ABSTRACT: The catalytically inactive Domains Rearranged Methyltransferase3 controls DNA methylation and regulates RNA polymerase V transcript abundance in Arabidopsis
Project description:RNA sequencing was performed on various W303 variants to determine effects of nucleosome repositioning on transcript abundance RNA sequencing was carried out in multiple backgrounds to determine effects of nucleosome repositioning in various contexts. For engineered chromatin remodeling factors, a catalytically inactive control was included.
Project description:DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DRM2 controls RNA-directed DNA methylation in a pathway that also involves the plant specific RNA Polymerase V (Pol V). The Arabidopsis genome also encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase DRM3. Here, we show that DRM3 has moderate effects on global RNA-directed DNA methylation and small RNA abundance throughout the genome, and DRM3 protein physically interacts with Pol V. In drm3 mutants, we observe a lower level of Pol V-dependent transcripts, even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts, and shed further light on the mechanism of RNA-directed DNA methylation.
Project description:Ppg1 phosphatase post-translationally regulates Far11. To identify Far11 phosphorylation sites regulated by Ppg1, we immunopurified Far11 from wild-type and Ppg1 catalytically inactive (Ppg1-H111N) cells. We used this immunopurified Far11 for proteomics and identified Ppg1 dependent Far11 phosphorylation sites.
Project description:ChIP-seq for H3K27me3 and Ring1B was performed in WT mESCs and mESCs containing catalytically inactive Ring1B (I53A mutant). Cells expressing catalytically inactive Ring1B maintain the spatial distribution of Ring1B and H3K27me3 but at reduced levels. These findings support the notion that PRC2 recruitment is, in part, dependent on H2A ubiquitination (H2AK119ub). Two biological replicates were performed for Ring1B and H3K27me3 ChIPs in WT and Ring1B I53A/I53A mouse ESCs. Input chromatin was sequenced for each replicate as a control for ChIP enrichment.
Project description:ChIP-seq for H3K27me3 and Ring1B was performed in WT mESCs and mESCs containing catalytically inactive Ring1B (I53A mutant). Cells expressing catalytically inactive Ring1B maintain the spatial distribution of Ring1B and H3K27me3 but at reduced levels. These findings support the notion that PRC2 recruitment is, in part, dependent on H2A ubiquitination (H2AK119ub).
Project description:DNA methylation is a mechanism of epigenetic gene regulation and genome defense conserved in many eukaryotic organisms. In Arabidopsis, the DNA methyltransferase DRM2 controls RNA-directed DNA methylation in a pathway that also involves the plant specific RNA Polymerase V (Pol V). The Arabidopsis genome also encodes an evolutionarily conserved but catalytically inactive DNA methyltransferase DRM3. Here, we show that DRM3 has moderate effects on global RNA-directed DNA methylation and small RNA abundance throughout the genome, and DRM3 protein physically interacts with Pol V. In drm3 mutants, we observe a lower level of Pol V-dependent transcripts, even though Pol V chromatin occupancy is increased at many sites in the genome. These findings suggest that DRM3 acts to promote Pol V transcriptional elongation or assist in the stabilization of Pol V transcripts, and shed further light on the mechanism of RNA-directed DNA methylation. For wildtype plants as well as drm3, drm2, and nrpe1 mutants ChIP-seq was carried out using an endogenous NRPE1 antibody given to us by the Craig Pikaard lab. Two biological replicates of ChIP-seq were also carried out using anti-Flag resin on wildtype and drm3 plants carrying a Flag epitope tagged version of NRPE1. Small RNA sequencing was carried out on Col, drm3, drm2, and nrpe1 plants. Finally, whole-genome bisulfite sequencing analysis was carried out on previously published datasets (as detailed below) which were realigned using a newer genome version and mapping protocol. As such the updated processed files are part of this submission. Please note that the drm2, drm3, and nrpe1 mutant libraries used in this study were previously published (GSE39901), as were the 2 other Col replicates used (GSE36129) as below and thus duplicated sample records were created for the convenient retrieval of the complete raw data from SRA; Bisulfite_seq-Col_1 - GSM881756 Bisulfite_seq-Col_2 - GSM1193638 Bisulfite_seq-drm3 - GSM981017 Bisulfite_seq-drm2 - GSM981015 Bisulfite_seq-nrpe1- GSM981040
Project description:FAM46C is one of the most frequently mutated genes in multiple myeloma (MM) and encodes a protein of unknown function. Using a combination of in vitro and in vivo approaches, we demonstrate that FAM46C encodes an active cytoplasmic non-canonical poly(A) polymerase, which enhances mRNA stability and gene expression. Moreover, we also found that the reintroduction of active FAM46C into MM cell lines, but not its catalytically-inactive mutant, leads to broad polyadenylation and stabilization of mRNAs strongly enriched with those encoding endoplasmic reticulum-targeted proteins and induced cell death. This is, to our knowledge, the first report that directly associates cytoplasmic poly(A) polymerase with carcinogenesis. Furthermore, our data suggest that the human genome encodes at least eleven non-canonical poly(A) polymerases with four FAM46 family members. Since FAM46 proteins are differentially expressed during development, these proteins may positively regulate transcript stability and translational rate in a tissue-specific manner.
Project description:In vitro differentiation of Nsd1-/- erythroblasts with retroviral expression of wildtype Nsd1, or a catalytically-inactive Nsd1N1918Q SET-domain
Project description:In vitro differentiation of Nsd1-/- erythroblasts with retroviral expression of wildtype Nsd1, or a catalytically-inactive Nsd1N1918Q SET-domain
Project description:Purpose: We aimed to determine whether the expression of either wild-type or catalytically inactive LSH, carrying a single point mutation in its ATP binding site (K237Q), could restore the levels and patterns of DNA methylation in Lsh-/- mouse embryonic fibroblasts (MEFs). Methods: Lsh-/- MEFs were transduced with lentiviral particles carrying empty pMSCV vector, pMSCV-LSH-3xFLAG and pMSCV-LSH K/Q-3xFLAG, respectively. Clonal cell lines were generated and tested for LSH expression. Two independent cell lines expressing wild-type LSH and two expressing LSH K/Q were used for further analyses and comparison with wild-type MEFs and Lsh-/- MEFs carrying the empty vector. Genomic DNA was purified from all six cell lines and methylated DNA immunoprecipitation (MeDIP) was performed as described in Weber et al., 2007, Nat Genetics. MeDIP libraries were generated and sequenced on Illumina HiSeq 2000 instrument. Results and conclusions: Our experiments demonstrate that the expression of wild-type LSH, but not the catalytically inactive LSH K/Q, in Lsh-/- MEFs leads to reestablishment of DNA methylation at repetitive sequences and unique developmentally regulated loci in a cell-autonomous manner. Analyses of DNA methylation upon expression of either wild-type or catalytically-inactive LSH in Lsh-/- mouse embryonic fibroblasts.
