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Epigenetics mediated gene

regulation in plants

Centre for Plant Biotechnology and Molecular

Biology

Kerala Agricultural University

Epigenetics – meaning and definition

Mechanisms

Implications

Applications

Conclusions

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Changes in gene expression that occur by a mechanism other than change to the DNA sequence

Gene-regulatory information that is not expressed in DNA sequences but transmitted from one generation (of cells or organisms) to the next

Coined by C. H. Waddington in 1942

Epigenetics means ‘above’ or ‘on top of genetics’

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What Does “Epigenetics” Mean?

Epigenetics describes phenomenon in which

genetically identical cells or organisms express their

genomes differently, causing phenotypic differences

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Genetically

identical cells or

individuals

Different epigenetic

modifications leading to

different expression patterns

Different

phenotypes

Molecular epigenetic mechanism Development of multicellular organisms occurs due to cells

differentiation by various programs of gene expression

Cells have own epigenetic signatures like

1. Genotype

2. Developmental history

3. Environmental influences and it is ultimately reflected in the

phenotype of the cells and the organism

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cytosine 5-methylcytosine

DNA can be covalently

modified by cytosine

methylation.

TTCGCCGACTAA

Methyl-

cytosine

.... DNA Methylation

DNA methylation in mammals mainly occurs on the

cytosine nucleotide in a CpG site

In plants the cytosine can be methylated at CpG, CpHpG,

and CpHpH sites, where H represents any nucleotide

DRM2, MET1, and CMT3: DNA methylases in plants

CMT3 protein is unique to the plant kingdom

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.... DNA Methylation

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Role in prokaryotes, as defence mechanism

•Escape from the restriction enzymes

•Protection from bacteriophages

In eukaryotes, it controls the mechanism of transposable

elements in the genome

Classes of methyl transferase

De novo class: Enzymes that create new methylation

mark on DNA

Eg. DNMT3a and DNMT3b

Maintainance class: Recognizes the methylation

marks on the parental strand of DNA and transfers new

methylation to the daughter strands after DNA

replication

Eg. DNMT1

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12(http://atlasgeneticsoncology.org/Educ/HeterochromEng.html)

2. Histone modification Histone: Several small, basic proteins most commonly found in

association with the DNA in the chromatin of eukaryotes

Packaging and ordering the DNA into structural unit called

nucleosomes

Histone modifications also known as epigenetic modifiers

Biological functions:

• In chromatin organization

• Gene expression

• DNA repair

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Writers: enzymes that add a mark

Erasers: enzymes that remove a mark

Readers: proteins that bind to and “interpret” the mark

14(Tarakhovsky,. Nature Immunology, 2010)

Writers' introduce histone marks (circles), 'erasers' take them out and 'readers'

can recognize a particular form of histone modification.

Histone modifications occur primarily on histone tails

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(Zeng et al., 2007)

Histone Modifications and Modifiers

Residue on the

histone tail

Type of modification Modifying enzyme

Lysine Acetylation HAT, KAT

Deacetylation HDAC

Lysine Methylation, HMT, KMT

Demethylation HDM, KDM

Lysine Unbiquitylation Ub ligase

Deubiquitylation Ub protease

Serine/

Threonine

Phosphorylation Kinase

Dephosphorylation Phosphatase

Arginine Methylation PRMT

Demethylation Deiminase

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HAT – Histone Acetylase KAT- Lysine Acetyl Transferase HDAC –Histone de- acetylase HMT-

Histone methyl transferase KMT- Lysine methyl transferase HDM-Histone de –methyl transferase

KDM – Lysine de-methyl transferase PRMT- Protein Arginine methyl transferase

Do not bind to DNA themselves

Can be recruited by:

Histone modifications (through chromodomains,

bromodomains, etc.)

Transcription factors

DNA damage

Act as transcriptional co-regulators

Enhance activities of transcriptional repressors or activators

Co-repressor: eg. HDACs

Co-activator: eg. HATs

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General roles of histone modifications

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(Moggs and Orphanides, Toxicological Sciences, 2004)

During DNA DamageIn Gene Regulation

(Wade , Hum. Mol. Genet. 2001)

HDAC

Also called post transcriptional gene silencing (PTGS)

Is a biological process in which RNA molecules inhibit

gene action

Andrew Fire and Craig C. Mello shared the Nobel Prize

in Physiology or Medicine - 2006

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20(Long et al., 2011, Genetics, 189, 1093–1102)

Methods for studying epigenetic modifications

• DNA methylation– Bisulfite sequencing

• Methylation Sensitive Amplification Polymorphism

(MSAP)

• Histone modification

• Chromatin immunoprecipitation (ChIP)

• DNA adenosine methylation identification (DamID)

• RNAi – Deep sequencing

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Bisulfite treatment differentiates cytosine and

methylcytosine

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Bisulfite treatment

TTCGCCGACTAA

No treatment

TTCGCCGACTAA

TTCGCCGAuTAA

TTCGCCGACTAA TTCGCCGATTAA

Methyl-cytosine

After bisulfite treatment,

unmethylated Cs are

read as T and so differ in

the treated and

untreated samples.

By contrast, methyl-C is

read as C and is the

same as the reference

sequence.

What is Stress?

Any external factor that exerts disadvantageous

influence on organisms

Strategies to minimize stress influence:

Tolerance, Resistance, Avoidance or Escape

Physiological alteration in metabolic pathways

Modification in gene expression pattern

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Plant materials and genotyping

Droughttolerant

DK151

Droughtsusceptible

IR64

1. Case study

Drought stress

treatment

Irrigated control Recovery

Irrigation was held at

55 d after transplanting

The stress was

maintained until leaves

of the treated plants

rolled completely and

their leaf relative water

content reached 70–

75%.

Field was irrigated at

weekly intervals

Stressed plants were

recovered by

rewatering

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Tissue sample for DNA isolation: Root and Leaf tissue collected from the drought-

stressed, well-watered, and recovered plants

Analysis of DNA Methylation-Sensitive Amplified

Polymorphisms (MSAPS)

Double enzyme combinations:

• EcoRI/MspI and EcoRI/ HpaII, used to digest the DNA samples

PCR is used to selectively amplify the DNA fragments

A set of 26 randomly selected differentially amplified

fragments were isolated, re-amplified, and purified cloned

with TA-cloning vector

The sequences obtained were analysed by NCBI BLAST

(http://blast.ncbi.nlm.nih.gov/Blast.cgi)

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type I Represents the band presence for both enzyme

combinations

type II Band presence only for EcoRI/HpaII

type III Band presence for EcoRI/MspI

type IV Represents the band absence for both enzyme

combinations

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Analysis of MSAP

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DNA methylation changes in leaves and roots of DK 151 and IR 64

Conclusion of experiment

Drought - induced genome-wide changes in DNA

methylation/ demethylation.

Large difference - DNA methylation/ demethylation

sites between drought tolerance DK151 and drought

sensitive IR64

Resistant – Highly methylated, retained methylation

on stress

Susceptible – Less methylated, lesser retention of

methylation

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MORPHOLOGICAL AND MOLECULAR

ANALYSIS OF GENETIC STABILITY IN

MICROPROPAGATED BANANA

(MUSSA SPP.) VAR. NENDAN

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2. Case study

m- Marker DNA (100 bp), 1- Mother plant, 2- SC 3rd, 3- SC 6th, 4- SC 8th, 5- SC10th,

6- SC 12th, 7- SC 14th, 8- SC16th 31

Subculture no. Hemi methylation

(%)

Internal full-

methylation

(%)

Total methylation

(%)

Mp 3.00 6.30 9.3

Sc3 3.20 9.20 12.4

Sc6 3.20 6.15 9.15

Sc8 3.42 9.36 12.78

Sc10 10.68 14.84 25.52

Sc12 16.89 26.79 43.68

Sc14 18.12 28.33 46.45

Sc16 18.27 30.43 48.7

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Initial culture shows less methylation

High variation occurs after 10th subculture and

onwards >48%

Advantage of MSAP is direct identification of

methylated sequence in the genome

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A. Better understanding on the physiological mechanisms

Epigenetic variation can causes heritable variation

DNA methylation majorly involve in plant defence against herbivorous

and pathogens

Heritable variation in plant growth responses to jasmonic acid and

salicylic acid

(Latzel et al., 2012)

B. Improving Plant Stress Tolerance

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Stress tolerance can be improved by the controlling

transposable elements

Plant phenotypic variation, improve long-term plant adaptation

to environmental challenges and, thus, increase productivity.

(Mirouze and Paszkowski, 2011)

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C. Evolutionary studies/ epigenetic diversity studies

Variation of ecologically important plant traits, root

allocation, drought tolerance and nutrient plasticity

Rapid evolution based on epigenetic variation alone should

thus be possible

(Zhang et.al., 2012)

D. Epigenetic mechanisms, yield, and heterosis

Hybrids are in general, less methylated than their parental

inbreds

Heterotic hybrids are less methylated than related nonheterotic

hybrids

Low-yielding inbreds are more methylated

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F. Transient expression of transgene

Single copy transgene into hypomethylated region

Careful design of transgene construct

Analysis of transformants at the molecular level38

Plant genomes have a distinct range of target sites

Ability to detect trait loci

To identify additional significant QTL associated with

important agronomic traits

(Long et al., 2011)

E. Epigenetic QTL mapping

39(Long et al., 2011, Genetics 189: 1093–1102)

CONCLUSIONS

Epigenetics involves DNA methylation, histone

modification and RNAi

DNA methylation effectively down-regulates/ up-

regulates gene activity by addition of a methyl group to

the five-carbon of a cytosine base

Epigenetic changes can be studied by

• Bisulfite treatment, MSAP

• DNA adenine methylase identification

• Chromatin immunoprecipitation

• Deep sequencing for RNAi

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.... CONCLUSIONS

Various application are:

• Better understanding of physiological mechanisms

• Improvement of plant stress tolerance

• Improvement of yield and heterosis

• Evolutionary and diversity studies

• QTL mapping

Disadvantage – Transient expression of transgene

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