Introductory Chapter: Epigenetics in Summary

*Rosaria Meccariello*

## **1. Definition**

In 1940 the developmental biologist Conrad H. Waddington firstly used the term "epigenetics" to describe "*the interaction of genes with their environment, which bring the phenotype into being*" [1]. Two years later, Conrad Waddington pointed out that "*It is possible that an adaptive response can be fixed without waiting for the occurrence of a mutation*" [2]. Thus, epigenetic modifications are heritable and reversible modifications that significantly affect gene expression without any change in the nucleotide sequence of DNA [3].

## **2. Molecular mechanisms**

Classically, epigenetic mechanisms include (i) the methylation of DNA, (ii) the imprinting, (iii) the remodeling of chromatin, and (iv) the production of noncoding RNA (ncRNA) [4, 5].

The methylation of DNA usually occurs at the 5-position of DNA cytosine (5mC) in the CpG islands located within the promoter region of specific genes; such a modification inhibits both the binding of transcription factors to DNA and affects the recruitment of proteins involved in chromatin remodeling [6, 7], thus causing gene silencing.

Genomic imprinting is a DNA methylation-dependent phenomenon, occurring during embryogenesis; it causes genes to be expressed from a parent of originspecific manner [8] and specifically interests at some genetic loci.

Nuclear DNA is structured in chromatin, an instructive DNA scaffold that can respond to external cues regulating DNA activity, composed of histone and nonhistone proteins [9]. Euchromatin, which is the transcriptionally active region of the DNA, represents the loosely folded part of the chromatin; heterochromatin, which is a transcriptionally poorly active region of the DNA, represents the tightly folded part of the chromatin [10]. Therefore, the transcription rate of genes is strongly affected by dynamic chromatin remodeling. In this respect, posttranslational modifications of histone tails like methylation and acetylation play critical roles, by affecting either the affinity of transcriptional factors for gene promoter region or the recruitment to chromatin of nonhistone protein, thus disturbing chromatin contacts [10]. Histone tail acetylation usually promotes the transcription and is a feature of euchromatin; by contrast, histone tail methylation has usually an inhibitory role for transcription and is a feature of heterochromatin.

The family of ncRNA includes a large set of RNAs like the well-known microRNA (miRNA) or the less known long noncoding RNA (lnRNA) and tRNA fragments (tRF) among others [11]. NcRNAs are involved in the control of gene expression and in the regulation of many biological functions in several tissues;

#### *Epigenetics*

their expression rate is affected by environmental cues; thus, their expression rate changes in health and disease. Furthermore, the detection of ncRNA in biological fluids makes them a possible epigenetic biomarker for the prognosis, the diagnosis, and the treatment of diseases [12–14].

Thus, an epigenetic machinery comprising various writers, readers, and erasers that have unique structures, functions, and modes of action like the *de novo* and maintenance DNA methyltransferases, histone acetyltransferases, deacetylases, methyltransferases and demethylases, or the ncRNA biosynthetic pathways has been identified in living organisms [13]. However, additional epigenetic mechanisms such as the delivery among tissues of epigenetic marks within extracellular vesicles, exosomes, or microvesicles are starting to emerge, providing evidence of upcoming communication pathways in which the products of specific cell types may affect the expression rate of specific RNAs in target tissues [15–17].

## **3. Epigenetics in health and disease**

In mammals, epigenetic signature is firstly defined in the embryo [18, 19], but this mark is deeply remodeled during the life course as a direct consequence of environmental cues and lifestyle which includes diet, stress, pollutants, smoking, endocrine-disrupting chemicals, physical activity, sedentary life, etc. Therefore, genome activity is epigenetically modulated under exogenous influence, and the environment-dependent changes in gene activity stably propagate from one generation of cells to the next one. Epigenetic changes impact genome functions, thus affecting health and disease status and also behavior; aging-related diseases, cancer, immunity and related disorders, obesity, metabolic disorders, infertility, and cardiovascular and neurological diseases represent only few examples of environmentally dependent diseases, and the literature in the field is growing up day by day [20–35].

Individual health or disease status strongly depends on epigenetic marks, but "parental experiences" may be epigenetically transmitted to the offspring, thus causing trans-generational epigenetic inheritance and affecting offspring health. Such a process requires the transmission of epigenetic marks through gametes and influences fertilization, embryo development, embryo gene expression, and phenotype [36]. Particularly interesting is the possibility that spermatozoa may use ncRNAs as carrier of paternal experiences, thus providing an "epigenetic memory" capable of affecting embryo development and health with consequences on adult offspring phenotype [13, 32, 33].

## **4. Conclusions and future perspectives**

Taken together, both environment and lifestyle deeply affect DNA functions, and their influence may be transmitted to the next generations with consequences on health status. However, experimental data point out that epigenetic marks, and in particular circulating ncRNAs, may represent upcoming biomarkers for the prevention, the diagnosis, and the treatment of diseases, due to the great potential laying in developing epigenetic therapies [37–39].

**3**

**Author details**

Napoli, Italy

Rosaria Meccariello

provided the original work is properly cited.

Dipartimento di Scienze Motorie e del Benessere, Università di Napoli Parthenope,

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

\*Address all correspondence to: rosaria.meccariello@uniparthenope.it

*Introductory Chapter: Epigenetics in Summary DOI: http://dx.doi.org/10.5772/intechopen.86541* *Introductory Chapter: Epigenetics in Summary DOI: http://dx.doi.org/10.5772/intechopen.86541*

*Epigenetics*

[20–35].

and the treatment of diseases [12–14].

**3. Epigenetics in health and disease**

offspring phenotype [13, 32, 33].

**4. Conclusions and future perspectives**

laying in developing epigenetic therapies [37–39].

their expression rate is affected by environmental cues; thus, their expression rate changes in health and disease. Furthermore, the detection of ncRNA in biological fluids makes them a possible epigenetic biomarker for the prognosis, the diagnosis,

Thus, an epigenetic machinery comprising various writers, readers, and erasers that have unique structures, functions, and modes of action like the *de novo* and maintenance DNA methyltransferases, histone acetyltransferases, deacetylases, methyltransferases and demethylases, or the ncRNA biosynthetic pathways has been identified in living organisms [13]. However, additional epigenetic mechanisms such as the delivery among tissues of epigenetic marks within extracellular vesicles, exosomes, or microvesicles are starting to emerge, providing evidence of upcoming communication pathways in which the products of specific cell types

In mammals, epigenetic signature is firstly defined in the embryo [18, 19], but this mark is deeply remodeled during the life course as a direct consequence of environmental cues and lifestyle which includes diet, stress, pollutants, smoking, endocrine-disrupting chemicals, physical activity, sedentary life, etc. Therefore, genome activity is epigenetically modulated under exogenous influence, and the environment-dependent changes in gene activity stably propagate from one generation of cells to the next one. Epigenetic changes impact genome functions, thus affecting health and disease status and also behavior; aging-related diseases, cancer, immunity and related disorders, obesity, metabolic disorders, infertility, and cardiovascular and neurological diseases represent only few examples of environmentally dependent diseases, and the literature in the field is growing up day by day

Individual health or disease status strongly depends on epigenetic marks, but "parental experiences" may be epigenetically transmitted to the offspring, thus causing trans-generational epigenetic inheritance and affecting offspring health. Such a process requires the transmission of epigenetic marks through gametes and influences fertilization, embryo development, embryo gene expression, and phenotype [36]. Particularly interesting is the possibility that spermatozoa may use ncRNAs as carrier of paternal experiences, thus providing an "epigenetic memory" capable of affecting embryo development and health with consequences on adult

Taken together, both environment and lifestyle deeply affect DNA functions, and their influence may be transmitted to the next generations with consequences on health status. However, experimental data point out that epigenetic marks, and in particular circulating ncRNAs, may represent upcoming biomarkers for the prevention, the diagnosis, and the treatment of diseases, due to the great potential

may affect the expression rate of specific RNAs in target tissues [15–17].

**2**

## **Author details**

Rosaria Meccariello Dipartimento di Scienze Motorie e del Benessere, Università di Napoli Parthenope, Napoli, Italy

\*Address all correspondence to: rosaria.meccariello@uniparthenope.it

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

## **References**

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[2] Waddington CH. Canalization of development and the inheritance of acquired characters. Nature;**150**:563-565

[3] Feinberg AP. Phenotypic plasticity and the epigenetics of human disease. Nature. 2007;**447**:433-440

[4] Kim JK, Samaranayake M, Pradhan S. Epigenetic mechanisms in mammals. Cellular and Molecular Life Sciences. 2009;**66**(4):596-612

[5] Cholewa-Waclaw J, Bird A, von Schimmelmann M, Schaefer A, Yu H, Song H, et al. The role of epigenetic mechanisms in the regulation of gene expression in the nervous system. The Journal of Neuroscience. 2016;**36**(45):11427-11434

[6] Holliday R. DNA methylation and epigenetic mechanisms. Cell Biophysics. 1989;**15**(1-2):15-20

[7] Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology. 2013;**38**:23-38

[8] Ferguson-Smith AC. Genomic imprinting: The emergence of an epigenetic paradigm. Nature Reviews. Genetics. 2011;**12**(8):565-575

[9] Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Research. 2011;**21**(3):381-395

[10] Javaid N, Choi N. Acetylation- and methylation-related epigenetic proteins in the context of their targets. Genes (Basel). 2017;**8**(8):196

[11] Palazzo AF, Eliza S, Lee ES. Noncoding RNA: What is functional and what is junk? Frontiers in Genetics. 2015;**6**:2

[12] Taft RJ, Pang KC, Mercer TR, Dinger M, Mattick JS. Non-coding RNAs: Regulators of disease. The Journal of Pathology. 2010;**220**:126-139

[13] Chianese R, Troisi J, Richards S, Scafuro M, Fasano S, Guida M, et al. In reproduction: Epigenetic effects. Current Medicinal Chemistry. 2018;**25**(6):748-770

[14] Kumar P, Kuscu C, Dutta A. Biogenesis and function of transfer RNA-related fragments (tRFs). Trends in Biochemical Sciences. 2016;**41**:679-689

[15] Bakhshandeh B, Kamaleddin MA, Aalishah KA. Comprehensive review on exosomes and microvesicles as epigenetic factors. Current Stem Cell Research & Therapy. 2017;**12**(1):31-36

[16] Qian Z, Shen Q, Yang X, Qiu Y, Zhang W. The role of extracellular vesicles: An epigenetic view of the cancer microenvironment. BioMed Research International. 2015;**2015**:649161

[17] Motti ML, D'Angelo S, Meccariello R. MicroRNAs, cancer and diet: Facts and new exciting perspectives. Current Molecular Pharmacology. 2018;**11**(2):90-96

[18] Seisenberger S, Peat JR, Hore TA, Santos F, Dean W, Reik W. Reprogramming DNA methylation in the mammalian life cycle: Building and breaking epigenetic barriers. Philosophical Transactions of the Royal Society of London. Series B, Biological Sciences. 2013;**368**:20110330

[19] Hogg K, Western PS. Refurbishing the germline epigenome: Out with the old, in with the new. Seminars

**5**

eCollection 2019

*Introductory Chapter: Epigenetics in Summary DOI: http://dx.doi.org/10.5772/intechopen.86541*

Annual Review of Pharmacology and

Toxicology. 2018;**58**:187-207

[28] Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the hallmarks of cancer. Science

2017;**357**(6348):pii: eaal2380

[30] Sen P, Shah PP, Nativio R, Berger SL. Epigenetic mechanisms and longevity and aging. Cell.

2016;**166**(4):822-839

2015;**9**:58

[29] Landgrave-Gómez J, Mercado-Gómez O, Guevara-Guzmán R.

Epigenetic mechanisms in neurological and neurodegenerative diseases. Frontiers in Cellular Neuroscience.

[31] Das L, Parbin S, Pradhan N, Kausar C, Patra SK. Epigenetics of reproductive infertility. Frontiers in Bioscience (Scholar Edition). 2017;**9**:509-535

[32] Stuppia L, Franzago M, Ballerini P, Gatta V, Antonucci I. Epigenetics and male reproduction: The consequences of paternal lifestyle on fertility, embryo development, and children lifetime health. Clinical Epigenetics. 2015;**7**:120

[33] Jenkins TG, Aston KI, James ER, Carrell DT. Sperm epigenetics in the study of male fertility, offspring health, and potential clinical applications. Systems Biology in Reproductive Medicine. 2017;**63**(2):69-76

[34] Crews D. Epigenetics and its implications for behavioral neuroendocrinology. Frontiers in Neuroendocrinology. 2008;**29**(3):344-357

2013;**25**(4 Pt 2):1279-1291

[36] Daxinger L, Whitelaw E. Understanding transgenerational

[35] Roth TL. Epigenetic mechanisms in the development of behavior: Advances, challenges, and future promises of a new field. Development and Psychopathology.

in Cell & Developmental Biology.

[20] Ling C, Rönn T. Epigeneticsin human obesity and type 2 diabetes.

[21] Renani PG, Taheri F, Rostami D, Farahani N, Abdolkarimi H, Abdollahi E, et al. Involvement of aberrant regulation of epigenetic mechanisms in the pathogenesis of Parkinson�s disease and epigenetic-based therapies. Journal of Cellular Physiology. 2019. DOI: 10.1002/jcp.28622. [Epub ahead of print]

[22] Rutten MGS, Rots MG, Oosterveer

[23] Kato M, Natarajan R. Epigenetics and epigenomics in diabetic kidney disease and metabolic memory. Nature Reviews. Nephrology. 2019. DOI: 10.1038/s41581-019-0135-6. [Epub

[24] Grova N, Schroeder H, Olivier JL, Turner JD. Epigenetic and neurological impairments associated with early life exposure to persistent organic pollutants. International Journal of Genomics. 2019;**2019**:2085496. DOI: 10.1155/2019/2085496. eCollection 2019

[25] Al-Hasani K, Mathiyalagan P, El-Osta A. Epigenetics, cardiovascular disease, and cellular reprogramming. Journal of Molecular and Cellular Cardiology. 2019;**128**:129-133

[26] Stylianou E. Epigenetics of chronic inflammatory diseases. Journal of Inflammation Research. 2018;**20**(12): 1-14. DOI: 10.2147/JIR.S129027.

[27] Richard L, Bennett RL, Licht JD. Targeting epigenetics in cancer.

ahead of print]

MH. Exploiting epigenetics for the treatment of inborn errors of metabolism. Journal of Inherited Metabolic Disease. 2019. DOI: 10.1002/ jimd.12093. [Epub ahead of print]

Cell Metabolism. 2019. pii: S1550-4131(19)30137-8

2015;**45**:104-113

*Introductory Chapter: Epigenetics in Summary DOI: http://dx.doi.org/10.5772/intechopen.86541*

in Cell & Developmental Biology. 2015;**45**:104-113

[20] Ling C, Rönn T. Epigeneticsin human obesity and type 2 diabetes. Cell Metabolism. 2019. pii: S1550-4131(19)30137-8

[21] Renani PG, Taheri F, Rostami D, Farahani N, Abdolkarimi H, Abdollahi E, et al. Involvement of aberrant regulation of epigenetic mechanisms in the pathogenesis of Parkinson�s disease and epigenetic-based therapies. Journal of Cellular Physiology. 2019. DOI: 10.1002/jcp.28622. [Epub ahead of print]

[22] Rutten MGS, Rots MG, Oosterveer MH. Exploiting epigenetics for the treatment of inborn errors of metabolism. Journal of Inherited Metabolic Disease. 2019. DOI: 10.1002/ jimd.12093. [Epub ahead of print]

[23] Kato M, Natarajan R. Epigenetics and epigenomics in diabetic kidney disease and metabolic memory. Nature Reviews. Nephrology. 2019. DOI: 10.1038/s41581-019-0135-6. [Epub ahead of print]

[24] Grova N, Schroeder H, Olivier JL, Turner JD. Epigenetic and neurological impairments associated with early life exposure to persistent organic pollutants. International Journal of Genomics. 2019;**2019**:2085496. DOI: 10.1155/2019/2085496. eCollection 2019

[25] Al-Hasani K, Mathiyalagan P, El-Osta A. Epigenetics, cardiovascular disease, and cellular reprogramming. Journal of Molecular and Cellular Cardiology. 2019;**128**:129-133

[26] Stylianou E. Epigenetics of chronic inflammatory diseases. Journal of Inflammation Research. 2018;**20**(12): 1-14. DOI: 10.2147/JIR.S129027. eCollection 2019

[27] Richard L, Bennett RL, Licht JD. Targeting epigenetics in cancer.

Annual Review of Pharmacology and Toxicology. 2018;**58**:187-207

[28] Flavahan WA, Gaskell E, Bernstein BE. Epigenetic plasticity and the hallmarks of cancer. Science 2017;**357**(6348):pii: eaal2380

[29] Landgrave-Gómez J, Mercado-Gómez O, Guevara-Guzmán R. Epigenetic mechanisms in neurological and neurodegenerative diseases. Frontiers in Cellular Neuroscience. 2015;**9**:58

[30] Sen P, Shah PP, Nativio R, Berger SL. Epigenetic mechanisms and longevity and aging. Cell. 2016;**166**(4):822-839

[31] Das L, Parbin S, Pradhan N, Kausar C, Patra SK. Epigenetics of reproductive infertility. Frontiers in Bioscience (Scholar Edition). 2017;**9**:509-535

[32] Stuppia L, Franzago M, Ballerini P, Gatta V, Antonucci I. Epigenetics and male reproduction: The consequences of paternal lifestyle on fertility, embryo development, and children lifetime health. Clinical Epigenetics. 2015;**7**:120

[33] Jenkins TG, Aston KI, James ER, Carrell DT. Sperm epigenetics in the study of male fertility, offspring health, and potential clinical applications. Systems Biology in Reproductive Medicine. 2017;**63**(2):69-76

[34] Crews D. Epigenetics and its implications for behavioral neuroendocrinology. Frontiers in Neuroendocrinology. 2008;**29**(3):344-357

[35] Roth TL. Epigenetic mechanisms in the development of behavior: Advances, challenges, and future promises of a new field. Development and Psychopathology. 2013;**25**(4 Pt 2):1279-1291

[36] Daxinger L, Whitelaw E. Understanding transgenerational

**4**

*Epigenetics*

**References**

Academic; 1940

[1] Waddington CH. Organizers and Genes. Cambridge: Cambridge

[2] Waddington CH. Canalization of development and the inheritance of acquired characters. Nature;**150**:563-565 what is junk? Frontiers in Genetics.

Pathology. 2010;**220**:126-139

2018;**25**(6):748-770

2016;**41**:679-689

2015;**2015**:649161

2018;**11**(2):90-96

[18] Seisenberger S, Peat JR,

Sciences. 2013;**368**:20110330

Hore TA, Santos F, Dean W, Reik W. Reprogramming DNA methylation in the mammalian life cycle: Building and breaking epigenetic barriers. Philosophical Transactions of the Royal Society of London. Series B, Biological

[19] Hogg K, Western PS. Refurbishing the germline epigenome: Out with the old, in with the new. Seminars

[13] Chianese R, Troisi J, Richards S, Scafuro M, Fasano S, Guida M, et al. In reproduction: Epigenetic effects. Current Medicinal Chemistry.

[14] Kumar P, Kuscu C, Dutta A. Biogenesis and function of transfer RNA-related fragments (tRFs). Trends in Biochemical Sciences.

[15] Bakhshandeh B, Kamaleddin MA, Aalishah KA. Comprehensive review on exosomes and microvesicles as epigenetic factors. Current Stem Cell Research & Therapy. 2017;**12**(1):31-36

[16] Qian Z, Shen Q, Yang X, Qiu Y, Zhang W. The role of extracellular vesicles: An epigenetic view of the cancer microenvironment. BioMed Research International.

[17] Motti ML, D'Angelo S, Meccariello R. MicroRNAs, cancer and diet: Facts and new exciting perspectives. Current Molecular Pharmacology.

[12] Taft RJ, Pang KC, Mercer TR, Dinger M, Mattick JS. Non-coding RNAs: Regulators of disease. The Journal of

2015;**6**:2

[3] Feinberg AP. Phenotypic plasticity and the epigenetics of human disease.

[4] Kim JK, Samaranayake M, Pradhan S. Epigenetic mechanisms in mammals. Cellular and Molecular Life Sciences.

[5] Cholewa-Waclaw J, Bird A, von Schimmelmann M, Schaefer A, Yu H, Song H, et al. The role of epigenetic mechanisms in the regulation of gene expression in the nervous system. The Journal of Neuroscience.

[6] Holliday R. DNA methylation and epigenetic mechanisms. Cell Biophysics.

[7] Moore LD, Le T, Fan G. DNA methylation and its basic function. Neuropsychopharmacology.

[8] Ferguson-Smith AC. Genomic imprinting: The emergence of an epigenetic paradigm. Nature Reviews.

Genetics. 2011;**12**(8):565-575

[9] Bannister AJ, Kouzarides T. Regulation of chromatin by histone modifications. Cell Research.

[10] Javaid N, Choi N. Acetylation- and methylation-related epigenetic proteins in the context of their targets. Genes

[11] Palazzo AF, Eliza S, Lee ES. Noncoding RNA: What is functional and

2011;**21**(3):381-395

(Basel). 2017;**8**(8):196

Nature. 2007;**447**:433-440

2009;**66**(4):596-612

2016;**36**(45):11427-11434

1989;**15**(1-2):15-20

2013;**38**:23-38

epigenetic inheritance via the gametes in mammals. Nature Reviews. Genetics. 2012;**13**(3):153-162

[37] Ahuja N, Sharma AR, Baylin SB. Epigenetic therapeutics: A new weapon in the war against cancer. Annual Review of Medicine. 2016;**67**:73-89

[38] Valdespino V, Valdespino PM. Potential of epigenetic therapies in the management of solid tumors. Cancer Management and Research. 2015;**7**:241-251

[39] Mau T, Yung R. Potential of epigenetic therapies in non-cancerous conditions. Frontiers in Genetics. 2014;**5**:438

**7**

**Chapter 2**

**Abstract**

paramount importance.

**1. Introduction**

Epigenetic Modifications in Plants

Plants face a plethora of biotic and abiotic stresses ranging from extreme temperatures to salinity, drought, nutritional deficiencies, chemical toxicity, and pathogen attacks. As a consequence, plants have acquired several sophisticated regulatory mechanisms that allow them to cope with such adverse conditions. Epigenetic regulation plays a key role in the mechanisms of plant response to the environment, without altering DNA sequences. Epigenetics refers to heritable alterations in chromatin architecture that do not involve changes in the underlying DNA sequence but alter gene expression through DNA methylation or histone modifications. The epigenetic regulation of the plant genome is a highly dynamic process that fine-tunes the expression of a pertinent set of genes under certain environmental or developmental conditions. Over the past two decades rapid advancements in the field of high throughput sequencing unveil epigenetic information at genome wide level in various plant species. In view of the adverse effects of global climatic change, utilizing epigenetic differences for developing improved crop varieties is of

**Keywords:** histone modification, DNA methylation, abiotic stress, chromatin

(RNAi) are exploited by plants in order to survive adverse conditions.

DNA methylation is a chemical modification, catalyzed by cytosine methyltransferases which involves addition of a methyl group in a DNA sequence onto the

Plants being sessile organisms are being constantly challenged by various biotic and abiotic stresses. In order to adapt themselves to the changing environments they need constant changes at molecular level. These efficient and effective controls are provided by epigenetic regulations which improve the survivability of plants by increasing their tolerance toward stress [1, 2]. It is now evident that heritable phenotypic variation does not need to be based on DNA sequence polymorphism [2, 3]. These epigenetic regulations involve different chemical modifications at molecular level that influence gene expression. Epigenetic as defined by Conrad Waddington, is "the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence" [4]. Today epigenetic refers mainly to the changes that do not relate to the changes in DNA sequence but to chemical modification that can be inherited from one generation to the next [5, 6]. Three types of epigenetic regulatory mechanisms viz. DNA methylation, histone modification and RNA interference

under Abiotic Stress

*Garima Singroha and Pradeep Sharma*

## **Chapter 2**

*Epigenetics*

2012;**13**(3):153-162

2016;**67**:73-89

2015;**7**:241-251

2014;**5**:438

epigenetic inheritance via the gametes in mammals. Nature Reviews. Genetics.

[37] Ahuja N, Sharma AR, Baylin SB. Epigenetic therapeutics: A new weapon in the war against cancer. Annual Review of Medicine.

[38] Valdespino V, Valdespino PM. Potential of epigenetic therapies in the management of solid tumors. Cancer Management and Research.

[39] Mau T, Yung R. Potential of epigenetic therapies in non-cancerous conditions. Frontiers in Genetics.

**6**

## Epigenetic Modifications in Plants under Abiotic Stress

*Garima Singroha and Pradeep Sharma*

## **Abstract**

Plants face a plethora of biotic and abiotic stresses ranging from extreme temperatures to salinity, drought, nutritional deficiencies, chemical toxicity, and pathogen attacks. As a consequence, plants have acquired several sophisticated regulatory mechanisms that allow them to cope with such adverse conditions. Epigenetic regulation plays a key role in the mechanisms of plant response to the environment, without altering DNA sequences. Epigenetics refers to heritable alterations in chromatin architecture that do not involve changes in the underlying DNA sequence but alter gene expression through DNA methylation or histone modifications. The epigenetic regulation of the plant genome is a highly dynamic process that fine-tunes the expression of a pertinent set of genes under certain environmental or developmental conditions. Over the past two decades rapid advancements in the field of high throughput sequencing unveil epigenetic information at genome wide level in various plant species. In view of the adverse effects of global climatic change, utilizing epigenetic differences for developing improved crop varieties is of paramount importance.

**Keywords:** histone modification, DNA methylation, abiotic stress, chromatin

### **1. Introduction**

Plants being sessile organisms are being constantly challenged by various biotic and abiotic stresses. In order to adapt themselves to the changing environments they need constant changes at molecular level. These efficient and effective controls are provided by epigenetic regulations which improve the survivability of plants by increasing their tolerance toward stress [1, 2]. It is now evident that heritable phenotypic variation does not need to be based on DNA sequence polymorphism [2, 3]. These epigenetic regulations involve different chemical modifications at molecular level that influence gene expression. Epigenetic as defined by Conrad Waddington, is "the study of mitotically and/or meiotically heritable changes in gene function that cannot be explained by changes in DNA sequence" [4]. Today epigenetic refers mainly to the changes that do not relate to the changes in DNA sequence but to chemical modification that can be inherited from one generation to the next [5, 6]. Three types of epigenetic regulatory mechanisms viz. DNA methylation, histone modification and RNA interference (RNAi) are exploited by plants in order to survive adverse conditions.

DNA methylation is a chemical modification, catalyzed by cytosine methyltransferases which involves addition of a methyl group in a DNA sequence onto the cytosine residue in a sequence specific manner, primarily within CpG dinucleotide [7, 8]. The added methyl group provides platform for attachment of various protein complexes that modifies the histone scaffolds resulting in altered gene expression.

In eukaryotic nuclei DNA is organized in the form of nucleosome where it is wrapped around by histone proteins. Histones comprise a family of highly conserved globular proteins whose N-terminal tails are exposed on the surface of the nucleosome octamer for chemical modifications. Histones offer a wealth of post-translational modifications (PTMs) that physically regulate the accessibility of the transcriptional machinery to certain genomic regions, making loci more or less permissive for transcription [9]. Histone modifications include acetylation, methylation, sumoylation, ubiquitination and phosphorylation of histone proteins. Acetylation and phosphorylation are mostly associated with induced gene expression while on the other hand modifications like sumoylation and biotinylation represses gene expression [10, 11]. Such modifications not only impinge on DNA accessibility, but also on the recruitment of specific proteins involved in several processes, including transcription, DNA replication and repair. Histone proteins are not only modified, but can also be replaced by histone variants with different physical properties, or released, in order to allow gene expression [12].

In epigenetic cross-talks diverse classes of noncoding RNA (e.g., small RNAs and long noncoding RNAs) can also modify chromatin structure and silence transcription through formation of RNA scaffolds mediating the recruitment of histone and DNA methyltransferases [13]. RNAi is a sequence specific gene regulation mechanism that acts as a barrier against viruses but also regulates gene expression. In plants RNA interference pathways are mediated by siRNA, miRNA and lncRNA (long non coding RNA). These RNAs are synthesized as 20–30 nucleotide, single stranded molecules from double stranded RNA precursors.

Activation of one or more of these pathways results into changes in chromatin architecture and impacts gene expression. Open chromatin form or closed

**9**

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

respective transcriptional and physiological implications.

**2. Different types of epigenetic modifications**

**2.1 DNA methylation modification**

**2.2 Histone modifications in plants**

sented in **Figure 1**.

chromatin conformation is associated with gene activation or silencing respectively and governs the onset of gene expression in cells under different developmental or environmental stimuli [14–16]. Transitions from open to close and crosstalk between different epigenetic mechanisms are vital to ensure proper cell function at different developmental stages and under abiotic stress conditions [17–19]. Different types of epigenetic modifications under abiotic stresses have been pre-

In the recent years, numerous studies performed toward the characterization of the epigenomic regulation of stress responses in plants have added to our understanding of how diverse abiotic stresses affect chromatin modifications, with their

DNA methylation arises as a result of addition of a methyl group to the nitrogenous base in the DNA strand in a sequence specific manner. DNA methylation occurs at the fifth carbon position of a cytosine ring. Methylation of cytosine leads to the generation of 5-methyl cytosine. On the basis of the target sequence, methylation is classified either as symmetrical or asymmetrical methylation. CG and CHG methylation are termed as symmetrical and CHH methylation as asymmetrical. In plants DNA methylation occurs in all three sequence contexts; the symmetric CG and CHG context and asymmetric CHH (H = A, C or T) context [20]. Plants methylate only some genes and this methylation is usually restricted to CGs located within the gene body while Transposable Element sequences tend to be methylated at most of their CG, CHG, and CHH sites. Methylation in transposable elements and promoter region of a gene leads to silencing on the other hand methylation inside gene body induce gene expression [21]. Thus DNA methylation results into following (i) methylcytosines in the gene body play an important role in regulating the gene expression and (ii) methylcytosines in repetitive sequences (transposable elements), are thought to prevent repetitive sequences from compromising normal genome function [20, 21]. Increased methylation of genomic DNA down regulates gene expression. Down regulated gene expression enable the plants to conserve energy for the sake of biotic or abiotic stress. In contrast, the reduction in methylation of resistance-related genes favors chromatin activation and the expression of

novel genes, which provides long-term or permanent resistance for stress.

In addition to DNA methylation, histone N-terminal tail modifications constitute an attractive area in epigenetics [22]. Plants contain several histone variants and enzymes that posttranslationally modify histones and influence gene regulation. Application of chromatin immunoprecipitation followed by deep sequencing has given insight into the genome-wide distribution of histone variants and histones bearing different posttranslational modifications [22, 23]. Histone proteins are wrapped around DNA and forms a highly compact structure called nucleosome. Nucleosomes are composed of histone octamers that comprise two copies each of H2A, H2B, H3, and H4. A total of 147 base pair of DNA sequence is wrapped around the histone core. The N termini of histone proteins called N terminal tails undergo various chemical modifications like methylation or acetylation. Such histone modifications are associated with either gene repression or gene activation [24, 25].

**Figure 1.** *Various types of epigenetic modifications under stress conditions.*

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

*Epigenetics*

cytosine residue in a sequence specific manner, primarily within CpG dinucleotide [7, 8]. The added methyl group provides platform for attachment of various protein complexes that modifies the histone scaffolds resulting in altered gene expression. In eukaryotic nuclei DNA is organized in the form of nucleosome where it is wrapped around by histone proteins. Histones comprise a family of highly conserved globular proteins whose N-terminal tails are exposed on the surface of the nucleosome octamer for chemical modifications. Histones offer a wealth of post-translational modifications (PTMs) that physically regulate the accessibility of the transcriptional machinery to certain genomic regions, making loci more or less permissive for transcription [9]. Histone modifications include acetylation, methylation, sumoylation, ubiquitination and phosphorylation of histone proteins. Acetylation and phosphorylation are mostly associated with induced gene expression while on the other hand modifications like sumoylation and biotinylation represses gene expression [10, 11]. Such modifications not only impinge on DNA accessibility, but also on the recruitment of specific proteins involved in several processes, including transcription, DNA replication and repair. Histone proteins are not only modified, but can also be replaced by histone variants with different

physical properties, or released, in order to allow gene expression [12].

stranded molecules from double stranded RNA precursors.

In epigenetic cross-talks diverse classes of noncoding RNA (e.g., small RNAs and long noncoding RNAs) can also modify chromatin structure and silence transcription through formation of RNA scaffolds mediating the recruitment of histone and DNA methyltransferases [13]. RNAi is a sequence specific gene regulation mechanism that acts as a barrier against viruses but also regulates gene expression. In plants RNA interference pathways are mediated by siRNA, miRNA and lncRNA (long non coding RNA). These RNAs are synthesized as 20–30 nucleotide, single

Activation of one or more of these pathways results into changes in chromatin architecture and impacts gene expression. Open chromatin form or closed

**8**

**Figure 1.**

*Various types of epigenetic modifications under stress conditions.*

chromatin conformation is associated with gene activation or silencing respectively and governs the onset of gene expression in cells under different developmental or environmental stimuli [14–16]. Transitions from open to close and crosstalk between different epigenetic mechanisms are vital to ensure proper cell function at different developmental stages and under abiotic stress conditions [17–19]. Different types of epigenetic modifications under abiotic stresses have been presented in **Figure 1**.

In the recent years, numerous studies performed toward the characterization of the epigenomic regulation of stress responses in plants have added to our understanding of how diverse abiotic stresses affect chromatin modifications, with their respective transcriptional and physiological implications.

## **2. Different types of epigenetic modifications**

#### **2.1 DNA methylation modification**

DNA methylation arises as a result of addition of a methyl group to the nitrogenous base in the DNA strand in a sequence specific manner. DNA methylation occurs at the fifth carbon position of a cytosine ring. Methylation of cytosine leads to the generation of 5-methyl cytosine. On the basis of the target sequence, methylation is classified either as symmetrical or asymmetrical methylation. CG and CHG methylation are termed as symmetrical and CHH methylation as asymmetrical. In plants DNA methylation occurs in all three sequence contexts; the symmetric CG and CHG context and asymmetric CHH (H = A, C or T) context [20]. Plants methylate only some genes and this methylation is usually restricted to CGs located within the gene body while Transposable Element sequences tend to be methylated at most of their CG, CHG, and CHH sites. Methylation in transposable elements and promoter region of a gene leads to silencing on the other hand methylation inside gene body induce gene expression [21]. Thus DNA methylation results into following (i) methylcytosines in the gene body play an important role in regulating the gene expression and (ii) methylcytosines in repetitive sequences (transposable elements), are thought to prevent repetitive sequences from compromising normal genome function [20, 21]. Increased methylation of genomic DNA down regulates gene expression. Down regulated gene expression enable the plants to conserve energy for the sake of biotic or abiotic stress. In contrast, the reduction in methylation of resistance-related genes favors chromatin activation and the expression of novel genes, which provides long-term or permanent resistance for stress.

#### **2.2 Histone modifications in plants**

In addition to DNA methylation, histone N-terminal tail modifications constitute an attractive area in epigenetics [22]. Plants contain several histone variants and enzymes that posttranslationally modify histones and influence gene regulation. Application of chromatin immunoprecipitation followed by deep sequencing has given insight into the genome-wide distribution of histone variants and histones bearing different posttranslational modifications [22, 23]. Histone proteins are wrapped around DNA and forms a highly compact structure called nucleosome. Nucleosomes are composed of histone octamers that comprise two copies each of H2A, H2B, H3, and H4. A total of 147 base pair of DNA sequence is wrapped around the histone core. The N termini of histone proteins called N terminal tails undergo various chemical modifications like methylation or acetylation. Such histone modifications are associated with either gene repression or gene activation [24, 25].

In plants methylation and deacetylation of H3K9 and H3K27 results into gene repression whereas acetylation and methylation of H3K4 and H3K36 is associated with gene activation and thus induces gene expression [26]. These covalent modifications in response to various environmental stresses regulates the transcription of wrapped DNA sequence by altering the packaging structure which either activates the DNA for the transcription or makes the structure more condensed so that transcription machinery is unable to reach it.

## *2.2.1 Histone acetylation/deacetylation*

Addition of acetyl group to the N terminal Lysine of histones results into transcriptional activation of a DNA sequence [27]. Acetylation of N terminal lysine causes reduction in the net positive charge of histone and as a result the electrostatic force of attraction between the negatively charged DNA and positively charged histone reduces which leads to the loosening of chromatin and transcriptional activation of DNA [28]. The addition of acetyl group to Lysine is catalyzed by histone acetyltransferases (HATs). Five types of HATs have been identified in eukaryotes viz. GNAT—GCN5-related N-terminal acetyltransferase; MYST—MOZ, Ybf2/Sas3, Sas2, and Tip60; CBP—CREB binding protein; TFII250—TATA binding proteinassociated factors and the nuclear hormone-related HATs family. Only specific lysines in a histone protein are acetylated. In different histone proteins different lysine residues undergo modifications for instance, lysine residues of H4 (K5, K8, K12, K16, and K20) and histone H3 (K9, K14, K18, K23, and K27) are subjected to acetylation modifications [29, 30].

#### *2.2.2 Histone methylation*

Arginine and Lysine amino acids in histone proteins undergo methylation. Different arginine and lysine residues in different histones undergo different types of methylation (R3 of H2A, R3, K20 of H4 and K4, K9, K27, K36, R2, and R17 of H3 etc.) and these residues can be mono, di or tri methylated. Usually, arginine undergoes mono- and dimethylation only while lysine can undergo mono, di and tri methylation. Methylation can either activate or deactivate a gene depending on the nature of residues methylated for example H3K4 trimethylation activates transcription on the other hand K9 and K27 dimethylation in H3 acts as a repressor [31]. Methylation affects the hydrophobicity of the histone and hence may change histone DNA interactions or may create binding site for various proteins which restricts the binding of transcription machinery and prevents transcription. Histone lysine methyltransferases (HKMT) and protein arginine methyltransferases (PRMT) catalyze methylation of lysine and arginine residues respectively [32].

#### **2.3 miRNA directed DNA methylation**

RNA directed DNA methylation (RdDM) is *de novo* cytosine methylation primarily within the region of RNA-DNA sequence identity. Although RdDM pathway can methylate all sequence contexts, but it specifically methylates CHH sequences. The reason for this is that symmetrical methylation is maintained by maintenance methylation each time the DNA is replicated whereas the CHH methylation at many silenced loci is dependent on RNA-guided *de novo* methylation [33].

The 24-nt siRNAs are generated by DNA dependent RNA polymerase Pol IV enzyme, in association with RNA-dependent RNA polymerase 2 (RDR2), and processed by dicer-like 3 (DCL3) [34]. One strand of the resulting 24-nt dsRNA fragments is loaded onto argonaute 4 (AGO4) leading to generation of a silencing

**11**

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

effector complex. DNA methylation at sites having sequence homology to the siRNA is dependent on, Pol V, which is a DNA dependent RNA polymerase that transcribes non-coding RNAs. Transcription of Pol V is facilitated by a chromatin remodeling protein which is defective in RNA-directed DNA methylation 1 (DRD1) [35]. KOW domain transcription factor1 (KTF1) which is an adaptor protein, mediates binding of AGO4 and AGO4-bound siRNAs onto the transcripts generated by Pol V forming a silencing effector. This effector acts as signal for DRM2 to introduce methylation at target sites [35]. Development of stress tolerant crop has successfully been achieved by the use of RNAi technology. Transgenic rice plants with tolerance to drought were developed by silencing of activated C-kinase1 receptor [36].

Due to the unpredictable climate change, crop plants are frequently exposed to a variety of abiotic stresses resulting in reduced crop productivity. Analysis of the stress-associated genes and their regulation in response to the stress can be utilized to enhance understanding of the plant's ability to adapt under changing climatic conditions. DNA methylation and/or histone modifications are influenced by abiotic/biotic factors resulting in the better adaptability of the plants to the adverse environmental conditions. Such epigenetic modifications provide a mechanistic basis for stress memory, which enables plants to respond more effectively and efficiently to the recurring stress as well as to prepare the offspring for potential future assaults.

Environmental stresses result in hyper or hypomethylation of DNA. Evidence implicates epigenetic mechanisms in modulating gene expression in plants under abiotic stress. Promoter and gene-body methylation plays important role in regulating gene expression in genotype and organ specific manner under salt stress conditions. Song et al. [37] observed that DNA methylation and histone modifications may have a combined effect on stress inducible gene as salinity stress was reported to affect the expression of various transcription factors in soybean. Ferreira et al., [38] emphasized that hypomethylation in response to salt stress may be correlated with altered expression of DNA demethylases. In another report [39] contrasting differences in cytosine methylation patterns were observed in salinity tolerant wheat cultivar SR3 and its progenitor upon salinity stress imposition. The responses of contrasting wheat genotypes under salt stress could be attributed to the altered expression levels of high affinity potassium transporters (HKTs) regulated through genetic and/or epigenetic mechanisms [40]. It was found that the coding region of high affinity potassium transporters (HKTs) showed variations in 5-mC content in the contrasting wheat genotypes. Salt stress significantly increased methylation level in wheat genotypes. Cytosine residues in CG context were all methylated, whereas increase in 5-mC was observed in CHG and CHH contexts in the shoot of a salt-sensitive wheat genotype under the stress. Variations in chromatin structure (facilitated through histone modifications) also play important role in salt tolerance. Kaldis et al. [41] reported that in *Arabidopsis thaliana* the transcriptional adaptor ADA2b (a modulator of histone acetyltransferases activity) is responsible for its hypersensitivity to salt stress. However, histone modifications are reversible and cross-talk between histone acetylation and cytosine methylation makes the plant responses more complex. Thus, salt stress affects genome-wide DNA methylation as well as histone modifications and these processes are linked to each other for

**3. Epigenetic changes in crops against abiotic stresses**

**3.1 Salt-induced epigenetic changes in crop plants**

synchronized action against salt stress [42].

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

*Epigenetics*

In plants methylation and deacetylation of H3K9 and H3K27 results into gene repression whereas acetylation and methylation of H3K4 and H3K36 is associated with gene activation and thus induces gene expression [26]. These covalent modifications in response to various environmental stresses regulates the transcription of wrapped DNA sequence by altering the packaging structure which either activates the DNA for the transcription or makes the structure more condensed so that

Addition of acetyl group to the N terminal Lysine of histones results into transcriptional activation of a DNA sequence [27]. Acetylation of N terminal lysine causes reduction in the net positive charge of histone and as a result the electrostatic force of attraction between the negatively charged DNA and positively charged histone reduces which leads to the loosening of chromatin and transcriptional activation of DNA [28]. The addition of acetyl group to Lysine is catalyzed by histone acetyltransferases (HATs). Five types of HATs have been identified in eukaryotes viz. GNAT—GCN5-related N-terminal acetyltransferase; MYST—MOZ, Ybf2/Sas3, Sas2, and Tip60; CBP—CREB binding protein; TFII250—TATA binding proteinassociated factors and the nuclear hormone-related HATs family. Only specific lysines in a histone protein are acetylated. In different histone proteins different lysine residues undergo modifications for instance, lysine residues of H4 (K5, K8, K12, K16, and K20) and histone H3 (K9, K14, K18, K23, and K27) are subjected to

Arginine and Lysine amino acids in histone proteins undergo methylation. Different arginine and lysine residues in different histones undergo different types of methylation (R3 of H2A, R3, K20 of H4 and K4, K9, K27, K36, R2, and R17 of H3 etc.) and these residues can be mono, di or tri methylated. Usually, arginine undergoes mono- and dimethylation only while lysine can undergo mono, di and tri methylation. Methylation can either activate or deactivate a gene depending on the nature of residues methylated for example H3K4 trimethylation activates transcription on the other hand K9 and K27 dimethylation in H3 acts as a repressor [31]. Methylation affects the hydrophobicity of the histone and hence may change histone DNA interactions or may create binding site for various proteins which restricts the binding of transcription machinery and prevents transcription. Histone lysine methyltransferases (HKMT) and protein arginine methyltransferases (PRMT) catalyze methylation of lysine and arginine residues respectively [32].

RNA directed DNA methylation (RdDM) is *de novo* cytosine methylation primarily within the region of RNA-DNA sequence identity. Although RdDM pathway can methylate all sequence contexts, but it specifically methylates CHH sequences. The reason for this is that symmetrical methylation is maintained by maintenance methylation each time the DNA is replicated whereas the CHH methylation at many

The 24-nt siRNAs are generated by DNA dependent RNA polymerase Pol IV enzyme, in association with RNA-dependent RNA polymerase 2 (RDR2), and processed by dicer-like 3 (DCL3) [34]. One strand of the resulting 24-nt dsRNA fragments is loaded onto argonaute 4 (AGO4) leading to generation of a silencing

silenced loci is dependent on RNA-guided *de novo* methylation [33].

transcription machinery is unable to reach it.

*2.2.1 Histone acetylation/deacetylation*

acetylation modifications [29, 30].

**2.3 miRNA directed DNA methylation**

*2.2.2 Histone methylation*

**10**

effector complex. DNA methylation at sites having sequence homology to the siRNA is dependent on, Pol V, which is a DNA dependent RNA polymerase that transcribes non-coding RNAs. Transcription of Pol V is facilitated by a chromatin remodeling protein which is defective in RNA-directed DNA methylation 1 (DRD1) [35]. KOW domain transcription factor1 (KTF1) which is an adaptor protein, mediates binding of AGO4 and AGO4-bound siRNAs onto the transcripts generated by Pol V forming a silencing effector. This effector acts as signal for DRM2 to introduce methylation at target sites [35]. Development of stress tolerant crop has successfully been achieved by the use of RNAi technology. Transgenic rice plants with tolerance to drought were developed by silencing of activated C-kinase1 receptor [36].

### **3. Epigenetic changes in crops against abiotic stresses**

Due to the unpredictable climate change, crop plants are frequently exposed to a variety of abiotic stresses resulting in reduced crop productivity. Analysis of the stress-associated genes and their regulation in response to the stress can be utilized to enhance understanding of the plant's ability to adapt under changing climatic conditions. DNA methylation and/or histone modifications are influenced by abiotic/biotic factors resulting in the better adaptability of the plants to the adverse environmental conditions. Such epigenetic modifications provide a mechanistic basis for stress memory, which enables plants to respond more effectively and efficiently to the recurring stress as well as to prepare the offspring for potential future assaults.

#### **3.1 Salt-induced epigenetic changes in crop plants**

Environmental stresses result in hyper or hypomethylation of DNA. Evidence implicates epigenetic mechanisms in modulating gene expression in plants under abiotic stress. Promoter and gene-body methylation plays important role in regulating gene expression in genotype and organ specific manner under salt stress conditions. Song et al. [37] observed that DNA methylation and histone modifications may have a combined effect on stress inducible gene as salinity stress was reported to affect the expression of various transcription factors in soybean. Ferreira et al., [38] emphasized that hypomethylation in response to salt stress may be correlated with altered expression of DNA demethylases. In another report [39] contrasting differences in cytosine methylation patterns were observed in salinity tolerant wheat cultivar SR3 and its progenitor upon salinity stress imposition. The responses of contrasting wheat genotypes under salt stress could be attributed to the altered expression levels of high affinity potassium transporters (HKTs) regulated through genetic and/or epigenetic mechanisms [40]. It was found that the coding region of high affinity potassium transporters (HKTs) showed variations in 5-mC content in the contrasting wheat genotypes. Salt stress significantly increased methylation level in wheat genotypes. Cytosine residues in CG context were all methylated, whereas increase in 5-mC was observed in CHG and CHH contexts in the shoot of a salt-sensitive wheat genotype under the stress. Variations in chromatin structure (facilitated through histone modifications) also play important role in salt tolerance. Kaldis et al. [41] reported that in *Arabidopsis thaliana* the transcriptional adaptor ADA2b (a modulator of histone acetyltransferases activity) is responsible for its hypersensitivity to salt stress. However, histone modifications are reversible and cross-talk between histone acetylation and cytosine methylation makes the plant responses more complex. Thus, salt stress affects genome-wide DNA methylation as well as histone modifications and these processes are linked to each other for synchronized action against salt stress [42].

### **3.2 Heat induced epigenetic changes in crop plants**

Naydenov, [43] reported that upregulated epigenetic modulators like DRM2, nuclear RNA polymerase D1 (NRPD1) and NRPE1 may be responsible for increased genome methylation in *Arabidopsis thaliana* under heat stress conditions. Heat stress related study in rice showed reduction in seed size which is controlled by OsFIE1 (fertilization independent endosperm). Folsom and coworkers [44] in their study reported that DNA methylation and histone (H3K9me2) methylation are the two major factors governing the expression of OsFIE1. It was found that under heat stress both DNA methylation as well as histone methylation showed a decline (DNA methylation declined by 8.8% and 6.6% with respect to CH and CHG context). Reduced methylation levels resulted into lower expression of OsFIE1 and lead to reduction in rice seed size. Histone modifications like acetylation have also been reported to occur under heat stress conditions. At high temperatures, a histone variant H2A.Z causes transcriptional changes in stress responsive genes [45]. Mutations in a gene *GCN5* that codes for histone acetyltransferase, resulted in impaired transcriptional activation of heat stress responsive genes like HSAF3 and MBF1c and lead to thermal susceptibility of *Arabidopsis thaliana* [46]. The duration of heat treatment also has diverse effects on the epigenetic mechanisms emphasizing complexity in the epigenetic regulation of heat stress [47].

#### **3.3 Epigenetic modifications in response to drought**

Drought stress conditions generally tend to increase demethylation. It is also observed that DNA methylation shows tissue specificity. In *Oryza sativa* drought induced a total of 12.1% methylation differences accounted across different tissues, genotype and developmental stages. The overall DNA methylation level at the same developmental stage was lesser in roots than in leaves indicating significant role of roots under water insufficiency [48]. Correlation between DNA methylation and drought stress tolerance has been shown in lowland and drought-tolerant rice cultivars. IR20, a drought susceptible variety, undergoes hypomethylation under drought conditions whereas the tolerant varieties "PMK3" and "Paiyur" showed hypermethylation. These changes in methylation pattern contributed to differential expression of stress responsive genes [49]. In another study conducted in rice it was illustrated that hypomethylation has significant role in the drought tolerant attribute of rice genotypes [50].

In several studies the abundance in transcript levels of drought responsive gene was correlated with changes in histone modification. Under drought conditions several histone alterations like acetylation, methylation, phosphorylation and sumoylation occurs [51]. Reports have documented that drought stress response is memorized through histone modification of various drought stress induced genes [52]. In a study in *A. thaliana* it was shown that an increase in H3K4 trimethylation and H3K9 acetylation on the promoter region and H3K23 and H3K27 acetylation on the coding regions is responsible for drought-induced expression of stressresponsive genes [53]. Under stress conditions, accumulation of transcripts of stress responsive genes was positively correlated with histone modifications H3K9ac and H3K4me3 as both are marks of an active state of gene expression [54].

#### **3.4 Epigenetic modifications in response to cold**

Upon imposition of cold stress HDACs are upregulated that results into deacetylation of H3 and H4 and successively heterochromatic tandem repeats get activated [55, 56]. This results into reduction of DNA methylation and histone (H3K9me2) methylation at the targeted region of maize genome [39, 57]. In a study conducted

**13**

resolution.

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

**4.1 Histone modifications**

*4.1.2 ChiP-Seq*

*4.1.3 ChiP PCR*

on the effect of cold on maize seedlings it was found that cold stress induced genome wide DNA methylation in root tissues except only in a 1.8-kb segment designated as ZmMI1. Under normal conditions ZmMI1 segment is methylated but under chilling conditions it is demethylated. This segment is representative of a

Even after 7 days of recovery, cold induced hypomethylation was not reverted back. In a similar study conducted by Saraswat et al. of DNA methylation pattern in cold grown maize 28 differentially amplified fragments were obtained. *In silico* analysis of these fragments revealed their role in several processes like photosynthesis, hormone regulation and in cold response [59]. A recent study in apple highlighted the importance of epigenetic changes in response to dormancy caused by low temperature. High chilling conditions decreased total methylation that lead

This technique is used to assay DNA–protein binding under *in vivo* conditions. This involves shearing genomic DNA into smaller fragments through sonication to generate fragments ranging 200–800 base pairs. Gentle formaldehyde treatment is given to crosslink proteins with DNA. Antibodies raised specifically for protein of interest are used to precipitate the protein-DNA complex. Precipitated DNA thus

Advancements in the field of next-generation sequencing have made it possible

to combines ChiP with next-generation sequencing technology such as Solexa. ChiP-Seq combines Chromatin immunoprecipitation and sequencing technologies

Immunoprecipitated DNA is amplified and quantified by real time PCR (RT-PCR) using TaqMan or Syber Green Technologies with specified primers for

Earlier studies focused on determining methylation status of the gene of interest. With the use of microarray hybridization technology DNA methylation has been scaled up to genome wide level. Next generation sequencing platforms are now being used for the construction of genomic maps of DNA methylation at single-base

Bisulfite treatment converts unmethylated cytosines to uracil, allowing for the identification of methylated cytosines by comparing a treated sample to a reference

analysis of specific genomic regions associated with particular histones.

stress responsive gene that plays role under stress conditions [58].

to reinitiation of active growth and subsequent fruit set in apple [60, 61].

**4. Techniques for deciphering epigenetic changes in plants**

obtained is released by acid treatment and amplified by PCR [62].

to decipher genome wide distribution of histone proteins [62].

**4.2 DNA methylation profiling in plants**

*4.2.1 Genome-wide bisulfite sequencing*

*4.1.1 Chromatin immunoprecipitation (ChiP) techniques*

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

*Epigenetics*

**3.2 Heat induced epigenetic changes in crop plants**

ing complexity in the epigenetic regulation of heat stress [47].

Drought stress conditions generally tend to increase demethylation. It is also observed that DNA methylation shows tissue specificity. In *Oryza sativa* drought induced a total of 12.1% methylation differences accounted across different tissues, genotype and developmental stages. The overall DNA methylation level at the same developmental stage was lesser in roots than in leaves indicating significant role of roots under water insufficiency [48]. Correlation between DNA methylation and drought stress tolerance has been shown in lowland and drought-tolerant rice cultivars. IR20, a drought susceptible variety, undergoes hypomethylation under drought conditions whereas the tolerant varieties "PMK3" and "Paiyur" showed hypermethylation. These changes in methylation pattern contributed to differential expression of stress responsive genes [49]. In another study conducted in rice it was illustrated that hypomethylation has significant role in the drought tolerant attribute of rice genotypes [50]. In several studies the abundance in transcript levels of drought responsive gene was correlated with changes in histone modification. Under drought conditions several histone alterations like acetylation, methylation, phosphorylation and sumoylation occurs [51]. Reports have documented that drought stress response is memorized through histone modification of various drought stress induced genes [52]. In a study in *A. thaliana* it was shown that an increase in H3K4 trimethylation and H3K9 acetylation on the promoter region and H3K23 and H3K27 acetylation on the coding regions is responsible for drought-induced expression of stressresponsive genes [53]. Under stress conditions, accumulation of transcripts of stress responsive genes was positively correlated with histone modifications H3K9ac and

H3K4me3 as both are marks of an active state of gene expression [54].

Upon imposition of cold stress HDACs are upregulated that results into deacetylation of H3 and H4 and successively heterochromatic tandem repeats get activated [55, 56]. This results into reduction of DNA methylation and histone (H3K9me2) methylation at the targeted region of maize genome [39, 57]. In a study conducted

**3.4 Epigenetic modifications in response to cold**

**3.3 Epigenetic modifications in response to drought**

Naydenov, [43] reported that upregulated epigenetic modulators like DRM2, nuclear RNA polymerase D1 (NRPD1) and NRPE1 may be responsible for increased genome methylation in *Arabidopsis thaliana* under heat stress conditions. Heat stress related study in rice showed reduction in seed size which is controlled by OsFIE1 (fertilization independent endosperm). Folsom and coworkers [44] in their study reported that DNA methylation and histone (H3K9me2) methylation are the two major factors governing the expression of OsFIE1. It was found that under heat stress both DNA methylation as well as histone methylation showed a decline (DNA methylation declined by 8.8% and 6.6% with respect to CH and CHG context). Reduced methylation levels resulted into lower expression of OsFIE1 and lead to reduction in rice seed size. Histone modifications like acetylation have also been reported to occur under heat stress conditions. At high temperatures, a histone variant H2A.Z causes transcriptional changes in stress responsive genes [45]. Mutations in a gene *GCN5* that codes for histone acetyltransferase, resulted in impaired transcriptional activation of heat stress responsive genes like HSAF3 and MBF1c and lead to thermal susceptibility of *Arabidopsis thaliana* [46]. The duration of heat treatment also has diverse effects on the epigenetic mechanisms emphasiz-

**12**

on the effect of cold on maize seedlings it was found that cold stress induced genome wide DNA methylation in root tissues except only in a 1.8-kb segment designated as ZmMI1. Under normal conditions ZmMI1 segment is methylated but under chilling conditions it is demethylated. This segment is representative of a stress responsive gene that plays role under stress conditions [58].

Even after 7 days of recovery, cold induced hypomethylation was not reverted back. In a similar study conducted by Saraswat et al. of DNA methylation pattern in cold grown maize 28 differentially amplified fragments were obtained. *In silico* analysis of these fragments revealed their role in several processes like photosynthesis, hormone regulation and in cold response [59]. A recent study in apple highlighted the importance of epigenetic changes in response to dormancy caused by low temperature. High chilling conditions decreased total methylation that lead to reinitiation of active growth and subsequent fruit set in apple [60, 61].

## **4. Techniques for deciphering epigenetic changes in plants**

### **4.1 Histone modifications**

#### *4.1.1 Chromatin immunoprecipitation (ChiP) techniques*

This technique is used to assay DNA–protein binding under *in vivo* conditions. This involves shearing genomic DNA into smaller fragments through sonication to generate fragments ranging 200–800 base pairs. Gentle formaldehyde treatment is given to crosslink proteins with DNA. Antibodies raised specifically for protein of interest are used to precipitate the protein-DNA complex. Precipitated DNA thus obtained is released by acid treatment and amplified by PCR [62].

#### *4.1.2 ChiP-Seq*

Advancements in the field of next-generation sequencing have made it possible to combines ChiP with next-generation sequencing technology such as Solexa. ChiP-Seq combines Chromatin immunoprecipitation and sequencing technologies to decipher genome wide distribution of histone proteins [62].

#### *4.1.3 ChiP PCR*

Immunoprecipitated DNA is amplified and quantified by real time PCR (RT-PCR) using TaqMan or Syber Green Technologies with specified primers for analysis of specific genomic regions associated with particular histones.

#### **4.2 DNA methylation profiling in plants**

Earlier studies focused on determining methylation status of the gene of interest. With the use of microarray hybridization technology DNA methylation has been scaled up to genome wide level. Next generation sequencing platforms are now being used for the construction of genomic maps of DNA methylation at single-base resolution.

#### *4.2.1 Genome-wide bisulfite sequencing*

Bisulfite treatment converts unmethylated cytosines to uracil, allowing for the identification of methylated cytosines by comparing a treated sample to a reference sample [63]. Bisulfite sequencing evaluates individual cytosines in a target sequence for essentially all cytosines in a genome (i.e. whole-genome bisulfate sequencing or WGBS).

## *4.2.2 Methylated DNA immunoprecipitation (MeDIP)*

Genomic DNA is fragmented and precipitated with 5-methylcytosine-specific antibody. The precipitated DNA is then analyzed by PCR or whole genome tiling microarrays [64, 65].

## *4.2.3 Reduced-representation bisulfite sequencing (RRBS)*

RRBS came into existence for the purpose of deciphering the mammalian methylome at low cost [66]. Bisulfite sequencing can be used for genomic fragments that are isolated with restriction enzymes thus providing single-nucleotide resolution of DNA methylation within each of the fragments. Availability of both sequence and methylation variation from same set of locus allows comparison of genetic and epigenetic differences. It is based on MspI restriction digestion and selection of (40 and 220 basepair) digested fragments for bisulfite conversion and sequencing [67]. RRBS has been adopted for plant population studies and can be applied to species for which no reference genomes are available [68, 69]. RRBS has also been used in oak populations [70] and *Brassica rapa* [71].

## *4.2.4 Shotgun bisulfite sequencing*

This combines bisulfite treatment of genomic DNA with next generation sequencing technology such as Solexa sequencing. The converted sequences are mapped to the reference genome sequence to identify methyl-cytosines [63, 72].

## **5. Conclusions**

In view of the increasing stress conditions experienced by plants due to global climatic changes, epigenetics is considered as an important regulatory mechanism that is influenced by environmental stimulus. This regulatory mechanism is of utmost significant importance in terms of its inheritance over generations. Advancements in the ultra-high-throughput techniques have revolutionized identification of epigenetic changes and improved our knowledge on effect of epigenetic changes on regulation of gene expression. Manipulation of DNA (de) methylation level at specific loci may allow us to regulate gene expression and the neighboring chromatin states, impacting cell physiology and biochemistry. Therefore, one of the possible, yet unexplored, ways to improve stress tolerance in crop plants may be to augment stress memory of the plants by targeted modification of the epigenome. Thus utilizing epigenetic variation for developing improved abiotic stress tolerant crop verities is an undertaking of paramount importance.

## **Acknowledgements**

Authors would like to thank Director ICAR-Indian Institute of Wheat and Barley Research.

**15**

**Author details**

Karnal, Haryana, India

provided the original work is properly cited.

Garima Singroha\* and Pradeep Sharma

ICAR-Indian Institute of Wheat and Barley Research (IIWBR),

\*Address all correspondence to: garima.singroha@gmail.com

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

DNA deoxyribonucleic acid RNAi ribonucleic acid interference PTMs post-transcriptional modifications

H3K9 histone 3, 9th lysine H3K27 histone 3, 27th lysine HATs histone acetyltransferases

nt nucleotide

AGO4 argonaute 4

HKMT histone lysine methyl transferases PRMT protein arginine methyl transferases

RDR2 RNA-dependent RNA polymerase 2 siRNAs small interfering ribonucleic acid

dsRNA double stranded ribonucleic acid HKTs high affinity potassium transporters

OsFIE1 fertilization independent endosperm

WGBS whole-genome bisulfate sequencing MeDIP methylated DNA immunoprecipitation RRBS reduced representation bisulfite sequencing

NRPD1 nuclear RNA polymerase D1

PCR polymerase chain reactions

The authors hereby declare that there is no conflict of interest.

**Conflict of interest**

**Abbreviations**

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium,

*Epigenetic Modifications in Plants under Abiotic Stress DOI: http://dx.doi.org/10.5772/intechopen.84455*

## **Conflict of interest**

*Epigenetics*

WGBS).

microarrays [64, 65].

sample [63]. Bisulfite sequencing evaluates individual cytosines in a target sequence for essentially all cytosines in a genome (i.e. whole-genome bisulfate sequencing or

Genomic DNA is fragmented and precipitated with 5-methylcytosine-specific antibody. The precipitated DNA is then analyzed by PCR or whole genome tiling

RRBS came into existence for the purpose of deciphering the mammalian methylome at low cost [66]. Bisulfite sequencing can be used for genomic fragments that are isolated with restriction enzymes thus providing single-nucleotide resolution of DNA methylation within each of the fragments. Availability of both sequence and methylation variation from same set of locus allows comparison of genetic and epigenetic differences. It is based on MspI restriction digestion and selection of (40 and 220 basepair) digested fragments for bisulfite conversion and sequencing [67]. RRBS has been adopted for plant population studies and can be applied to species for which no reference genomes are available [68, 69]. RRBS has also been used in

This combines bisulfite treatment of genomic DNA with next generation sequencing technology such as Solexa sequencing. The converted sequences are mapped to the reference genome sequence to identify methyl-cytosines

In view of the increasing stress conditions experienced by plants due to global climatic changes, epigenetics is considered as an important regulatory mechanism that is influenced by environmental stimulus. This regulatory mechanism is of utmost significant importance in terms of its inheritance over generations. Advancements in the ultra-high-throughput techniques have revolutionized identification of epigenetic changes and improved our knowledge on effect of epigenetic changes on regulation of gene expression. Manipulation of DNA (de) methylation level at specific loci may allow us to regulate gene expression and the neighboring chromatin states, impacting cell physiology and biochemistry. Therefore, one of the possible, yet unexplored, ways to improve stress tolerance in crop plants may be to augment stress memory of the plants by targeted modification of the epigenome. Thus utilizing epigenetic variation for developing improved abiotic stress tolerant crop verities is an undertaking of paramount

Authors would like to thank Director ICAR-Indian Institute of Wheat and Barley

*4.2.2 Methylated DNA immunoprecipitation (MeDIP)*

*4.2.3 Reduced-representation bisulfite sequencing (RRBS)*

oak populations [70] and *Brassica rapa* [71].

*4.2.4 Shotgun bisulfite sequencing*

[63, 72].

**5. Conclusions**

**14**

importance.

Research.

**Acknowledgements**

The authors hereby declare that there is no conflict of interest.

## **Abbreviations**


## **Author details**

Garima Singroha\* and Pradeep Sharma ICAR-Indian Institute of Wheat and Barley Research (IIWBR), Karnal, Haryana, India

\*Address all correspondence to: garima.singroha@gmail.com

© 2019 The Author(s). Licensee IntechOpen. This chapter is distributed under the terms of the Creative Commons Attribution License (http://creativecommons.org/licenses/ by/3.0), which permits unrestricted use, distribution, and reproduction in any medium, provided the original work is properly cited.

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**21**

**Chapter 3**

**Abstract**

A Landscape of Epigenetic

Regulation by MicroRNAs to

the Hallmarks of Cancer and

Activity to Tumor Regression

*Gabriel Cardial Tobias, João Lucas Penteado Gomes,* 

*Ursula Paula Renó Soci, Tiago Fernandes* 

and their potential role in clinical applications for cancer.

tumor progression, microRNAs

**Keywords:** epigenetic, cancer, hallmarks of cancer, cachexia, physical activity,

In the last decades, there has been a remarkable advance in the treatment of most

types of cancer, improving the patient's prognosis [1]. However, cancer remains the second major cause of death in the world and major cause of death in the rich countries [2, 3]. Cancer consists in a set of diseases characterized by the progressive accumulation of mutations in the cell. These mutations provide changes in

**1. Introduction: hallmarks of cancer, genetics and epigenetics**

*and Edilamar Menezes de Oliveira*

Cachexia: Implications of Physical

In the last decades, there has been a remarkable advance in the treatment of most types of cancer, improving the patient's prognosis. During cancer progression, tumor cells develop several biological changes to support initiation, proliferation, and resistance to death. Nearly 50–80% of all oncologic patients experience rapid weight loss that is related to ~20% of cancer-related deaths. Cancer cachexia is a syndrome characterized by loss of skeletal muscle mass, anorexia, and anemia. A lot of effort in scientific investigation has contributed to the understanding of cancer processes, in which epigenetic changes, as microRNAs, can influence cancer progression. Therefore, useful strategies to control the cancer-induced epigenetic changes in the tumor cells can have a key role in a clinical perspective to decrease the cancer development and aggressiveness. Physical activity has been proposed as a suitable tool to manage tumor growth and cachexia and to improve the deleterious sequelae experienced during cancer treatment. Although the molecular mechanisms involved in these responses are poorly understood, this chapter aims to discuss the role of microRNAs in the cancer-induced epigenetic changes and how physical activity could influence the epigenetic control of tumor cells and cachexia

## **Chapter 3**

*Epigenetics*

2016;**13**:322-324

DOI: 10.1111/mec.13230

of reduced representation bisulfite sequencing libraries for genome-scale DNA methylation profiling. Nature Protocols. 2011;**6**(4):468-481

[68] Trucchi E, Mazzarella AB, Gilfillan GD, Romero MT, Paun O. BsRADseq screening DNA methylation in natural populations of non-model species. Molecular Ecology. 2016;**25**:1697-1713

[69] van Gurp TP, Wagemaker NCAM, Wouters B, Vergeer P, Ouborg JNJ, Verhoeven KJF. epiGBS: Referencefree reduced representation bisulfite sequencing. Nature Methods.

[70] Platt A, Gugger PF, Pellegrini M, Sork VL. Genome-wide signature of local adaptation linked to variable CpG methylation in oak populations. Molecular Ecology. 2015;**24**:3823-3830.

[71] Chen X, Ge X, Wang J, Tan C, King GJ, Liu K. Genome wide DNA methylation profiling modified reduced representation bisulphate sequencing in *Brassica rapa* suggests that epigenetic

modifications play a key role in

[72] Lister R, O'Malley RC, Tonti-Filippini J, Gregory BD, Berry CC, Millar AH, et al. Highly integrated single-base resolution maps of the epigenome in *Arabidopsis*. Cell.

in Plant Sciences. 2015;**6**:836

2008;**133**:523-536

polyploidy genome evaluation. Frontiers

**20**
