Epigenetics and Reproductive Health

By Academic Press

Epigenetics and Reproductive Health, a new volume in the Translational Epigenetics series, provides a thorough overview and discussion of epigenetics in reproduction and implications for reproductive medicine. Twenty international researchers discuss epigenetic mechanisms operating during the formation of male and female gametes, fertilization and subsequent embryo and placental development, particularly in mammals and transgenerational epigenetic inheritance. This volume also addresses aberrant epigenetic changes influencing male and female infertility, pregnancy related disorders, and those potentially linked to therapeutic manipulations and assisted reproductive technologies. Emphasis is placed on identifying biomarkers for early detection of aberrant epigenetic mechanisms.

Later chapters examine the possibility of correcting these epigenetic dysfunctions, as well as current challenges and next steps in research, enabling new translational discoveries and efforts towards developing therapeutics.

• Thoroughly examines the influence of aberrant epigenetics during gametogenesis and embryogenesis, affecting parents, gametes and embryos, offspring and future generations
• Explores health outcomes for reproductive senescence, endocrine disruption, testicular cancer, prostrate cancer, breast cancer, ovarian, cancer, endometrial cancer and cervical cancers
• Features chapter contributions from international researchers in the field

• Language: English
• Publisher: Academic Press
• Release date: Sep 12, 2020
• ISBN: 9780128197547

Book preview

Epigenetics and Reproductive Health

First Edition

Editor

Trygve Tollefsbol

Table of Contents

Cover image

Title page

Translational Epigenetics Series

Dedication

Copyright

Contributors

Acknowledgment

Introduction to epigenetics: basic concepts and advancements in the field

Section I. Spermatogenesis, oogenesis and fertility

Chapter 1. Epigenome reprogramming in the male and female germ line

Introduction

Reprogramming of mouse germ cells

Epigenome reprogramming during the development of human germ cells

Concluding remarks

Glossary

Chapter 2. Genomic imprinting

Introduction

Genomic imprinting in placentation

Genomic imprinting in post-natal development

Genomic imprinting in disease conditions

Pregnancy-related disorders

Genetic and environmental influences on genomic imprinting

Genetic influences

Environmental influences

Assisted reproductive technologies induced defects

Concluding remarks

Chapter 3. Chromatin remodeling of the male genome during spermiogenesis and embryo development

Introduction

Nuclear remodeling in sperm

From being sperm nucleus to paternal pronucleus

Is sperm merely a vector for the paternal genome?

Father's extra-genomic contribution to the zygote

Chapter 4. Epigenetic regulation in stem cells

History of stem cell research

Types of stem cells

Induced pluripotent stem cells (iPSCs)

Mechanism on the self-renewal of PSCs

Core transcription factors

Signal transduction pathways

LIF-STAT pathway

Wnt pathway

TGF-β pathway

Epigenetics

DNA methylation

Histone modification

Chromatin remodeling

Three-dimensional structure of genome

Conclusion

Chapter 5. Aberrant epigenetics and reproductive disorders

Epigenetic alterations and reproductive system

Role of epigenetic alterations in female infertility

Premature ovarian failure

Male infertility and DNA methylation

Conclusion

Section II. Pregnancy/ developmental/ placental epigenetics

Chapter 6. Epigenetic reprogramming in the embryo

Introduction

Epigenetics, a brief overview

Epigenetic marks that are relevant to preimplantation development

Epigenetic reprogramming in the zygote and preimplantation embryo: an overview

The gametes and their epigenetic characteristics

The zygote: a hotbed of epigenetic activity

Detailed mapping of methylation changes in preimplantation development

Mediators of DNA methylation in gametes and preimplantation embryos

The subcortical maternal complex is a key effector of epigenetic programming in the oocyte/early embryo

Demethylation events in the zygote and preimplantation embryo

Species-specific differences in reprogramming

RNA as a mediator of epigenetic events in gametes and preimplantation embryos

Conclusions

Chapter 7. Epigenetic regulation during placentation

The placenta

Epigenetic mechanisms in placental development

Disturbed placental epigenetics

Conclusions and future perspectives

Chapter 8. Epigenetic modulation during pregnancy and pregnancy related disorders

Introduction

Epigenetic regulation of placenta during pregnancy

Epigenetics and pathogenesis of pregnancy disorders

Conclusion

Chapter 9. Epigenetic involvement in fetal and neonatal origins of late-onset disease

Introduction

Fetal origins of late-onset disease and the underlying epigenetic mechanism

Neonatal origins of late-onset disease and the underyling epigenetic mechanism

Transgenerational epigenetic inheritance

Medical and educational intervention in early life to correct epigenetic dysfunctions

Conclusion

Section III. Epigenetic e lifestyle, aging and environmental influence

Chapter 10. Impact of environmental chemicals and endocrine disruptors on mammalian germ cell epigenome

Introduction

Epigenetic effects of EDCs on male germ cell development

Epigenetic effects of EDCs on female germ cell development

Transgenerational epigenetic inheritance

Conclusions and research perspectives

Chapter 11. Influence of nutrition on reproductive health through epigenetic mechanisms

Introduction

Nutrition and reproductive health

Macronutrients

Micronutrients

Nutrition and developmental origins of health and disease (DOHaD)

One carbon metabolism

Epigenetics

Maternal nutrition, epigenetics and reproductive health

Gestational diabetes mellitus (GDM)

Preterm birth (PTB)

Intrauterine growth restriction (IUGR)

Paternal nutrition, epigenetics and reproductive health

Conclusion

Chapter 12. Influence of stress and lifestyle on epigenetic modifications

Introduction

The influence of stress during prenatal life on epigenetic modifications of the offsprings

Epigenetic changes of the HPA axis related genes in offsprings upon exposure to prenatal stress

Epigenetic changes of the spermatozoa due to the paternal lifestyle

Epigenetics and assisted reproductive technologies (ART)

Conclusions

Chapter 13. Aging of male and female gametes

Introduction

Aging in sperm

Aging in oocytes

Conclusion

Section IV. Reproductive cancer and epigenetics

Chapter 14. Testicular and prostate cancers

Introduction

Male reproductive cancers

Genetic and epigenetic interplay in reproductive cancers

Epigenetic regulation of testicular cancer

Epigenetic regulation of prostate cancer

Clinical implication of testicular and prostate cancer epigenetics

Conclusions

Chapter 15. Emerging patterns and implications of breast cancer epigenetics: an update of the current knowledge

Introduction

Conclusion

Chapter 16. Ovarian & endometrial cancers

Epidemiology of endometrial and epithelial ovarian cancer

Epigenetics of endometrial and epithelial ovarian cancer

Epigenetic therapy in endometrial and ovarian cancer

ncRNA transcripts in ovarian cancer and endometrial cancer

Conclusion

Chapter 17. Epigenetic aberrations in cervical cancer

Introduction

Epigenetic alterations in cervical cancer

Influence of epigenetic alterations on cancer hallmarks

Therapeutics

Conclusion

Section V. Epigenetics in diagnosis, prognosis and therapy

Chapter 18. Natural molecules as epigenetic modifiers in reproduction

Introduction

Folic acid (FA)

Players in the endocannabinoid system

Role of ECS in female reproductive system

Role of ECS in male reproductive system

Epigenetic effects on reproduction by cannabinoids

Multigenerational effects of cannabis

Conclusion

Chapter 19. Epigenetics in diagnosis, prognosis and therapy: therapies targeting the epigenome

Introduction

Types of epigenetic mechanisms

Epigenetics in diagnosis

Role of epigenetics in various diseases

Epigenetics in cancer: toward simplified therapies

Epigenetic treatments: reprogrammers & targeted therapy

Epigenetic regulation of autophagy in cancer: a new approach to curb cancer?

Future perspective & conclusion

Index

Translational Epigenetics Series

Trygve O. Tollefsbol, Series Editor

Transgenerational Epigenetics

Edited by Trygve O. Tollefsbol, 2014

Personalized Epigenetics

Edited by Trygve O. Tollefsbol, 2015

Epigenetic Technological Applications

Edited by Y. George Zheng, 2015

Epigenetic Cancer Therapy

Edited by Steven G. Gray, 2015

DNA Methylation and Complex Human Disease

By Michel Neidhart, 2015

Epigenomics in Health and Disease

Edited by Mario F. Fraga and

Agustin F. F Fernández, 2015

Epigenetic Gene Expression and Regulation

Edited by Suming Huang, Michael Litt and

C. Ann Blakey, 2015

Epigenetic Biomarkers and Diagnostics

Edited by Jose Luis García-Gim_enez, 2015

Drug Discovery in Cancer Epigenetics

Edited by Gerda Egger and

Paola Barbara Arimondo, 2015

Medical Epigenetics

Edited by Trygve O. Tollefsbol, 2016

Chromatin Signaling and Diseases

Edited by Olivier Binda and

Martin Fernandez-Zapico, 2016

Genome Stability

Edited by Igor Kovalchuk and

Olga Kovalchuk, 2016

Chromatin Regulation and Dynamics

Edited by Anita G€ond€or, 2016

Neuropsychiatric Disorders and Epigenetics

Edited by Dag H. Yasui, Jacob Peedicayil and

Dennis R. Grayson, 2016

Polycomb Group Proteins

Edited by Vincenzo Pirrotta, 2016

Epigenetics and Systems Biology

Edited by Leonie Ringrose, 2017

Cancer and Noncoding RNAs

Edited by Jayprokas Chakrabarti and

Sanga Mitra, 2017

Nuclear Architecture and Dynamics

Edited by Christophe Lavelle and

Jean-Marc Victor, 2017

Epigenetic Mechanisms in Cancer

Edited by Sabita Saldanha, 2017

Epigenetics of Aging and Longevity

Edited by Alexey Moskalev and

Alexander M. Vaiserman, 2017

The Epigenetics of Autoimmunity

Edited by Rongxin Zhang, 2018

Epigenetics in Human Disease, Second Edition

Edited by Trygve O. Tollefsbol, 2018

Epigenetics of Chronic Pain

Edited by Guang Bai and

Ke Ren, 2018

Epigenetics of Cancer Prevention

Edited by Anupam Bishayee and

Deepak Bhatia, 2018

Computational Epigenetics and Diseases

Edited by Loo Keat Wei, 2019

Pharmacoepigenetics

Edited by Ramón Cacabelos, 2019

Epigenetics and Regeneration

Edited by Daniela Palacios, 2019

Chromatin Signaling and Neurological Disorders

Edited by Olivier Binda, 2019

Transgenerational Epigenetics, Second Edition

Edited by Trygve Tollefsbol, 2019

Nutritional Epigenomics

Edited by Bradley Ferguson, 2019

Prognostic Epigenetics

Edited by Shilpy Sharma, 2019

Epigenetics of the Immune System

Edited by Dieter Kabelitz, 2020

Stem Cell Epigenetics

Edited by Eran Meshorer and

Giuseppe Testa, 2020

Epigenetics Methods

Edited by Trygve Tollefsbol, 2020

RNA-Based Regulation in Human Health and Disease

Edited by Rajesh Pandey, 2020

Histone Modifications in Therapy

Editor by Pedro Castelo-Branco and

Carmen Jeronimo, 2020

Dedication

With immense gratitude, we dedicate this book to the memory of our mentor late Dr Harbans Singh Juneja who introduced us to research in the area of neuroendocrinology and this exciting world of the epigenome and its role in reproductive physiology.

Copyright

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ISBN: 978-0-12-819753-0

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Contributors

Ummet Abur

Department of Medical Genetics, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey

Department of Multidisciplinary Molecular Medicine, Health Sciences Institute, Ondokuz Mayıs University, Samsun, Turkey

Ahmed Hamed Arisha,     Department of Physiology, Faculty of Veterinary Medicine, Zagazig University, Zagazig, Egypt

Brooke Armistead,     Michigan State University, Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Grand Rapids, MI, United States

Kenneth I. Aston,     Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, United States

N.H. Balasinor,     Neuroendocrinology, ICMR- National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Barbara Benassi,     Laboratory of Health and Environment, ENEA, Rome, Italy

Douglas T. Carrell,     Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, United States

Shrijeet Chakraborti,     Leighton Hospital, Mid Cheshire Hospitals NHS Foundation Trust, Crewe, Cheshire, United Kingdom

Eugenia Cordelli,     Laboratory of Health and Environment, ENEA, Rome, Italy

Kinjal Dave,     Mother and Child Health, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be) University, Pune, Maharashtra, India

Sharvari Deshpande,     Neuroendocrinology, ICMR- National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Sascha Drewlo,     Michigan State University, Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Grand Rapids, MI, United States

Anthony R. Gostick,     Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, United States

Sezgin Gunes

Department of Multidisciplinary Molecular Medicine, Health Sciences Institute, Ondokuz Mayıs University, Samsun, Turkey

Department of Medical Biology, Faculty of Medicine, Ondokuz Mayıs University, Samsun, Turkey

Nojan Hafizi,     Near East University, Institute of Health Sciences, Department of Medical Biology and Genetics, Nicosia, Cyprus

Jinlian Hua,     College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, China

John Huntriss,     Discovery and Translational Science Department (DTSD), University of Leeds, Leeds, West Yorkshire, Great Britain

Arif Hussain,     School of Life Sciences, Manipal Academy of Higher Education, Dubai, United Arab Emirates

Hiroki Ikeda,     Nara Medical University, Department of Embryology, Kashihara, Nara, Japan

Emma R. James,     Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, United States

Timothy G. Jenkins,     Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, United States

Eugenia Johnson,     Michigan State University, Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Grand Rapids, MI, United States

Sadhana Joshi,     Mother and Child Health, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be) University, Pune, Maharashtra, India

Shama Prasada Kabekkodu,     Department of Cell and Molecular Biology, Manipal School of Life Sciences, Manipal Academy of Higher Education, Manipal, Karnataka, India

Leena Kadam,     Department of Obstetrics and Gynecology, Wayne State University School of Medicine, Detroit, MI, United States

Jyotdeep Kaur,     Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Hisato Kobayashi,     Nara Medical University, Department of Embryology, Kashihara, Nara, Japan

Hamid-Reza Kohan-Ghadr,     Michigan State University, Department of Obstetrics, Gynecology and Reproductive Biology, College of Human Medicine, Grand Rapids, MI, United States

Takeo Kubota

Faculty of Child Studies, Seitoku University, Chiba, Japan

Graduate School of Teacher Education, Seitoku University, Chiba, Japan

Kazuki Kurimoto,     Nara Medical University, Department of Embryology, Kashihara, Nara, Japan

Aatish Mahajan,     Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Sweta Nair,     Neuroendocrinology, ICMR- National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Lakshmi Natarajan,     Independent Researcher, NJ, United States

Francesca Pacchierotti,     Laboratory of Health and Environment, ENEA, Rome, Italy

Priyanka Parte,     Department of Gamete Immunobiology, ICMR-National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Aniket G. Patankar,     Department of Gamete Immunobiology, ICMR-National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Sahar Qazi,     Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, Delhi, India

Beenish Rahat,     Division of Intramural Research, Eunice Kennedy Shriver National Institute of Child Health and Human Development, National Institutes of Health, Bethesda, MD, United States

Albert Salas-Huetos,     Andrology and IVF Laboratories, Department of Surgery, University of Utah School of Medicine, Salt Lake City, UT, United States

Sabita N. Saldanha,     Alabama State University, Department of Biological Sciences, Montgomery, AL, United States

Divika Sapehia,     Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Ashok Sharma,     Department of Biochemistry, All India Institute of Medical Sciences, New Delhi, Delhi, India

Shefina Silas,     School of Life Sciences, Manipal Academy of Higher Education, Dubai, United Arab Emirates

Isha Singh,     Department of Gamete Immunobiology, ICMR-National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Parampal Singh,     Department of Biochemistry, Postgraduate Institute of Medical Education and Research, Chandigarh, India

Madhumitha Kedhari Sundaram,     School of Life Sciences, Manipal Academy of Higher Education, Dubai, United Arab Emirates

Deepali Sundrani,     Mother and Child Health, Interactive Research School for Health Affairs (IRSHA), Bharati Vidyapeeth (Deemed to be) University, Pune, Maharashtra, India

Padmanaban S. Suresh,     School of Biotechnology, National Institute of Technology, Calicut, Kerala, India

Burak Tatar

Department of Gynecologic Oncology, Health Sciences University Samsun Research and Training Hospital, Samsun, Turkey

Department of Multidisciplinary Molecular Medicine, Health Sciences Institute, Ondokuz Mayıs University, Samsun, Turkey

Shilpa Thakur,     National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda, MD, United States

Sanu Thankachan,     School of Biotechnology, National Institute of Technology, Calicut, Kerala, India

Pinar Tulay

Near East University, Faculty of Medicine, Department of Medical Genetics, Nicosia, Cyprus

Near East University, DESAM Institute, Nicosia, Cyprus

Eva Tvrdá,     Department of Animal Physiology, Faculty of Biotechnology and Food Sciences, Slovak University of Agriculture in Nitra, Tr. A. Hlinku 2, Nitra, Slovakia

Thejaswini Venkatesh,     Department of Biochemistry and Molecular Biology, Central University of Kerala, Kasaragod, Kerala, India

Juqing Zhang,     College of Veterinary Medicine, Shaanxi Centre of Stem Cells Engineering & Technology, Northwest A&F University, Yangling, Shaanxi, China

Acknowledgment

The Editorial team acknowledges the contributions by all the authors and co-authors to the chapters resulting in a comprehensive book on Epigenetics and Reproductive Health. We wish to thank the entire support team of Elsevier for the smooth publication of the book. We also thank Megan Ashdown for efficient coordination with the authors and editors.

Introduction to epigenetics: basic concepts and advancements in the field

Dipty Singh ∗ , Kumari Nishi ∗ , Kushaan Khambata ∗ , and N.H. Balasinor,     Neuroendocrinology, ICMR- National Institute for Research in Reproductive Health, Mumbai, Maharashtra, India

Abstract

Reproduction is central to the beginning of new lives and inheritance of traits across generations. The genetic as well as other non-genetic factors in germ cells like epigenetics govern the process of reproduction and inheritance of traits from parents to offspring. Epigenetics plays a crucial role in germ cell as well as embryo development. DNA methylation, histone modifications, higher-order chromatin organization such as histone acetylation, methylation, phosphorylation, non-coding RNA and RNA modifications are key epigenetic mechanisms regulating reproductive health and fertility. The understanding of epigenetic factors in disease conditions is crucial and advancements in techniques to assess these factors have led to better understanding of many disease etiology. In this chapter, different epigenetic mechanisms and techniques to assess the epigenetic changes have been discussed in details. Moreover, the single cell epigenomics, an emerging technique to identify epigenetic regulatory mechanisms at single cell level and advancements of epigenome editing technology have been reviewed.

Keywords: Epigenetics, Epigenetic modifications, Techniques for epigenetic analysis, Single cell epigenomics, Epigenome manipulation

Introduction

∗  Equal contribution.

Reproduction is a fundamental process of replicating life which also contributes toward heritability of traits from one generation to the next [1]. The heritability is not only governed by genetic information harbored in gametes but also dictated by non-genetic factors such as epigenetics. Germ cell development and early embryo development are the crucial reproductive events when epigenetic landscape is architectured or maintained [2]. The developmental exposure to certain lifestyle and environmental factors may affect the phenotype of the next generation through remodeling of the epigenetic blueprint of gametes [3]. It is well understood that genetic factors contribute to risk of many diseases affecting human health. Evidence is slowly accumulating that genetic factors are not the only repository of all information in the health status of an organism; other external factors may also influence health via epigenetic modulations. Similarly, along with the genetic factors, epigenetic factors also influence reproductive health and fertility [3]. Therefore, understanding epigenetic modifications, basic cellular physiology and the underlying cause of a disease condition becomes important [4]. New advancements in mapping human epigenome has helped researchers to understand disease etiology in past three   decades. This provides possibilities to interpret epigenetic code and develop better treatment strategies. This chapter presents an overview of key epigenetic mechanisms available techniques for epigenetic analysis and recent advances in epigenetic technology.

Epigenetics

Definition

Although all of more than 200 cell types in humans have the same DNA sequences, yet they exhibit different gene expression profiles and phenotypes. The phenotypic changes that occur in the cells during the course of development in a multicellular organism were originally described as an epigenetic landscape by developmental biologist Conrad Waddingtion. Later, Holliday defined epigenetics as nuclear inheritance which is not based on differences in DNA sequence. In its more modern version, epigenetics is molecularly and mechanistically described as the sum of modifications to the chromatin template that come together to establish and propagate different patterns of gene expression and silencing from the same genome. If genetics can be considered as words, epigenetics instructs how these words are read. Alternatively, if groups of genes can be thought of as computer hardware, epigenetic control can be compared with computer software. Thus, epigenetics is an additional regulatory layer that provides insights into how cellular events are coordinated [5].

Epigenetic changes to the chromatin are brought about by three main mechanisms; namely, DNA methylation, post-translational histone modifications and non-coding RNAs. We attempt to provide a brief overview of these concepts in this chapter.

DNA methylation

Since its discovery more than 35 years ago [6], DNA methylation at the cytosine residues is recognized as one of the prime epigenetic mechanisms regulating gene expression. Cytosine residues are converted by the addition of a methyl group to 5-methylcytosine (5mC) in the DNA template. It mainly occurs in dinucleotide sequence 5′CpG3′ (i.e. CpG abbreviated for cytosine and guanine separated by phosphate group in the DNA). It can be transmitted by both the DNA strands, from mother to daughter cells during DNA replication and, can thus be inherited through cell division. Since DNA methylation patterns are heritable, they provide an epigenetic marking for the genome that is stable through multiple cell divisions, and thus contribute to form a cellular memory.

CpGs are often found in clusters, and are called CpG islands. They are usually enriched at non-coding regions (e.g. centromeric heterochromatin) and interspersed repetitive elements (e.g. retrotransposons). CpG islands are also commonly found in the upstream region in the gene promoters where they regulate gene expression [7]. The methyl moiety of cytosines lies in the major groove of the DNA helix where many DNA-binding proteins interact with DNA. Thus, DNA methylation results in attraction or repulsion of various DNA-binding proteins. The binding of methyl-CpG binding domain (MBD) proteins to methylated CpGs recruits repressor complexes, (like HDACs, which abrogate activating histone acetylation marks) to the methylated promoter regions causing transcriptional silencing. Conversely, CpG methylation inhibits binding of certain transcriptional regulators like CTCF, thereby preventing transcription. Thus, in general, DNA methylation brings about gene repression or silencing and plays a crucial role in cellular differentiation, X-chromosome inactivation and genomic imprinting [8]. An important function of DNA methylation is to protect the genomic integrity by silencing transposable elements and ensure genomic stability. Although DNA methylation patterns are transmitted from cell to cell during cell division, they are not permanent. DNA methylation patterns can change throughout the lifetime of an individual. These changes can be a physiological response to environmental stimuli or may be associated with pathological processes like oncogenic transformation and aging [5].

DNA methylation: establishment and erasure

In mammals, DNA methylation is brought about by DNA methyltransferases (Dnmts), which include three proteins belonging to two families that are structurally and functionally distinct. The first family includes the maintenance methyltransferase Dnmt1, which preferentially methylates hemimethylated CpG dinucleotides (i.e. DNA methylated at CpG in one of the two strands). Thus Dnmt1 is present at the replication fork and is responsible for semiconservative replicating DNA methylation patterns. The other family consists of de novo methyltransferases Dnmt3a and 3b, which lay down de novo DNA methylation patterns in early embryo development. The functions of Dnmt3a and 3b are guided by Dnmt3l, which itself lacks active methyltransferase activity, but is essential for sequence specific de novo methylation activities. DNA methylation marks can be removed by active demethylation process, which involves a family of DNA hydroxylases called ten-eleven translocation (Tet) proteins or by a passive demethylation process by inhibition of Dnmt1 during cell division [9].

The methyl group on CpG can be oxidized by Tet enzymes to 5-hydroxymethylcytosine (5hmC), which is another DNA modification found across the genome specifically at the transcription start sites and are enriched in the active chromatin regions, and are thus involved in regulation of gene expression. The Tet family of proteins can further oxidize 5hmC into 5-carboxylcytosine (5caC) and 5-formylcytosine (5fC) utilizing ATP. These are less stable marks and activate base excision repair pathway, which ultimately leads to removal of the modified base and returns the 5mC to an unmethylated cytosine and thus promotes transcriptional activation [9].

Histone modifications

In multicellular organisms, the DNA is packaged into a nucleoprotein complex called chromatin. The chromatin consists of DNA wrapped around a core of highly basic proteins called histones. The fundamental unit of the chromatin is called a nucleosome, which consists of 146 base pairs (bp) of DNA wrapped approximately twice around an octamer of core histones. Each nucleosome consists of two of each core histones, H2A, H2B, H3 and H4. In each nucleosome, the DNA and histone octamer core are associated with H1 linker protein. The histone core is bound to DNA by non-covalent ionic interactions between positively charged residues on the histone proteins and phosphate groups on the DNA [10,11].

Higher-order chromatin organization

These nucleosomes form the building blocks of higher order chromatin structure. In its completely unfolded confirmation, the chromatin structures are visualised microscopically as 11-nm polymers with a bead-on-string appearance. According to the solenoid model of chromatin fiber, the nucleosomes are arranged into a helical array of about six to eight nucleosomes per turn with the histone H1 on the inside of the fiber. The linker DNA is bent to connect each nucleosome with the next one along the helical filament. This results in a more compacted 30 nm transcriptionally incompetent chromatin conformation. The chromatin can then be organized into larger looped domains (300–700 nm). The most condensed chromatin structure is formed at the time of chromosome formation in the metaphase of mitosis or meiosis, to permit faithful segregation of the genome [5].

Chromatin is a highly dynamic structure, and exists in many conformations. It is historically classified as euchromatin and heterochromatin. Euchromatin or active chromatin consists of coding and regulatory (e.g., promoters and enhancers) regions of the genome. It exists in an open, decompacted confirmation, favoring active transcription or poised for gene expression. Heterochromatin refers to the inactive regions of the genome existing in a closed, highly compacted state. It contributes to the great majority of the genome; and comprises of non-coding sequences and repetitive elements (e.g., retrotransposons, satellite repeats and LINEs). Heterochomatin can exists in two states: the permanently silenced constitutive heterochromatin, which generally found at pericentric and subtelomeric regions or as facultative heterochromatin, in which genes can be derepressed during a specific cell cycle or developmental stage [5].

The core histones are some of the most evolutionarily conserved eukaryotic proteins. They consist of a basic N-terminal domain and a histone-fold C-terminal domain. The histone-fold or globular domain heterodimerizes with a second histone (H3 with H4, H2A with H2B); and wraps DNA around to form nucleosome. The basic N-terminal tail domain lies outside the nucleosome and does not have any defined structure. Many residues in the histone tails, particularly in histones H3 and H4, are important sites of post-translational modifications (PTMs); although some residues in the more structured globular domains are also targeted. These modifications promote nucleosomal, and hence, chromatin variability. Acetylation and methylation of core histones, especially H3 and H4, were among the first covalent modifications studied. Besides these, several other histone modifications have been identified; namely, phosphorylation, ubiquitination, sumoylation, ADP-ribosylation, biotinylation, crotonylation, among others [5]. We shall take a look at some of them in a little detail.

Histone acetylation

Histone can be acetlyated at the ε-amino groups of lysine (K) residues located at the N-termini. Although, all core histones can be acetylated in vivo, acetylation of H3 and H4 are most extensively studied [12]. H3 can be acetylated at lysine positions 9, 14, 18 and 23; while the lysine positions of 5, 8, 12 and 16 can be acetylated for H4. Addition of acetyl groups neutralizes the basic charge of histone tails and thus decreases affinity for DNA. It also alters histone-to-histone interactions between neighboring nucleosomes, and the interaction of histones with other regulatory proteins. Thus, histone acetylation creates an open chromatin environment favorable for gene transcription and is characteristically found in euchromatin regions [13]. Histone acetylation is highly dynamic and controlled by the opposing action of two types of enzymes: histone acetyltransferases and histone deacetylases (HDACs).

Histone methylation

Histone can be methylated on the side chains of lysines (K) and arginines (R) residues. Unlike histone acetylation, methylation does not affect the charge of the histone protein. Additionally, lysines residues can be mono-, di- or tri-methylated, whereas arginines may be mono-, symmetrically or asymmetrically di-methylated [14]. This enhances the level of complexity offered by this modification. Several lysine residues on positions 4, 9, 27, and 36 of H3 and lysine 20 of H4, are preferred sites of methylation [15]. Sites of arginine methylation include H3R2, H3R8, H3R17, H3R26 and H4R3 (Young et al., 2010).

As opposed to acetylation, which usually results in transcriptional activation, histone methylation can signal either activation or repression, depending on the sites of methylation. For example, methylation at H3K4 results in gene activation, while H3K27 methylation leads to gene silencing. Thus, these modifications are usually found in the regulatory regions of the genes like the promoter and enhancer elements. Methylation at H3K9 position leads to a closed chromatin conformation and is consequently associated with heterochomatin regions of the genome. Histone lysine methylation is also involved in diverse set of biological processes, such as heterochromatin-mediated transcriptional silencing, DNA damage response and X chromosome inactivation. Histone lysine methylation is tightly regulated by the enzymes methyltransferases (KMTs) and demethylases (KDMs) in order to maintain cell fate and genomic stability.

Other histone modifications

Histone Phosphorylation: occurs on serine, threonine and tyrosine residues mainly in the N-terminal histone tails. Histone phosphorylation confers a negative charge to the histone, resulting in a more open chromatin conformation. It is therefore associated with gene expression and is involved in DNA damage repair and chromatin remodelling [16].

ADP ribosylation: Histones can be reversibly mono- and poly-ADP ribosylated on glutamate and arginine residues, conferring a negative charge to the histone, and thus contribute to a relaxed chromatin state. These modifications increase upon DNA damage and are involved in the DNA damage response pathway [17].

Ubiquitylation and sumoylation: Histone ubiquitylation involves addition of large ubiquitin moiety (76-amino acid polypeptide) to lysine residues. Mono-ubiquitylation of histones can bring about either gene activation and repression, whereas polyubiquitinylation labels them for proteolytic degradation. Sumoylation, like ubiquitylation, results in the covalent attachment of small ubiquitin-like modifier molecules to histone lysines. Sumoylation can occur on all four core histones and inhibits acetylation or ubiquitylation of lysine residues, thereby causing gene repression [14].

The Histone Code- its writers, readers and erasers

The covalent modifications occurring at multiple and specific sites on the histones give rise to remarkable nucleosomal diversity. Different combinations of histone modifications can modify the chromatin structure, which in turn regulate and determine the changes in gene expression. This concept was put forth as the Histone Code Hypothesis. According to this hypothesis, histones modifications provide a signaling platform to alter the chromatin states in order to bring about gene activation or silencing. The enzymes which establish these histone modifications are collectively referred to as writers of the histone code. They include enzymes like HATs, HMTs, histone kinases among others. In this scenario, the enzymes which remove the histone modifications like the HDACs, KDMs, are called erasers of histone codes. Besides altering the chromatin states, these modifications also serve as binding or recognition sites for the recruitment of several effector proteins termed as readers of the histone codes, which in turn recruit other co-regulator complexes to bring about further changes in the chromatin structure and hence the gene expression [18].

Non-coding RNAs

The Human Genome Project revealed that only a small fraction of the human transcriptome (2%–5%) encoded for proteins while the functions of rest of the transcripts were unknown. Recent advances in sequencing technologies have revealed that in fact, three-quarters of the genome is transcribed [19] (with some studies estimating more than 90%). Although this view is hotly debated, it has challenged the long standing view point that most of the genome is not transcribed and considered as non-functional junk DNA. In recent years, the classification of RNA classes has undergone a major change, with it can be broadly classified into: protein-coding messenger RNAs and noncoding RNAs. The category of noncoding RNAs have now expanded beyond the well-known ribosomal and transfer RNAs (rRNA and tRNA), to include small interfering RNA (siRNA), microRNA (miRNA), circular RNA (circ RNA), PIWI-interacting RNAs (piRNAs), long non-coding RNAs (lncRNAs), promoter-associated RNAs (PARs), enhancer RNAs (eRNAs) and many others [20]. The functions of these ncRNAs have been under intense investigations and they are now known to play many important structural and regulatory roles. More importantly, noncoding RNAs have now emerged as a separate epigenetic mechanism regulating gene expression. The ncRNAs provide a scaffold for chromatin-remodelling and -modifying enzyme complexes to bring about changes in the chromatin states through cis or trans mechanisms. They can also allow recruitment of factors involved in silencing (e.g. co-repressor) or activation of gene transcription. These ncRNAs are thought to provide the sequence specificity to guide the chromatin modifying complexes to their sites of action. As functions of more ncRNAs are being uncovered, they represent a key layer of epigenetic regulation [21].

RNA modifications

RNA can be covalently modified by vast array of chemical additions to both its sugar and nucleotide groups [22]. The additions to the sugar backbone mainly protects the RNA molecule from degradation, the modifications at the nucleotide base confers novel regulatory functions. Specifically, the posttranslational addition of methyl group to N(6) position of adenosine (m(6)A) is the most common RNA modification in the coding and non-coding RNA. It occurs predominantly in the 3′UTRs and stop codons. RNA methylation controls various steps of mRNA metabolism, microRNA mediated decay, pre-microRNA processing, RNA poly adenylation. It also affects RNA secondary structure and regulates alternative splicing for a subset of mRNAs and lncRNAs, and it is also involved in translation and RNA degradation [22,23]. This modification is brought about by the METTL3 RNA methyltransferase complex and is erased by FTO (fat-mass and obesity associated) RNA demethylase. The dynamic activity of these enzymes regulates the level of this modification, which plays a crucial role in development, metabolism, and fertility [24].

Techniques for epigenetic analysis

The primary methods in epigenetics engaged specific nuclease enzymes (e.g., restriction enzymes, DNase I, MNase) and later identification of DNA and histone modifying enzymes produced a big new data in the field. Past few years have witnessed development of new techniques which directly assess chromatin modification, interacting proteins, patterns of DNA methylation and nucleosomal occupancy. Active emergence of these new technologies to assess genome-wide DNA methylation patterns, chromatin structure in modern era has accelerated the speed of understanding of epigenetic mechanisms.Various methods developed to detect and measure epigenetic marks and assess their functions depending on genome wide or loci specific approaches have been discussed below.

DNA methylation

A lot of advancements have been made in DNA methylation detection technologies starting from southern blot using restriction endonucleases to microarray-based epigenomics and methylation specific polymerase chain reaction to next generation sequencing based targeted or whole genome bisulfite sequencing [41,70]. The choice of method is important to get an unbiased answer to the research question. The selection of method depends on the study aim, amount and quality of DNA sample, availability of reagents and instruments, its cost effectiveness, how robust and simple the method is to meet the sensitivity and specificity criteria. Another criterion for choosing the method depends on whether information being sought is genome-wide or specific locibased. The method can also be chosen based on whether the candidate genes are known or not [4]. The methods used for DNA methylation analysis have been enlisted in Table 1.

Table 1

Histone modifications and chromatin remodelling

In past three decades a significant progress has been made in genome wide characterization of histone modifications and chromatin remodelling [43,50]. The major developments made in histone modification chromatin analysis such as improved high-throughput sequencing in combination with chromatin immunoprecipitation assay (ChIP) and DNA microarray (DNA chip) i.e. ChIP-on-chip, Chromatin Interaction Analysis by Paired-End Tag Sequencing (ChIA-PET), ChIP-Sequencing (ChIP-Seq) has helped in unraveling the human epigenome [47].

(1) ChIP: Chromatin immunoprecipitation, a powerful tool to analyze protein-DNA interactions allows to study the dynamics of histone methylation. The principle behind ChIP is the enrichment of the specific portion of DNA of interest (antigen) by immunoprecipitation followed by amplification of the enriched region by PCR so as to obtain sufficient quantity of the enriched fraction. Further, analysis is done by southern blotting, PCR or genome wide methods [53,54].

(2) ChIP-on-chip: There are two types of ChIP-on-chip method based on microarray contents i.e. (1) Promoter tiling arrays and (2) Genome tiling arrays. In promoter tiling arrays the probes are designed on specific genomic elements or promoters which may lead to loss of some relevant regions. The genome tiling array employs probes that cover entire genome thus, allowing global genome-wide analysis. Earlier, the ChIP-on-chip method was used to analyze histone modifications in yeast and Drosophila melanogaster. Recently, ChIP-on-chip was successful inanalysing histone modifications in human genome [29,64].

(3) ChIP-seq: Another method of analyzing histone methylation as well as chromatin remodelling is ChIP-seq which combines ChIP with next generation sequencing procedures. The ChIP-seq involves repair of DNA ends and ligation to a pair of adapters. The DNA is amplified and bound to the flow cell surface containing oligonucleotides. The adapter sequences ligated to DNA are recognized by these oligonucleotides. The genome analyzer reads each DNA sequence during solid-phase PCR processing and the resulting reads are mapped to a reference genome to find coordinates [26,63]. This technique enabled researchers to overcome low resolution and high noise problem arising from ChIP-chip technique [25].

(4) Mass Spectrometry: The mass spectrometry (MS) can be used for quantitative analysis of protein expression and differential expression of protein modifications [28,66]. Recently, to address the issue of inability of MS to map the modification patterns to specific promoter regions a combinatorial technique namely chromatin affinity purification along with MS (ChAP-MS) and Chromatin Proteomics (ChroP)/ChIP-MS) was developed. Therefore, now functionally distinct chromatin regions can be analyzed for the histone marks and binding proteins simultaneously [28,66]. Mass spectrometry strategies have been divided in to three categories on the basis of portion of histone analyzed viz. bottom-up, middle-down and top down. In traditional ‘bottom up’ approach the protease enzymes are used to cleave the target protein into smaller peptides (5–20 aa) before MS analysis. The analysis of intact protein is done by top down approach, while the middle-down approach which is a modified version of top-down method is used for the characterization of large peptides containing less than 50 N-terminal amino acid residues of histone tails [57].

(5) Chromosome Conformation Capture Technologies: Different chromosome conformation capture technologies have been developed to study three-dimensional structure of whole genome. These include chromosome conformation capture (3C), chromosome conformation capture on-chip (4C), Chromosome Conformation Capture Carbon Copy (5C), combined 3C-ChIP-cloning (6C), Genome Conformation Capture (GCC), and (ChIA-PET [37,62]. The 3C protocol involves crosslinking of cells by formaldehyde, cell lysis by hypotonic buffer and protease inhibitors, solubilization and digestion of chromatin by sodium dodecyl sulfate (SDS) followed by chromatin ligation under dilute conditions using ligase. Further, reverse cross linking and purification is done to generate 3C libraries. The 3C libraries are used to generate 5C libraries which are used to study and quantify 3D organization of chromatin at a particular locus at higher resolution and throughput [36,37].

(6) (ChIA-PET: The drawback of ChIP-chip technique is the designing of microarrays. To overcome this issue another strategy namely Chromatin Interaction Analysis by paired end tag ((ChIA-PET) a 3C based technique was developed. This strategy incorporates immunoprecipitated DNA tags cloned into a plasmid library andsequenced [45,46].

Methods to analyze methylation of RNA and ncRNA-species

N6-methyladenosine (m6A) and other RNA modifications like N1-Methyladenosine (m¹A), 2′-O-Methylation (2′OMe/Nm) and 5-Methylcytosine (m⁵C) can be analyzed by purification of RNA by established protocols and detection of the type of RNA methylation [59]. The analysis and characterization of RNA depends on various factors such as modification types abundance, and the RNA sequence [56,59]. Various approaches to understand the RNA methylation are listed below.

(1) Radioisotope incorporation assays: The radioisotope incorporation assay strategies utilize incorporating radioactive isotopes into RNA to estimate RNA methylation. The methyl donor, s-adenosyl-methionine is labeled with tritium and addition of radioactive methyl group from donor to nucleoside takes place by methyltransferase activity which is measured by scintillation [27,52,56].

(2) Thin-layer chromatography: Two dimensional thin layer chromatography is another way of identifying most of RNA modifications. Two dimensional separation of RNA disperses nucleotides across cellulose substrate as per their charge and hydrophobicity. The drawback of 2D-TLC is that it provides a general transcriptome wide methylation status.The imaging of pattern with ultraviolet light can be then done. Site-specific cleavage and radioactive labeling followed by ligation-assisted extraction and thin-layer chromatography (SCARLET) can be used to study stoichiometry when the sequence is known [49].

(3) Mass spectrometry: The nucleotides are identified on the basis of mass-to-charge ratio by a standard comparative method using MS. Though the MS approach is similar to chromatography-based protocols, it does not require radioisotope or labeling. The MS strategies involve large amount of RNA and a priori-sequence information, which is a major drawback for MS based strategies [56,60].

(4) Bisulphite sequencing: Deamination of cytosine to uracil on sodium bisulphite treatment leads to a mutation in thymine when reverse transcription takes place and depicted in the final sequencing dataset. The methylated cytosine does not undergo deamination. This is the strategy behind bisulphite sequencing and provides information about RNA methylation to single base pair resolution. The drawback in bisulphite sequencing is the requirement of large RNA quantities and resistance of neighboring modifications as well as double stranded regions to bisulphite treatment [56,67].

(5) Antibody-based methods: Commercial antibodies against methylated RNA residues e.g. m⁶A, m¹A and m⁵C are available. Moreover, antibodies specific to modifications along with NGS are being used to estimate m⁵C, m¹A and m⁶A. The most common and established strategy to map these modification is methylated RNA immunoprecipitation sequencing (MeRIP-seq) [23].N6-Methyladenosine using m⁶A Crosslinking Immunoprecipitation Sequencing (m⁶A-CLIP Seq/mi-CLIP) is immunoprecipitation based method for high resolution mapping of as small as 1μg of poly(A)-selected mRNA for m⁶A modification [32,48].

Analysis of epigenetic modulating enzymes and their functions

Most common methods to study the expression and analysis of epigenetic modulators viz. DNMTs, HDACs and MeCPs are western blotting, ELISA, immunoprecipitation (ChIP) assays or co-immunoprecipitation. The co-immunoprecipitation method is generally used to study the interactions between epigenetic modulators [34]. Further, in vivo imaging methods are being used to study HDAC inhibitor pharmacokinetics [40], HDAC direct binding [38], HDAC activity [58]; Yeh et al., 2013; [61].

Single cell epigenomics

The present knowledge of epigenetics has been derived from bulk measurements in population of cells which majorly associated it with active or repressed transcriptional states [72]. Such generalizations more often lead to an ambiguous answer of many complex biological questions. Epigenetic regulations can be more precisely studied at the single-cell level, where intercellular differences can be investigated and a deeper understanding of cellular functions and dysfunctions can be achieved [71,72]. Recent advances in single cell technologies convincingly demonstrated that seemingly homogenous cell population have difference in gene expression which can possibly be due to heterogeneity at epigenetic level. The emerging technology of single cell epigenomics is a powerful method to understand the gene regulation and associated molecular pathologies. This exciting technology may refine our existing understanding of epigenetic regulations [73]. However, the real potential of single-cell epigenetic studies can be appreciated through parallel evaluation of genomic, transcriptional, and epigenetic information. Collating different components of the epigenome into multi-omics measurements can add new layers of molecular connections among the genome and its functional output.

Methodologies for single cell epigenetic analysis

Prerequisite of single cell epigenetic analysis is isolation of single cells from culture or dissociated tissue followed by cell lysis. This can be performed by means of manual manipulations, droplet encapsulation or fluorescence-activated cell sorting (FACS). Recently, developed microfluidics systems can also be used in which cells are trapped in chambers where lysis and RNA-seq library preparation can be done subsequently [72].

DNA methylation and other modification

Today several DNA modifications—like methylation (5mC), hydroxymethylation (5hmC), and formylcytosine (5fC)—can be probed in a single-cell by sequencing at single-nucleotide resolution [71]. Initially, genome-wide 5mC measurements in a single cell were performed using reduced representation bisulfite sequencing (scRRBS) method which was based on enrichment of CpG dense regions (such as CpG islands) and restriction digestion. Though it allows the measurement of lager fraction of promotor region CpG sites, it does not cover many important regulatory regions [72]. Further, technological developments in single-cell whole-genome methylation sequencing are based on a post-bisulfite adapter-tagging (PBAT) method in which bisulfite modification is performed before library preparation.

Another innovative approach for generating single-cell libraries using microfluidics has recently emerged. As compared to other methods, this technology has significantly increased the library preparation throughput, where cell specific barcoding is also performed before pooling the adapter-tagged fragments. This technology enables methylation measurements in ∼50% of the CpG sites in single cell. This has allowed the detection of high variability between individual cell in distal enhancer methylation which is not usually captured by scRRBS. For single cell analysis of hydroxymethylated cytosine (5hmC), the currently established methods such as TET-assisted bisulfite sequencing (TAB-seq) and AbaSI (restriction enzyme) coupled with sequencing (Aba-seq) could potentially be adapted [72].

Histone modifications and transcription factor binding

Histone marks are mapped by chromatin immunoprecipitation followed by sequencing (ChIP-seq), however performing ChIP-seq at the single-cell level is very challenging [71]. The single cell ChIP-seq has the limitation of background noise of nonspecific antibody pull-down, which increases with decreasing levels of target antigen. Recently, this has been substituted by micrococcal nuclease (MNase) digestion and barcoding to be effectively performed on thousands of cells. This approach uses droplet-based microfluidics technology and processes large numbers of cells parallelly [72].

Protein–DNA interactions in single cells can be studied by DamID, where a cell line expresses low levels of a fusion protein of Escheriichia coli deoxyadenosine methylase (Dam) and the protein of interest [74]. This Dam based technique methylates DNA on adenine residues next to sites of protein binding which is further cut by the methylation-sensitive restriction enzyme DpnI and ligation of sequencing adapters. Presently, this technique is limited by poor resolution (100 kb), however future optimizations may enable mapping of transcription factor binding sites in single cells. Additionally, single-cell DamID could also be used in genome-wide analysis of histone modifications by using Dam fusion with specific histone readers or modifiers [72,74].

Chromatin structure and chromosome organization

The single cell evaluation for chromatin structure is based on the assay for transposase-accessible chromatin (ATAC-seq). This technique uses a Tn5 transposase enzyme for tagmentation which is a process of DNA fragmentation and adapter sequences attachment simultaneously. ATAC-seq gives single-cell resolution by applying a combinatorial indexing strategy, where tagmentation is carried out on 96 pools of a few thousand nuclei, and a unique barcode to every pool is introduced [72,74]. The second method for single-cell ATAC-seq has also been described by using a commercially available microfluidics device, which carry out the transposition reaction on individual cells. The resolution has been largely increased by using this combinatorial indexing method, which maps an average of 70,000 reads per cell. Ultimately, study of open chromatin genomic regions has been done in single cells by applying a DNase-seq approach to map regions that are DNaseI hypersensitive. Single cell DNase-seq provides an improved resolution of 300,000 mapped reads per cell, although with a very low mapping proficiency (2%) and even lower throughput [72].

Technological advancement has now made possible to assess chromosome conformation at the single-cell level with a HiC-based method in addition to chromatin organization. Hi-C, is considered as an extension of chromosome conformation capture (3C), which is capable of identifying genome-wide long range interactions (Berkum et al., 2010). Though single-cell HiC is presently limited in its resolution but still allows depiction of the individual chromosome organization such as compartmentalization and interchromosomal interactions [72,74].

Epigenome manipulation and editing

The epigenome of eukaryotic cells is highly complex and strongly correlated with central cellular processes. Their dysregulation manifests in aberrant gene expression and disease. The epigenome editing holds a great promise of enhancing knowledge of epigenetics in gene regulation and enabling manipulation of cell phenotype for research or therapeutic purposes [75,79]. Recent advancements in genome engineering technologies use highly specific DNA-targeting tools to precisely manipulate epigenetic changes in a locus-specific manner, generating diverse epigenome editing platforms.

Epigenetic manipulation techniques

Classic myriad of genetic techniques such as gene knockouts and individual domain deletions, point mutations, inducible expression constructs, ectopic expression of vectors, targeted knockdowns of a transcript, and various screens for gain or loss of function that manipulate genome structure or gene expression facilitate mimicking of the perturbation of discrete components of the epigenome [75]. These techniques have contributed greatly for creating the foundation of our current epigenetics knowledge. However, these methodologies also lead to global effects on the epigenome that may confound the experimental results [79].

Small-molecule inhibitors

A subset of small-molecule inhibitors that can manipulate epigenetic marks are being applied in research and as anticancer