Whole Genome Bisulfite Sequencing of CLDN6High vs CLDN6Low Sorted EpiSCs and APSD, and WT vs Pbx1-KO EpiSCs
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PROVIDER: PRJEB70786 | ENA |
REPOSITORIES: ENA
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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).
2021-07-21 | E-MTAB-9775 | biostudies-arrayexpress
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. To pinpoint the role of PBX proteins in the development of pre-somitic mesoderm (PSM), wild-type (WT) and Pbx1/Pbx2 double-mutant (Pbx1/2-DKO) EpiSCs were differentiated in vitro to PSM. Single cells from different time-points (WT: EpiSCs, 6 hours, 12 hours, 24 hours, 36 hours, 48 hours; Pbx1/2-DKO: 24 hours, 36 hours, 48 hours) were FACS-sorted into 384-well capture plates, and single-cell RNA-sequencing was performed using MARS-seq (Jaitin D.A. et al., Science, 343, 776-779 (2014)). Two wells were left empty in each plate, as a no-cell control during data analysis.
2021-07-21 | E-MTAB-9774 | biostudies-arrayexpress
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: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.
2021-07-21 | E-MTAB-9773 | biostudies-arrayexpress
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). NMPs reside in the caudal lateral epiblast and tailbud, from where they sustain axial elongation. 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. To pinpoint the role of PBX proteins in the development of pre-somitic mesoderm, single-cell RNA sequencing (scRNA-seq) was performed on cells isolated from the tailbuds of control, Pbx1/Pbx2 compound (Pbx1/2-com) and Pbx1/Pbx2 double-mutant (Pbx1/2-DKO) embryos at embryonic days 8.5 and 9.0. Single cells from at least three embryos per genotype and stage were FACS-sorted into 384-well capture plates, and scRNA-seq was performed using MARS-seq (Jaitin D.A. et al., Science, 343, 776-779 (2014)). As a spike-in internal control for batch-effect correction, 71 EpiSCs cultivated in vitro were sorted into each plate, together with 311 cells from embryonic tailbuds. Two wells were left empty in each plate, as a no-cell control during data analysis.
2021-07-21 | E-MTAB-9785 | biostudies-arrayexpress
Project description:We compared the transcriptomes of EpiSCs maintained in the presence or absence of Wnt pathway inhibitor IWP2. We screened also our gene expression data for potential markers for genuine EpiSCs, maintained in the presence of Wnt inhibition and compared with ESC expression data. We compared the transcriptomes of EpiSCs maintained in the presence or absence of IWP2. The high level of Wnt-induced differentiation occurring in conventional EpiSC cultures may have interfered with the analysis of their characteristics. By applying Wnt inhibitors we are now able to establish the properties of genuine EpiSCs.
2014-12-24 | GSE62155 | GEO
Project description:Epiblast stem cells (EpiSCs) were derived from the epiblast or the ectoderm (epi/ect) of pre-gastrula stage to late-bud stage mouse embryos. To identify if the EpiSCs retain any original stage specific characteristics or which developmental stage of epi/ect they most closely related to, we performed microarray analysis to compare the gene expression profile of multiple EpiSC lines with that of epi/ect of 7 different stages.
2013-09-15 | GSE46227 | GEO
Project description:ChIP-seq of PBX1, PBX2 and LEF1 in wild-type and Pbx1/Pbx2 double-knockout EpiSCs differentiated in vitro to pre-somitic mesoderm in the first 48 hours of differentiation.
| PRJEB41392 | ENA
Project description:Developmental transcription factors act in networks, but how these networks achieve cell- and tissue specificity is still poorly understood. We here explored pre-B-cell leukemia homeobox 1 (PBX1) in adult neurogenesis combining genomic, transcriptomic, and proteomic approaches. ChIP-Seq analysis uncovered PBX1 binding to a wide range of different genes. Integration of PBX1 ChIP-seq with ATAC-seq data predicted interaction partners, which were subsequently validated by mass-spectrometry. Spatial transcriptomics revealed distinct temporal expression dynamics of Pbx1 and interacting factors. Among these were class I bHLH proteins TCF3, TCF4 and TCF12. RNA-seq upon Pbx1, Tcf3 and Tcf4 knockdown identified proliferation and differentiation associated genes as shared targets. Neuronal differentiation was reduced upon depletion of either factor, suggesting functional cooperation between PBX1 and TCF3/4. Notably, while physiological PBX1-TCF interactions have not yet been described, chromosomal translocation resulting in genomic TCF3::PBX1 fusion characterizes a subtype of acute lymphoblastic leukemia. Introducing Pbx1 into Nalm6 cells, a pre B-cell line expressing TCF3 but lacking PBX1, upregulated leukemogenic genes including BLK and NOTCH3, arguing that functional PBX1-TCF cooperation likely extends to hematopoietic contexts. Our study hence uncovers a PBX1-TCF module orchestrating the balance between progenitor cell proliferation and differentiation in adult neurogenesis with implications for leukemia etiology.
2024-09-16 | PXD048222 | Pride
Project description:Pre B cell leukemia homeobox 1 (Pbx1) regulates the balance between self-renewal and differentiation of hematopoietic stem cells, and maintains proto-oncogenic transcriptional pathways implicated in several tumors. Its aberrant expression was found in a subset of myeloproliferative neoplasms (MPN) patients bearing the JAK2V617F mutation. To investigate if Pbx1 contributes to MPN, and to explore its potential as therapeutic target, we generated a new mouse strain, that we called JP, by crossing a known JAK2V617F inducible knock-in MPN model with a Pbx1 conditional-ko. In JP mice we can simultaneously activate the human JAK2 mutation and delete Pbx1. Typical MPN features, such as thrombocytemia and granulocytosis, did not develop in the absence of Pbx1. Erythrocytosis, initially displayed by JP mice, gradually resolved over time. Moreover, splenic myeloid metaplasia and in vitro cytokine independent growth were rescued by Pbx1 inactivation. Through RNA-Sequencing we analyzed molecular pathways downstream of Pbx1 and involved in MPN maintenance in stem and progenitor cells. The aberrant transcriptome in the MPN model compared to wild-type was rescued by the absence of Pbx1. Our results demonstrate that inhibition of the Pbx1-driven transcriptional program is beneficial in MPN. Modulation of Pbx1 activity by direct targeting or by targeting its downstream mediators might thus represent a novel therapeutic approach.
2021-10-21 | GSE153482 | GEO