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Epigenetics and Crop Improvement
Introduction
Epigenetics is the branch of biology which studies the causal interactions between genes
and their products, which bring the phenotype into being” Waddington 1942. It is
dynamic process that plays a key role in normal cell growth and differentiation.
“ Changes in gene expression inherited from cell to cell, not caused by DNA.” Holliday,
1996 .
“The study of mitotically and/or meiotically heritable changes in genes function that
cannot be explained by changes in DNA sequence” Russo et al, 1996.
“The structural adaptation of chromosomal regions so as to register, signal or perpetuate
altered activity states” Bird 2007.
“Phenotypic variation that is not attributable to genetic variation”. Champagne 2010.
What is Epigenetics?
The term epigenetics refers to heritable changes in gene expression (active versus
inactive genes) that does not involve changes to the underlying DNA sequence; a change
inphenotype without a change in genotype. Epigenetic change is a regular and natural
occurrence but can also be influenced by several factors including age, the
environment/lifestyle, and disease state. Epigenetic modifications can manifest as commonly
as the manner in which cells terminally differentiate to end up as skin cells, liver cells, brain
cells, etc. Or, epigenetic change can have more damaging effects that can result in diseases
like cancer. At least three systems including DNA methylation, histone modification
and non-coding RNA (ncRNA)-associated gene silencing are currently considered to initiate
and sustain epigenetic change.[1] New and ongoing research is continuously uncovering the
role of epigenetics in a variety of human disorders and fatal diseases.
The Evolving Landscape of Epigenetic Research: A Brief History
What began as broad research focused on combining genetics and developmental biology
by well-respected scientists including Conrad H. Waddington and Ernst Hadorn during the
mid-twentieth century has evolved into the field we currently refer to as epigenetics. The
term epigenetics, which was coined by Waddington in 1942, was derived from the Greek
word “epigenesis” which originally described the influence of genetic processes on
development. During the 1990s there became a renewed interest in genetic assimilation. This
lead to elucidation of the molecular basis of Conrad Waddington’s observations in which
environmental stress caused genetic assimilation of certain phenotypic characteristics
in Drosophila fruit flies. Since then, research efforts have been focused on unraveling the
epigenetic mechanisms related to these types of changes. Currently, DNA methylation is one
of the most broadly studied and well-characterized epigenetic modifications dating back to
studies done by Griffith and Mahler in 1969 which suggested that DNA methylation may be
important in long term memory function. The renewed interest in epigenetics has led to new
findings about the relationship between epigenetic changes and a host of disorders including
various cancers, mental retardation associated disorders, immune disorders, neuropsychiatric
disorders and pediatric disorders.
EPIGENETIC GENE REGULATION
Two major machanism
. Modification at the DNA level
1. cytosine methylation
Histone modification - the histone code
1. Histone acetylation
2. Histone methylation
3. Histone phosphorylation
4. Histone ubiquitilation
5. Different types of histones
DNA methylation;
Occurs after DNA replication and during cell differentiation. Involves addition of a methyl
group (-CH3) to cytosine by methyltransferases. Takes place almost exclusively on cytosine
bases adjacent a guanine base, a combination called CpG dinucleotide. Many CpG cluster in and
near promoter sequences of genes, called Cp islands.
•
DNA structure is maintained from generation to generation.This structure is modified by
base methylation in nearly all cells and organisms.It occurs in the cells of fungi, plants,
non-vertebrates and vertebrates.In vertebrates, 3-6% of DNA cytosine is methylated. No
methylation in many insects and single-celled eukaryotes. In plants, 30% of DNA
cytosine is methylated.
•
HOW DNA METHYLATION HELP THE GENE TURNED OFF?
EXAMPLE FOR DNA METHYLATION IN HONEYBEES

Honeybees grow to be either queens or workers depending on whether they are fed royal
jelly or beebread. Despite they are genetically identical at the larvae level, honeybee
queens fed pure royal jelly are markedly different from workers. The different honeybee
phenotype occurs through epigenetic changes in DNA methylation patterns induced by
the different type of honey. Science 2008;319:1827

Comparative genomic hybridization for methylated DNA


Mapping chromosomal regions with differential DNA methylation in MZ twins by
using comparative genomic hybridization for methylated DNA. Competitive
hybridization onto normal metaphase chromosomes of the AIMS products generated
from 3- and 50-year-old twinpairs.

Examples of the hybridization of chromosomes 1, 3, 12, and 17 are displayed. The 50year-old twin pair shows abundant changes in the pattern of DNA methylation observed
by the presence of green and red signals that indicate hypermethylation and
hypomethylation events, whereas the 3-year-old twins have a very similar distribution of
DNA methylation indicated by the presence of the yellow color obtained by equal
amounts of the green and red dyes.
Role of DNA methylation
Long term silencing of gene.
Silencing of repetitive elements ( eg: transposons).
Role in X-chromosome inactivation.
In the establishment and maintenance of imprinted genes.
Suppresses the expression of viral genes and other deletorious elements that have been
incorporated into the genome of the host over time.
Histone modification
•
In Eukaryotes, DNA is wrapped around histone proteins and forms nucleosome.
•
Nucleosome is a basic unit of chromatin
•
•
Chromatin can be highly packed or loosely packed, and correlated to the gene expression
levels.
•
Modifications in histone proteins affects the structure of Chromatin
Heterochromatin = Highly packed form
Euchromatin = Loosely packed form
Low gene expression in heterochromatin in euchromatin is vice versa.
Histone Acetylation/Deacetylation
Histone acetylation occurs by the enzymatic addition of an acetyl group (COCH3) from
acetyl coenzyme A. The process of histone acetylation is tightly involved in the regulation of
many cellular processes including chromatin dynamics and transcription, gene silencing, cell
cycle progression, apoptosis, differentiation, DNA replication, DNA repair, nuclear import, and
neuronal repression. The modifying enzymes involved in histone acetylation are called
histone acetyltransferases (HATs) and they play a critical role in controlling histone H3 and H4
acetylation. More than 20 HATs have been identified which can be classified into five families:
GNAT1, MYST, TAFII250, P300/CBP, and nuclear receptor coactivators such as
ACTR. Histone H3 acetylation may be increased by inhibition of histone deacetylases (HDACs)
and decreased by HAT inhibition. Histone deacetylaces (HDACs) catalyze the hydrolytic
removal of acetyl groups from histone lysine residues.
An imbalance in the equilibrium of histone acetylation has been associated with tumorigenesis
and cancer progression. Detecting whether histone H3 is acetylated at its lysine residues would
provide useful information for further characterization of acetylation patterns or sites, thereby
leading to a better understanding of epigenetic regulation of gene activation as well as the
development of HAT-targeted drugs. Similar to HATs, HDACs play a critical role in various
cellular processes involving histone H3 and H4. So far, at least 4 classes of HDACs have been
identified. Class I HDACs include 1, 2, 3, and 8. Class II HDACs are comprised of 4, 5, 6, 7, 9,
and 10. Class III enzymes, known as sirtuins, require NAD+ cofactors and include SIRTs 1-7.
The Class IV enzyme, which contains only HDAC11, has features of both Class I and II. HDAC
inhibition displays significant effects on apoptosis, cell cycle arrest, and differentiation in cancer
cells. HDAC inhibitors are currently being developed as anticancer
agents
Histone Methylation/Demethylation
Histone methylation is defined as the transfer of one, two, or three methyl groups from Sadenosyl-L-methionine to lysine or arginine residues of histone proteins by histone
methyltransferases (HMTs). HMTs control or regulate DNA methylation through chromatindependent transcriptional repression or activation. In the cell nucleus, when histone methylation
occurs, specific genes within the DNA complexed with the histone may be activated or
silenced. Several different histone methyltransferases exist that are specific for the lysine or
arginine residue which they modify. On histone H3 for example, SET1, SET7/9, Ash1, ALL-1,
MLL, ALR, Trx, and SMYD3 are histone methyltransferases that catalyze methylation of
histone H3 at lysine 4 (H3-K4) in mammalian cells. ESET, G9a, SUV39-h1, SUV39-h2,
SETDB1, Dim-5, and Eu-HMTase are histone methyltransferases that catalyze methylation of
histone H3 at lysine 9 (H3-K9) in mammalian cells. G9a and polycomb group enzymes such as
EZH2 are histone methyltransferases that catalyze methylation of histone H3 at lysine 27 (H3K27)
in
mammalian
cells. Both
H3-K9
and
H3-K27
methylation
mediatesheterochromatin formation and also participates in silencing gene expression at
euchromatic sites. Increased global H3-K27 methylation is also found to be involved in some
pathological processes such as cancer progression.
On the other hand, arginine methylation of histones H3 and H4 promotes transcriptional
activation and is mediated by a family of protein arginine methyltransferases (PRMTs). There
are 9 types of PRMTs found in humans but only 7 members are reported to methylate histones.
They can mediate mono or dimethylation of arginine residues. Based on the position of the
methyl group addition, PRMTs can be classified into type I (CARM1, PRMT1, PRMT2,
PRMT3, PRMT6, and PRMT8) and type II (PRMT5 and PRMT7). Type II PRMTs are found to
be strongly implicated in diseases like cancer.[3] For example, PRMT5 plays a role in the
repression of certain tumor suppressor genes such as RB tumor suppressors while PRMT7
overexpression is observed in breast cancer. Detection of activity and inhibition of type II
PRMTs as well as other HMTs would be important in elucidating mechanisms of epigenetic
regulation of gene activation and silencing, as well as benefiting cancer diagnostics and
therapeutics.
Histone demethylation is the removal of methyl groups in modified histone proteins via
histone demethylases. These demethylases have been found to have potential oncogenic
functions and involvement in other pathological processes. The discovery of histone
demethylases demonstrates that histone methylation is not a permanent modification but rather a
more dynamic process. Two major families of demethylases have been discovered: Lysine
specific demethylase 1 (LSD1) and Jumonji domain containing (JmjC domain) histone
demethylases (JMJD2, JMJD3/UTX and JARIDs). The specific amino acid residue and degree of
methylation determines the demethylation enzyme. For example, on histone H3, mono- and dimethylated lysine 4 are demethylated by LSD1 (BHC110, KDM1) and tri-methylated lysine 4 by
JARID (1A-1D); di- and tri-methylated lysine 27 are demethylated by JMJD3 and UTX
(KDM6A) and mono- and di-methylated lysine 9 are demethylated by JMJD1 and tri-methylated
lysine 9 is demethylated by JMJD2. Inhibition of histone demethylases may lead to histone remethylation at specific residues important for chromatin dynamics and gene expression.
Furthermore, detection of the activity and inhibition of these enzymes would be important in
elucidating mechanisms of epigenetic regulation of gene activation and silencing and may
benefit cancer diagnostics and therapeutics.
Histone phosphorylation (H3)
 Histones are phosphorylated during mitosis.
 Histones are also phosphorylated by signal transduction pathways like the ERK pathway
in response to external signals. It is not known how (and if) this phosphorylation
contributes to gene expression.
Histone ubiquitylation
•
Addition and removal of Ub (a LARGE moiety) to histone tails – Functions largely
unknown in vertebrates.
•
ubiquitylation
•
H2A K119: repression
•
H2B K120: activation
•
H3 and H4: DNA repair (CUL4)
de-ubiquitylation – Recrutiment of other proteins in yeast
H2A
Dub (PCAF)
H2B
Ubp8 (SAGA)
Functions: transcription elongation, polycomb repression
Transposon silencing
Transposons; DNA sequence which is able to relocate itself within a genome. it is responsible
for the mutation to large scale genome rearrengements.
Methyltransferase mutants accumlate the mutations caused by transposon activity over the
generations. Kakutani, 1996
Four classical epigenetic phenomena
•
Position-Effect Variegation (PEV) (H.J. Muller, 1930)
•
Paramutation (R.A. Brink, 1958)
•
X-chromosome Inactivation (M.F. Lyon, 1961)
Position effect variegation (PEV) in Drosophila
Transgenes located near heterochromatic regions undergo mitotically stable changes in
expression due to changes in chromatin organization. An inversion placed the white gene
close to the pericentric heterochromatin .Sectors of drosophila white gene off (white eye
color) result from heterochromatic regions expanding to include the white gene (Grelon &
Elgin, Nature 447:399
Paramutation: The interaction of two alleles of the same locus, resulting in a heritable
change of one allele that isinduced by the other allele.
•
The mop1 (mediator of paramutation1) mutation
 The mop1 mutations reactivate silenced Mutator elements.
 Plants carrying mutations in the mop1 gene also stochastically exhibit pleiotropic
developmental phenotypes.
 Mop1 is an RNA-dependent RNA polymerase gene (RDRP), most similar to the RDRP
in plants that is associated with the production of short interfering RNA (siRNA)
targeting chromatin.
 It was proposed the mop1 RDRP is required to maintain a threshold level of repeat RNA,
which functions in trans to establish and maintain the heritable chromatin states
associated with paramutation.
X choromosome inactivation :
 The effects of epigenetics have been known for many years.
 Lyon Hypothesis from 1961.
 Renamed the Lyon Law in 2011.
 Inactive X chromosome is heavily epigenetically modified.
B – orange, b - black
One of the genes controlling fur color is on the X chromosome
Female mammals are genetic mosaics. Random X inactivation early in embryonic development
leads to patchworks of skin cells expressing each allele
Genomic imprinting:
Genomic imprinting is a genetic phenomenon by which certain genes are expressed in a parentof-origin-specific manner. It is an inheritance process independent of the classical Mendelian
inheritance. Imprinted genes are either expressed only from the allele inherited from the mother
(eg. H19 or CDKN1C), or in other instances from the allele inherited from the father (eg. IGF2).
Forms of genomic imprinting have been demonstrated in insects, mammals and flowering plants.
Beckwith-Wiedemann Syndrome
•
Activation of the maternal Igf2 gene during egg formation or very early in development
causes Beckwith-Wiedemann Syndrome (BWS).
•
Symptoms:
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overgrowth.
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increased risk of cancer, especially during childhood.
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Imprinted genes are under greater selective pressure than normal genes. This is because
only one copy is active at a time. Any variations in that copy will be expressed. There is
no "back-up copy" to mask its effects.
•
Ligers and Tigons: A male lion and a female tiger produce a liger - the biggest of the big
cats. A male tiger and a female lion produce a tigon, a cat that is about the same size as
its parents.
Epigenetic diseases
Angelman/Prader-Willi
•
Commonly referred to as genomic imprinting.
•
Imprinting is not the cause of these syndromes but is responsible for the unique
presentation of these diseases.
•
1.Prader willi syndrome 2. Angleman syndrome
Epigenetics and cancer
 DNA repair is a critical process to maintain genomic fidelity.
 Loss of DNA repair is thought to be a major contributor to the development of cancer.
 Epigenetic changes involving DNA repair genes are thought to be a major early step in
cancer progression.
 ~13% of sporadic breast cancers and 5-30% of ovarian cancers present with
hypermethylation of the BRCA1 gene.
 40-90% of sporadic colorectal cancer has hypermethylation of the MGMT gene (O6methylguanine methyltransferase).
Epigenetic Modifications May Drive Cognitive Decline
 Chromatin remodeling in the hippocampus is necessary for stabilizing long term
memories.
 Aged mice have lower H4K12 acetylation.
 HDAC inhibitor restores H4K12 acetylation and improved memory function
 from Sweatt, Science 328, 701 (2010.
Epigenetics in medicine
 Epigenetics drugs such as azacitidine can treat leukemia.
 But epigenetics drugs are not specific and can harm other areas of the genome.
 It can also reexamine existing drugs.
 Epigenetics can help us master stem cells.
Epigenetics: Applications in crop improvement
Improvement in Nutritional value
By using the RNAi technology the genes can be expressed at specific stage of the crop. It
has been used in many crops for nutritional improvement like in cotton it was used to develop
low gossypol lines, Increasing grain amylase content in wheat, Bringing down BOAA (βoxalylamino alanine-L-alanine) levels from Lathyrus Fruit quality in tomato, Coffee with very
low caffeine content .(Angaji et al. 2010).
Dosage compensation or parental imprinting
Epigenetic studies have been conducted to know its implications in controlling the
contribution of parents in seeds or endosperm development.
•
It was observed that the characteristics of seeds in Arabidopsis were significantly affected
due to changes in parental genome dosage (Scott et al. 1998).
•
This could be very useful in heterosis breeding to manipulate contribution of
parents.
Increasing stress tolerance in plants

These technologies could be effectively used for the down regulation of the stress related
genes for drought, salinity, heat, cold etc.

In rice the RNA mediated disruption of some genes resulted in the drought tolerance at
vegetative and reproductive stages (Manavalan et al. 2012).
Improvement in virus resistance in plants

RNAi technology has been fruitfully used in banana for production of varieties resistant
to Banana Bract Mosaic Virus (BBrMV) and in barley for Barley Yellow Dwarf Virus
(BYDV).

In addition to these applications, epigenetic research has greater role in manipulating
plant architecture, flower colour, flowering time, in forestry for wood quality etc.
References
 Berger, S.L. et. al. 2009. An operational definition of epigenetics. Genes Dev. 23, 781-783.
 Kaati, G. et. al. 2002. Cardiovascular and diabetes mortality determined by nutrition during
parents' and grandparents' slow growth period. Eur. J. Hum. Genet. 10 682-688.
 Esteller, M. et. al. 2000. Promoter hypermethylation and BRCA1 inactivation in sporadic
breast and ovarian tumors. J Natl Cancer Inst. 92, 564-569.
 Shen, L. et. al. 2005. MGMT promoter methylation and field defect in sporadic colorectal
cancer. J Natl Cancer Inst. 97, 1330-1338.
 Garcia-Manero, G. 2012. Can we improve outcomes in patients with acute myelogenous
leukemia? Incorporating HDAC inhibitors into front-line therapy. Best Pract Res Clin
Haematol. 25, 427-435.
 Fraga, M.F. et al. 2005. Epigenetic differences arise during the lifetime of monozygotic
twins. Proc Natl Acad Sci U S A. 102,10604-10609.
 Roth, T.L. et. al. 2009. Lasting epigenetic influence of early-life adversity on the BDNF
gene. Biol Psychiatry. 65 760-769.
 Mirouze M, Paszkowski J (2011) Epigenetic contribution to stress adaptation in plants. Curr
Opin Plant Biol 14:267–274
 Morales-Ruiz T, Ortega-Galisteo AP, Ponferrada-Marin MI, Martinez- Macias MI, Ariza
RR, Roldan-Arjona T (2006) DEMETER and REPRESSOR OF SILENCING 1 encode 5methylcytosine DNA glycosylases. Proc Natl Acad Sci USA 103:6853–6858
 Muthamilarasan M, Prasad M (2013) Plant innate immunity: an updated insight in defense
mechanism. J Biosci. doi:10.1007/ s12038-013-9302-2
 Penterman J, Zilberman D, Huh JH, Ballinger T, Henikoff S, Fischer RL (2007) DNA
demethylation in the Arabidopsis genome. ProcNatl Acad Sci USA 104:6752–6757
 Ramsahoye BH, Biniszkiewicz D, Lyko F, Clrk V, Bird AP, Jaenisch R (2000) Non-CpG
methylation is prevalent in embryonic stem cells and may be mediated by DNA
methyltransferase 3a. Proc Natl Acad Sci USA 97:5237–5242
 Rodrı´guez-Negrete EA, Carrillo-Tripp J, Rivera-Bustamante RF (2009) RNA silencing
against geminivirus: complementary action of posttranscriptional gene silencing and
transcriptional gene silencing in host recovery. J Virol 83:1332–1340
 Sabbah M, Raise M, Tal M (1995) Methylation of DNA in NaCladapted cells of potato.
Plant Cell Rep 14:467–470
 Sahu PP, Rai NK, Chakraborty S, Singh M, Chandrappa PH, Ramesh B, Chattopadhyay D,
Prasad M (2010) Tomato cultivar tolerant to Tomato leaf curl New Delhi virus infection
induces virus-specific short interfering RNA accumulation and defence-associated host gene
expression. Mol Plant Pathol 11: 531–544
 Sahu PP, Rai NK, Puranik S, Roy A, Khan M, Prasad M (2012) Dynamics of defenserelated components in two contrasting genotypes of tomato upon infection with Tomato
Leaf Curl New Delhi Virus. Mol Biotechnol 52:140–150
 Santos AP, Serra T, Figueiredo DD, Barros P, Lourenc¸o T, Chander S, Oliveira MM, Saibo
NJ (2011) Transcription regulation of abiotic stress responses in rice: a combined action of
transcription factors and epigenetic mechanisms. OMICS 15:839–857
 Shaik R, Ramakrishna W (2012) Bioinformatic analysis of epigenetic and micro-RNA
mediated regulation of drought responsive genes in rice. PLoS ONE 7(11):e49331
 Sharma N, Sahu PP, Puranik S, Prasad M (2012) Recent advances in plant-virus interaction
with emphasis on small interfering RNAs (siRNAs). Mol Biotechnol. doi: 10.1007/s12033012-9615-7
 Song Y, Ji D, Li S, Wang P, Li Q, Xiang F (2012) The dynamic changes of DNA
methylation and histone modifications of salt responsive transcription
 Vassoler, F.M. 2013. Epigenetic inheritance of a cocaine-resistance phenotype. Nat.
Neurosci. 16, 42-47.
 Qian Y, Cheng X, Liu Y, Jiang H, Zhu S, Cheng B (2010) An epigenetic change in rice
cultivars under water stress conditions. Plant Cell Rep 29:1365–1376