Chromatin Signaling and Diseases

Chromatin Signaling and Diseases covers the molecular mechanisms that regulate gene expression, which govern everything from embryonic development, growth, and human pathologies associated with aging, such as cancer. This book helps researchers learn about or keep up with the quickly expanding field of chromatin signaling.

After reading this book, clinicians will be more capable of explaining the mechanisms of gene expression regulation to their patients to reassure them about new drug developments that target chromatin signaling mechanisms. For example, several epigenetic drugs that act on chromatin signaling factors are in clinical trials or even approved for usage in cancer treatments, Alzheimer's, and Huntington's diseases. Other epigenetic drugs are in development to regulate various class of chromatin signaling factors. To keep up with this changing landscape, clinicians and doctors will need to stay familiar with genetic advances that translate to clinical practice, such as chromatin signaling.

Although sequencing of the human genome was completed over a decade ago and its structure investigated for nearly half a century, molecular mechanisms that regulate gene expression remain largely misunderstood. An emerging concept called chromatin signaling proposes that small protein domains recognize chemical modifications on the genome scaffolding histone proteins, facilitating the nucleation of enzymatic complexes at specific loci that then open up or shut down the access to genetic information, thereby regulating gene expression. The addition and removal of chemical modifications on histones, as well as the proteins that specifically recognize these, is reviewed in Chromatin Signaling and Diseases. Finally, the impact of gene expression defects associated with malfunctioning chromatin signaling is also explored.

• Explains molecular mechanisms that regulate gene expression, which governs everything from embryonic development, growth, and human pathologies associated with aging
• Educates clinicians and researchers about chromatin signaling, a molecular mechanism that is changing our understanding of human pathology
• Explores the addition and removal of chemical modifications on histones, the proteins that specifically recognize these, and the impact of gene expression defects associated with malfunctioning chromatin signaling
• Helps researchers learn about the quickly expanding field of chromatin signaling

• Language: English
• Publisher: Academic Press
• Release date: Aug 6, 2016
• ISBN: 9780128026090

Book preview

Chromatin Signaling and Diseases

Editors

Olivier Binda

Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom

Martin Ernesto Fernandez-Zapico

Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN, United States

Table of Contents

Cover image

Title page

Translational Epigenetics Series

Copyright

List of Contributors

Bases of Chromatin Signaling and Their Impact on Diseases Pathogenesis

Section I. Histone Mark Writers

Chapter 1. Histone Acetyltransferases, Key Writers of the Epigenetic Language

Introduction

Functional and Mechanistic Impact of Histone Acetylation

Identification of the First Histone Acetyltransferases, a Historical Perspective

Yeast Histone Acetyltransferases Belong to Three Different Families

Three Families of Metazoan Histone Acetyltransferases and Their Roles in Animal Development

Role of Histone Acetyltransferases in the Pathogenesis of Human Diseases

Conclusions and Future Directions

List of Acronyms and Abbreviations

Glossary

Chapter 2. Impacts of Histone Lysine Methylation on Chromatin

Chromatin Regulation and Posttranslational Modifications

Histone Lysine Methyltransferases

Histone H3K4 Methylation

Histone H3K9 Methylation

Histone H3K27 Methylation

Histone H3K36 Methylation

Histone H3K79 Methylation

Histone H4K20 Methylation

Monomethylation of H4K20 Promotes Mitotic Chromatin Condensation and DNA Replication

Emerging Roles of Non-canonical Histone Lysine Methylation

Conclusions

List of Acronyms and Abbreviations

Chapter 3. The Role of Histone Mark Writers in Chromatin Signaling: Protein Arginine Methyltransferases

Introduction

General Properties of Protein Arginine Methyltransferases

Mammalian Protein Arginine Methyltransferases and Their Effects on Gene Expression

Conclusions

List of Acronyms and Abbreviations

Glossary

Chapter 4. Histone Kinases and Phosphatases

Introduction

Histone Phosphorylation: Basic Concepts

Histone Kinases in Nucleosome Packing and Assembly

Histone Kinases and Cell Division

Histone Kinases in DNA Repair and Replication

Histone Kinases and Programmed Cell Death

Histone Kinases and Transcription

Histone Phosphatases

Conclusions

List of Acronyms and Abbreviations

Glossary

Section II. Histone Mark Readers

Chapter 5. The Bromodomain as an Acetyl-Lysine Reader Domain

Lysine Acetylation and the Bromodomain

Discovery of the Bromodomain as an Acetyl-Lysine Recognition Module

Biological Functions of Bromodomain Proteins

Bromodomain Proteins in Human Disease Pathways

Small Molecular Inhibitors of Bromodomains

Conclusion

List of Acronyms and Abbreviations

Chapter 6. Chromo Domain Proteins

Introduction

The Chromo Domain Structure

Varieties of Chromo Domains

Chromo Shadow Domain

Nucleic Acid–Binding Chromo Domains

Posttranslational Modifications and Chromo Domain Transactions

Targeting Chromo Domains for Therapeutics

Chromo Domain Association With Human Disease

Conclusion

List of Acronyms and Abbreviations

Chapter 7. The Role of PHD Fingers in Chromatin Signaling: Mechanisms and Functional Consequences of the Recognition of Histone and Non-histone Targets

Introduction

The History of the Plant Homeodomain Finger

The Structural Basis of Histone Recognition

The Mechanism of Plant Homeodomain Fingers in the Combinatorial Readout of Patterns of Histone Post-Translational Modifications

Non-Histone Targets

Understanding the Role of the Plant Homeodomain Finger in the Function of Its Host Protein

Plant Homeodomain Fingers in Disease

Conclusion

Chapter 8. Tudor Domains as Methyl-Lysine and Methyl-Arginine Readers

Introduction

The Aromatic Cage: Molecular Basis of Methyl-Lysine and Methyl-Arginine Recognition

Tudor Domains Interacting With Methylated Lysine-Containing Peptides

Tudor Domains Interacting With Methylated Arginine-Containing Peptides

Conclusion

Section III. Histone Mark Erasers

Chapter 9. Histone Deacetylases, the Erasers of the Code

Introduction

Histone Modifications and Gene Transcription

Histone Deacetylase Families and Classes

Histone Deacetylases Structures and Catalytic Mechanisms

Histone Deacetylases as Modulators of the Epigenome

Histone Deacetylases Biology

Conclusion

List of Abbreviations

Chapter 10. Lysine Demethylases: Structure, Function, and Disfunction

Introduction

Families: Functional and Structural Features

Transcriptional Output and Regulation

Physiological Role

KDM-Associated Diseases

KDM Inhibitors as New Epigenetic Drugs

Conclusion

Section IV. Chromatin Signaling

Chapter 11. Variation, Modification, and Reorganization of Broken Chromatin

Introduction

Histone Modifications in DSB Repair

Histone Variants in DSB Repair

Chromatin Remodeling Enzymes in DSB Repair

Conclusion

Chapter 12. Crosstalk Between Histone Modifications Integrates Various Signaling Inputs to Fine-Tune Transcriptional Output

Introduction

Crosstalk Between Histone Modifications and Their Consequences

Histone Marks and Recruitment of Chromatin Factors

Histone Lysine Methylation and Acetylation

Histone Ubiquitination and Methylation: a Trans Effect

Histone Phosphorylation

Histone-Modifying Complexes Have Multiple Catalytic Roles and Functions

Histone Arginine Methylation

Histone Tail Cleavage Exhibits the Ultimate Irreversible Removal of Histone Modifications

Histone Modification at Enhancers

Conclusion

List of Acronyms and Abbreviations

Glossary

Chapter 13. Signaling and Chromatin Networks in Cancer Biology

Introduction

Regulation of Epithelial-Mesenchymal-Transition

Chromatin Remodeling in the Regulation of Cancer Cell Plasticity

The Epithelial-Mesenchymal-Transition Transcription Factor Machinery

The Role of Posttranscriptional Regulation of Epithelial-Mesenchymal-Transition

Perspective

Section V. Chromatin Dynamics in Normal and Disease Conditions

Chapter 14. Crosstalk Between DNA Methylation and Chromatin Structure

Introduction

Crosstalk Between DNA Methylation and Chromatin Structure

Significance of Altered DNA-Chromatin Crosstalk in Disease

Conclusions

List of Abbreviations

Chapter 15. Epigenetic Regulation of Endoplasmic Reticulum Stress

Introduction

Conclusions

Acronyms

Chapter 16. Chromatin Signaling in Aging and Cellular Senescence

Aging, Cellular Senescence, and Chromatin: an Introduction

Nucleosomal Modifications in Senescence and Aging

Histone Posttranslational Modifications During Cellular Senescence and Aging

Modulation of Lifespan by Experimental Alteration of Chromatin Modifiers in Animal Models

Age-Dependent Regulation of the Chromatin-Metabolism Connection

Effect of Telomere Shortening on Telomeric Chromatin

Conclusion

List of Acronyms and Abbreviations

Chapter 17. Chromatin Dynamics and Epigenetics of Stem Cells and Stem-Like Cancer Cells

Introduction

Nuclear Architecture

Chromatin Structure

Histone Modifications and Variants

Chromatin Remodeling

Chromatin Dynamics During Embryo Development

Chromatin Dynamics During Lineage Differentiation

Cancer Stem-Like Cells: Historical Perspective

Epigenetic Factors Regulating Tumorigenesis and Cancer Stem Cells

Histone Modifications and Variants in CSCs

Chromatin Remodeling in CSCs

Conclusion

Chapter 18. Altered Chromatin Signaling in Cancer

Introduction

Epigenetic Readers

Polycomb and Trithorax

Metabolic Regulation of Epigenetic Signaling

Conclusion

Chapter 19. Impact of Chromatin Changes in Pathogenesis of Infectious Diseases: A Pathogen View

Parasites

Yeast

Conclusion

Chapter 20. Chromatin Remodeling and Epigenetic Reprogramming in Chronic Disease and Cancer in the Liver and Pancreas

Introduction

Development of Pancreas and Liver

Epigenetics of Liver Diseases

Epigenetics of Pancreatic Diseases

Future Directions: Clinical Potential of Targeting Chromatin-Remodeling Proteins

Chapter 21. Pharmacological and Therapeutic Targeting of Epigenetic Regulators

Introduction

Targeting the DNA Methylation Pathway

The RNA World

Histone Acetylases and Deacetylases

Histone Methylases and Demethylases

Chromatin Readers

Concluding Remarks

Chapter 22. Use of Chromatin Changes as Biomarkers

Introduction

Concept 1: Altered DNA Methylation Landscapes as Biomarkers of Human Disease

Concept 2: Aberrant microRNA Expression in Disease States

Concept 3: Disease-Specific Histone Posttranslational Modifications Function as Biomarkers

Concept 4: Tissue Surrogate Epigenetic Biomarkers

Conclusion

List of Acronyms and Abbreviations

Glossary

Chapter 23. Regulation of Host Chromatin by Bacterial Metabolites

Introduction

The Commensal Bacterial Metabolome

Dietary Carcinogens Alter Chromatin

Bile Acids: Microbial Bile-ome and Nuclear Receptors

Estrogens: Microbial Estrabolome and Chromatin Modulation

Ellagic Acid and Histones Methylation

Short Chain Fatty Acids (SCFAs), G Protein–Coupled Receptors, and Histone Deacetylases

Indoles and Nuclear Receptors

Polyketides

Bacterial Nucleomodulins

Miscellaneous

Non-Mammalian Systems

Plants

Lessons to Apply from Host Intermediary Metabolism and Chromatin

Metabolite Mining for Chromatin Modulation

Conclusion

List of Acronyms and Abbreviations

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. 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-Giménez, 2015

Drug Discovery in Cancer Epigenetics

Edited by Gerda Egger and Paola Barbara Arimondo, 2015

Medical Epigenetics

Edited by Trygve O. Tollefsbol, 2016

Copyright

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List of Contributors

Kim Barroso,     Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France

Olivier Binda,     Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom

Maria Victoria Botuyan,     Mayo Clinic, Rochester, MN, United States

Anna L. Chambers,     University of Bristol, Bristol, United Kingdom

Eric Chevet,     Centre de Lutte Contre le Cancer Eugène Marquis, Rennes, France

Jocelyn Côté,     University of Ottawa, Ottawa, ON, Canada

Florence Couteau,     Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC, Canada

Jean-François Couture,     University of Ottawa, Ottawa, ON, Canada

Jessica A. Downs,     University of Sussex, Brighton, United Kingdom

Joel C. Eissenberg,     Saint Louis University School of Medicine, St. Louis, MO, United States

Maite G. Fernandez-Barrena

University of Navarra, Pamplona, Spain

University Clinic Navarra, Pamplona, Spain

Instituto de Salud Carlos III, Pamplona, Spain

Instituto de Investigación Sanitaria de Navarra (IdiSNA), Pamplona, Spain

Martin Ernesto Fernandez-Zapico,     Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN, United States

Raquel Fueyo,     Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Parc Científic de Barcelona (PCB), Barcelona, Spain

María Alejandra García,     Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Parc Científic de Barcelona (PCB), Barcelona, Spain

Alexandre Gaspar-Maia,     Icahn School of Medicine at Mount Sinai, New York, NY, United States

John Haddad,     University of Ottawa, Ottawa, ON, Canada

Nasim Haghandish,     University of Ottawa, Ottawa, ON, Canada

Elisabeth Hessmann,     University Medical Center Göttingen, Göttingen, Germany

Jonathan M.G. Higgins,     Newcastle University, Newcastle upon Tyne, United Kingdom

Ryan A. Hlady,     Mayo Clinic, Rochester, MN, United States

Timothy C. Humphrey,     University of Oxford, Oxford, United Kingdom

Steven A. Johnsen,     University Medical Center Göttingen, Göttingen, Germany

Alexander Koenig,     University Medical Center Göttingen, Göttingen, Germany

María Julia Lamberti,     National University of Río Cuarto, Rio Cuarto, Córdoba, Argentina

Sylvain Lanouette,     University of Ottawa, Ottawa, ON, Canada

Andrew Liss,     Massachusetts General Hospital, Boston, MA, United States

Gwen A. Lomberk,     Mayo Clinic, Rochester, MN, United States

Frédérick A. Mallette

Maisonneuve-Rosemont Hospital Research Centre, Montréal, QC, Canada

Université de Montréal, Montréal, QC, Canada

Sridhar Mani,     Albert Einstein College of Medicine, INC, New York, NY, United States

Marian A. Martínez-Balbás,     Instituto de Biología Molecular de Barcelona (IBMB), Consejo Superior de Investigaciones Científicas (CSIC), Parc Científic de Barcelona (PCB), Barcelona, Spain

Georges Mer,     Mayo Clinic, Rochester, MN, United States

Emma A. Morrison,     University of Iowa, Carver College of Medicine, Iowa City, IA, United States

Catherine A. Musselman,     University of Iowa, Carver College of Medicine, Iowa City, IA, United States

Sankari Nagarajan,     University Medical Center Göttingen, Göttingen, Germany

Christopher L. Pin

University of Western Ontario, London, ON, Canada

Children's Health Research Institute, London, ON, Canada

Keith D. Robertson,     Mayo Clinic, Rochester, MN, United States

Andrea Ropolo,     Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC – CONICET – Universidad Nacional de Córdoba, Córdoba, Argentina

María Roqué

National University of Cuyo, Mendoza, Argentina

National Council of Scientific and Technological Research (CONICET) Mendoza, Argentina

Natalia Belén Rumie Vittar,     National University of Río Cuarto, Rio Cuarto, Córdoba, Argentina

Güenter Schneider,     Technical University of Munich, Munich, Germany

Ana Sevilla,     The New York Stem Cell Foundation Research Institute, New York, NY, United States

Steven G. Smith,     Icahn School of Medicine at Mount Sinai, New York, NY, United States

Maria Carolina Touz,     Instituto de Investigación Médica Mercedes y Martín Ferreyra, INIMEC – CONICET – Universidad Nacional de Córdoba, Córdoba, Argentina

Raul Urrutia,     Mayo Clinic, Rochester, MN, United States

Laura Vargas-Roig,     National Council of Scientific and Technological Research (CONICET) Mendoza, Argentina

Renzo Emanuel Vera,     National University of Río Cuarto, Rio Cuarto, Córdoba, Argentina

Nikolaus A. Watson,     Newcastle University, Newcastle upon Tyne, United Kingdom

Xiang-Jiao Yang,     McGill University, Montreal, QC, Canada

Pamela Zhang,     University of Ottawa, Ottawa, ON, Canada

Ming-Ming Zhou,     Icahn School of Medicine at Mount Sinai, New York, NY, United States

Bases of Chromatin Signaling and Their Impact on Diseases Pathogenesis

O. Binda¹,  and M.E. Fernandez-Zapico²,     ¹Northern Institute for Cancer Research, Newcastle University, Newcastle upon Tyne, United Kingdom,     ²Schulze Center for Novel Therapeutics, Division of Oncology Research, Mayo Clinic, Rochester, MN, United States

Despite their fundamental importance in several human pathologies, the molecular mechanisms regulating access to genetic information remain incompletely defined. The last decade has seen the identification of three major types of factors regulating chromatin dynamics and biology. These include factors that deposit small chemical moieties on the scaffolding histone proteins that condense the genome within the nucleus of the cell and are thus referred to as writers. Complementary to writers are erasers, which are factors that remove chemical modifications from the histones. To interpret these various modifications, there is a third family of factors called readers, which physically interact with modified histones to interpret the epigenetic information. These readers recruit enzymes to open or close the structure of the genome, thereby regulating access to genetic information. Aberrant access to genetic information is involved in the development of various pathologies, including aging, diabetes, cancer, and neurological and cardiovascular disorders. The aim of this book is to highlight our current understanding of the chromatin-based mechanisms regulating the access to genetic information in the context of human pathologies.

Chromatin Structure and Dynamics

Genetic information is organized similarly to the binary code of informatic data (code lines) that is stored on disks and organized into folders, which are often encoded or packaged to decrease space requirements (Fig. 1). To access the informatic data, users utilize a disk reader and run programs, which unpack instructions specific to each program. In a similar fashion, the genetic information (code lines), encoded by the four nucleotides constituting the genome, is stored in the nucleus of the cell (disk) and organized in chromosomes (folders), which are compacted to fit within the nuclear compartment. The cellular machinery utilizes factors such as proteins called readers, which associate with the structure of the genome, and enzymatic activities (programs) to decipher the genetic information.

Figure 1  Concept of chromatin signaling.

Informational Technology (IT) data is organised in folders and stored on a disk. To access this information, the user utilise a disk reader. An analoguous process is used by cellular machinery. Specifically, genetic information is stored in the cell nucleus and organised in chromosomes with the help of histone proteins. Histone sequences protruding outside the nucleosome are modified by enzymes called writers and erasers to allow or restrain access to genetic information. These histone marks are recognised by readers, which then nucleate enzymatic activities to open or close the chromatin fiber.

In cells, the genetic material of eukaryotes is highly compacted within the nucleus. For example, the genome of a normal human diploid cell is composed of about three billion base pairs of deoxyribonucleic acid (DNA), which if put side-by-side would add up to about 1  m in length. Moreover, the whole genome is confined within a nucleus that is only 6–10  μm wide, which is about one-third of the width of a fine human hair (if we did the math right!). These strands of genomic DNA are organized in 22 pairs of autosomal chromosomes and two sexual chromosomes (46 chromosomes overall). Each chromosome is compacted as a protein-nucleic acid hybrid macro-molecule essentially composed of two copies of each core histone (H2A, H2B, H3, and H4), constituting the histone octamer, around which 146 base pairs of genomic DNA are spooled to form a nucleosome, the basic repeating unit of chromatin. The nucleosomes are spread along the length of the genome resembling a pearl necklace. In addition, the linker histone H1 interacts with the DNA spooled around the histone octamer, stabilizing the nucleosome. H1 also interacts with linker DNA between nucleosomes to promote higher order chromatin organization. Nucleosomes can also be rearranged by chromatin remodeling enzymatic activities to increase (heterochromatin) or decrease (euchromatin) the nucleosome density. Densely packed nucleosomes prevent access of DNA-binding factors to genetic elements, while loosely packed nucleosomes allow access to genetic elements [1].

Gene Expression

Due to its highly condensed nature, the inherent structure of chromatin is refractory to DNA transactions (such as transcription, replication, and repair), which require access to DNA sequences. To facilitate access to genetic information by cellular machinery, chromatin is opened and nucleosomes are displaced to form euchromatin.

Transcription factors bind to specific DNA sequences and serve as landing pads to stabilize enzymatic activities at promoters to facilitate gene expression. The transcriptional machinery is composed of general transcription factors and accessory factors. The general transcription factors (GTF) include the TATAA box binding protein (TBP), a number of TBP-associated factors, and the RNA polymerase II enzyme, which synthesize mRNA strands from the genetic information. In addition, a number of sequence-specific DNA-binding factors associate with regulatory elements of genes, such as enhancers and promoters, to activate transcription by the general machinery. The GTF are nucleated at the core promoter by TBP, while activators are found in the vicinity and facilitate the formation of the preinitiation complex (PIC) by interacting with GTF, modifying chromatin, or remodeling nucleosomes.

The GTF are essential for basal transcription, but for high level of expression and regulated activation of genes, other transcription factors are required. These can act in various ways, such as facilitating the assembly and reassembly of the PIC, histone modification to loosen chromatin compaction, and nucleosome remodeling to reposition histones along the chromatin axis to facilitate access to genetic information. Many transcriptional activation domains interact with components of the basal transcriptional machinery to activate transcription. For instance, transactivation by the transcription factor E2F1 relies on physical association of its acidic transactivation domain with TBP and TFIIH [2]. E2F1 also associates with the acetyltransferases p300/CBP, GCN5/PCAF, and TIP60 to acetylate histones H3 and H4 at target promoters and enhances transcription in late G1 following mitogenic stimulation [3].

Chromatin Signaling

The structure of the nucleosome was resolved almost two decades ago [1], and it became evident that the amino terminal portion of each histone (also called histone tails) is protruding outside of the structure, making these amino acid sequences readily available for posttranslational modifications, including arginine methylation, lysine acetylation, lysine methylation, and phosphorylation. Similar to classical cell signaling, whereby posttranslational events serve as landing pads for protein–protein interactions and signal transduction, histone modifications (also called histone marks) serve as landing pads that stabilize proteins (readers) on the chromatin. Thus, the term chromatin signaling was coined to highlight the similarity between classical signal transduction and signaling mediated by posttranslational modifications of the chromatin.

The posttranslational modifications of DNA and histones are broadly referred to as epigenetic modifications or inheritable alterations that do not change the sequence of the genome. These include lysine acetylation, lysine methylation, arginine methylation, serine phosphorylation, SUMOylation, citrullination, and ubiquitination. The enzymes that add (writers) and remove (erasers) to these modifications will be discussed in subsequent chapters, but briefly, they include lysine methyltransferases (KMT), which catalyze the transfer of methyl moieties from the methyl donor S-adenosylmethionine to the ε amine on the side chain of lysine such that the amino acid can either be mono-, di-, or trimethylated. The KMT activities are counteracted by lysine demethylases (KDM), which remove the methyl groups by either flavin-dependent demethylases or 2-oxoglutarate-dependent JmjC subfamilies, which both employ oxidative mechanisms. The outcome of lysine methylation is dependent on the state and site of methylation. For instance, trimethylation of histone H3 on lysine 4 (H3K4me3) is usually associated with actively transcribed promoters, while methylation on lysine 9 (H3K9me3) is associated with transcriptionally silenced chromatin. But, monomethylation of lysine 9 (H3K9me1) is found at transcribed genes.

Similar to KMT, histone acetyltransferases (HATs) modify the lysine side chain by transferring the acetyl moiety from acetyl-coenzyme A to the ε amine. The effect of HAT is reversed by histone deacetylases (HDAC), which include a subfamily of enzymes called sirtuins, presumably involved in aging. Unlike methylation, lysine acetylation is more straightforward; acetylated histones are found at euchromatin, and hypoacetylated histones are found at heterochromatin. Although readers that interpret acetylation states do exist, such as bromodomain proteins, it is generally accepted that the acetyl group also neutralizes the positive charge of the side chain of the lysine residues within the histone tails, therefore diminishing the interaction with the negatively charged DNA, resulting in a loosening and opening of the chromatin structure. Histone methylation occurs also on arginine residues, which can be monomethylated, or either symmetrically or asymmetrically dimethylated by arginine methyltransferases. Other modifications include phosphorylation of the histone variant H2AX, which is induced in response to damages to DNA and signals for repair. Although broadly referred to as epigenetic modifications, histone marks are not necessarily truly epigenetic in nature. At least, the mechanisms of histone marks inheritance remain poorly understood. However, there are strong links between DNA methylation and the maintenance of at least some histone marks, such as H3K9me3 and H3K27me3, thus providing an inheritance mechanism for the transmission of these histone marks.

Role of Chromatin Signaling in Human Pathologies

Aberrant access to genetic information, via chromatin modification, remodeling, epigenetic changes, and whatnot, leads to expression of genes otherwise silenced and silencing of genes that should be active. A classic example of aberrant gene expression in pathological condition was reported a long time ago. In cancer cells, tumor suppressor genes are hypermethylated, while oncogenes are hypomethylated, leading to cancer phenotypes.

A classic example of the contribution of epigenetics in human pathologies was first described 50  years ago [6] as a severe neurological disorder afflicting young females and causing mental retardation. The pathology called Rett syndrome is caused by mutation of the methyl-CpG binding protein 2 (MECP2) gene on the X chromosome [7]. Rett syndrome patients harbor symptoms including mental retardation, microcephaly, impaired social interactions, deficient communication skills, breathing abnormality, cardiac dysrhythmia, and reduced life span, all associated with mutations of the MECP2 gene. Interestingly, targeted disruption of Mecp2 in the brain or specific regions of the brain of animal models recapitulates most Rett symptoms [8], highlighting the neurological malfunctions of Mecp2 as the culprit in Rett development. Similar to MECP2, the nuclear receptor-binding SET domain family of H3K6 methyltransferases are involved in developmental defects such as Sotos (NSD1) and Wolf-Hirschhorn (NSD2) syndromes as well as several forms of cancer [9,10]. Recent studies have identified a key mutation in the histone variant H3.3  at lysine 27 (H3.3K27M) as a driver in highly aggressive pediatric gliomas [4,5,14].

Book Opening Remarks

Herein, we have compiled and organized, in a hopefully comprehensible manner, an exhaustive amount of knowledge on the current vision of the role chromatin signaling and epigenetic mechanisms play in human pathologies. A roster of internationally renowned academics from around the world will take you on a trip to the center of the cell to explore the genome, its regulation, and how human pathologies emerge from aberrant interpretation of the genetic information. Topics to be covered include histone modifying enzymes, how these marks are interpreted by readers, the crosstalk between epigenetic events, signaling pathways involving communication with chromatin, and the role these play in human pathologies ranging from aging to stem-like cancer cells. Here is a brief overview of our textbook. Enjoy!

Histone modifications such as acetylation and methylation were first detected over half a century ago [11,12]. Histone acetyltransferase activities were the histone modifying enzymes identified with the cloning of p300/CBP, a cellular factor that associates with the viral oncoprotein E1A, in the 1990s. Lysine acetylation is consequently the most studied histone posttranslational modification. It is generally accepted that negatively charged acetyl moieties neutralize the positively charged side chain of lysine residues, weakening the histone-DNA interactions, and thus the opening of the chromatin structure and activation of transcription. However, the acetyl-lysine histone marks can also serve as landing pads for histone mark readers such as proteins containing a bromodomain, which facilitate the recruitment of enzymatic activities that modulate gene expression. In the opening chapter, Chapter 1, Xiang-Jiao Yang from McGill University will enlighten us on the biochemistry of HAT activities and the various roles HAT plays in cancer, developmental biology, and neurodegenerative disorders.

Although lysine methylation was detected on histones over half a century ago, the catalytic activities that deposit the various methylation marks on histones were identified only at the beginning of the new millennium. While lysines can either be acetylated or methylated, the two modifications are quite distinct in character and function. Unlike acetylation, methylation comes in three states (ie, mono-, di-, and trimethylation). Jean-François Couture from the University of Ottawa and his colleagues provide in Chapter 2 a detailed structural perspective of the most predominant lysine methyltransferases, their activities, specificities, functions, and a brief description of the role they play in human pathologies, but mainly cancer.

Histones can be methylated on lysines, but also on the other positively charged amino acid arginine. Similar to lysine methylation, arginine methylation comes in three states, mono- and symmetrical or asymmetrical dimethylation. In Chapter 3, Jocelyn Côté from the University of Ottawa will discuss the seven protein arginine methyltransferases (PRMT) known in human cells.

Phosphorylation is a posttranslational modification that can be found on serine, threonine, and tyrosine amino acids. It is by far the most studied posttranslational modification and, with the seminal work of Tony Pawson in the 1990s on the SH2 phospho-binding module [13], it set the stage for the field of cellular signaling. In Chapter 4, Nikolaus A. Watson and Jonathan M.G. Higgins from Newcastle University will focus on the role of serine/threonine phosphorylation of histone H3 during cell cycle progression and its central function in the segregation of sister chromatids during mitosis.

Posttranslational modifications alter the physico-chemical properties of proteins by adding a charge (phosphorylation), neutralizing a charge (lysine acetylation), or increasing the size and hydrophobicity (lysine methylation) of the amino acid concerned. But, as was discovered with phosphorylation in cell signaling contexts, posttranslational modifications also facilitate protein–protein interactions, acting as molecular switches. In the following chapters, commencing with Chapter 5 on the bromodomain family of histone mark readers, Steven G. Smith and Ming–Ming Zhou from the Icahn School of Medicine at Mount Sinai will tell us how these readers recognize acetylated lysines and concretize the biological functions of this important posttranslational modification.

Back in 2000, the Thomas Jenuwein group identified the chromodomain of the heterochromatin protein HP1α as the first of an increasingly larger family of histone mark readers that specifically recognize methylated lysine. In Chapter 6, Joel C. Eissenberg from Saint Louis University will discuss the molecular mechanism utilized by chromodomains to recognize methylated lysines, about chromodomain-containing proteins, and how their normal functions are altered in pathological states.

In the previous chapter, we learned that methylated lysines are recognized by chromodomains through an aromatic cage, a feature found in many other histone mark reader domains, including the plant homeodomain (PHD). PHDs are C4HC3 zinc finger motifs that form two zinc-binding clusters and are found in hundreds of human proteins, including the inhibitor of growth (ING) family of tumor suppressors. In Chapter 7, Emma A. Morrison and Catherine A. Musselman from the University of Iowa inform us about various PHD-containing proteins and how some PHDs associate with methylated lysines, while others associate strictly with unmodified histone tails.

In Chapter 8, Maria Victoria Botuyan and Georges Mer from Mayo Clinic tell us about Tudor domains, another form of readers that interact with methylated histones.

To regulate chromatin signaling, enzymatic activities such as deacetylases and demethylases can respectively remove acetyl and methyl groups from histones. In Chapter 9, Gόenter Schneider and his colleagues from Universidad Nacional de Río Cuarto tell us about the various histone deacetylases found in eukaryotes and their roles in development.

Lysine methylation was believed for a long time to be an irreversible modification. However, about a decade ago the first enzymes that remove methyl groups from histones were identified. Chapter 10, the second chapter of the section on erasers, is presented by Marian A. Martínez-Balbás and her colleagues from the Instituto de Biología Molecular de Barcelona, who inform us about lysine demethylases and the roles they play in cancer and neurological disorders.

In the next section, we move away from predominantly biochemical aspects to look at various biological roles chromatin play in the cell. In Chapter 11, Timothy C. Humphrey (University of Oxford), Jessica A. Downs (University of Sussex), and Anna L. Chambers (University of Bristol) explain how damages to DNA are repaired in the context of chromatin.

In Chapters 12 and 14, Sankari Nagarajan and Steven A. Johnsen (University Medical Center Göttingen) and María Roqué and Laura Vargas-Roig (Universidad de Cuyo) respectively will describe how the crosstalk among histone marks or these posttranslational medications and DNA methylation help coordinate transcriptional response and how its deregulation could lead to pathological conditions like cancers.

Alexander Koenig and colleagues from the University Medical Center Göttingen will discuss in Chapter 13 how signaling networks control chromatin dynamics and impact the development of tumors, with an emphasis on pancreatic cancer and the role of epithelial to mesenchymal transition and gain of stem cell characteristics in tumor progression and prognosis. In Chapter 17, Alexandre Gaspar-Maia and Ana Sevilla from The New York Stem Cell Foundation Research Institute will describe the role of chromatin signaling in stem cell biology with a focus on the epigenetic changes during stem cell commitment and highlight the processes deregulated in stem-like cancer cells.

Endoplasmic reticulum stress and senescence are central processes regulating the pathogenesis of multiple diseases including cancer. In Chapter 15, Kim Barroso and Éric Chevet from Université de Rennes and in Chapter 16 Florence Couteau and Frédérick A. Mallette from Université de Montréal provide evidence of the epigenetic mechanisms controlling the aforementioned cellular processes.

These genetic-based drivers of cancer have been well studied, but they do not account for all of the phenotypic and molecular alterations demonstrated by cancer cells. Some tumor suppressor and oncogenes involved with tumor development have aberrant expression and function not due to genetic causes within these genes but rather due to the effects of epigenetic mechanisms regulating their expression. Andrew Liss (Massachusetts General Hospital) in Chapter 18 provides an overview of the main epigenetic pathway dysregulated in cancer development and its contribution to tumor initiation and progression.

In Chapter 19, Andrea Ropolo and Maria Carolina Touz (Universidad Nacional de Córdoba) review the latest finding on the role epigenetic changes in parasites controlling their life cycle and as well as the interaction with the host.

Maite G. Fernandez-Barrena (University of Navarra) and Christopher L. Pin (University of Western Ontario) discuss in Chapter 20 the pathway controlling nonmalignant diseases of pancreas and liver. Related to these GI-related organs, in Chapter 23, Sridhar Mani from Albert Einstein College of Medicine discusses the epigenetic changes modulated by the intestinal microbiome.

Finally, Chapter 21 (Raul Urrutia and Gwen A. Lomberk) and Chapter 22 (Ryan A. Hlady and Keith D. Robertson) discuss the translational significance of the targeting and use as biomarker of chromatin signaling pathways.

References

[1] Luger K, Mäder A.W, Richmond R.K, Sargent D.F, Richmond T.J. Crystal structure of the nucleosome core particle at 2.8 Å resolution. Nature. 1997;389(6648):251–260.

[2] Pearson A, Greenblatt J. Modular organization of the E2F1 activation domain and its interaction with general transcription factors TBP and TFIIH. Oncogene. 1997;15(22):2643–2658.

[3] Taubert S, Gorrini C, Frank S.R, et al. E2F-dependent histone acetylation and recruitment of the Tip60 acetyltransferase complex to chromatin in late G1. Mol Cell Biol. 2004;24(10):4546–4556.

[4] Schwartzentruber J, Korshunov A, Liu X.-Y, et al. Driver mutations in histone H3.3 and chromatin remodelling genes in paediatric glioblastoma. Nature. 2012;482(7384):226–231.

[5] Wu G, Broniscer A, McEachron T.A, et al. Somatic histone H3 alterations in pediatric diffuse intrinsic pontine gliomas and non-brainstem glioblastomas. Nat Genet. 2012;44(3):251–253.

[6] Rett A. On a unusual brain atrophy syndrome in hyperammonemia in childhood. Wiener medizinische Wochenschrift. 1966;116(37):723–726.

[7] Amir R.E, Van den Veyver I.B, Wan M, Tran C.Q, Francke U, Zoghbi H.Y. Rett syndrome is caused by mutations in X-linked MECP2, encoding methyl-CpG-binding protein 2. Nat Genet. 1999;23(2):185–188.

[8] Guy J, Hendrich B, Holmes M, Martin J.E, Bird A.P. A mouse Mecp2-null mutation causes neurological symptoms that mimic Rett syndrome. Nat Genet. 2001;27(3):322–326.

[9] Morishita M, di Luccio E. Cancers and the NSD family of histone lysine methyltransferases. Biochim Biophys Acta. 2011;1816(2):158–163.

[10] Li Y, Trojer P, Xu C.-F, et al. The target of the NSD family of histone lysine methyltransferases depends on the nature of the substrate. J Biol Chem. 2009;284(49):34283–34295.

[11] Allfrey V, Faulkner R, Mirsky A. Acetylation and methylation of histones and their possible role in the regulation of RNA synthesis. Proc Natl Acad Sci USA. 1964;51(5):786–794.

[12] Murray K. The occurrence of ε-N-methyl lysine in histones. Biochemistry. 1964;3:10–15.

[13] Ellis C, Moran M, McCormick F, Pawson T. Phosphorylation of GAP and GAP-associated proteins by transforming and mitogenic tyrosine kinases. Nature. 1990;343(6256):377–381.

[14] Fang D, Gan H, Lee J.H, Han J, Wang Z, Riester S.M, et al. The histone H3.3K36M mutation reprograms the epigenome of chondroblastomas. Science. June 10, 2016;352(6291):1344–1348.

Section I

Histone Mark Writers

Outline

Chapter 1. Histone Acetyltransferases, Key Writers of the Epigenetic Language

Chapter 2. Impacts of Histone Lysine Methylation on Chromatin

Chapter 3. The Role of Histone Mark Writers in Chromatin Signaling: Protein Arginine Methyltransferases

Chapter 4. Histone Kinases and Phosphatases

Chapter 1

Histone Acetyltransferases, Key Writers of the Epigenetic Language

X.-J. Yang     McGill University, Montreal, QC, Canada

Abstract

Lysine acetylation refers to the transfer of the acetyl moiety from acetyl coenzyme A to the ε-amino group of a lysine residue. Specific lysine residues, enriched at the N-terminal tails of core histones H2A, H2B, H3, and H4, are subject to this modification. Each core histone contains multiple lysine residues for acetylation, and the functional impact is site specific. Due to its polymeric nature, chromatin is a unique substrate in that locus-specific histone acetylation dictates its impact on expression of genes. This modification is reversible, and the forward reaction is catalyzed by histone acetyltransferases (HATs). Due to historical reasons, these enzymes have been known as HATs, even though they also target many nonhistone proteins. Since the initial molecular identification of HATs in the mid-1990s, a dozen human proteins have been well demonstrated to possess intrinsic HAT activities. This chapter provides an overview of histone acetylation and the well-characterized yeast and metazoan HATs, especially their molecular interaction with noncatalytic subunits within multiprotein complexes and their important roles in regulating animal development. Also included in this chapter is a survey about direct genetic links of human HATs to cancer, developmental disorders, neurodegenerative diseases, and other pathological conditions.

Keywords

Bromodomain; Chromatin structure and dynamics; Gcn5 (general control nonderepressible 5); Histone acetyltransferases (HATs); Lysine acetyltransferase; PHD and YEATS domains

Outline

Introduction

Functional and Mechanistic Impact of Histone Acetylation

Identification of the First Histone Acetyltransferases, a Historical Perspective

Identification of a Histone Deposition-Related Histone Acetyltransferase

Identification of Transcription-Related Histone Acetyltransferases

Yeast Histone Acetyltransferases Belong to Three Different Families

The GNAT Family of Histone Acetyltransferases

The MYST Family of Histone Acetyltransferases

Rtt109 as a Unique Fungal-Specific Histone Acetyltransferase

General Principles About Yeast Histone Acetyltransferases

Three Families of Metazoan Histone Acetyltransferases and Their Roles in Animal Development

The GNAT Family of Histone Acetyltransferases and Their Roles in Animal Development

p300, CBP, and Their Functions in Different Developmental Processes

MYST Proteins, Multisubunit Complexes, and Functions in Animal Development

Role of Histone Acetyltransferases in the Pathogenesis of Human Diseases

GNATs in Cancer, Neurodegenerative Disorders, and Genetic Diseases

p300 and CBP in Rubinstein–Taybi Syndrome and Different Types of Cancer

MYST Proteins in Cancer and Developmental Disorders

Conclusions and Future Directions

List of Acronyms and Abbreviations

Glossary

References

Introduction

According to the genetic code, mRNA is translated into newly synthesized proteins, composed of 20 common amino acids. Maturation of the newly synthesized proteins and functional regulation of the mature proteins require posttranslational modifications, which occur mainly in a subgroup of the 20 common amino acids, including serine, threonine, tyrosine, proline, arginine, and lysine. Among them, lysine is unique in that it is subject to multiple modifications such as acetylation, methylation, ubiquitination, sumoylation, and hydroxylation. As a result, for a lysine residue at the same position in a protein, these modifications are mutually exclusive and provide an antagonistic mechanism of regulation for different types of lysine modifications. Lysine acetyl transferase (HAT) transfers the acetyl moiety from acetyl coenzyme A to the ε-amino group of a lysine residue and converts it to acetyl-lysine (Fig. 1.1) [1,2]. As it targets the ε-amino group of the side chain, this modification is also known as N-ε-acetylation. It is different from N-α-acetylation, which is typically co-translational and targets lysine or other residues located at the amino terminus of proteins. Histone acetylation was first discovered by Allfrey and colleagues in 1964 [3], and the chemical nature was defined as N-ε-acetylation in 1968 [4]. Notably, the significance of this initial discovery was only widely recognized and subsequently proven in the 1990s [5,6]. Proteomic studies have revealed that thousands of mammalian proteins contain acetyl-lysine [5,6], indicating that the lysine acetylome is comparable to the phosphoproteome.

Figure 1.1  Cartoon illustrating acetylation and deacetylation at a lysine residue. A histone acetyltransferase (HAT) catalyzes the transfer of the acetyl moiety (in red) from acetyl-coenzyme A to the ε-group of a lysine residue, whereas a histone deacetylase (HDAC) of the Rpd3 superfamily removes the acetyl group from an acetyl lysine residue, releasing acetate. Notably, sirtuins utilize a catalytic mechanism that is completely different from what is illustrated here [5] .

In eukaryotic cells, lysine acetylation is reversible, and its level is dynamically controlled by antagonistic actions of lysine acetyltransferases and deacetylases (Fig. 1.1). A majority of these enzymes were initially identified as ones targeting histones and have thus been known as histone acetyltransferases (HATs) and deacetylases [7,8]. This chapter focuses on HATs as the histone mark writers, and the deacetylases will be discussed in Chapter 9.

Functional and Mechanistic Impact of Histone Acetylation

In eukaryotic cells, nuclear DNA is elegantly organized into arrays of nucleosomes, each of which contains 20–90  bp linker DNA and a nucleosomal core with ∼146  bp DNA wrapping around a histone octamer [9]. While histone H1 binds to the linker DNA, the histone octamer comprises two copies of core histones H2A, H2B, H3, and H4 [9]. These four core histones consist of an N-terminal tail and a C-terminal histone fold domain (Fig. 1.2) [9]. The histone fold domains have well-defined structures and are sufficient for formation of the histone octamer [9,10]. By contrast, the N-terminal tails are motile and not required for histone octamer formation [9,10]. At the amino acid sequence level, the tails are almost invariant from yeast to humans [9], suggesting the importance in vivo. Related to this, these tails are subject to diverse modifications, such as acetylation, methylation, and phosphorylation [11], indicating important regulatory roles. A systematic nomenclature system was proposed in 2005 for different histone marks and has been widely used [12]. For example, acetylation of histone H3 at K4 is referred to as H3K4ac, whereas mono-, di-, and trimethylation of this residue are denoted H3K4me1, H3K4me2, and H3K4me3, respectively. As covered in the current and other chapters in this book, different histone modifications form the very basis for chromatin signaling.

Acetylation occurs at multiple lysine residues at the N-terminal tails of core histones. For example, histone H3 is acetylated at K4, 9, 14, 18, 23, 27, and 36, whereas histone H4 is acetylated at K5, 8, 12, and 16 (Fig. 1.2). Although located within the histone fold domains, K56 of histone H3 and K91 of histone H4 are also acetylated (Fig. 1.2). Notably, functional consequences of acetylation at different lysine residues are different. For example, H3K56ac, H4K5ac, H4K12ac, and H4K91ac (Fig. 1.2) are present in newly synthesized histones and important for chromatin assembly during DNA replication and repair. By contrast, acetylation at other lysine residues (Fig. 1.2) is found exclusively in assembled chromatin. In addition to association with newly synthesized histones, H4K5ac and H4K12ac are present in assembled chromatin [13]. In a majority of cases, histone hyperacetylation is linked to transcriptionally active chromatin and leads to gene activation [11]. One exception is H4K12ac, which is sometimes associated with densely packed chromatin [13]. In addition to core histones, linker histones are acetylated, but much less is known about it. It has shown that histone H1.4 is acetylated at K34 to promote gene activation [14]. Thus, generally speaking, histone acetylation leads to gene activation.

Figure 1.2  Schematic diagram showing acetylation of histones H3 and H4. Through their histone fold domains, core histones interact with each other and form a histone octamer. The N-terminal tails of the four core histones are heavily modified by acetylation, methylation, phosphorylation (not shown here), and other modifications. Ac , acetylation; me3 , trimethylation. For simplicity, only one-half of the histone octamer and the N-terminal tails of histones H3 and H4 (but not those of histones H2A and H2B) are shown here. For some acetylation sites, specific HATs are shown.

At least four mechanisms are involved in regulating chromatin structure and activating gene expression by histone acetylation. Firstly, this modification neutralizes the positive charge of the lysine side chain, which in turn affects the interaction of histones with the negatively charged DNA backbone in chromatin [9]. This is the classical mechanism and has been frequently referred in textbooks. Secondly, acetylation makes the same residue unavailable to other histone modifications such as methylation. For example, acetylation of H3K9 and H3K27 prevents their methylation (Fig. 1.2). Thirdly, acetylation promotes incorporation of specific histone variants into nucleosomes. For example, while H4K16ac stimulates formation of nucleosomes containing the histone H2A variant H2A.Z [15] and the histone H3 variant H3.3 [16], H3K56ac stimulates deposition of H2A.Z into chromatin by an adenosine triphosphate (ATP)-dependent chromatin-remodeling complex [17]. Both histone variants are frequently associated with transcriptionally active chromatin. Finally, acetylation generates specific docking sites for structural modules such as the bromodomain (∼110-residue domain originally identified in Drosophila brahma) [18], PHD (plant homeodomain-linked) zinc finger [19], and YEATS (Yaf9, Enl, Af9, Taf14, and Sas5) domain [20] (Fig. 1.3). These domains, in turn, promote the recruitment of chromatin modifiers and ATP-dependent chromatin remodelers to open up densely packed chromatin, thereby increasing the accessibility to the general transcription machinery and RNA polymerases for transcriptional activation (discussed in detail in Chapter 5). These four mechanisms should also apply to other chromatin-templated nuclear processes, such as DNA replication, repair, and recombination.

Identification of the First Histone Acetyltransferases, a Historical Perspective

The initial report of histone acetylation and subsequent definement of its chemical nature as lysine acetylation [3,4] spurred ensuing research interests in histone acetylation and the responsible enzymatic activities. In the 1970s, 1980s, and early 1990s, histone acetylation was the most widely studied histone modification. In 1992, immunostaining with antibodies specific to histone H4 isoforms acetylated at K5, K8, K12, or K16 identified interesting patterns on polytene chromosomes from Drosophila larvae [13]. H4K5ac and H4K8ac are distributed in overlapping bands throughout euchromatic chromosome arms, whereas H4K12ac is enriched in heterochromatin. H4K16ac is present at numerous sites on the transcriptionally hyperactive X chromosome in male larvae, but not on any X chromosomes in female cells [13]. These results provided correlative support for the role of histone acetylation in gene regulation. However, molecular identity of the responsible enzymes remained elusive and hindered the efforts to test this important link directly.

Figure 1.3  Schematic representation showing that acetylation generates specific docking sites for different protein modules including the bromodomain, PHD fingers, and YEATS domain. In addition to the acetyl-lysine residue shown here, the surrounding sequence controls the binding specificity, so acetylation is necessary but not sufficient for specific recognition by the protein domains.

Biochemical fractionation experiments in various laboratories concluded that there are two types of acetyltransferases: one acetylates newly synthesized histones H3 and H4 in the cytoplasm for histone deposition during chromatin assembly, whereas the other is nuclear and transcription related [21]. That set the stage for answering a key question in the chromatin field: which proteins are responsible for histone acetylation? The question was important because the molecular identification of such proteins would make it possible to directly investigate functions of these enzymes in regulating chromatin organization and gene expression. As described in the following brief historical account about the identification of two types of HATs involved in chromatin assembly and gene regulation, the answer to this important question was finally obtained in the mid-1990s. However, that was three decades after the initial discovery of histone acetylation in 1964 [3], thereby illustrating nicely that a scientific breakthrough takes years and sometimes decades of persistent research efforts involving many investigators in different laboratories. In addition, model organisms, such as the budding yeast Saccharomyces cerevisiae (which is the one used for wine making, baking, and brewing) and the ciliated protozoan Tetrahymena, made key contributions. The stories involved also reiterate the importance of basic fundamental research in leading the way to subsequent translational research for developing drugs to treat human diseases.

Identification of a Histone Deposition-Related Histone Acetyltransferase

The first acetyltransferase responsible for histone acetylation was not cloned until 1995. Using histone H4 peptides (containing the N-terminal 21 or 28 residues) as substrates, Sternglanz and colleagues screened acetyltransferase activity in fractionated protein extracts from a collection of 250 mutant strains of the budding yeast [22]. Luckily, 1 of the 250 mutant strains showed ∼40% reduction in one particular protein fraction, leading to the cloning of Hat1 (histone acetyltransferse 1) [22]. Importantly, bacterially expressed Hat1 protein showed acetyltransferase activity, supporting that the observed enzymatic activity is intrinsic to Hat1; consistent with this, Hat1 possesses two signature motifs for acetyl coenzyme A binding. Moreover, Hat1 targets histone H4K12 [22], a histone mark known to be important for histone deposition during chromatin assembly [23], suggesting that Hat1 regulates this nuclear process. In October of 1996, Gottschling and colleagues performed biochemical purification of a major cytoplasmic HAT activity from the budding yeast and identified a complex containing two subunits, Hat1 and Hat2 [24]. Interestingly, Hat2 is a noncatalytic subunit containing WD40 (dipeptide WD-containing ∼40-residue motif) repeats and able to stimulate the acetyltransferase activity of Hat1 toward H4K12 [24]. Although H4K12 is the preferred site, Hat1 alone also targets H4K5; association with Hat2 makes Hat1 almost exclusively active toward H4K12 [24]. This was the first example that a noncatalytic subunit interacts with an HAT and regulates its enzymatic activity and substrate specificity. Also in October 1996, Stillman's group showed that human HAT2, a homolog of yeast Hat2, is a subunit of a multiprotein complex required for chromatin assembly and that this complex contains histone H4 acetylated at K5, K8, and K12 [25]. Together, these studies established that Hat1 forms a heterodimeric complex with Hat2 for regulating specific acetylation of histone H4 and controlling histone deposition in chromatin assembly.

Identification of Transcription-Related Histone Acetyltransferases

While important for deposition of newly synthesized histones into chromatin, the discovery of Hat1 did not provide support for the potential link of histone acetylation to gene regulation, as was initially proposed in 1964 [3]. Different from the approaches leading to the Hat1 discovery, Allis and colleagues sought to purify a transcription-associated HAT. For this, they utilized protein extracts prepared from macronuclei of the protozoan Tetrahymena. As the expression level of such an HAT was considered to be very low, Allis and colleagues developed an in-gel acetyltransferase activity assay to facilitate the purification. With this assay, they identified a 55-kDa protein in partially purified protein fractions [26]. Subsequent cloning of the 55-kDa protein revealed an unexpected homology to yeast Gcn5 (general control nonderepressible 5) and identified two acetyl coenzyme A-binding signature motifs with similarity to those in Hat1 [27]. Reported in March 1996, this unexpected discovery of Tetrahymena Gcn5 as an HAT was exciting because its yeast homolog had been shown to be important for gene activation [28,29]. Moreover, Gcn5 is highly homologous to two human proteins [30,31]. In July 1996, Nakatani and colleagues reported that both human proteins possess intrinsic acetyltransferase activity toward histone H3 and proposed that targeted histone acetylation at specific chromatin loci leads to gene-specific transcriptional activation [31]. Two months later, Allis' group described that yeast Gcn5 acetylates histone H3 at K14 [32]. This is in stark contrast to Hat1, which acts upon histone H4 at K12, and to a lesser extent, at K5. Together, these studies established that Gcn5 and its mammalian homologs are HATs with a direct role in gene regulation.

The exciting link of histone acetylation to gene regulation received further support from at least three other lines of research published in the same year. Firstly, using a specific histone deacetylase inhibitor identified by the Yoshida group [33], Schreiber's group affinity-purified histone deacetylase 1 and showed, in April 1996, that it is homologous to yeast Rpd3 (reduced potassium dependency 3) [34]. This echoed the excitement in the discovery of Gcn5 as an HAT because Rpd3 was known to regulate gene expression [35]. Moreover, Seto and colleagues reported in 1996 that an Rpd3 mammalian homolog interacts directly with the transcriptional repressor YY1 (Yin-Yang 1) and represses transcription [36], supporting that targeted histone deacetylation leads to gene-specific transcriptional repression.

Secondly, two research groups described a family of putative HATs in September 1996. While performing genetic screening for yeast mutant strains with epigenetic silencing defects, Pillus and colleagues identified Sas2 (something about silencing 2) and Sas3 as two homologous proteins important for gene silencing [37]. Independently, Housman's group characterized the balanced chromosome translocation t(8;16)(p11;p13), associated with acute myeloid leukemia, and identified two genes located at the breakpoints [38]. The gene on chromosome 8p11 encodes a novel protein named MOZ (monocytic leukemia zinc finger protein), whereas the one on chromosome 16p13 encodes CBP [cyclic adenosine monophosphate (cAMP) response element-binding (CREB)-binding protein] [38]. Importantly, sequence comparison of Sas2, Sas3, and MOZ led to identification of a homologous domain [37,38]. This domain also displays sequence similarity to TIP60 [trans-activator of transcription (Tat)-interactive protein of 60  kDa], which was reported a few months earlier and shown to act as a coactivator for HIV Tat-dependent transcription [39]. This homologous domain was thus named the MYST (MOZ, Ybf2/Sas3, Sas2, and TIP60) domain [37,38]. Strikingly, it possesses a recognizable sequence block with similarity to one of the two acetyl coenzyme A-binding signature motifs that Hat1 and Gcn5 share [37,38]. Based on this, Pillus, Housman, and their colleagues proposed that MYST domain proteins form a new family of HATs [37,38].

Finally, Nakatani's and Kouzarides' groups demonstrated in November and December 1996, respectively, that p300 and CBP are also HATs [40,41]. Different from yeast Gcn5 and its mammalian homologs, p300 and CBP are promiscuous and acetylate all four core histones [40,41]. Histones H4, K5, 8, 12, and 16 are acetylated by CBP [40]. Intriguingly, neither p300 nor CBP display any sequence similarity to Hat1 and Gcn5, indicating that p300 and CBP form a new family of HATs. These discoveries were exciting because just a few years earlier, Goodman's and Livingston's laboratories had established that p300 and CBP function as two paralogous transcriptional coactivators for DNA-binding transcription factors such as CREB [42,43]. In response to stimuli by different hormones and neurotransmitters, the classical second messenger cAMP activates cAMP-dependent protein kinase (also known as protein kinase A), which in turn phosphorylates CREB at a single conserved serine residue to promote interaction with CBP and stimulate transcription [42]. In fact, CREB was identified as a transcription factor required for regulating the expression of the important peptide hormone somatostatin [44]. Moreover, cAMP-responsive elements were shown to be crucial for long-term memory formation in Aplysia, an extremely large sea slug [45]; this and related studies about the molecular basis of memory would eventually lead to a Nobel prize to be awarded to Eric Kandel in 2000. Thus, CBP was considered to be a novel signal-dependent transcriptional coactivator, important in diverse cellular processes. Different from CBP, p300 was identified as a cellular protein targeted by the adenoviral oncoprotein E1A [43]. Furthermore, the CBP gene was known to be mutated in Rubinstein–Taybi syndrome [46] and be rearranged in t(8;16)(p11;p13) associated with acute myeloid leukemia [38]. Together, these results not only strengthened the link of histone acetylation to gene regulation, but also made a direct link of abnormal histone acetylation to genetic disease and cancer. Along with the initial discoveries of Hat1 as a cytoplasmic HAT and Tetrahymena Gcn5 and its mammalian homologs as transcription-related HATs, the identification of an Rpd3 human homolog as the first histone deacetylase, MYST proteins as putative HATs and the known transcriptional coactivators p300 and CBP as HATs ushered in a new era of histone acetylation research, which has subsequently established this modification as a major histone mark for chromatin signaling [11,47]. In addition, these initial studies spurred research interests in modification of nonhistone proteins by lysine acetylation [48,49]. It is now known that lysine acetylation is as important as, if not more so than, other major posttranslational modifications such as phosphorylation, ubiquitination, and sumoylation [5,6].

Yeast Histone Acetyltransferases Belong to Three Different Families

Since 1996, various proteins have been discovered to possess HAT activity. In budding yeast, in addition to Hat1, Gcn5, Sas2, and Sas3, four other proteins were shown to have such activity: Esa1 (essential Sas2-related acetyltransferase 1) [50], Elp3 (elongator protein 3) [51,52], Eco1 (establishment of cohesion 1) [53], and Rtt109 (regulator of Ty1 transposition gene product 109) [54–58]. Thus, there are eight yeast proteins with relatively well-characterized HAT activity (Table 1.1). Among them, Elp3 and Eco1 share sequence motifs with Gcn5 and Hat1, so these four proteins are grouped into the GNAT (Gcn5-related N-acetyltransferase) superfamily. Esa1 is highly homologous to Sas2 and Sas3, so they form the MYST family in the budding yeast. They also share a classical acetyl coenzyme A-binding motif with GNATs, so strictly speaking, these three MYST proteins also belong to the GNAT superfamily; however, because of their distinct MYST domains, these three proteins have been considered to form an independent family. The fungal-specific protein Rtt109 is unique and shows no sequence similarity to GNATs or MYST proteins, so it forms a third family [54–58].

The GNAT Family of Histone Acetyltransferases

As described above, Hat1 forms a heterodimeric complex with Hat2, and acting as a noncatalytic subunit, Hat2 regulates the catalytic properties such as enzymatic activity and substrate specificity of Hat1 [59]. Hat2 binds to histone H3 in an H3/H4 heterodimer and facilitates acetylation of histone H4 at K12 by Hat1 [60]. Similar themes about complex formation and subunit–subunit interaction frequently occur with other HATs. Of relevance, Gcn5 forms two distinct complexes with multiple subunits [61]. While Gcn5 acetylates free histone H3 but not nucleosomal histone H3, its complexes are able to acetylate nucleosomal histone H3 efficiently [61], reiterating that noncatalytic subunits regulate enzymatic properties of the catalytic subunit. No evidence indicates that Eco1 forms a multisubunit complex. Different from Hat1 and Gcn5, Eco1 acetylates a cohesin subunit, instead of histones, to regulate sister chromatid separation. Elp3 is a subunit of the elongator complex important for transcriptional elongation and has been shown to acetylate histones H3 and H4 [52]. Unexpectedly, Elp3 also catalyzes tRNA modification in the anticodon [62], raising the unaddressed question whether this enzyme has dual roles in histone acetylation and tRNA modification in vivo.

Table 1.1

Comparison of Human HATs With Their Budding Yeast Orthologs

Note: RTS, Rubinstein–Taybi syndrome; ?, no KAT name has been given.

Like Hat1 and Gcn5, all three MYST proteins form multisubunit complexes. Sas2 is the catalytic subunit of a trimeric complex, and the two noncatalytic subunits are required for Sas2 to efficiently acetylate histone H4 at K16 [63]. Deletion of the Sas2 gene greatly reduces the H4K16ac level, indicating that Sas2 is the major HAT targeting H4K16 [64]. Interestingly, H4K16 acetylation by Sas2 serves as a histone mark to prevent the spreading of heterochromatin through mechanisms that remain to be fully characterized [64,65]. This modification promotes incorporation of the histone H2A variant H2A.Z into chromatin at subtelomeric regions [15] and is also associated with nucleosomes containing the histone H3 variant H3.3 [16]. Together, these studies provide molecular insights into how Sas2 demarcates the boundary between transcriptionally active euchromatin and transcriptionally inert heterochromatin.

The MYST Family of Histone Acetyltransferases

Sas3 forms a multisubunit complex and targets H3K14 [66,67]. Genetic studies in the budding yeast S. cerevisiae and the fission yeast Schizosaccharomyces pombe revealed that Sas3 is important for acetylating H3K14 [66,68]. One subunit possesses a proline-tryptophan-tryptophan-proline tetrapeptide ​(PWWP)-containing domain for recognizing H3K36me and recruiting the Sas3 complex to chromatin [67]. In addition to Sas3, Gcn5 contributes to H3K14 acetylation in vivo [66,68]. Different from Sas2 and Sas3, Esa1 mainly acetylates histones H2A and H4 [50]. For histone H4, Esa1 acetylates K5, 8, 12, and 16 [50]. Esa1 is unique in that it is essential for yeast growth [50]. This is different from Sas2, Sas3, Hat1, and Gcn5, which are not essential for yeast survival. Esa1 is the catalytic subunit of a 13-protein complex important for transcriptional regulation [69]. In addition, a trimeric submodule contains a chromodomain to recognize H3K36me3, which recruits the Esa1 complex to travel with elongating RNA polymerase II and regulate nucleosome recycling during transcriptional elongation [70]. Thus, Esa1, Sas2, and Sas3 have distinct functions in vivo.

Rtt109 as a Unique Fungal-Specific Histone Acetyltransferase

As the sole member of the third family, Rtt109 is crucial for yeast survival when exposed to DNA-damaging agents [54–58]. As discussed with most members of the GNAT and MYST families, Rtt109 forms complexes with noncatalytic subunits [55–58]. Two histone chaperones bind Rtt109 to form distinct complexes, one targeting H3K56 and the other recognizing H3K9 and H3K27 [55–58,71]. It is H3K56ac that is essential for DNA-damaging response [72,73]. As shown in Fig. 1.2, different from many other histone acetylation sites, this histone mark is located within the histone fold domain. H3K56 is able to form an electrovalent bond with the phosphate group of the DNA in the nucleosomal core, and acetylation may disrupt the formation of this bond [72,73]. A study indicated that H3K56ac enhances the binding to the double pleckstrin homology (PH) domain of Rtt106 (regulator of Ty1 transposition gene product 106), a fungal-specific histone chaperone that escorts newly synthesized H3 and H4 histones into chromatin during DNA replication and transcription [74], indicating that this histone mark forms a specific docking site for the PH domain of Rtt106 to promote chromatin assembly.

General Principles About Yeast Histone Acetyltransferases

From the above description, it is clear that there are at least five principles that can be generalized from studies of yeast HATs. First, in the budding yeast, HATs are divided into three families: GNATs, MYST proteins, and Rtt109 (Table 1.1). Second, members of these three families have different functions (ie, clear division of labor) to maintain histone acetylome in this unicellular model organism (Fig. 1.2). Third, acetylation of lysine residues at different positions on histones has distinct