Current Developments in Biotechnology and Bioengineering: Functional Genomics and Metabolic Engineering

Current Developments in Biotechnology and Bioengineering

Functional Genomics and Metabolic Engineering

Editors

Paramasamy Gunasekaran

Santosh Noronha

Ashok Pandey

Table of Contents

Cover image

Title page

Copyright

List of Contributors

About the Editors

Preface

Part 1. Functional Genomics

1. Functional Epigenomics

1.1. Introduction

1.2. Chromatin: The Epigenetic Center

1.3. The Epigenetic Machinery

1.4. Epigenetics in Diseases

1.5. Functional Epigenomics—The Definition

1.6. Approaches to Reading the Alternate Genomic Code

1.7. Modern Methods to Manipulate the Epigenome

1.8. Conclusion

2. Functional Metagenomics: Exploring Nature's Gold Mine

2.1. Introduction

2.2. A Two-Way Approach

2.3. Metagenomic DNA Recovery

2.4. Functional Metagenomics: Toward Community Understanding

2.5. Major Discoveries

2.6. Summary and Future Perspective

3. Functional Genomics of the Extremophilic Bacteria and Archaea

3.1. Introduction

3.2. Comparative and Functional Genomics of Thermophilic Bacteria

3.3. Comparative and Functional Genomics of Psychrophilic Bacteria

3.4. Comparative and Functional Genomics of Acidophilic Bacteria

3.5. Comparative and Functional Genomics of Alkaliphilic Bacteria

3.6. Comparative and Functional Genomics of Radiation-Tolerant Bacteria

3.7. Functional Genomics Versus Traditional Gene-by-Gene Approach

3.8. Comparative Genomics, Proteomics, and Metabolomics

3.9. Conclusions

4. Functional Genomics of Riboflavin Transport: Genes and Regulatory Mechanisms

4.1. Introduction

4.2. Chromosomal Location and Genome Organization of Riboflavin Transporters

4.3. Transport Mechanism and Regulation

4.4. Pathophysiology

4.5. Conclusion

5. Functional Genomics of MicroRNAs

5.1. Introduction

5.2. MicroRNAs—Biogenesis and Activity

5.3. Screening of MicroRNA Expression

5.4. MicroRNAs as Biomarkers

5.5. MicroRNA Therapeutics

5.6. MicroRNAs as Tools for Generating Transgenic Plants

5.7. Future Prospects

6. Functional Genomics of Pathogenesis

6.1. Introduction

6.2. Array-Based Approaches

6.3. Mutagenesis-Based Approaches

6.4. RNA Sequencing in Microbial Pathogenesis

6.5. Proteome Analysis

6.6. Databases, Computational Tools, and Comparative Analysis

6.7. Future Perspective

7. Next-Generation Sequencing Methods

7.1. Introduction

7.2. Methods of Next-Generation Sequencing

7.3. Comparison of Sequencing Methods and Their Applications

7.4. Future Perspectives

Part 2. Metabolic Engineering

8. In Silico Approaches to Metabolic Engineering

8.1. Introduction

8.2. Kinetic Modeling

8.3. Constraint-Based Analysis Methods

8.4. Methods Based on Graph-Theoretic/Network Analysis

8.5. Challenges and Future Perspectives

9. Building Metabolic Models From First Principles

9.1. Introduction

9.2. Building a Constraint-Based Model

9.3. Solving a Constraint-Based Model

9.4. Limitations of Constraint-Based Models

9.5. A Phylogeny of Model Methods

9.6. A Few Applications in Escherichia coli and Other Organisms

9.7. Conclusion

10. Redesigning Cofactor Availability: An Essential Requirement for Metabolic Engineering

10.1. Introduction

10.2. Green Chemistry Methods for Cofactor Regeneration

10.3. Cofactor Balance at the Genome Level

10.4. Case Study of Use of Multiple Strategies for Cofactor Regeneration for Similar Bioprocesses

10.5. In Silico Design Approaches to Altering the Cofactor Specificity of Single Enzymes and Rerouting or Redesigning Altered Pathways

10.6. Cofactor Engineering as a Tool for Flux Redistribution

10.7. Future Prospects

10.8. Conclusion

11. Sugar Co-utilization in Microorganisms

11.1. Introduction

11.2. Sequential Utilization of Mixed Sugars and Issues

11.3. Strategies for Co-utilization of Mixed Sugars in Various Microorganisms

11.4. Conclusion

12. Metabolic Engineering of Saccharomyces cerevisiae for Synthesis of Ephedrine Alkaloids

12.1. Introduction

12.2. Scope of Metabolic Engineering for Ephedrine Synthesis

12.3. Microbial Consortium

12.4. Use of Synthetic Biology Tools for the Assembly of the Pathway

12.5. Conclusions

Index

Copyright

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List of Contributors

P.K. Agarwal,     Gennova BioPharmaceuticals, Pune, Maharasthra, India

B. Ashokkumar,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

A. Badri,     Indian Institute of Technology Madras, Chennai, Tamil Nadu, India

D. Choudhury,     Indian Institute of Technology Bombay, Mumbai, Maharasthra, India

A. Dasgupta,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

K. Gandhimathi,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

P. Gunasekaran,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

A.H. Iyer,     Indian Institute of Technology-Kanpur, Kanpur, Uttar Pradesh, India

Kirti Jain,     Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

Kunal Jain,     Sardar Patel University, Vallabh Vidyanagar, Gujarat, India

M. Jaya,     PSG Institute of Medical Sciences and Research, Coimbatore, Tamil Nadu, India

J. Johnson,     Sardar Patel University, Vallabh Vidyanagar, Gujarat, India

K. Krishnan,     Tamil Nadu Agricultural University, Coimbatore, Tamil Nadu, India

D. Madamwar,     Sardar Patel University, Vallabh Vidyanagar, Gujarat, India

S. Majumdar,     Indian Institute of Technology-Kanpur, Kanpur, Uttar Pradesh, India

D. Mehta,     University of Delhi South Campus, New Delhi, India

S.B. Noronha,     IIT Bombay, Mumbai, Maharasthra, India

M.K. Prajapat,     Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

J. Rajendhran,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

T. Rajesh,     The University of New South Wales, Sydney, NSW, Australia

K. Raman,     Indian Institute of Technology Madras, Chennai, Tamil Nadu, India

S. Ramasamy,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

J. Ranjani,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

S. Saini,     Indian Institute of Technology Bombay, Mumbai, Maharashtra, India

M. Saravanan,     Indian Institute of Technology-Kanpur, Kanpur, Uttar Pradesh, India

K. Satpute,     IIT Bombay, Mumbai, Maharasthra, India

T. Satyanarayana,     University of Delhi South Campus, New Delhi, India

P. Singh,     Indian Institute of Technology-Kanpur, Kanpur, Uttar Pradesh, India

A. Sivakumar,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

A. Srinivasan,     Indian Institute of Technology Madras, Chennai, Tamil Nadu, India

G.A. Swaminathan,     Embio Limited, Mumbai, Maharasthra, India

T. Udhayabanu,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

V. Uppada,     Guru Ghasidas University, Bilaspur, Chattisgarh, India

P. Varalakshmi,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

G. Velmurugan,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

J.C. Yacob,     Madurai Kamaraj University, Madurai, Tamil Nadu, India

About the Editors

P. Gunasekaran

Professor P. Gunasekaran is a senior professor of Microbiology in India. He has 33  years of teaching and research experience in microbiology, biotechnology, and genomics and is currently guiding eight Ph.D. students at MKU. He has published 150 research articles in national and international journals with a cumulative impact factor of 250, and his research articles have been cited 1923 time in various journal articles with an h-index of 23 and i10 index of 50. In addition, he has contributed 28 book chapters to books published by leading publishers. Professor Gunasekaran has presented his research findings at more than 200 national and international conferences and serves as the editor of three international journals: Applied Biotechnology and Biotechnology (USA), Journal of Microbiology and Biotechnology (South Korea), and Indian Journal of Microbiology (India). In addition, he has served as a guest editor of special issues in leading Indian journals such as Current Science, Journal of Scientific Research, Indian Journal of Biotechnology, and Indian Journal of Experimental Biology. He has received several medals, awards, and honors in India and abroad for his immense research contributions. Major recognitions include an Outstanding Service Award from the International Board of the American Society for Microbiology—2011.

Santosh Noronha

Dr. Santosh Noronha is a biochemical engineer by training who has evolved multidisciplinary interests. He has focused on understanding various metabolic and regulatory aspects of microbial systems, toward rationally manipulating their productivity for the production of therapeutics. He is an assistant professor in the Department of Chemical Engineering, Indian Institute of Technology, Mumbai, India.

Ashok Pandey

Professor Ashok Pandey is Eminent Scientist at the Center of Innovative and Applied Bioprocessing, Mohali (a national institute under the Department of Biotechnology, Ministry of Science and Technology, Government of India), and former chief scientist and head of the Biotechnology Division at the CSIR’s National Institute for Interdisciplinary Science and Technology at Trivandrum. He is an adjunct professor at Mar Athanasios College for Advanced Studies Thiruvalla, Kerala, and at Kalasalingam University, Krishnan Koil, Tamil Nadu. His major research interests are in the areas of microbial, enzyme, and bioprocess technology, which span various programs, including biomass to fuels and chemicals, probiotics and nutraceuticals, industrial enzymes, solid-state fermentation, etc. He has more than 1100 publications and communications, which include 16 patents, 50+ books, 125 book chapters, and 425 original and review papers, with an h index of 75 and more than 23,500 citations (Google Scholar). He has transferred several technologies to industries and has been an industrial consultant for about a dozen projects for Indian and international industries.

Professor Pandey is the recipient of many national and international awards and fellowships, which include Elected Member of the European Academy of Sciences and Arts, Germany; Fellow of the International Society for Energy, Environment and Sustainability; Fellow of the National Academy of Science (India); Fellow of the Biotech Research Society, India; Fellow of the International Organization of Biotechnology and Bioengineering; Fellow of the Association of Microbiologists of India; honorary doctorate degree from the Université Blaise Pascal, France; Thomson Scientific India Citation Laureate Award, United States; Lupin Visiting Fellowship; Visiting Professor at the Université Blaise Pascal, France, the Federal University of Parana, Brazil, and the École Polytechnique Fédérale de Lausanne, Switzerland; Best Scientific Work Achievement Award, Government of Cuba; UNESCO Professor; Raman Research Fellowship Award, CSIR; GBF, Germany, and CNRS, France fellowships; Young Scientist Award; and others. He was chairman of the International Society of Food, Agriculture and Environment, Finland (Food & Health) during 2003–04. He is the Founder President of the Biotech Research Society, India (www.brsi.in); International Coordinator of the International Forum on Industrial Bioprocesses, France (www.ifibiop.org); chairman of the International Society for Energy, Environment & Sustainability (www.isees.org); and vice president of the All India Biotech Association (www.aibaonline.com). Professor Pandey is editor-in-chief of Bioresource Technology, Honorary Executive Advisor of the Journal of Water Sustainability and Journal of Energy and Environmental Sustainability, subject editor of the Proceedings of the National Academy of Sciences (India), and editorial board member of several international and Indian journals, and also a member of several national and international committees.

Preface

This is the second volume in a series being brought out by Elsevier on Current Developments in Biotechnology and Bioengineering (Editor-in-Chief: Ashok Pandey), and it covers advances in the areas of functional genomics and metabolic engineering. It seeks to address and survey frontier research issues that have an impact on our understanding of cellular and genetic regulation, with the ultimate intent of understanding and implementing rational strain-level interventions that result in improved industrial bioprocesses. Students looking to enter into this endeavor will find substantially detailed material that helps them come up to speed; more experienced practitioners in the field will find this to be a convenient reference source. In this context, this book seeks to bridge the gap between introductory textbooks at one extreme and original research articles at the other.

There are 12 chapters presented in this volume, in two sections; a brief overview of these contributions is provided below. The first section provides surveys of work in the domain of functional genomics. In the first contribution, Majumdar et al. survey aspects of functional epigenetics, including its involvement in several diseases, and suggest approaches toward the characterization of epigenetic changes on a genome. This sets up the intriguing possibility of the manipulation of the epigenome itself. The second chapter by Johnson et al. surveys advances made in the domain of functional metagenomics. In addition to massively parallel sequencing of metagenomes, the customization of other omics technologies now offers the potential for identification of individual traits of interest.

Mehta and Satyanarayana provide an overview of the application of functional genomics toward providing a detailed understanding of the evolution, physiology, and adaptation of extremophilic bacteria. Udhayabanu and others provide, in the next chapter, a review of the functional genomics of the transport of riboflavin, an essential micronutrient. A description of how riboflavin transporter malfunction arises and the resultant clinical indications is also provided.

Velmurugan and others next survey the biology of microRNAs (miRNAs); these small single-stranded RNA molecules play a significant role in regulation by facilitating posttranslational silencing. This promises to be a valuable approach for the development of low-cost diagnostics, as well as for therapeutic intervention in human disease conditions. In the next contribution, Ranjani and coworkers look into the application of functional genomics techniques for elucidation of pathogenesis mechanisms and in particular the nature of host–pathogen interactions. The set of known pathogen genomes is limited; transcriptome and proteome analyses provide leads relevant to the inference of relevant signaling pathways. The applicability of transposon mutagenesis approaches toward the identification of host–pathogen interactions and genes associated with infection is also discussed. In the final contribution in this section, Rajesh and Jaya discuss advances in next-generation sequencing technologies.

The second section covers aspects of computational approaches to metabolic engineering and of a few case studies reviewing experimental efforts recently described in the field. Badri and coworkers start this section with a review of in silico approaches that have been deployed; these methods attempt to predict targets for rational strain development by combining network-based information with kinetic and thermodynamic constraints. Jain and others next elaborate further on constraint-based model development and identify applications of such approaches in studies on metabolism, evolution, and drug discovery. In the next chapter, Uppada et al. survey various approaches toward ensuring the availability of cofactors in synthetic engineering efforts: efficient regeneration of cofactors is critical for cyclic and continuous use of enzymes, particularly when the metabolic engineering effort is directed toward the synthesis of metabolites themselves as products, or in biotransformations.

Cofactor optimization concepts and examples of their successful implementation are also discussed. Choudhury and Saini next review aspects of sugar co-utilization in microorganisms. This issue is of immense importance given the need to efficiently utilize all sugars present in hydrolyzed lignocellulosic biomass toward ensuring the cost-effectiveness of schemes being developed for the production of biofuels and other platform chemicals. The major efforts are oriented toward deregulating the extensive carbon catabolite repression mechanisms that industrially preferred hosts have evolved. A specific challenge remains the co-utilization of arabinose and xylose, both pentoses, with glucose. In the final contribution, Uppada and coworkers describe efforts that have been taken toward the engineering of efficient synthesis pathways for the production of ephedrine alkaloids in Saccharomyces cerevisiae. This process, for the synthesis of what is a chiral intermediate, is one of the few whole-cell biotransformations undertaken at very large scales commercially. They describe attempts to manipulate yeast metabolism and the associated product and by-product formation reactions using strain, enzyme, and cofactor engineering.

The successful completion of this volume has been made possible by the timely cooperation of the contributors to this volume. We thank them for their efforts. We hope that readers will enjoy going through the chapters and find them useful. We thank Dr. Kostas Marinakis, Book Acquisition Editor; Ms. Anneka Hess; and the entire production team at Elsevier for their help and support in bringing out this volume. Without their commitment, efficiency, and dedicated work, this volume could not have ever been accomplished.

Editors

Paramasamy Gunasekaran

Santosh Noronha

Ashok Pandey

Part 1

Functional Genomics

Outline

1. Functional Epigenomics

2. Functional Metagenomics: Exploring Nature’s Gold Mine

3. Functional Genomics of the Extremophilic Bacteria and Archaea

4. Functional Genomics of Riboflavin Transport: Genes and Regulatory Mechanisms

5. Functional Genomics of MicroRNAs

6. Functional Genomics of Pathogenesis

7. Next-Generation Sequencing Methods

1

Functional Epigenomics

S. Majumdar, P. Singh, A.H. Iyer,  and M. Saravanan∗     Indian Institute of Technology-Kanpur, Kanpur, Uttar Pradesh, India

Abstract

In eukaryotic cells, DNA is present as a compact nucleoprotein complex called chromatin. Chromatin compromises DNA accessibility for the diverse DNA-dependent cellular processes. Hence, timely remodeling of the chromatin structure to enhance DNA availability is one of the key factors in gene expression. Chromatin remodeling is in turn influenced by the epigenetic machinery that modifies DNA and histones. Epigenetic modifications are capable of regulating gene expression by modulating DNA access rather than altering the DNA sequence. Several diseases are associated with altered gene expression levels, implying a possible role for epigenetic modifications. With the emergence of high-throughput techniques, numerous critical diseases have been associated with aberrant epigenetic modifications and mutations in the epigenetic machinery. However, it was realized that regulation of gene expression relies on cross talk between diverse epigenetic marks across the genome. Subsequently, a lot of effort has been focused on developing high-throughput techniques that could precisely identify the characteristic epigenetic pattern for a particular disease. In this chapter we discuss the diverse epigenetic modifications and how these modifications influence the expression of genes associated with specific diseases. Further, we introduce the available techniques to characterize epigenetic changes at the genome level and also present certain recent advances in the development of epigenetic therapies.

Keywords

Cancer epigenetics; Chromatin remodeling; DNA methylation; Epigenetics; Functional epigenomics; Histone modification; Transcription

1.1. Introduction

Diversity driven by inheritance and evolution is the essence of life. For decades researchers have attempted to comprehend these aspects for a holistic understanding of living organisms. In the year 1859, the revolutionary theory of evolution through natural selection by Charles Darwin changed the whole paradigm of the field [1]. The theory essentially comprised three components: variation, inheritance, and competition for survival. Darwin defined inheritance as the transfer of adaptations (both wanted and unwanted) from one generation to the next. Ever since, innumerable efforts were focused on understanding how these adaptations are transferred across generations. A crucial turning point was the year 1953, when Watson and Crick revealed the double helical structure of DNA and clearly showed that this molecule is solely responsible for the inheritance of genetic traits [2]. Another remarkable discovery was in the year 1961, when Marshall Nirenberg deciphered the genetic code [3]. It could successfully explain one of the fundamental aspects of Darwinian theory, which is variation. The genetic code is based on five nucleotides, A, C, G, T, and U, which combine to generate 64 codons (triplets), which in turn code for 20 different amino acids. The variations prevalent in a population were attributed to changes in these codons, which would then code for a different amino acid and reflect on the properties of the final protein being built. Overall, the above studies established the flow of genetic information within an organism from DNA to RNA to protein.

It was difficult, however, to believe that the immense diversity of life could be attributed to something so simple. The field of epigenetics added another dimension to our view of the DNA world. Epigenetics refers to variations in the activity of a gene without altering its nucleotide sequence. The term epigenetics was coined in the year 1942 by Conrad Hal Waddington [4]. He proposed that development is an epigenetic process wherein a phenotype not only is a manifestation of genetic processes but also depends on environmental interactions. Ever since, extensive effort has been focused on refining the concept of epigenetics, which revealed that epigenetic modifications involve chemical alterations of chromatin affecting gene expression.

Overall, epigenetics is the study of chemical reactions that systematically modulate the expression of certain sections of the genome at calculated times and also involves identification of factors that regulate this process. It adds a novel dimension to the current picture of evolution by considering the possibility of the transfer of information gathered from the environment by parents to the offspring in the form of epigenetic modifications, along with the genetic code. Hence, epigenetics generates the possibility of prompt response to an environmental stimulus. The epigenome, which refers to the comprehensive set of epigenetic modifications in the genome, would be flexible to environmental changes, which would manifest as a variance in the expression of genes without any revision to the nucleotide sequence. There is increasing evidence of epigenetic changes associated with disease states, the first among them being cancer [5]. Hence, understanding the epigenome in relation to diseases is of immense priority in the field of biomedical research as well. Functional epigenomics, which involves the study and manipulation of epigenetic changes at the global level employing high-throughput approaches or pharmacological molecules to alter the epigenetic state, is thus the need of the hour.

1.2. Chromatin: The Epigenetic Center

In eukaryotes the DNA occurs as a highly compacted nucleoprotein complex, the chromatin, which accommodates the DNA inside the nucleus and also protects it from damaging agents. The chromatin is the systematic arrangement of nucleosomes, consisting of a core of histone octamer wrapped by DNA. The histone octamer comprises two molecules each of H2A, H2B, H3, and H4 (histone proteins) [6]. All four histone proteins have a highly unstructured N-terminal tail and a core histone fold [6]. The histones assemble into octamers with a dyad symmetry via the histone folds in a head-to-tail orientation [7]. Further, the DNA associated with the histone octamer is stabilized through hydrogen bonds between the phosphodiester backbone and the amino acid residues [6]. The nucleosomes form an 11-nm fiber, which appears like beads on a string—this represents the first level of compaction. These nucleosomes are arranged superhelically around a central axis such that the faces of the octamers are arranged adjacent to one another while the DNA lies on the more accessible surface with the linker DNA buried in the core [8]. This 30-nm fiber forms loops around a central protein structure termed the nuclear scaffold, conforming to additional compaction. Further, the chromatin thread compacts to form discrete structures termed chromosomes in association with structural maintenance proteins such as condensin and cohesin (Fig. 1.1).

The organization of DNA into chromatin restricts its accessibility. In the nucleus two forms of chromatin exist, one is less condensed transcriptionally active euchromatin and the second is more condensed transcriptionally inactive heterochromatin [9]. The euchromatin and heterochromatin are dynamically regulated within the cell based on the cell cycle stage, cell type, cellular environment, etc., which thereby modulate the expression levels of the genes.

Figure 1.1  Higher order organization of chromatin. The DNA helix wraps around a histone octamer to form nucleosomes. The nucleosomes arrange themselves around an axis to form a 30-nm solenoid fiber. Further packaging takes place by formation of loops of the 30-nm fiber over a protein scaffold. During M phase, Structural Maintenance of Chromosomes (SMC) proteins impart further compaction that leads to the formation of a distinctly visible chromosome.

1.3. The Epigenetic Machinery

The nucleosome is the center of epigenetic modifications. The histones comprising the nucleosomes have an N-terminal unstructured tail, which is prone to modifications. Acetylation and/or methylation of lysine residues and phosphorylation of serine residues are some of the commonly encountered modifications. Acetylation is usually associated with enhanced transcription, whereas the effects of the other modifications are poorly understood. Apart from nucleosome modifications, epigenetic changes also include the process of chromatin remodeling, nucleosome repositioning, DNA methylation (at CpG), and regulation mediated by small noncoding RNAs. Chromatin remodelers alter the compaction of the chromatin, rendering the DNA accessible for a host of cellular processes. Enzymes involved in histone modification work in consort with the chromatin remodelers, whereby certain histone modifications recruit a particular remodeling complex to the chromatin. Further, DNA methylation recruits methyl-CpG-binding domain (MBD)-containing proteins, which form complexes with the histone-modifying enzymes. Overall, there is an intricate cross talk within the epigenetic machinery (DNA methylation, histone modification, and chromatin remodeling), which imparts remarkable cellular diversity.

1.3.1. DNA Methylation

DNA methylation occurs primarily at two nucleotides, adenine and cytosine, which leads to suppression of gene expression. X-chromosome inactivation, carcinogenesis, suppression of repetitive elements, and genomic imprinting are associated with DNA methylation.

Cytosine methylation at and beyond CpG islands has been widely investigated. Methylation of cytosine at these sites leads to gene silencing. CpG islands are about 200  bp in length, having a GC content of ∼50% [10]. They are associated with almost 60% of human gene promoters, usually in the unmethylated state; however, they acquire methylation in specific tissues during early development [11]. Methylation-induced gene silencing forms the basis of genome imprinting wherein hypermethylation of one parental allele leads to monoallelic expression [12], e.g., X-chromosome inactivation.

Various mechanisms hinder gene expression upon DNA methylation. MBD proteins bind to methylated DNA and recruit chromatin remodelers and histone modifiers that initiate further compaction of chromatin into inactive heterochromatin [13,14]. Apart from this DNA methylation might impede the recruitment of DNA-binding proteins thereby inhibiting gene expression [15]. Unmethylated DNA promotes gene expression by indirectly recruiting histone methyl transferases, which create methylated histone (H3K4 methylation)-rich domains. Further, these domains recruit proteins that alter chromatin structure for specific gene expression [16].

DNA methylation extends beyond the CpG islands to regions in its vicinity, having lower CpG density, referred to as CpG island shores. Methylation at these sites has shown strong correlation with transcriptional inactivation. Studies suggest that tissue-specific methylation is associated with CpG island shores rather than CpG islands [17,18].

DNA methylation beyond CpG dinucleotides, at CHH and CHG sites (H represents A, C, or T) has been reported in stem cells. CHH and CHG methylation is enriched at the gene bodies (associated with gene expression), whereas it is depleted at sites that recruit enhancers [19]. Studies suggest that the non-CpG methylation levels are lower during differentiation, whereas in induced pluripotent cells, they revert to their normal levels. This might indicate a role for non-CpG methylation to maintain pluripotency [19,20].

The DNMT family of enzymes catalyzes DNA methylation, employing S-adenosylmethionine as a cofactor. The DNA Methyl Transferases (DNMT) family includes DNMT1, DNMT2, DNMT3A, DNMT3B, and DNMT3L. However, only DNMT1, DNMT3A, and DNMT3B possess methyltransferase activity. These are further grouped into de novo DNMTs and maintenance DNMTs. The de novo class has been implicated in generating a methylation pattern during embryo development [13]. The expression of this group of DNMTs is restricted to differentiated cells. DNMT3L is expressed at the time of gametogenesis and is vital for maternal genomic imprinting despite being inactive [21]. It colocalizes and interacts with the other DNMT3 members (DNMT3A and DNMT3B) in the nucleus, acting as a general stimulating factor [22,23].

The most predominant DNMT expressed at its highest level during the cell cycle (S phase) is DNMT1. Apart from de novo activity it exhibits selectivity for hemimethylated DNA. It often participates in methylating the hemimethylated DNA obtained during semiconservative replication. Its affinity toward newly synthesized DNA is imparted by its interacting partner, proliferating cell nuclear antigen [24].

It has been proposed that the de novo and maintenance DNMTs may not have discrete distribution of function [25]. DNMT1 is responsible for maintaining methylation in actively dividing cells. Nevertheless, DNMT3A and DNMT3B, although associated with the nucleosomes comprising methylated DNA [26], would methylate sites at the replication fork overlooked by DNMT1. DNMT2 contains all the prototypical catalytic motifs of DNMTs but exhibits no DNA methylase activity.

1.3.2. Histone Modification

The discovery of histone modifications by Vincent G. Allfrey in 1968 [27] and further contributions from Turner and O'Neil in the mid-1990s [28] enhanced our understanding of the complexity of temporal and tissue-specific gene expression regulated by chromatin structure. Histones undergo numerous posttranslational modifications, which are usually confined to the unstructured N-terminal histone tails. Acetylation, methylation, phosphorylation, SUMOylation, and ubiquitination are some of the modifications that render histones competent for regulating diverse cellular processes like DNA replication, transcription, DNA repair, recombination, chromosome compaction, etc. [29]. In contrast, histone modifications play a rather indirect role in gene activation or silencing [30].

Histone acetylation is the best-studied posttranslational modification of histones. Acetylation negates the positive charge on the lysine residues, which reduces the affinity of histone for DNA, forming highly decondensed euchromatin leading to transcriptional activation [31,32]. Histone acetyltransferases (HATs) catalyze the transfer of an acetyl group from the cofactor acetyl-CoA to histones H3 and H4. Twenty HATs have been identified, which have been classified into five families, namely, MYST, GNAT1, P300/CBP, nuclear receptor coactivators, and TAFII250 [33]. Any imbalance in the histone acetylation equilibrium has been associated with cancer.

In contrast to HATs, histone deacetylases (HDACs) are the enzymes that catalyze histone deacetylation (removal of an acetyl group) and assist in the formation of heterochromatin. Inhibitors against HDACs are being developed as anticancer drugs [34].

Based on transcription levels, the human genome can be segregated into heterochromatin and euchromatin (Fig. 1.2). Euchromatin is actively transcribed and possesses distinctly higher levels of acetylated and trimethylated H3K4, H3K79, and H3K36 histones [35]. It has been proposed that actively transcribing genes can be identified based on their histone modification pattern. Transcriptionally active genes are marked by higher amounts of H3K27ac, H3K4me3 (trimethylation), H4K20me1, and H2BK5ac at the promoter and H4K20me1 and H3K79me1 within the gene body [36].

Histone methyltransferases (HMTs) methylate arginine or lysine residues of histones. Histone methylation modulates chromatin architecture, which consecutively regulates DNA methylation, affecting transcription levels. Overall, when histone methylation occurs in the cell, the genes associated with the modified histones might be either activated or repressed. The HMTs are specific toward the lysine or arginine residues that they modify. H3K9 and H3K27 methylation is a critical modification leading to heterochromatinization and gene silencing [37].

Figure 1.2  Dynamic interconversion of euchromatin and heterochromatin. The highly condensed heterochromatin is transcriptionally inactive, whereas the loosely condensed or relaxed euchromatin is transcriptionally active. As shown, epigenetic modifications such as histone deacetylation and DNA methylation along with corepressor complexes condense the euchromatin to heterochromatin. The reversed process is brought about by histone acetylation and phosphorylation, DNA demethylation, along with coactivator complexes. The black flag indicates histone modification and green (gray in print versions) flags denote DNA methylation.

A very different kind of histone modification has been reported. Histone H3 undergoes clipping at its N-terminal tail at Ala21, such that 21 amino acids from the tail along with the associated modifications are removed. This process is inhibited by H3K4me [38].

The histone modifications occur at more than one site on the histones. The overall response of these modifications depends on the intricate cross talk between them. This cross talk could be between modifications from near and distant sites [39–41]. It is the pattern of histone modifications in a nucleosome