RNA-seq analysis of EpiSCs undergoing differentiation induced by EB formation
Ontology highlight
ABSTRACT:
INSTRUMENT(S): Illumina HiSeq 2500
ORGANISM(S): Mus musculus
SUBMITTER: Kenjiro Adachi
PROVIDER: E-MTAB-5343 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:To characterize the reprogramming of epiblast stem cells (EpiSCs) into embryonic stem cells (ESCs) induced by Esrrb, we performed microarray analysis of Tet-on Esrrb EpiSCs after treatment with doxycycline (Dox).
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Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). WNT signalling sustains both NMP expansion and PM differentiation, but the mechanism by which it distinguishes between these alternative fates is unknown. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX/HOX complexes establish a permissive chromatin landscape for de novo recruitment of the WNT-effector LEF1 on PM genes, including the master regulator Mesogenin1 (Msgn1). To assess the PBX-dependent changes in chromatin accessibility during PM differentiation, we performed ATAC-seq of wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to pre-somitic mesoderm (PSM) at different time-points (EpiSCs, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours). To assess the direct consequence of PBX/HOX binding on chromatin accessibility, we employed the CRISPR/Cas9 technology to generate lines carrying base-pair substitutions on the Msgn1 promoter (pMsgn1-mut) that abrogate the recruitment of PBX/HOX complexes, and we performed ATAC-seq of pMsgn1-mut EpiSCs differentiated in vitro to PSM at different time-points (12 hours, 24 hours, 36 hours, 48 hours).
2021-07-21 | E-MTAB-9776 | biostudies-arrayexpress
Project description:To investigate the molecular mechanisms underlying the reprogramming of epiblast stem cells (EpiSCs) into embryonic stem cells (ESCs) induced by Esrrb, we performed ChIP-seq analysis of Esrrb, Nanog, Oct4, and Sox2 in Tet-on Esrrb EpiSCs after treatment with doxycycline (Dox).
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Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX proteins play a fundamental role in promoting the expression of PM genes, including the master regulator Mesogenin1 (Msgn1). To address the role of PBX proteins in PM differentiation, RNA-seq was performed on wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to pre-somitic mesoderm (PSM) at different time-points (12 hours and 24 hours). To establish the direct transcriptional regulation of Msgn1 by PBX/HOX, we employed the CRISPR/Cas9 technology to generate lines carrying base-pair substitutions on the Msgn1 promoter (pMsgn1-mut) that abrogate the recruitment of PBX/HOX complexes, and we performed RNA-seq of pMsgn1-mut EpiSCs differentiated in vitro to PSM at 24 hours. All differentiation experiments were performed in biological triplicates with WT and Pbx1/2-DKO lines, and in biological duplicates with pMsgn1-mut lines.
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Project description:Epiblast stem cells (EpiSCs) are pluripotent cells that can be isolated and cultured from post implantation embryos. In contrast to embryonic stem cells (ESCs), systematic studies to investigate the genes that maintain pluripotency in EpiSCs have not been reported. Here we combine a genome-wide RNAi screen with genetic interaction, protein localization and protein-level dependency studies to delineate connectivity between factors that control Oct4 expression in EpiSCs and compare the role of these factors to their function in ES cells. We demonstrate the power of this integrative approach by the identification of Tox4 as an interactor of PP1 (Protein Phosphatase 1) and Paf1C, a complex that acts in multiple aspects of RNAPII regulation. Our results indicate that Tox4 cooperates with Paf1C and PP1 to influence the phosphorylation status of the RNAPII CTD tail during transcription and that this function is vital for maintenance of pluripotent cell identity. RNA-seq of Tox4 knockdown in mouse EpiSCs
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Project description:EpiSCs (epiblast stem cells) are murine pluripotent stem cells derived from the epiblast of a post-implantation embryo. These cells are primed to differentiate towards embryonic germ layers and are a useful model to study these early developmental events. PGR (progesterone receptor) is a nuclear receptor described mainly in the context of fertility and breast cancer. We noticed that it is also a TF that is expressed in primed EpiSCs, but not in naive mESCs and is further upregulated during mesoderm specification. We hypothesized that it is involved in differentiation to primitive streak and mesoderm progenitors. To test this hypothesis we generated EpiSCs with a genetic knockout of PGR using CRISPR-Cas9-mediated knock-in of an antibiotic resistance cassette. Differentiation of the wild-type and knockout cells and comparison of the different cell types allowed us to conclude that PGR is important for acquiring extraembryonic mesoderm fate and modulates cardiac differentiation, specifically it ensures that correct pools of FHF (first heart field) and SHF (second heart field) are being produced. Our study sheds light on previously unappreciated role of PGR in early embryonic development.
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Project description:In vertebrates, body axis elongation is fuelled by bipotent neuromesodermal progenitors (NMPs), which support the development of both spinal cord and paraxial mesoderm (PM). WNT signalling sustains both NMP expansion and PM differentiation, but the mechanism by which it distinguishes between these alternative fates is unknown. HOX transcription factors have been historically implicated in axial elongation, with their sequential activation playing a fundamental role in timing PM development. PBX1 and PBX2 are obligate anterior HOX cofactors, and therefore they represent prominent candidates for controlling the distinct response to individual HOX factors. In our work, we have demonstrated that PBX proteins generate the DNA-binding context that allows recruitment of the WNT-effector LEF1, and consequently promote the expression of PM genes. Here, we wanted to identify the chromatin binding pattern of PBX1, PBX2 and LEF1 during the first 48 hours of pre-somitic mesoderm (PSM) in vitro differentiation, by performing chromatin immunoprecipitation followed by deep sequencing (ChIP-seq) at different time-points (EpiSCs, 6 hours, 12 hours, 24 hours, 48 hours). To address the impact of PBX loss on LEF1 recruitment to chromatin, we additionally performed ChIP-seq in wild-type (WT) and Pbx1/Pbx2 double-knockout (Pbx1/2-DKO) EpiSCs differentiated in vitro to PSM at different time-points (EpiSCs, 12 hours, 24 hours, 48 hours).
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