Methytransferases and RNAi Pathways
DNA methyltransferase
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I am interested in examining the conservation of these enzymes and short interfering RNA across plant genomes. I will examine paralogs and orthologs of these genes and compare and contrast the elements of both de novo and maintenance methylation pathways. I would also love to isolate possible methylation sites across the Vaccinium corymbosum genome. I hope to find differences in methylation pathways between plant species at both the protein and epigenetic levels. A phylogenetic analysis from the methylation pathway standpoint will follow.
- METHYLTRANSFERASE1 (MET1)
- CHROMOMETHYLASE3 (CMT3)
- DOMAINS REARRANGED METHYLTRANSFERASE1 (DRM1) and (DRM2)
- DOMAINS REARRANGED METHYLTRANSFERASE3 (DRM3)
MET1 is responsible for maintaining CG methylation.
Chromomethylase3 maintains methylation in non-Cg sequences redundantly with DRM1/2 (Henderson et. al, 2006).
DRM2 is the primary enzyme implicated in the RNAi mediated methylation pathway. The enzyme constantly targets CHH sites (Widman et. al, 2009). DRM2 is expressed at higher levels than DRM1 and is the main enzyme involved in de novo methylation (Meyer, 2005).
Henderson et. al (2006) discovered that DRM3 is required for non-CG DNA methylation. DRM3 is a catalytically mutated paralog of DRM2. The activity of DRM2 is dependent on DRM3 and a UBA binding domain (Henderson et. al, 2010). Henderson et. al (2006) tested two T-DNA insertion strains (drm3-1 and drm3-2), finding a reduction of methylation of MEA-ISR. After performing a southern blot on the Msp1 (methyl-sensitive restriction enzyme) digested MEA-ISR repeat and sequencing samples of this DNA using the bisulfite method, the researchers concluded that DRM3 is necessary for proper DRM2 functioning.
(A) Southern blots hybridized for the MEA-ISR repeat using DNA digested with the methyl-sensitive restriction enzyme MspI. The 4.3 kb band and 2 kb bands represent digested and undigested DNA respectively. (B) Graphical representation of bisulfite sequencing at MEA-ISR with % cytosine methylation shown for each genotype. CG sequence contexts are shaded black, CHG are shaded grey and white represents CHH. The 95% confidence limits shown are given by the Wilson score interval. (C) Graphical representation of bisulfite sequencing at the methylated promoter repeats of the FWA endogene with % cytosine methylation shown for each genotype and shaded according to sequence context as in (B). (D) Quantitative PCR measurement of AtSN1 genomic DNA following restriction endonuclease digestion with DNA methylation sensitive enzyme HaeIII. The value on the y-axis represents the ratio of DNA measured by quantitative PCR with and without HaeIII digestion. The data represents three biological replicates and the error bars represent standard error.
Other proteins/RNA involved in RNAi and chromatin processing
- NUCLEAR RNA POLYMERASE D1 (NRPD1)
- NUCLEAR RNA POLYMERASE E1 (NRPE1)
- RNA DEPENDENT RNA POLYMERASE2 (RDR2)
- DICER-LIKE3 (DCL3)
- ARGONAUTE4 (AGO4)
- DEFECTIVE IN RNA DIRECTED DNA METHYLATION 1 (DRD1)
- DEFECTIVE IN MERISTEM SILENCING1 (DMS1)
- INVOLVED IN DE NOVO 2 (IND2)
- SUPPRESSOR OF VARIEGATION 3-9 HOMOLOGUE 2 (SUVH2) (SUVH2 homolog)
- SUPPRESSOR OF VARIEGATION 3-9 HOMOLOGUE 9 (SUVH9)
Other Links and Resources
http://www.ncbi.nlm.nih.gov/pmc/articles/PMC197300/
http://www.mcdb.ucla.edu/Research/Jacobsen/Labwebsite/PDFPublication/DRMgenePNAS.pdf
http://www.ncbi.nlm.nih.gov/pubmed/19384058
http://www.plosgenetics.org/article/info%3Adoi%2F10.1371%2Fjournal.pgen.1001182;jsessionid=35538162FE4C85BD00D0578063186ED0.ambra01
http://books.google.com/books?id=aBlXT_Y7JCwC&pg=PA3&lpg=PA3&dq=drm1+methylation&source=bl&ots=Cy4nSsNyPE&sig=ei_5_6t6j9cLBObpxE7coRyO1OI&hl=en&ei=5vlHTcagB4XVgQet56WfBg&sa=X&oi=book_result&ct=result&resnum=6&sqi=2&ved=0CDkQ6AEwBQ#v=onepage&q=drm1%20methylation&f=false
http://www.pnas.org/content/107/19/8689.full.pdf
http://www.plosbiology.org/article/info:doi/10.1371/journal.pbio.0000067
http://www.mcdb.ucla.edu/research/jacobsen/labwebsite/PDFPublication/MethylationConservation.pdf
