BRACHYURY orchestrates distinct mesoderm and endoderm gene regulatory networks in differentiating human embryonic stem cells
ABSTRACT: The transcription factor BRACHYURY (T, BRA) is one of the first markers of gastrulation and lineage specification in mammals. Despite its wide use and importance in stem cell and developmental biology, its genomic targets are largely unknown. Here, we used differentiated human embryonic stem cells to study the role of BRA in Bmp4-induced mesoderm and Activin-induced endoderm progenitors by ChIP-seq. We show that BRA has distinct genome-wide binding landscapes in these two populations. Our data illuminate the function of BRA in the context of human embryonic development and show that the regulatory role of BRA is context-dependent. ChIP-seq of BRACHYURY (T, BRA) in two cell types: endoderm and mesoderm progenitors derived from human embryonic stem cells after 36 hours of growth in chemically-defined media (described in Bernardo et al., Cell Stem Cell, 2011, 9:144-155). Input DNA samples are included as a control.
Project description:Transcriptional profiling of H9 hESCs (wild type and BRACHYURY shRNA knockdown) differentiated for 36h or 72h in chemically-defined culture media supplemented with FGF2, LY294002 and either BMP4 or ActivinA.
Project description:Transcriptional profiling of hESCs differentiated towards an endodermal fate in chemically-defined media (3 days) compared to clones subjected to EOMES knockdown (via shRNA) under the same conditions.
Project description:Transcriptional profiling of hESCs in chemically-defined culture media compared to hESCs differentiated for 36h in the additional presence of FGF, LY294002 and either BMP or ActivinA.
Project description:Understanding the molecular mechanisms controlling early cell fate decisions in mammals is a major objective towards the development of robust methods for the differentiation of human pluripotent stem cells into clinically relevant cell types. Here, we used human embryonic stem cells (hESCs) to study specification of definitive endoderm in vitro. Using a combination of whole genome expression and ChIP-seq analyses, we established a hierarchy of transcription factors regulating endoderm specification. Importantly, pluripotency factors, namely NANOG, OCT4 and SOX2 have an essential function in this network by actively directing differentiation. Indeed, these transcription factors control the expression of EOMES, which marks the onset of endoderm specification. In turn, EOMES interacts with SMAD2/3 to initiate the transcriptional network governing endoderm formation. Together, these results provide for the first time a comprehensive molecular model connecting the transition from pluripotency to endoderm specification during mammalian development. ChIP-Seq of Eomesodermin binding in human embyonic stem cells, differentiated towards an endodermal fate for 48h in chemically-defined culture media. Includes an input DNA control. Supplementary file GSE26097_README.txt contains descriptions of the raw data files and processed data files.
Project description:The key notochord transcription factor Brachyury was ectopically expressed in Ciona embryos under the control of the FoxAa cis-regulatory region (which drives expression in neural, endodermal and mesenchymal lineages in addition to notochord). Misexpression of Brachyury induced 925 genes compared to a control reporter plasmid (Bra>GFP). There was only modest overlap with a set of notochord-enriched genes previously identified by RNAseq of flow-sorted notochord cells, indicating that Brachyury is not a notochord master regulator gene as strictly defined. Overall design: Fertilized dechorionated eggs were electroporated with either FoxAa>Venus-Bra plasmid or control plasmid (Bra<GFP). For each of the replicates, 400 experimental and 400 control embryos were collected at stage 19.5 (7.5hpf at 21.5C). Triple biological replicates of purified RNA were analyzed by RNAseq and transcript enrichment was calculated using the Cufflinks software suite
Project description:To test our hypothesis that human embryonic stem cell-derived endothelial cells(hESC-ECs) represent their in vivo counterparts, we performed transcriptional profiling experiments in which we compared the gene expression profiles of d6 CD34+KDR+ cells, d12-14 hESC-ECs and human umbilical vein endothelial cells(HUVECs). Significantly upregulated genes were identified using significance analysis of microarrays (SAM), by comparing each of these populations to undifferentiated hESCs. Differentially regulated genes from each of these populations were examined using overlap analysis;to identify which genes are uniquely expressed in each cell type and which are expressed in common, followed by functional annotation clustering of the conserved core group of genes.
Project description:Comparison of H9 human embryonic stem cells, grown in chemically defined and animal-product free conditions, with their differentiated progeny - mesendodermal progenitors arisen as the result of changes made to chemically defined hESC growth conditions. Contributes to a larger work on the use of chemically defined growth conditions to direct hESC differentiation.
Project description:Single-cell RNA-seq (scRNA-seq) on nocodazole and DMSO treated cells before and after differentiation into endoderm. hPSC colonies were treated with DMSO or 100ng/ml nocodazole for 16 hours and induced to differentiate into definitive endoderm for three days. Single cells were subsequently collected in either undifferentiated conditions (Und) or after 3 days of endoderm differentiation (Endoderm) and then sorted onto 384 well plates for Smart-Seq2 processing.