore, under the joint efforts of BMP and LIF, ERK activity is maintained at an appropriate level to keep mouse ESCs in self-renewal and undifferentiated state. The results have an important influence on understanding the molecular mechanism of cell fate determination in mouse ESCs and lay a foundation for further research in regenerative medicine (Li et al. 2012b). After the publication, the findings were introduced in Nature Chemical Biology and commented on F1000.

3.3 Epigenetic Mechanisms Related to Cell Differentiation and Transdifferentiation, Ontogeny, and Diseases 3.3.1 Discovery of the Key Factors to Promote the Transdifferentiation of Somatic Cells to Hepatocytes Our scientists have demonstrated that with a lentiviral system, adult mouse tail-tip fibroblasts were successfully induced into functional hepatocyte-like (iHep) ells by overexpression of transcription factors FOXA3, HNFLA, and GATA4, and inactivation of p19. They discovered that the activation of p53 was the key inhibitory mechanism of dedifferentiation reprogramming (Fig. 3.3). iHep cells could be integrated into the mouse liver and acquire hepatocyte functions in vivo. Additionally, tumors were not found in Fah − / − mice or nonobese diabetic/ severe combined immunodeficient (NOD/SCID) mice with transplanted iHep cells, which has further suggested the safety of iHep cells (Huang et al. 2011). With the transcription factors FOXA3, HNF1A, and HNF4A as well as SV40 Large T, human embryonic fibroblasts have been converted to expandable human hepatocyte-like cells (hiHeps). hiHeps have the gene expression profile similar to human primary human hepatocytes and display functions characteristic of hepatocytes in vitro, especially a good biliary excretion capability, which could be used for the assessment of biliary drug clearance in drug discovery. The work has been reported in Cell Stem Cell and SciBX, which believe that hiHeps overcome the hurdle of the proliferation arrest of differentiated cells, and that the functional hepatocytes obtained via transdifferentiation have made an exciting step toward the hepatocytes needed for drug research and treatment. It has also been recommended by F1000 with the comment that using the method in the

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research, functional hepatocytes can be generated on a large scale, a very important progress in the whole field. It has been selected as one of “Cell Press Papers of the Year 2014 China.” The further research has produced hiHeps at clinical scales and developed a bioartificial liver device implanted with induced human functional hepatocytes to successfully treat miniature pigs with acute liver failure (ALF) (Shi et al. 2016). The survival rate of the treated pigs was significantly higher than that of the control group, and all the serum biochemical parameters and inflammatory indexes returned to normal levels, which has provided a solid foundation for the clinical application of hiHeps. In January 2016, through cooperation with Nanjing Drum Tower Hospital, our scientists successfully treated the first case of acute liver failure and saved a patient suffering from hepatitis B for 40 years who had a sudden acute liver failure. After treatment with a hiHep bioartificial liver, all the liver function indexes were significantly improved without any adverse effects. This case marks the successful completion of the first clinical treatment with the novel hiHep bioartificial liver. Promoting the clinical application of the hiHep bioartificial liver will greatly advance the transformation of scientific research achievements in China.

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3.3.2 Discovery of the Epigenetic Mechanisms and Cell Signaling Pathways Inducing the Differentiation and Transdifferentiation from Embryonic Stem Cells or Somatic Cells to Neurons Histone modification is closely related to gene transcription regulation and plays an important role in neurogenesis. At the epiblast stage, inhibition of histone deacetylase activity can significantly suppress the neural differentiation of embryonic stem cells and promote mesendoderm differentiation, suggesting an important role of histone deacetylation in the neurodevelopment. The study has discovered that histone deacetylase 1 (HDAC1) specifically bound at the Nodal gene, and histone deacetylase inhibitors could significantly upregulate the expression of Nodal. The activation of Nodal signals promoted mesendoderm fate and inhibited neural fate commitment, which has indicated that HDAC1 ensures neural fate commitment by repressing Nodal signaling at the epiblast stage (Liu et al. 2015). The acetylation of H3K9 gradually declined during day 0–4 of the neural differentiation of embryonic stem cells in vitro, and increased during day 4–8. Correspondingly, during the neural differentiation, the enrichment of H3K9 acetylation decreased in the pluripotency-related genomic locus while increased in the neurodevelopment-related locus. Histone deacetylase inhibitors can inhibit the activity of HDAC3 to promote the expression of pluripotency genes and maintain the pluripotency of human embryonic stem cells. During the neural differentiation, histone deacetylase inhibitor relieves the inhibitory activities of HDAC1/5/8 and thereby promotes early neurodevelopmental gene expression (Qiao et al. 2015a). Histone methylation is also involved in the regulation of neural differentiation. The study has identified KIAA1718 (KDM7A) as a dual-specificity histone demethylase for H3K9 and H3K27. The transcription level of Fgf4 gene is positively regulated through the demethylation of H3K9me2 and H3K27me2, to mediate the neural differentiation of embryonic stem cells. The enzyme has been demonstrated to be related to the neurodevelopment in chick embryos (Huang et al. 2010). Additionally, DNA demethylation is found to play an important role in neurogenesis. The study has indicated that AF9 is necessary and sufficient for hESC neural differentiation and neurodevelopmental gene activation. DNA dioxygenase TET2 could interact with AF9, and they colocalized in 5-hydroxymethylcytosine (5hmC)-positive neurons. Upon recognizing AAC-containing motifs and binding at target gene promoters, AF9 recruited TET2 to occupy their common downstream neurodevelopmental gene loci to direct 5mC-to5hmC conversion, which was followed by sequential activation of the expression of these neurodevelopmental genes and hESC neural commitment (Qiao et al. 2015b). The treatment of nervous system disorders with neural stem cells is a research hotspot in the field of regenerative medicine. Inducing the transformation of somatic cells into neural stem cells indicates that somatic cells can also be directly converted to neural stem cell-like somatic stem cells without first going through a pluripotent state, which provides a rich source of cells for cell therapy. Our scientists have successfully used specific factors (Pax6, Ngn2, Hes1, Id1, Ascl1, Brn2, c-Myc, and

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Klf4) to induce the transdifferentiation of sertoli cells to neural stem cells. These induced neural stem cells (iNSCs) expressed normal neural stem cell markers, and the expression of the whole genome was also highly similar to that of normal neural stem cells. Functionally, iNSCs could maintain self-renewal and differentiate into neurons with various electrophysiological functions, such as dopaminergic neurons, γ-Aminobutyric acid neurons, and acetylcholinergic neurons. Importantly, the iNSCs could survive normally and establish synaptic connections with surrounding neurons, after they were introduced into the dentate gyrus of hippocampus in the adult brain of mice. The study has indicated that iNSCs could become a cell resource for clinical treatment of neurodegenerative diseases and new drug screening (Sheng et al. 2012). The paper has been selected as one of “The Top 100 Most Cited Chinese Papers Published in International Journals 2012,” ranking 13th. It has been found that overexpression of the catalytic domain of demethylase TET2 in neural progenitor cells (NPCs) could activate the astrogliogenic program and simultaneously inhibit the neurogenic program. The transcription factor OLIGO2 could directly bind itself to the promoter of TET2 gene to regulate its expression. During the early stages of embryonic neural development, the transcription factor Ngn1 could bind itself to the promoter of a brain-enriched microRNA, miR-9, to suppress the activation of the Jak-Stat pathway, to inhibit astrogliogenesis (Zhao et al. 2015a).

3.3.3 Discovery of Important Disease-related Epigenetic Modifications Rett syndrome (RTT) is caused by mutations in the X-linked Mecp2 gene. The mouse models are quite different from clinical patients, so it is difficult to use them to promote the research of pathogenesis and the development of new treatment methods. Nonhuman primates, such as rhesus and cynomolgus macaques, are ideal experimental animals to study brain development and neurological disorders because they share many similarities of genetic backgrounds and brain structures with humans. In early 2014, our scientists, for the first time in the world, reported successful TALEN-mediated mutagenesis of Mecp2 gene in monkey models. The results have been published in Cell Stem Cell (Liu et al. 2014). The further study has shown that unlike rodent models, Mecp2 mutant monkeys have a series of pathological and behavioral features resembling clinical manifestations of RTT patients. Given their incomparable advantages over rodents, they would have a profound impact on the pathogenetic studies as well as development of therapeutic interventions for RTT in future (Chen et al. 2017). Our scientists have found that a lymphocyte lineage-restricted transcription factor, Aiolos, alters the higher-order chromatin structure of the p66Shc gene to disrupt enhancer–promoter interactions and silence p66Shc transcription, and therefore promotes cancer cells to bypass anoikis and performs distant metastasis. They have

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presented a new idea that carcinoma cells “co-opt” a lymphocyte lineage-restricted transcription factor to mimic the lymphocyte properties of low adhesion and anoikis resistance to realize distant metastasis. They have also demonstrated the important role of higher-order chromatin structure as a type of epigenetic regulation in tumorigenesis (Li et al. 2014b). After its publication, it has been selected as a featured article. In the same issue, Steven Frisch, a pioneer in the anoikis research, wrote a preview on the paper and commented that it “provides provocative evidence that lung cancer cells utilize a hematopoietic transcription factor, Aiolos, to drive both anoikis resistance and anchorage independence.” The article of “Update in Lung Cancer in 2014” in American Journal of Respiratory and Critical Care Medicine, published by Professor Powell of ISMMS in the USA, said that “the discovery of the mechanism of tumor cells using hematopoietic cell signaling opens up a new promising direction for the study on lung cancer pathogenesis.” Their research has obtained two Chinese invention patents, one of which was transferred to Beijing Biocytogen Co., Ltd. in 2015 for achievement transformation. Within the DNA methyltransferase (DNMT) family, DNMT3A and DNMT3B are responsible for the de novo methylation. While DNMT3A is frequently mutated in hematological malignancies, DNMT3B is rarely mutated. Our scientists have studied its role in the acute myeloid leukemia (AML) in an MLL-AF9-induced AML mouse model. The deletion of DNMT3B accelerated MLL-AF9 leukemia progression by increasing the percentage of leukemia stem cells (LSCs) and enhancing leukemic cell cycle progression. Depletion of DNMT3B and DNMT3A synergistically promoted leukemia development. The study has provided new insights into the roles of DNA methyltransferases in leukemia development (Zhang et al. 2016). Enhancers are important regulatory elements controlling gene expression. Our scientists have found that the enhancers jointly marked by H3K4me3 and H3K27Ac might be overactive, and the complex containing RACK7 and KDM5C, two potential tumor suppressors, is a negative regulator of the overactivation. RACK7, a potential chromatin reader, and KDM5C, a histone demethylase, together act as a “brake” of active enhancers by controlling the dynamics between H3K4me1 versus H3K4me3 at active enhancers. Loss of such an enhancer surveillance mechanism can lead to altered cell behaviors, which may contribute to tumorigenesis (Shen et al. 2016). With the TAB-Seq sequencing method, our scientists have explored the reprogramming models and rules of 5mC and 5hmC in ccRCC at single-nucleotide resolution, and found that loss of 5hmC but not 5mC was a sensitive prognostic marker for kidney cancer. The nude mice experiment demonstrated that restoring 5hmC levels suppressed tumor growth, indicating that the 5hmC was more likely to be a driving force for cell reprogramming in kidney cancer than a concomitant phenomenon. This suggests that 5hmC can function as a novel epigenetic marker for prognostic monitoring of renal cell carcinoma and a therapeutic target (Chen et al. 2016b). It has been found that stabilization of histone demethylase PHF8 mediated by the deubiquitinase USP7 confers cellular resistance to DNA damage and promotes breast carcinogenesis (Wang et al. 2016). The results have been published in the Journal of Clinical Investigation and selected in “Editor’s Picks” of the issue. Additionally,

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mutations in PHF8 have been found to play an important role in the diagnosis of X-linked mental retardation and other diseases (Yu et al. 2010). Previous studies have found that the transcriptional mediator orchestrates the basal transcription machinery with RNA polymerase II for precise transcriptional control. The mediator complex is known to regulate transcription initiation and elongation, but its roles in histone modifying enzymes and protein post-translational modification have not been reported. The study has revealed that the mediator subunit Med23 specifically regulates H2B mono-ubiquitination (H2Bub) to control the transcription of specific genes, which plays an important role during the cell fate determination. It has demonstrated a new mechanism of transcription elongation regulation and the new function in cell fate determination. The results have been published in a well-known international journal as the cover article, and highly praised by peers. The tumor suppressor FoxO1 is a key protein to induce autophagy. FoxO1 in the cytoplasm binds itself to the histone deacetylase SIRT2 to stay inactivated. In response to stress, FoxO1 is acetylated by dissociation from SIRT2 and becomes activated. The activated FoxO1 specifically binds itself to ATG7, a key autophagy protein, to induce the autophagy process. Both animal experiments and the study on the clinical tumor specimens have indicated that cytosolic FoxO1-induced autophagy may be a critical factor in the anti-neoplastic effect of FoxO1. This finding has linked the histone deacetylase with epigenetic modifications to autophagy and anticancer activity. It has been published in Nature Cell Biology (Zhao et al. 2010). Telomere regulation and the mitochondrial function regulation related to cell metabolism were long considered as two independent pathways for cell aging. TIN2 is recruited to telomeres and associates with multiple telomere regulators including TPP1. TPP1 interacts with TIN2N-terminus and controls TIN2 localization. Our scientists have discovered that the telomeric protein TIN2, besides its important roles in the telomere functions, is post-translationally processed in mitochondria and regulates mitochondrial oxidative phosphorylation. Reducing TIN2 expression by RNAi knockdown inhibited glycolysis and reactive oxygen species (ROS) production and enhanced ATP levels and oxygen consumption in cells. TIN2 could locate in the telomeres and the mitochondria, as regulated by TPP1. These results have suggested a direct link between telomeric proteins and metabolic control, providing another important mechanism by which telomeric proteins regulate cancer and aging. The study has been published in Molecular Cell as the cover article, titled “Mitochondrial Localization of Telomeric Protein TIN2 Links Telomere Regulation to Metabolic Control.” Craniofacial bones are derived from the development and differentiation of cranial neural crest cells, involving the processes of their fate determination, migration, and differentiation. Abnormalities in any link could result in craniofacial deformity. Several BMP ligands are expressed in the pharyngeal pouches originated from the endoderm in the pharyngeal region, and their antagonist Noggin3 is expressed in the chondrogenic progenitors next to the pouch. The study has found that miR-92a is expressed in the Zebrafish pharyngeal region and acts to maintain the signaling of BMP, a protein involved in the pharyngeal cartilage formation, to ensure its normal development. This work has not only elucidated the important role of miR-92a in the

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cartilage development, but also further indicated that BMP signals must be strictly regulated during pharyngeal cartilage formation, and its activity higher or lower than the physiological level would lead to serious developmental defects (Ning et al. 2013). The discovery has been recommended by F1000 which believes that it has found a new molecular mechanism leading to craniofacial deformity that is worthy of writing in textbooks.

3.4 Establishment of Epigenetic Maps to Reveal the Characteristics and Rules of Epigenetic Modifications During the Embryonic Development The epigenetic maps have been established with high-throughput data collection to reveal the characteristics of epigenetic modifications during the early embryonic development of different species and the rules of genetic evolution, which has enriched our understanding of the origins and evolution of epigenetic networks.

3.4.1 Discovery of Distribution and Change Rules of Genome-Wide Histone Modifications in Pre-implantation Embryos Epigenetic remodeling is critical in the reprogramming of highly specialized gametes during fertilization and embryo development. The changes in these epigenetic modifications are key points to the activation of the embryonic genome and the first cell lineage differentiation. The post-transcriptional modification of histone directly regulates activation or silencing of gene expression. In early studies with immunofluorescent staining methods, most histone modifications underwent obvious changes during the pre-implantation embryo development, and abnormal expression or deletion of some enzymes regulating histone modification led to abnormal embryonic development or even death of pre-implantation embryos. These studies have indicated that the changes in the epigenetic modifications have critical roles during the early embryonic development. However, it remains poorly understood how the histone modifications in pre-implantation embryos distribute and change in the genome and how they regulate the expression of embryonic genes and the first differentiation of the cell fate. By using the latest ultra-low- input micrococcal nuclease-based native chromatin immunoprecipitation (ULI- NChIP) method, the study has detected the changes of histone H3K4me3 and H3K27me3, which are associated with gene activation and repression respectively, during the stages of mouse pre-implantation embryo development. This has been the first systematic genome-wide map of histone modifications in mouse pre-implantation embryos. It has revealed the establishment of

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H3K4me3 and HK27me3 modifications during the pre-implantation embryo development and found that the broad H3K4me3 domain plays an important role in gene expression regulation during the pre-implantation development (Liu et al. 2016). It is worth mentioning that another study funded by this MRP has been published in the same issue of Nature. This “back-to-back” paper has reported a noncanonical form of H3K4me3 in the untranscripted oocytes, one-cell and early two-cell embryos before the genome activation, which exists as broad peaks in nonpromoter regions, including the intergenic regions. Through downregulation of H3K4me3 in oocytes by overexpression of the demethylase KDM5B, the researchers have unexpectedly found that noncanonical H3K4me3 is necessary for the genome silencing (but not activation) during oogenesis (Zhang et al. 2016). The comment released in the same issue of Nature, titled “Developmental Biology: Panoramic Views of the Early Epigenome,” has pointed out that these studies detail changes in histone modifications after the fertilization and during the early embryonic development, the chromatin openness on the regulation of gene expression, and how epigenetic information is inherited from parents to progeny. These findings are of great significance for the research of abnormal embryonic development and the improvement of the success rate of assisted reproductive technology, which will benefit patients with recurrent abortion, embryo damage, and infertility. They symbolize that China has achieved research results with world influence in related fields. The study has been selected as one of China’s top 10 research advances in life sciences in 2016 by China Union of Life Science Societies.

3.4.2 Establishment of Single-Cell Transcriptome Sequencing and Analysis Tools The single-cell RNA-sequencing (SCRS) is a powerful technique to analyze genomic expression in a single-cell or micro RNAs. Compared with the microarray technology, SCRS can detect more transcriptomes in a more sensitive manner. It can analyze multiple transcripts of the same gene and their corresponding protein types and also detect new splice sites in known genes. It features high accuracy and low noise. SCRS helps to accurately display the detailed changes during cell programming and reprogramming differentiation. In recent years, researchers have begun to use this technique to overcome the bottleneck of small initial sample size. Through the self-developed single-cell transcriptome analysis technique and weighted gene co-expression network analysis (WGCNA), our scientists have uncovered molecular properties of CD133+/GFAP-ependymal (E) cells in the adult mouse forebrain neurogenic zone. Prominent hub genes of the gene network unique to ependymal CD133+/GFAP-quiescent cells were enriched for immune-responsive genes, as well as genes encoding receptors for angiogenic factors. Administration of vascular endothelial growth factor (VEGF) activated CD133+ ependymal neural stem cells (NSCs), lining both the lateral and the fourth ventricles and, together with basic

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fibroblast growth factor (bFGF), elicited subsequent neural lineage differentiation and migration. This study has revealed the existence of dormant ependymal NSCs throughout the ventricular surface of the central nervous system (CNS), as well as the abundant signals after CNS injury for their activation (Luo et al. 2015). With both patch-clamp recording and the single-neuron transcriptome analyses, another study has identified the molecular markers of neuronal maturation and their relations with energy metabolism (Chen et al. 2016c). The hepatic stem cell lineage and the activation mechanisms have been revealed with the single-cell transcriptome analysis.

3.4.3 Establishment of Genome-Wide Methylation Profiles to Reveal the Inheritance and Evolution Rules of Epigenetic Modifications With the support of this MRP, our scientists, using MethylC-Seq, have surveyed the silkworm Bombyx mori that have a low level of methylation and established the single-base resolution methylome of its silk gland (Xiang et al. 2010). The study has generated the first insect epigenome, confirmed the existence of epigenetic mechanism in insects and its important functional significance, clarified the long-standing fuzzy understanding of insect epigenetic system, and inspired researchers to rethink the research of insect DNA methylation and its functions. Since its publication, it has been cited for over 170 times. It has been reported in Nature China as a highlight, and in Nature Asia–Pacific as a research highlight titled “Threading Together a Map of Silkworm DNA Modifications.” It has also received a review in Nature China titled “Epigenetics, Few and Far Between”, writing that “this work shows that the evolution of insects can be understood from the perspective of epigenetics, and the research results provide valuable data for exploring the effect of epigenetics on silkworm domestication.” Since the publication, the study has been cited in three English monographs—Epigenetic Genetic Regulation and Epigenomics, Insect Molecular Biology and Biochemistry, and Honeybee Neurobiology and Behavior: A Tribute to Randolf Menzel. DNA methylation, as important epigenetic mechanisms regulating gene expression, influences a series of biological processes, such as cell fate determination, development, and homeostasis maintenance of tissues and organs. Its modifications, including 5-methylcytosine (5mC), N6-methyladenine (6 mA), and N4methylcytosine (4mC), have been found in genomic DNA from bacteria and eukaryotes. 5mC and its derivatives during demethylation, 5hmC, are thought to be the type of methylated base in mammalian genomic DNA. Unlike 5mC, 6 mA is present in prokaryotes and some lower eukaryotes, especially bacteria, of high abundance. It plays important roles in controlling a number of biological functions, such as DNA replication and repair, gene expression, and host–pathogen interactions. Our scientists have shown that 6 mA is present in Drosophila genome (Zhang et al.

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2015), and that the 6 mA modification is precisely regulated by the Drosophila TET homolog, DNA 6 mA demethylase (DMAD), during early embryogenesis. The study has uncovered a new DNA modification in eukaryotes, making it an original breakthrough in epigenetic research. Using MethylC-Seq, our scientists have measured genome-wide DNA methylomes at single-base resolution in zebrafish gametes and early embryos (Fig. 3.4) and studied the rule of their inheritance from parents to offspring (Jiang et al. 2013). The study has found that paternal DNA stably maintains the sperm methylome; for maternal DNA, the oocyte methylome is gradually discarded and reprogrammed to a pattern similar to that of the sperm methylome; the sperm methylome facilitates the epigenetic regulation of embryogenesis. This discovery has challenged the traditional view that “the majority of information for the early embryonic development regulation is stored in egg, and sperm carries just one set of DNA.” This study on the zebrafish methylation inheritance has illustrated that besides DNA that can be inherited, the epigenetic information (DNA methylome) can also be completely inherited into the offspring. Upon its publication, Cell released a preview titled “Beyond DNA: Programming and Inheritance of Parental Methylomes.” The inheritance of the epigenetic information into the offspring means that the variation of epigenetic information may play an important role in regulating animal development, phenotypes, and even diseases, just as that of genetic information does. After the study was published, 1/4 of its citations come from the field of evolution, and it has triggered the new reflection whether epigenetic information plays a driving role in evolution. Meanwhile, the study on the DNA methylation reprogramming in mammals has reported that the oxidation products of the methylation exist in both maternal Erasing the whole Setting sperm-oocytemethylome in PGCs

Protection of genomic imprinting

Active depletion of 5mC

Paternal

de novo methylation

Maternal

Paternal Maternal 5hmC+5fC

2-cell 4-cell

blastocyst/ICM

E6.5

E7.5

E13.5 PGCs

Fig. 3.4 Active demethylation of maternal and paternal genomes in mammals (Wang et al. 2014a)

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and paternal genomes during their early development, so both DNA methylomes go through active demethylation. This discovery has changed the long-standing understanding that the maternal genome is demethylated through passive dilution during the early embryonic development in mammals.

Chapter 4

Outlook Gang Pei, Yongfeng Shang, Fanglin Sun, Zuoyan Zhu, Runsheng Chen, Xiaofeng Cao, and Zhen Xi

Epigenetics has emerged since the late 1980s. In the twenty-first century, with advances in technological means, this new discipline is developing in an unprecedentedly rapid speed. Increasingly more biophysicists, developmental biologists, chemists, bioinformaticians, and geneticists are participating in the exploration of epigenetics. Focusing on the international frontier and development trends of this discipline, they promptly apply the latest research ideas and technical means of related disciplines to epigenetic research and have greatly improved our comprehensive understanding of the epigenetic system. With the promotion of this MRP, Chinese scholars have made great progress in the areas of molecular basis of epigenetics and theoretical innovation of cell reprogramming and contributed significantly to the comprehensive leapfrog development of the epigenetic research in China.

G. Pei (B) Tongji University, Shanghai, 200000, China e-mail: [email protected] Y. Shang Hangzhou Normal University, Hangzhou, Zhejiang, China F. Sun Tongji University, Shanghai, China Z. Zhu Institute of Hydrobiology, Chinese Academy of Sciences, Wuhan, Hubei, China R. Chen Institute of Biophysics, Chinese Academy of Sciences, Beijing, China X. Cao Institute of Genetics and Developmental Biology, Chinese Academy of Sciences, Beijing, China Z. Xi Nankai University, Tianjin, China © Zhejiang University Press 2023 G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming, Reports of China’s Basic Research, https://doi.org/10.1007/978-981-19-7419-9_4

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At present when the cell reprogramming research sees rapid development, there are major breakthroughs in both principle and technology in many directions, with emerging hotspots and fast changes. In addition to the Yamanaka factors OSKM, scientists have found more pluripotency reprogramming factors, such as NANOG, PRDM14, SALL4, ESRRB, UTFL1, TET2, and GLIS1. To prevent the exogenous genes from integrating into the genome of target cells and improve the safety of the system, our scientists have achieved considerable progress in somatic cell reprogramming and cell transdifferentiation with non-viral vectors, RNAs, transmembrane proteins, and small chemical molecules. Meanwhile, the epigenetic research in cell reprogramming witnesses rapid development. The successful primate somatic cell nuclear transfer depends on the use of epigenetic factors. However, current research focuses on the discovery and functional verification of key factors, and the understanding on the chromosome dynamics in reprogrammed cells, changes in cell epigenomes and the regulated transcriptomes, and mechanisms for determining cell fate transitions remains elusive. In this context, our scientists should seize the opportunities, continue to give full play to the interdisciplinary advantages, and apply the technologies, strategies, and experience accumulated in the research of biomacromolecular structures, single-cell omics, and gene editing to the research of cell reprogramming and epigenetics. Centering on the frontier questions in life sciences, we should develop new technologies and methods and promote the focuses in the epigenetic research to progress from a single molecule to multiple molecules, from a single type to multi-type modifications, from qualitative to quantitative, from simple to complex systems. We should improve the efficiency and quality of the cell reprogramming processes, make qualitative leaps in the theory development and technological innovation in life sciences, open up new directions and bring a bright future for the treatment of human diseases.

4.1 Directions to Be Strengthened in China’s Epigenetic Research We need to strengthen the following weak research directions in epigenetics in China. (1) We should enhance the research on epigenetic regulation of cell differentiation, tissue homeostasis, and mechanisms of organ development and regeneration; and focus on elucidating the molecular mechanisms that epigenetic factors integrate changes in intracellular and extracellular environment and signals to regulate cell fate. (2) We should boost the research on the pathogenic mechanisms of epigenetic regulation in relevant major diseases, establish disease-related epigenome maps, and enhance interdisciplinary cooperation with clinical medicine. (3) We should utilize the advantages of species diversity in China to develop new model organisms. We should study the mechanisms of epigenetic factors on learning, memory, social animal behaviors, and intergenerational inheritance of

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acquired traits, discover new epigenetic phenomena, and explore the regulation mechanisms of epigenetic factors on the genome stability and biological traits from the perspective of species evolution. (4) We should strengthen the research on the composition and dynamic changes of higher-order chromatin structures in nucleus, advance further development of the single cell technologies, promote the interdisciplinary cooperation with physics, materials chemistry, and computational biology, and elucidate the general principles of the assembly of higher-order chromatin structures and the regulation mechanisms of their dynamic changes.

4.2 Strategic Needs of the Epigenetic Research in China Epigenetic regulation plays a decisive role in the development of complex conditions such as cancer, diabetes, mental illness, nervous system diseases, and reproductive system diseases. The orderly responses of individual life to environmental factors (including those of nutrition, physical chemistry, and psychology) largely depend on the effective operation of epigenetic regulation networks. Epigenetic regulation also plays an important role in plant development, resistance, and formation of heterosis. The basic biology research in epigenetics helps to better understand the pathogenesis of relevant complex diseases, means and intensity of individual responses to environmental changes, and regulation mechanisms of plant development and breeding, and lays a foundation for us to develop new treatment methods and original drugs for specific diseases, improve individual survival status, and enhance the quality of agricultural production and food security. Through the implementation of this MRP, China has cultivated and established an internationally leading research team covering all major directions of epigenetic research, whose various achievements prove their strength in the field of epigenetic research. In future, it is necessary for China to continue supporting these researchers and keep the epigenetics team stable. This is not only crucial for China to maintain its international status in this field, but also to support the development of national economy and people’s livelihood.

4.3 Conceptions and Suggestions for Further Research Based on the achievements and summaries of this MRP, the research group puts forward the following research conceptions. The convergence between chromatin biology and physics, materials chemistry, and computational biology needs to be facilitated to make breakthroughs in methodology, so as to realize in situ real-time measurement and tracking of chromatin morphology and higher-order chromatin structures at the single cell level, and carry out the epigenome research at the single cell level. This is of significance to the studies

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on the establishment of epigenetic information, cell fate regulation, and responses to environmental changes. The convergence between epigenetics and chemistry, pharmacy, and computational biology needs to be deepened to screen, identify, and synthesize a series of small molecule drugs targeting epigenetic regulators, and promote the clinical treatment of specific diseases. These small molecule drugs are important for both basic research and clinical application. The convergence between epigenetics and mathematics and computational biology needs to be deepened to perform systematic analyses and simulations of epigenomes and epigenetic regulation networks, and, combined with the interactions with other regulation networks in cells, to elucidate the significance of epigenetic regulation on individual development and responses to environmental changes from the perspective of system biology. The convergence between epigenetics and neuroscience, physiology, and bioinformatics needs to be deepened to discover new epigenetic phenomena, explore new epigenetic regulation mechanisms, and expand the current research field. On the basis of the important research directions supported by this MRP, the research group advises to enhance attention to and support for the following directions to continuously increase China’s international position in the epigenetic research. (1) Research on cellular higher-order chromatin structures, especially the analysis of their composition and dynamic changes in the cells of distinct differentiation states and different types. China’s overall research level of higher-order chromatin structures, especially the higher-order structure of 30-nm chromatin consisting of nucleosomes, has been in the forefront of the world. The 30-nm chromatin structure has been reported to be a twist of repeating tetranucleosomal structural units. The gap between such units provides a window for epigenetic regulation, which makes it possible to study epigenetic regulation mechanisms and explain the basic epigenetic questions. Strengthening the research in this direction can maintain our advantages in this field. (2) Expansion of studies on the mechanisms and functions of newly discovered epigenetic modifications. Our scientists have made major progress in discovering new epigenetic regulators, for example, the RNA m6A modification. The next step is to strengthen the investment in this direction. Meanwhile, we should promote the studies on the mechanisms and functions of newly discovered epigenetic modifications, possible interactions between DNA and RNA epigenetic modifications, and the epigenetic regulation mechanisms of cell differentiation, tissue homeostasis, organ development and regeneration. We should also develop new model organisms and explore the regulation mechanisms of epigenetic factors on the genome stability and biological traits from the perspective of species evolution. (3) Development of new technologies, theories, and methods. It is suggested to develop and optimize epigenome editing technologies and single-cell epigenetic information measurement technology of epigenetic transcriptomes, expand the

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bioinformatic algorithms of epigenetic networks, and study the network interactions between epigenomics and transcriptomics at the single-cell level. We should strengthen the research on the pathogenic mechanisms of epigenetic regulation in relevant major diseases and enhance interdisciplinary cooperation with clinical medicine. (4) Research on the dynamic influences of OSKM and other pluripotency factors on cell epigenomics and transcriptomics at the single-cell level during the human cell reprogramming. We should improve the efficiency of cell reprogramming, the quality of reprogrammed cells, and the stability and homogeneity of reprogramming system, to lay the foundation for regenerative medicine based on cell reprogramming and the clinical application of iPSC and transdifferentiated cells. (5) Research on the epigenetic responses and memory of environment signals to explore the mechanisms. The epigenetic systems in organisms can make cells with the same genome present different epigenomes and transcriptomes in different environmental conditions, to differentiate into different morphology with divergent functions. It has been found that histone modifications are inheritable and subject to the feedback regulation by epigenetic modifying enzymes, which indicates the existence of epigenetic response and memory systems in organisms. However, we do not know much about them.

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Index

C Cell programming and reprogramming, 14 Cell signaling pathways, 35

G Gene expression, 38

M Modifications modifying enzymes, 13 D DNA methylation, 43

E Epigenetic modifications, 40

R Reader proteins, 13

Y Yamanaka factors, 31

© Zhejiang University Press 2023 G. Pei (ed.), Epigenetic Mechanisms of Cell Programming and Reprogramming, Reports of China’s Basic Research, https://doi.org/10.1007/978-981-19-7419-9

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