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Protein and histone binding profiles from mouse ES cells during differentiation and after Kdm6a RNAi treatment.

ABSTRACT: Here we show binding and occupancy profiles for KDM6A, H3K27me3 and H3K4me3 to address the epigenetic regulation of a subset of Rhox genes, Rhox6 and 9, in female and male ES cells during differentiation. To further address a functional role for KDM6A in the epigenetic regulation of Rhox6 and 9, binding profiles for female ES cells treated with a control siRNA and siRNA specific for Kdm6a are shown. We report that two members of the Rhox cluster, Rhox6 and 9, are regulated by de-methylation of histone H3 at lysine 27 by KDM6A, a histone demethylase with female-biased expression. Our results are consistent with other homeobox genes in that Rhox6 and 9 are in bivalent domains prior to embryonic stem cell differentiation and thus poised for activation. In female mouse ES cells KDM6A is specifically recruited to Rhox6 and 9 for gene activation, a process inhibited by Kdm6a knockdown. In contrast, KDM6A occupancy at Rhox6 and 9 is low in male ES cells and knockdown has no effect on expression. Our study implicates Kdm6a, a gene that escapes X inactivation, in the regulation of genes important in reproduction, suggesting that KDM6A may play a role in the etiology of developmental and reproduction-related effects of X chromosome anomalies. ChIP-chip was used to analyze the binding profiles of KDM6A, H3K27me3, and H3K4me3 during differentiation in female and male ES cells. Additionally, ChiP-chip of KDM6A binding in control treated and siRNA treated ES cells is presented.

ORGANISM(S): Mus musculus

SUBMITTER: Joel Berletch

PROVIDER: E-GEOD-45390 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Expression data from mouse ES cells after control RNAi (scramble siRNAs) or RNAi specific for Kdm6a treatment.

Project description:To address the functional role of KDM6A in the regulation of Rhox genes, male and female mouse ES cells were transfected with a mixture of three small interfering RNA duplexes, each of which targets a different region of Kdm6a mRNA. We found that Kdm6a knockdown in mouse ES cells caused a decrease in expression of a subset of Rhox genes, Rhox6 and 9. Furthermore, Rhox6 and 9 expression was decreased in female ES cells but not male ES cells indicating that KDM6A regulates Rhox gene expression in a sexually dimorphic manner. Mouse ES cells were treated by Invitrogen scramble siRNA duplexes or specific siRNA duplexes and used for RNA extraction and hybridization on Affymetrix microarrays. Four RNA samples from two independent double-RNAi treatments and two single-RNAi treatment in undifferentiated female ES cells PGK12.1, and RNA samples from two single-RNAi treatments in undifferentiated male ES cells WD44 were assayed for expression changes by arrays.

2013-03-13 | E-GEOD-45034 | biostudies-arrayexpress

Expression data from mouse ES cells after control RNAi (scramble siRNAs) or RNAi specific for Kdm6a treatment.

2013-03-13 | GSE45034 | GEO

Protein and histone binding profiles from mouse ES cells during differentiation and after Kdm6a RNAi treatment.

Project description:Here we show binding and occupancy profiles for KDM6A, H3K27me3 and H3K4me3 to address the epigenetic regulation of a subset of Rhox genes, Rhox6 and 9, in female and male ES cells during differentiation. To further address a functional role for KDM6A in the epigenetic regulation of Rhox6 and 9, binding profiles for female ES cells treated with a control siRNA and siRNA specific for Kdm6a are shown. We report that two members of the Rhox cluster, Rhox6 and 9, are regulated by de-methylation of histone H3 at lysine 27 by KDM6A, a histone demethylase with female-biased expression. Our results are consistent with other homeobox genes in that Rhox6 and 9 are in bivalent domains prior to embryonic stem cell differentiation and thus poised for activation. In female mouse ES cells KDM6A is specifically recruited to Rhox6 and 9 for gene activation, a process inhibited by Kdm6a knockdown. In contrast, KDM6A occupancy at Rhox6 and 9 is low in male ES cells and knockdown has no effect on expression. Our study implicates Kdm6a, a gene that escapes X inactivation, in the regulation of genes important in reproduction, suggesting that KDM6A may play a role in the etiology of developmental and reproduction-related effects of X chromosome anomalies.

2013-03-22 | GSE45390 | GEO

Expression data from undifferentiated and differentiated mouse female ES cells PGK12.1

Project description:Affymetrix 430 2.0 mouse arrays were used for expression analyses in undifferentiated and differentiated PGK12.1 ES cells. We found that the X:autosome expression ratios calculated from the mean expression values of X-linked and autosomal genes from microarrays was ~1.4 in undifferentiated female ES cells and then decreased to 1.2 in PGK12.1 cells after 15-day embryoid body differentiation. Thus, a substantial level of X upregulation is already evident in these ES cells prior to differentiation. Our findings based on Affymetrix expression arrays are consistent with microarray analysis from other labs and our RNA-seq analysis of mouse female PGK12.1 ES cells. Mouse female ES cells PGK12.1 were differentiated by the EB (embryoid body) differentiation protocol. The presence of two active X chromosomes in undifferentiated female ES cells and one active X in 15-day differentiated cells was verified by Xist RNA FISH. Total RNA was prepared from undifferentiated and 15-day differentiated PGK12.1 and was used for expression array analyses to examine exprssion of the X chromosome during differentiation.

2013-02-15 | E-GEOD-44251 | biostudies-arrayexpress

Polycomb-dependent H3K27me1 and H3K27me2 regulate active transcription and enhancer fidelity

Project description:These data include the genome wide location of different histone modifications by ChIP sequencing in mouse ES cells, and RNA Seq data generated from wild type and EED KO mouse ES cells and knocked down for unrelated protein and Setd2 protein. ChIP-Seq: Immuno-precipitation of formaldehyde cross-linked chromatin prepared from wild type mouse E14 ES cells, wild type E36 ES cells, EED KO E36 ES cells, wild type Embryoid bodies (Ebs), EED KO Embryoid bodies (Ebs EED KO) using specific antibody against different histone modifications. RNA-Seq: Total RNA extracted from wild type E36 ES cells, EED KO E36 ES cells, wild type E36 Embryoid bodies (Ebs), EED KO Embryoid bodies (Ebs EED KO), E14 Ctrl KD, E14 Setd2 KD.

2013-12-05 | E-GEOD-51006 | biostudies-arrayexpress

Extended self-renewal and accelerated reprogramming in the absence of Kdm5b [RNA-Seq]

Project description:ES cell pluripotency is thought to be regulated in part by H3K4 methylation. However, it is unclear how H3K4 demethylation contributes to ES cell function and participates in iPS cell reprogramming. Here, we show that KDM5B, which demethylates H3K4, is important for ES cell differentiation, and presents a barrier to the reprogramming process. Depletion of Kdm5b leads to an extension in the self-renewal of ES cells in the absence of LIF. Transcriptome analysis revealed the persistent expression of pluripotency-genes and underexpression of developmental genes during differentiation in the absence of Kdm5b, suggesting that KDM5B plays a key role in cellular fate changes. We also observed accelerated reprogramming of differentiated cells in the absence of Kdm5b, demonstrating that KDM5B is a barrier to the reprogramming process. Expression analysis revealed that mesenchymal master regulators associated with epithelial-to-mesenchymal transition (EMT) are downregulated during reprogramming in the absence of Kdm5b. Moreover, global analysis of H3K4me3/2 revealed that enhancers of fibroblast genes are rapidly deactivated in the absence of Kdm5b, and genes associated with EMT lose H3K4me3/2 during the early reprogramming process. These findings provide functional insight into the role for KDM5B in regulating ES cell differentiation and as a barrier to the reprogramming process. RNA-Seq of undifferentiated and embryoid body (EB) differentiated murine shLuc and shKdm5b ES cells

2013-10-14 | E-GEOD-47124 | biostudies-arrayexpress

Expression data from mouse ES cells after control RNAi (scramble siRNAs) or specific RNAi (siRNAs for specific genes) treatment

Project description:To address the functional role of MOF in mammalian X upregulation, male and female mouse ES cells were transfected with a mixture of three small interfering RNA duplexes, each of which targets a different region of Mof mRNA. We found that MOF knockdown in mouse ES cells caused a greater drop in expression of X-linked genes compared to autosomal genes, as measured by expression array analyses. The strongest effect was observed on medium-expressed X-linked genes. We next examined components of the two known MOF protein complexes, MSL1 (male-specific lethal1) and NSL1 (nonspecific lethal1). Knockdown of MSL1 but not NSL1 in undifferentiated female ES cells PGK12.1 specifically caused a decrease in expression levels of X-linked genes. Cells co-transfected with both MOF and MSL1 siRNAs had similar expression changes to MSL1 knockdown alone, indicating that these components probably operate within the same complex but are not additive. Our findings that key components of the MSL but not NSL complex play a role in upregulation of mammalian X-linked genes in ES cells. Mouse ES cells were treated by Invitrogen scramble siRNA duplexes or specific siRNA duplexes and used for RNA extraction and hybridization on Affymetrix microarrays. Six RNA samples from two independent double-RNAi treatments and one single-RNAi treatment in undifferentiated female ES cells PGK12.1, and RNA samples from two single-RNAi treatments in undifferentiated male ES cells WD44 or E14 were assayed for expression changes by arrays. RNA samples from three MSL1RNAi treatments, two MOF/MSL1RNAi treatments and three NSL1RNAi treatments in undifferentiated female ES cells PGK12.1 were assayed by arrays.

2013-02-15 | E-GEOD-44252 | biostudies-arrayexpress

Jarid2 methylation is required for PRC2 functioning during ES cells differentiation

Project description:We report that Jarid2 is methylated at K116 by the polycomb complex PRC2. We analyzed Jarid2 versus Methylated Jarid2 genomic localization by ChIP-seq. We also study H3K27 enrichment in Jarid2 knock out ES cells and in cells rescued for Jarid2 or a mutant that cannot be methylated. ChIP-seq for H3K27me3 was perfomed in undiffrentiated ES cells as well as after 8 days of diffrentiation toward Embroyid Bodies (Hanging Drop method). Experiment 1: Jarid2 vs Jarid2me ChIP-seq in undifferentiated mouse ES cells (E14). Experiment 2: H3K27me3 ChIP-seq in Jarid2 -/- ES cells (peng, 2009, Cell), or the same cells rescue with a BAC expressing JARID2 or a mutant of K116 to A. H3K27me3 Chip-seq were perfomed in undifferentiated ES cells and in EB after 8 days of differentiation (hanging Drop method).

2015-01-22 | E-GEOD-55630 | biostudies-arrayexpress

Architectural protein subclasses shape 3-D organization of genomes during lineage commitment

Project description:Understanding the topological configurations of chromatin can reveal valuable insights into how the genome and epigenome act in concert to control cell fate during development. Here we generate high-resolution architecture maps across seven genomic loci in embryonic stem cells and neural progenitor cells. We observe a hierarchy of 3-D interactions that undergo marked reorganization at the sub-Mb scale during differentiation. Distinct combinations of CTCF, Mediator, and cohesin show widespread enrichment in architecture at different length scales. CTCF/cohesin anchor long-range constitutive interactions that might form the topological basis for invariant sub-domains. Conversely, Mediator/cohesin together with pioneer factors bridge short-range enhancer-promoter interactions within and between larger sub-domains. Knockdown of Smc1 or Med12 in ES cells results in disruption of spatial architecture and down-regulation of genes found in cohesin-mediated interactions. We conclude that cell type-specific chromatin organization occurs at the sub-Mb scale and that architectural proteins shape the genome in hierarchical length scales. Analysis of higher-order chromatin chromatin architecture in mouse ES cells and ES-derived NPCs. Analysis of CTCF and Smc1 occupied sites in ES-derived NPCs.

2013-05-24 | E-GEOD-36203 | biostudies-arrayexpress

Geminin represses mesendoderm cell fate acquisition in embryoid bodies

Project description:Geminin is a small nucleoprotein that neuralizes ectoderm in the Xenopus embryo. Geminin promotes neural fate acquisition of mouse embryonic stem cells: Geminin knockdown during neural fate acquisition decreased expression of neural precursor cell markers (Pax6, Sox1), while increasing expression of Pitx2, Lefty1 and Cited2, genes involved in formation of the mouse node. Here we differentiated mouse embryonic stem cells into embryoid bodies to study Geminin's ability to repress primitive streak mesendoderm fate acquisition. We used microarrays to define the sets of genes that are regulated by Geminin during cell fate acquisition in embryoid bodies, using Dox-inducible Geminin knockdown or overexpression mouse embryonic stem cell lines. ES cell lines for Geminin over-expression (GemOE) were treated without or with Dox from day 3 to day 5 of EB differentiation and were collected on days 4 or 5 for microarray analysis. Gem knockdown (KD) ES cell lines were treated without or with Dox from day 0 to day 4 of EB differentiation and were collected on day 4 for microarray analysis.

2014-07-01 | E-GEOD-39676 | biostudies-arrayexpress

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