Unknown,Transcriptomics,Genomics,Proteomics

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Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

ABSTRACT: Early mouse development is accompanied by dynamic changes in chromatin modifications, including G9a-mediated histone H3 lysine 9 dimethylation (H3K9me2), which is essential for embryogenesis. Here we show that H3K9me2 directs repression of 2-cell stage specific genes in nascent embryos to facilitate preimplantation development. Thereafter, genome-wide accumulation of H3K9me2 is crucial for postimplantation development, and coincides with redistribution of EZH2-dependent histone H3 lysine 27 trimethylation (H3K27me3). Loss of G9a or EZH2 results in upregulation of distinct gene sets involved in processes such as cell cycle regulation as well as germline and embryonic development. Accumulation of H3K9me2 not only occurs at promoters and gene bodies, but also extends to active enhancer elements to promote their developmentally-linked silencing. This epigenetic mechanism is important for priming gene regulatory networks in epiblast cells undergoing rapid cell proliferation in preparation for critical cell fate decisions. Examination of 2 histone modification in 3 cell types and transcriptional analysis of G9a and Ezh2 KO epiblasts and Whole genome bisulfite sequencing in two cell types

ORGANISM(S): Mus musculus

SUBMITTER: Charles Bradshaw

PROVIDER: E-GEOD-70355 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:DNA methylation is an epigenetic modification that plays critical roles in gene silencing, development, and the maintenance of genome integrity. In Arabidopsis, DNA methylation is established by DOMAINS REARRANGED METHYLTRANSFERASE 2 (DRM2) and is targeted by 24 nt small interfering RNAs (siRNAs) through a pathway termed RNA-directed DNA methylation (RdDM)1. This pathway requires two plant-specific RNA polymerases: Pol-IV, which functions to initiate siRNA biogenesis and Pol-V, which functions in the downstream DNA methyltransferase targeting phase of the RdDM pathway to generate scaffold transcripts that recruit downstream RdDM factors1,2. To understand the mechanisms controlling Pol-IV targeting we investigated the function of SAWADEE HOMEODOMAIN HOMOLOG 1 (SHH1)3,4, a Pol-IV interacting protein3. Here we show that SHH1 acts upstream in the RdDM pathway to enable siRNA production from a large subset of the most active RdDM targets and that SHH1 is required for Pol-IV occupancy at these same loci. We also show that the SHH1 SAWADEE domain is a novel chromatin binding module that adopts a unique tandem Tudor-like fold and functions as a dual lysine reader, probing for both unmethylated K4 and methylated K9 modifications on the histone 3 (H3) tail. Finally, we show that key residues within both lysine binding pockets of SHH1 are required in vivo to maintain siRNA and DNA methylation levels as well as Pol-IV occupancy at RdDM targets, demonstrating a central role for methyl H3K9 binding in SHH1 function and providing the first insights into the mechanism of Pol-IV targeting. Given the parallels between methylation systems in plants and mammals1,5, a further understanding of this early targeting step may aid in our ability to control the expression of endogenous and newly introduced genes, which has broad implications for agriculture and gene therapy. For wild type plants (ecotype Columbia) and RdDM mutants whole-genome small RNA (sRNA-seq) and bisulfite sequencing (BS-seq) was performed. The Col and nrpe1 BS-seq libraries were previously reported (GSE39247) and so are not part of this submission. In addition, two replicates of whole genome chromatin immunoprecipitation (ChIP-seq) was performed on wild type (ecotype Columbia) plants as a negative control with experimentals consiting of nrpd1 mutant plants carrying a C-terminally epitope tagged (3XFLAG) NRPD1. Whole-genome bisulfite sequencing and small RNA sequencing was also performed on shh1 mutant plants transformed with the wild-type SHH1 protein-coding construct as well as multiple constructs containing point mutations. For these complementation libraries a separate shh1 mutant and Col control line were sequenced (“complementation replicates”).

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Chromatin dynamics and the role of G9a in gene regulation and enhancer silencing during early mouse development

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