DNA Methylation in Plants

1. DNA Methylation in Plants Tianyin Zhou 2010.7.8

2. Methylation Pattern • Methylation can be categorized into three steps: CG, CHG and CHH (H = A,C or T) • Methylation in flowering plants exist in all three sequence contexts (CG predominiates animals) • Arabidopsis (observed 24%CG, 6.7%CHG, 1.7%CHH) • CHG sites, strong tendency for symmetrical m • <5% of expressed genes have m-promoter • 1/3 genes are methylated in ORF • Gene Bodies contain almost exclusively CG • Preference for Exons (similar to animials) • Enriched exons in nucleosome context • Nucleosome guide DNA methylation?

3. de novo DNA Methylation • Most transcript  ncRNA • ncRNA plays a key role in establishing epigenetics architecture • RNA-directed DNA methylation (RdDM) • ncRNA 24nt siRNA by RNAi machinery • siRNA is used to target the regions to be methylated by sequence homology

4. Three different kinds proteins are required: • Conserved DNA methyltransferases • Plant-specific proteins • Pol IV, Pol V • Nucleolar-associated proteins • DICER-LIKE3 • AGO4, binds siRNA • Steps: • 1.dsRNA--< overlap of two different strands transcript, inverted repeats, hair-pin • 2.DCL3--< clip • 3.AGO4 load 24nt si-RNA • 4.Pol V transcription facilitate m • 5.Nascent RNA transcript  follow steps a, amplifying the m signals

5. Some notes • RdDM accounts for 30% of de novo m in plants • Also mediate the active demethylation • Can spread m along a chromosome, resulting in a silenced state • Polymorphisms in transposon insertions and repeats  natural epigenetics variation

6. Replication of Methylation sites For CG, two homologous : MET1-plants DNMT1-mammals Passive mechanism After DNA replication, new strand is not methylated. A set of enzymes, recognize semi-methylated sites, remethylated

7. Replication of Methylation sites • For non-CpGmethylation • Might be asymmetric, passive mechanism does not apply • Seems to require an active maintenance signal after DNA replication • si-RNA is hypothesized provide such signals • Thus methylation can maintain remarkable stability throughout mitosis and meiosis

8. Evidence from Paramutation Paramutation is an interaction between two alleles of a single locus, resulting in an heritable change of one allele that is induced by the other. Colour-arrows – tandem-repeat regions Recessive mutation With the aid of MOPI and siRNA signal Penetrance 100%, dominant!!

9. Hypothesis for non-CpG Replication Suppose we have a diploid plant • ---------M--------- • --------------------- Replica Replica Active RNA signals • --------------------- • --------------------- • ---------M--------- • --------------------- • ---------M--------- • --------------------- • ---------M--------- • ---------------------

10. Methylation of Arabidopsis Telomeres • Chromosome termini consist of telomeric and subtelomeric repeats • Exception: 8 of 10 Arabidopsis subtelomeric regions have no repeats • Bound by a specific set of telomere binding proteins • Protect the telomere from DNA repair pathway • Form a specialized type of heterochromatin, important for stability • Arabidopsis telomere do not silence genes located in subtelomeiric regions ,why?

11. A Brief Review of Nucleosome • Nucleosome is the buliding-block of chromosomes • Consist of 8 protein subunits, called histones, 2 H2A, 2H2B, 2H3 and 2 H4 • DNA is wound to bind 8 histones

12. Histone Modification Side chain of Histone could be modified Modification changes the conformation and charge of nucleosome Eventually alter the nucleosome-nucleosome and DNA-nucleosome interaction

13. Telomeres exhibit Feature of Intermediate Heterochromatin Highly Condensed DNA is inaccessible to trascription factor Represent the mammalian telomere structure • Arabidopsis: High transcriptional activity near telomeres ? What is its structure of telomeres ? H3K27me1, H3K9me2 – mark of heterochromatin H3K4me3 – mark of euchromatin

14. Telomere is Transcription Active • Identified telomericDNA-containing transcripts and siRNA • Telomeric repeats for plants: TTTAGGG • Detected Telometric repeat-containing RNAs(TERRA) with proble CCCTAAA • Detected antisense telomeric transcripts(ARRET)) with proble TTTAGGG • Both strand of telomere is transcribed (In mammals only UUAGGG telomeric transcript is detected) • TERRA and ARRET are not uniformly distributed • Transcription level is low, detected by RT-PCR

15. Telomere transcripts->siRNA-?>Methylation Indication: Presence of TERRA and ARRET  dsRNA->siRNA They do detect siRNA Bisulphite sequencing shows that the over 40% cytosines in these telomeric repeats are methylated, with preferential enrichment at the third cytosine in the CCCTAA The RdDMpathyway mediates methylation of telomeric DNA???

16. The RdDM pathway mediates methylation of telomeric DNA Methylation of telomeric DNA in wild-type and RdDM-deficient plants

17. Function of MethylatedTelomeric DNA • Facilitate the heterochromatin formation by stabilizing interactions between heterochromatin factors and telomeric DNA • Not solely responsible for heterochromatin formation at teromeres • The loss of methylation significant loss of heterochromatin mark

18. Some Implication • Telomeric DNA may act as a promoter and initiate transcription • Short stretches of a telomeric sequence were found in numerous Arabidopsis promoters • RdDM may play a role in maintaining heterochromatin at the boundary between telomeres and adjacent euchromatic genes • The formation of Heterochromatin in particular mediated by heavily DNA methylation might contribute to the Gene Silencing in Plants • TERRA found in Human may also promote heterochromatin formation through RdDM pathway

19. Thank you