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Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability

ABSTRACT: This SuperSeries is composed of the SubSeries listed below.

ORGANISM(S): Mus musculus

PROVIDER: GSE57577 | GEO | 2015/06/22

SECONDARY ACCESSION(S): PRJNA246726

REPOSITORIES: GEO

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Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability

Project description:This SuperSeries is composed of the SubSeries listed below. Refer to individual Series

2015-06-22 | E-GEOD-57577 | biostudies-arrayexpress

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (Bisulfite-Seq)

Project description:Histone modifications and DNA methylation represent two distinct modes of varying epigenetic landscapes, but whose exact interrelationship remains unclear. Previous studies have shown that histone H3 lysine 4 trimethylation (H3K4me3) inhibits the binding of de novo DNA methyltransferases (Dnmt) through the ATRX-DNMT3-DNMTL (ADD) domain, thus protecting H3K4me3 marked CpG islands (CGI) from DNA methylation. In addition to H3K4me3, we identified antagonistic relationship between H3T3 phosphorylation and the binding of the ADD domain to the unmodified H3 N-terminus. To assess the physiological relevance of these restrictions, we engineered the wild-type ADD domain of Dnmt3a (WT) to permit additional binding to either H3K4me3 (WWD) or H3T3ph (R) and stably introduced FLAG-tagged, full-length normal or mutant Dnmt3a2 into ESCs lacking all Dnmts (TKO; triple knock-out of Dnmt1, Dnmt3a, and Dnmt3b) using the PiggyBac transposon system. For each WT-, WWD-, and R-Dnmt3a2, we generated bulk and clonally-derived ESC lines. We then employed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify the genomic distribution of full-length WT-, WWD-, R-Dnmt3a2, and the H3K4me3 distribution. In parallel, we quantitatively measured genome-wide CpG (cytosine) methylation at base-pair resolution using an enhanced form of reduced representation bisulfite sequencing (RRBS), and performed RNA-seq to assess transcription in matched ESC lines. Examination of DNA methylation levels in Dnmt TKO-ESCs expressing wild-type/mutant Dnmt3a2.

2015-06-22 | E-GEOD-57576 | biostudies-arrayexpress

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (RNA-Seq)

Project description:Histone modifications and DNA methylation represent two distinct modes of varying epigenetic landscapes, but whose exact interrelationship remains unclear. Previous studies have shown that histone H3 lysine 4 trimethylation (H3K4me3) inhibits the binding of de novo DNA methyltransferases (Dnmt) through the ATRX-DNMT3-DNMTL (ADD) domain, thus protecting H3K4me3 marked CpG islands (CGI) from DNA methylation. In addition to H3K4me3, we identified antagonistic relationship between H3T3 phosphorylation and the binding of the ADD domain to the unmodified H3 N-terminus. To assess the physiological relevance of these restrictions, we engineered the wild-type ADD domain of Dnmt3a (WT) to permit additional binding to either H3K4me3 (WWD) or H3T3ph (R) and stably introduced FLAG-tagged, full-length normal or mutant Dnmt3a2 into ESCs lacking all Dnmts (TKO; triple knock-out of Dnmt1, Dnmt3a, and Dnmt3b) using the PiggyBac transposon system. For each WT-, WWD-, and R-Dnmt3a2, we generated bulk and clonally-derived ESC lines. We then employed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify the genomic distribution of full-length WT-, WWD-, R-Dnmt3a2, and the H3K4me3 distribution. In parallel, we quantitatively measured genome-wide CpG (cytosine) methylation at base-pair resolution using an enhanced form of reduced representation bisulfite sequencing (RRBS), and performed RNA-seq to assess transcription in matched ESC lines. Examination of mRNA profiles in Dnmt TKO-ESCs expressing wild-type/mutant Dnmt3a2.

2015-06-22 | E-GEOD-57575 | biostudies-arrayexpress

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (ChIP-Seq)

Project description:Histone modifications and DNA methylation represent two distinct modes of varying epigenetic landscapes, but whose exact interrelationship remains unclear. Previous studies have shown that histone H3 lysine 4 trimethylation (H3K4me3) inhibits the binding of de novo DNA methyltransferases (Dnmt) through the ATRX-DNMT3-DNMTL (ADD) domain, thus protecting H3K4me3 marked CpG islands (CGI) from DNA methylation. In addition to H3K4me3, we identified antagonistic relationship between H3T3 phosphorylation and the binding of the ADD domain to the unmodified H3 N-terminus. To assess the physiological relevance of these restrictions, we engineered the wild-type ADD domain of Dnmt3a (WT) to permit additional binding to either H3K4me3 (WWD) or H3T3ph (R) and stably introduced FLAG-tagged, full-length normal or mutant Dnmt3a2 into ESCs lacking all Dnmts (TKO; triple knock-out of Dnmt1, Dnmt3a, and Dnmt3b) using the PiggyBac transposon system. For each WT-, WWD-, and R-Dnmt3a2, we generated bulk and clonally-derived ESC lines. We then employed chromatin immunoprecipitation followed by high-throughput DNA sequencing (ChIP-seq) to identify the genomic distribution of full-length WT-, WWD-, R-Dnmt3a2, and the H3K4me3 distribution. In parallel, we quantitatively measured genome-wide CpG (cytosine) methylation at base-pair resolution using an enhanced form of reduced representation bisulfite sequencing (RRBS), and performed RNA-seq to assess transcription in matched ESC lines. Examination of wild-type/mutant Dnmt3a2 and matched H3K4me3 distributions in TKO-ESCs.

2015-06-22 | E-GEOD-57574 | biostudies-arrayexpress

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (Bisulfite-Seq)

2015-06-22 | GSE57576 | GEO

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (RNA-Seq)

2015-06-22 | GSE57575 | GEO

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability (ChIP-Seq)

2015-06-22 | GSE57574 | GEO

Engineering of a Histone-Recognition Domain in Dnmt3a Alters the Epigenetic Landscape and Phenotypic Features of Mouse ESCs.

Project description:Histone modification and DNA methylation are associated with varying epigenetic "landscapes," but detailed mechanistic and functional links between the two remain unclear. Using the ATRX-DNMT3-DNMT3L (ADD) domain of the DNA methyltransferase Dnmt3a as a paradigm, we apply protein engineering to dissect the molecular interactions underlying the recruitment of this enzyme to specific regions of chromatin in mouse embryonic stem cells (ESCs). By rendering the ADD domain insensitive to histone modification, specifically H3K4 methylation or H3T3 phosphorylation, we demonstrate the consequence of dysregulated Dnmt3a binding and activity. Targeting of a Dnmt3a mutant to H3K4me3 promoters decreases gene expression in a subset of developmental genes and alters ESC differentiation, whereas aberrant binding of another mutant to H3T3ph during mitosis promotes chromosome instability. Our studies support the general view that histone modification "reading" and DNA methylation are closely coupled in mammalian cells, and suggest an avenue for the functional assessment of chromatin-associated proteins.

| S-EPMC4491196 | biostudies-literature

Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability

Project description:Engineering of a histone-recognition domain in Dnmt3a alters the epigenetic landscape of ESCs revealing changes in lineage specification and chromosomal stability

| PRJNA246726 | ENA

Floxin, a resource for genetically engineering mouse ESCs.

Project description:We describe a method for the highly efficient and precise targeted modification of gene trap loci in mouse embryonic stem cells (ESCs). Through the Floxin method, gene trap mutations were reverted and new DNA sequences inserted using Cre recombinase and a shuttle vector, pFloxin. Floxin technology is applicable to the existing collection of 24,149 compatible gene trap cell lines, which should enable high-throughput modification of many genes in mouse ESCs.

| S-EPMC2895430 | biostudies-literature

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