Poster abstracts
Poster number 20 submitted by Sarah Choudury
From Active to Silenced: Investigating How Repressive Chromatin Modification Is Initiated at Transposons
Sarah G. Choudury (MCDB, CMBP, Molecular Genetics Dept), Andrea D. McCue (Molecular Genetics Dept), Alissa Cullen (Molecular Genetics Dept)
Abstract:
In order to maintain genome integrity, fungi, plants, and animals modify transposable element (TE) chromatin to epigenetically repress TE activity. DNA methylation is critical for inhibiting the activity of TEs. Once established at TEs, DNA methylation can be propagated by methyltransferases. However, the mechanism by which DNA methylation and epigenetic silencing are originally targeted to TEs is not well understood. Our lab has identified a pathway in Arabidopsis that acts to direct de novo cytosine DNA methylation to transcriptionally active TEs, and is thus partially responsible for the initiation of transcriptional silencing at these loci. This pathway utilizes endogenous 21-22 nucleotide (nt) small interfering RNAs (siRNAs) which result from the post-transcriptional degradation of TE mRNAs. The siRNAs direct Argonaute (AGO) proteins to chromatin which then triggers de novo DNA methylation. Previous studies from our lab genetically identified AGO6 as the key effector protein in this pathway of DNA methylation of active TEs. Previous experiments in our lab utilized immunoprecipitation (IP) of AGO6 followed by mass spectrometry (MS) analysis to identify AGO6-interacting proteins. I set out to better understand the mechanism of the silencing initiation pathway by screening the candidate AGO6-interactors for a role in methylation using bisulfite DNA sequencing of methylation patterns. Three candidate AGO6-interacting proteins were found to be required for initiation of methylation. I further characterized their respective mutants by phenotype, DNA methylation pattern, Northern blot, Western blot, and a cytoplasmic enriched Western blot. Based on my results, it appears that all three proteins act downstream of siRNA production, and, while they do not effect overall accumulation of AGO6 protein, one newly identified protein, a P-Loop domain ATPase, functions to localize AGO6 to the nucleus where it targets repressive chromatin modification to TE sequences.
Keywords: TE silencing, transposable elements, arabidopsis