Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures [ChIP-Seq]

ABSTRACT: The limited number of in vivo germ cells poses an impediment to genome-wide studies. Here, we applied a small-scale ChIP-Seq method on purified mouse fetal germ cells to generate genome-wide maps of four histone modifications (H3K4me3, H3K27me3, H3K27ac and H2BK20ac), facilitating the identification of active and repressed cis-regulatory elements in germ cells in vivo. Comparison of active chromatin state between somatic, embryonic stem cells (ESC) and germ cells revealed promoters and enhancers needed for stem cell maintenance and germ cell development. The nuclear receptor Nr5a2 motif is enriched at a subset of cis-regulatory regions and we confirm its role in germ cell differentiation. Interestingly, germ cells have comparatively more H3K27me3-marked sites that are absent in ESC and other somatic cell types. These repressed regions are enriched for retrotransposons and MHC genes and this indicates that these loci are specifically silenced in germ cells. Together, our study provides the first genome-wide histone modification maps of in vivo germ cells and revealed the molecular chromatin signatures unique to germ cells. Germ cells were FACS-purified from gonadal single cell suspension based on Pou5f1-GFP expression. ChIP-seq of Histone modification was done for two timepoints in this study: E11.5 (male/female), E13.5 (male). For E13.5 timepoint, two biological replicates were analyzed. In order to validate small scale ChIP-seq method limited number of ES cells were used to check consistency of ChIP-seq data.

ORGANISM(S): Mus musculus

SUBMITTER: Vibhor Kumar

PROVIDER: E-GEOD-38164 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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Project description:The winged helix protein FOXA2 and the nuclear receptor PPARg are highly conserved, regionally-expressed transcription factors that regulate networks of genes controlling complex metabolic functions. Cistrome analysis for FOXA2 in mouse liver and PPARg in mouse adipocytes has previously produced consensus binding sites that are nearly identical to those used by the factors in human cells. Despite this conservation of the canonical binding motif, we report here that the great majority of specific binding regions for FOXA2 in human liver and for PPARg in human adipocytes are not in the orthologous locations to the mouse genome. Nevertheless, gene-centric analysis reveals strong shared transcription factor occupancy near genes in tissue-specific metabolic pathways that are functionally conserved across species. Genes with only species-specific binding sites fail to show enrichment for these pathways. Thus, the biological functions of transcription factors that control specific metabolic functions are highly shared across species. Two TFs, FOXA2 and PPARg, were studied for genome-wide conservation of binding between mouse and human in specific tissues/cell-types (liver for FOXA2, adipocytes for PPARg). The number of replicates for each TF was chosen to obtain a comparable number of reads between the TFs and species. Human FOXA2 ChIP-seq was performed on two biological replicates of human liver samples, in three technical replicates each. Input DNA was also collected and sequenced from both biological samples. Mouse FOXA2 ChIP-seq was performed on four biological replicates of mouse liver samples. The ChIP and sequencing were repeated on two of these biological replicates to create technical replicates for additional sequence reads. Input DNA was sequenced from three additional mouse livers. Human PPARg ChIP-seq was performed on a human adipocyte cell-line (SGBS) differentiated in two replicate cultures. Input DNA was also collected and sequenced from one culture. Mouse PPARg ChIP-seq was performed on 3T3-L1 cells differentiated into adipocytes in culture in a single replicate, and this sequence data was pooled with existing data previously generated by the same lab, already available in GEO (GSE21314). A standard pool of input DNA sample sequence from multiple mouse tissue was used for analyzing the Mouse PPARg ChIP-seq data.

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In vivo epigenomic profiling of germ cells reveals germ cell molecular signatures [ChIP-Seq]

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