EPIGENETICS & HETEROSIS

Models of Heterosis

Heterosis : a mystery mechanism

• Sum total of all genetic interactions in F1

hybrid cant explain every aspects of heterosis

• Emphasis to non genetic interactions

• Epigenetic effects: regulate gene expression cell fate and non mendelian inheritance

Epigenetics

spatio-temporally

change

heritable changes

No DNA sequence changes

same genotype -diverse

phenotypes

epialleles -identical DNA different

epigenetic states

inheritance of acquired

characteristics

DNA and chromatin

level

epigenetic marks –deviate from Mendelian

inheritance

Mechanisms

• DNA methylation

• Histone modifications & Chromatin remodeling

• RNAi pathway (including RNA directed DNA methylation, RdDM).

Dna methylation

• covalent addition of methyl groups to the bases of a DNA molecule- 5’ positions of cytosine residues

• catalyzed by DNA Methyltransferases

• In all sequence contexts the DOMAINS REARRANGED METHYLATION 2 (DRM2) gene-product plays a major role in establishment of mC

• Important for the silencing of active transposons

• Symmetric methylation in CpG contexts is maintained by the methyltransferase METHYLTRANSFERASE 1 (MET1).

• Cytosine methylation in CpHpG contexts is maintained by a CHROMOMETHYLASE 3 (CMT3) and the H3K9me2 methyltransferase, KRYPTONITE (KYP)

• Asymmetric cytosine methylation -de novo methylation - RNA directed DNA methylation (RdDM) in which the methyltransferase DRM2 methylatesCpHpH motifs

• Active demethylation can also occur through the action of DNA glycosylase-ligases such as DEMETER (DME)

Dna methylation

Histone modification

• covalent modification of histone proteins• Usually on their N-terminal tails• Causes nucleosome rearrangement, chromatin

remodeling and altered transcriptional potential.• Key histone modifications:methylation and

acetylation, especially of lysine (Lys, K) residues (which are abundant on histone N-terminal tails)

• orchestrated by complexes of histone lysine methyltransferases and demethylases(HKMT’s and HDM’s), and acetylases and deacetylases(HAT’s and HDAC’s)

• Histone modification marks can act as binding sites for different chromatin remodeling enzyme complexes

• Can lead to the formation of stable epigenetic loops involving feedback between DNA methylation and histone modification.

Histone modification

sRNAs

• Small RNA molecules : short (20–27 nucleotide, nt) non-coding RNA’s

• can regulate gene expression and also act as an RNA-based immune system to counteract against foreign viral RNA or transposons

• sRNA-mediated processes include transcriptional gene silencing (TGS) and post-transcriptional gene silencing(PTGS)

• Plant sRNAs - two major classes: the microRNAs (miRNA) and small interfering RNAs (siRNA)

• act to maintain genome stability by silencing transposonsand help to protect against viral RNA invasion

• RdDM can direct de novo DNA methylation and heterochromatin formation

sRNAs

Pre-miRNA

mi RNA genes

RNA pol III

Mature miRNA (20-27 nt)

DCL1

RISC + AGO1

PTGS

dsRNAs

DCL2,3,4

Mature siRNA (20-24 nt)

• As RdDM can direct DNA methylation and heterochromatin formation

• speculated that sRNAs could also regulate epigenetic changes associated with heterosis

• sRNA levels show substantial variation between parental inbred lines and their F1 hybrid or allopolyploid offspring

sRNAs

• Rice cultivar Nipponbare (Oryza sativa ssp japonica) and 93-11 (O. sativa ssp indica) and their reciprocal F1 hybrids (Nipponbare/93-11 and 93-11/ Nipponbare)

• soil under 16-h-light/8-h-dark conditions at 288C in a greenhouse.• After 4 weeks, seedling shoots at the four-leaf stage were harvested• frozen in liquid nitrogen, and stored at –8oC for DNA and total RNA

isolation or processed directly after harvesting for ChIP (chromatin immuno precipitation) assay.

• Total RNA was isolated - mRNA, sRNA• Chromatin from seedling shoots was immunoprecipitated with antibodies

against H3K4me3,H3K9ac , or H3K27me3

Case study 1

• integrated comparative analysis of the epigenome and overall transcriptional output (including mRNA and small RNAs) for two rice subspecies and their reciprocal hybrids.

• small RNA transcriptome differed in composition and expression between hybrid offspring and their inbred parents.

• genome-wide integrated maps of mRNA transcripts, DNA methylation, and three select histone modifications (H3K4me3, H3K9ac, and H3K27me3)

• high-throughput Illumina/Solexa 1G sequencing• Both reciprocal hybrids used in this study show significant

growth vigor compared with their parents

• Reciprocal hybrids to study maternal effects

• Variation in epigenetic regulation in hybrids and parents : methylation and histonemodification upregulated in hybrids

• Most of the variation in methylation patterns

• small RNA predominantly down regulated in hybrids irrespective of cross

Case study 1

• Intraspecific hybrids between the Arabidopsis thaliana accessions C24 and Landsberg erecta have strong heterosis- rosette diameter and biomass

• The reciprocal hybrids show a decreased level of 24-nt small RNA (sRNA)

• The genomic regions with reduced 24-nt sRNA levels were largely associated with genes

• flanking regions indicating a potential effect on gene expression.

• altered 24-nt sRNA levels that showed correlated changes in DNA methylation and expression levels

• suggest that such epigenetically generated differences in gene activity may contribute to hybrid vigor and that the epigenetic diversity between ecotypes

• increased allelic (epi-allelic) variability that could contribute to heterosis.

Case study 2

• The hybrids differed greatly from the parents in their sRNA populations-marked reduction in 24-nt sRNA

• In generations beyond the F1, an increasing number of loci may show “balancing” interactions between parental epialleles, leading to loss of epiallelic diversity.

• Hybrids- methylation levels increased• Even though siRNAs play a role in maintenance of

CHG methylation, it was affected to a lesser extent than CHH methylation,

Case study 2

• Three sets of triploid hybrids all with Oryza sativa ssp. japonica, cv. Nipponbare (genome AA, 2n = 2x = 24) as the maternal parent and three different wild tetraploid rice species as the paternal parents

• Embryo rescue • Amplified fragment length polymorphism (AFLP) and

methylationsensitive amplified fragment length polymorphism (MSAP)

• RT PCR to access the transcriptome activity

• All three sets of F1 triploid hybrids showed stochastic genetic and epigenetic changes at the molecular level

• 50% genetic and DNA methylation-altered loci are landed in genic regions

• suggests that the rapid genomic changes-emergent novel phenotypes of the hybrids

• DNA methylation is unevenly distributed across plant genomes, with heterochromatic regions, being predominantly constituted by high copy-number

• TEs and their derivatives are more heavily methylatedthan euchromatic regions

Case study 3

• Results:(i) all three triploid hybrids are stable in both chromosome

number and gross structure(ii) stochastic changes in both DNA sequence and

methylation occurred in individual plants of all three triploid hybrids, but in general methylation changes occurred at lower frequencies than genetic changes

(iii) alteration in DNA methylation occurred to a greater extent in genomic loci flanking potentially active TEs than in randomly sampled loci

(iv) transcriptional activation of several TEs commonly occurred in all three hybrids but transpositional events were detected in a genetic context-dependent manner.

Case study 3

• Short communication

• sRNAs play an important role in gene and genome regulation

• MicroRNAs (miRNAs) and trans-acting small interfering RNAs (tasiRNAs) regulate coding transcript levels

• small interfering RNAs (siRNAs) guide DNA methylation and stable chromatin modifications predominantly at Tes and other repeat sequences

• These epigenetic marks keep TEs repressed, thereby limiting potentially detrimental transposition

• Epigenetic and sRNA differences between and within species are relatively poorly described compared with genetic and transcriptome variation

• Tes can be activated in interspecific hybrids, accompanied by changes in DNA methylation

• TEs have been proposed to contribute to transgressivephenotypes through several mechanisms (Tenaillon et al, 2010)

• Also seen in hybrids between domesticated tomato (Solanum lycopersicum) and a wild relative (S. pennellii)

• Arabidopsis• Maize

Case study 4

• Genome-wide approaches to the study of hybrid vigor have identified epigenetic changes in the hybrid nucleus in Arabidopsis

• (Arabidopsis thaliana), maize (Zea mays), and rice (Oryza sativa)• DNA methylation associated with 24-nucleotide small interfering• RNAs exhibits transallelic effects in hybrids of Arabidopsis and other

species• epigenetically induced changes in gene• expression may contribute to hybrid vigor, the link between the

transcriptional changes and the hybrid phenotype is not• confirmed

• These ecotypes differ in their methylation, suggesting that the observed vigor could result from interactions between the two similar genomes

• the different epigenomes of the ecotypes to produce new patterns of transcription in the hybrids

• De novo methylation involves 24-nucleotide (nt) small interfering RNAs (siRNAs) acting as a guiding molecule for DOMAINS REARRANGED METHYLTRANSFERASE2 (DRM2),

• The high variability of the epigenome may enable plants to adapt to different environmental stress conditions, providing a heritable yet flexible system whereby information can be passed on to the next generation

• In a number of hybrid systems including Arabidopsis intra- and interspecific hybrids, and in rice, tomato (Solanum lycopersicum), and maize hybrids, a decrease in 24-nt siRNA levels has been observed,

• suggesting a change in epigenetic controls may be common to all hybrids

Case study 5

• The changes to the hybrid methylome occur through two related processes, trans chromosomal methylation (TCM) and trans chromosomal demethylation (TCdM),

• The uniform phenotype of F1 hybrid plants is lost in subsequent generations

• F2 populations showing heterogeneity in phenotypic traits such as biomass and flowering time

• It is probable that segregation of epigenetic• marks and changes to the plant’s epigenome may also• contribute to the phenotypic heterogeneity in F2 populations.• Changes in transcription levels of genes in defense response

pathways of hybrids could be related to changes in their epigenomes.

Case study 5