Unknown,Transcriptomics,Genomics,Proteomics

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Transcriptional gene silencing mediated by interplay of MOM1 and Pol V

ABSTRACT: In plants, multiple levels of epigenetic control suppress transposon movement and permit somatic as well as transgenerational transmission of gene expression patterns. A variety of factors are involved that establish and/or maintain epigenetic marks, such as covalent modification of DNA and histones. Alteration of epigenetic marks leads to the enhancement or release of transcriptional gene silencing (TGS). TGS regulator MOM1 is special in this respect since it silences transcription by an unknown mechanism operating without obvious changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and the plant-specific RNA Polymerase V (Pol V). Pol V transcribes heterochromatic loci and silences them. Although its biochemical properties have been studied; it is still not clear how Pol V is targeted to heterochromatin. We now provide evidence that Pol V is required for MOM1-mediated suppression of transcription at a subset of its chromosomal targets. Thus, Pol V interacts with MOM1 in the control of gene silencing. Interestingly, functional relationships between mutations in MOM1 and Pol V genes range from enhancement to independence or even suppression at different target loci. 4 samples: wt, mom1, nrpe1, mom/nrpe1 with 3 replicates each

ORGANISM(S): Arabidopsis thaliana

SUBMITTER: Etienne Buche

PROVIDER: E-GEOD-17940 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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MOM1 and Pol-IV/V interactions regulate the intensity and specificity of transcriptional gene silencing.

Yokthongwattana Chotika C Bucher Etienne E Caikovski Marian M Vaillant Isabelle I Nicolet Joël J Mittelsten Scheid Ortrun O Paszkowski Jerzy J

The EMBO journal 20091112 2

It is commonly observed that onset or release of transcriptional gene silencing (TGS) correlates with alteration of repressive epigenetic marks. The TGS regulator MOM1 in Arabidopsis is exceptional since it regulates transcription in intermediate heterochromatin with only minor changes in epigenetic marks. We have isolated an enhancer of the mom1 mutation that points towards regulatory interplay between MOM1 and RNA polymerase-V (Pol-V). Pol-V transcribes heterochromatic loci, which seems to be ...[more]

PMID: 19910926

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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:Epigenetic gene silencing is of central importance to maintain genome integrity and is mediated by an elaborate interplay between DNA methylation, histone posttranslational modifications and chromatin remodeling complexes. DNA methylation and repressive histone marks usually correlate with transcriptionally silent heterochromatin, however there are exceptions to this interdependence. In Arabidopsis, mutation of MORPHEUS MOLECULE 1 (MOM1) causes transcriptional derepression of heterochromatin independently of changes in DNA methylation. More recently, two Arabidopsis homologs of mouse Microrchidia (MORC) have also been implicated in gene silencing and heterochromatin condensation without altering genome-wide DNA methylation patterns. In this study, we show that AtMORC6 physically interacts with AtMORC1 and with its close homologue AtMORC2 in two mutually exclusive protein complexes. RNA-seq analysis of high-order mutants indicates that AtMORC1 and AtMORC2 act redundantly to repress a common set of loci. We also examined the genetic interactions between AtMORC6 and MOM1 pathways. Although AtMORC6 and MOM1 control the silencing of a very similar set of genomic loci, we observed synergistic transcriptional regulation in the mom1/atmorc6 double mutant, suggesting that these epigenetic regulators act mainly by independent silencing mechanisms. RNA-seq libraries were prepared for two suites of mutants to allow direct comparisons between mutants within each set. The two sets consisted of the following samples: Set_1) A wildtype (Col) control, the morc1 mutant, the morc2 mutant, the morc1 morc2 double mutant, the morc6 mutant, and the morc1 morc2 morc6 triple mutant ; Set_2) A wildtpe (Col) control, the morc6 mutant, the mom1 mutant, and the mom1 morc6 double mutant. For each sample, two biological replicates were performed (denoted "bio_replicate_1" or "bio_replicate_2"). Whole genome bisulifte libraries were sequenced from material grown in parallel.

Project description:Silencing pathways prevent transposable element (TE) proliferation and help to maintain genome integrity through cell division. Silenced genomic regions can be classified as either euchromatic or heterochromatic, and are targeted by genetically separable epigenetic pathways. In plants, the RNA-directed DNA methylation (RdDM) pathway targets mostly euchromatic regions, while CMT methyltransferases are mainly associated with heterochromatin. However, many epigenetic features - including DNA methylation patterning - are largely indistinguishable between these regions, so how the functional separation is maintained is unclear. The linker histone H1 is preferentially localised to heterochromatin and has been proposed to restrict RdDM from encroachment. To test this hypothesis, we followed RdDM genomic localisation in an H1 mutant by performing ChIP-seq on the largest subunit, NRPE1, of the central RdDM polymerase (Pol V). Loss of H1 resulted in heterochromatic TE enrichment of NPRE1. Increased NRPE1 binding was associated with increased chromatin accessibility in h1, suggesting that H1 restricts NRPE1 occupancy by compacting chromatin. However, RdDM occupancy did not impact H1 localization, demonstrating that H1 hierarchically restricts RdDM positioning. H1 mutants experience major symmetric (CG and CHG) DNA methylation gains, and by generating an h1/nrpe1 double mutant, we demonstrate these gains are largely independent of RdDM. However, loss of NRPE1 occupancy from a subset of euchromatic regions in h1 experienced corresponding loss of methylation in all sequence contexts, while at ectopically bound heterochromatic loci, NRPE1 deposited methylation specifically in the CHH context. Additionally, we found that H1 restricts the occupancy of the methylation reader and activator complex component, SUVH1, indicating that H1’s regulatory control of methylation pathways is not limited to RdDM. Together, the results support a model whereby H1 helps maintain the exclusivity of heterochromatin by preventing encroachment from other competing pathways.

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Transcriptional gene silencing mediated by interplay of MOM1 and Pol V

Publications

MOM1 and Pol-IV/V interactions regulate the intensity and specificity of transcriptional gene silencing.

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