RNA-seq of WT, Mbd3-/- and rescue hiPSC during neuroectodermal differentiation and of WT and Mbd3-/- in mEpiSC during neuroectodermal differentiation
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PROVIDER: PRJEB36525 | ENA |
REPOSITORIES: ENA
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Project description:Differentiation of mammalian pluripotent cells involves large-scale changes in transcription and chromatin remodellers are essential to initiate, establish and maintain a new gene regulatory network. In this study, we investigated the structure and function of the nucleosome remodeller and deacetylase (NuRD) complex in human induced pluripotent stem cells (hiPSCs) and in mouse epiblast stem cells (mEpiSCs). We studied gene expression changes at the self-renewal state (0 days) and at 3, 6 and 12 days of neuroectodermal differentiation in WT, Mbd3-/- and rescue hiPSCs and at 2, 4 and 8 days of neuroectodermal differentiation in mEpiSCs. Sequencing libraries were prepared using the NEXTflex Rapid Directional RNA-seq kit (Illumina) or SMARTer® Stranded Total RNA-Seq Kit v2—Pico Input Mammalian (Takara Bio) and sequenced on the Illumina platform at the CRUK Cambridge Institute Genomics Core facility (Cambridge, UK). Illumina sequence files were converted into FASTQ format. The short sequence reads (75 nucleotides) were aligned to the Human reference genome (hg38; http://genome.ucsc.edu/) or to the Mouse reference genome (mm10; http://genome.ucsc.edu/) and assigned to genes using BWA (Li & Durbin, 2009). We used the Subread package (R statistical tool; http://www.r-project.org/) to count aligned reads. Differentially expressed genes were identified using R package edgeR (Chen, Lun, & Smyth, 2016).
2020-03-30 | E-MTAB-8753 | biostudies-arrayexpress
Project description:We analyzed by BS-seq the methylation pattern of CTR and YAP knockdown cells in undifferentiated (T0) embryonic stem cells (ESCs) vs cells differentiated toward neuroectodermal fate at day 4 of differentiation (T4). Two biological duplicates for each condition.
2020-12-08 | GSE157704 | GEO
Project description:Methylation pattern profile of mouse embryonic stem cells upon knockdown of YAP, during neuroectodermal differentiation
| PRJNA662504 | ENA
Project description:The Nucleosome Remodeling and Deacetylation (NuRD) complex is a crucial regulator of cellular differentiation. Two members of the Methyl-CpG-binding domain (MBD) protein family, MBD2 and MBD3, are known to be integral, but mutually exclusive subunits of the NuRD complex. Several MBD2 and MBD3 isoforms are present in mammalian cells, resulting in distinct MBD-NuRD complexes. Whether these different complexes serve distinct functional activities during differentiation is not fully explored. Based on the essential role of MBD3 in lineage commitment, we systematically investigated a diverse set of MBD2 and MBD3 variants for their potential to rescue the differentiation block observed for mouse embryonic stem cells (ESCs) lacking MBD3. While MBD3 is indeed crucial for ESC differentiation to neuronal cells, it functions independently of its MBD domain. We further identify that MBD2 isoforms can replace MBD3 during lineage commitment, however with different potential. Full-length MBD2a only partially rescues the differentiation block, while MBD2b, an isoform lacking an N-terminal GR-rich repeat, fully rescues the Mbd3 KO phenotype. In case of MBD2a, we further show that removing the methylated DNA binding capacity or the GR-rich repeat enables full redundancy to MBD3, highlighting the synergistic requirements for these domains in diversifying NuRD complex function.
2023-05-23 | GSE199541 | GEO
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression. Identification of MBD3 localization in human breast cancer cell lines
2013-10-31 | E-GEOD-44737 | biostudies-arrayexpress
Project description:To obtain a comprehensive and quantitative view on MBD2 vs MBD3-NuRD complex stoichiometry, we performed biotin co-immunoprecipitations in Mbd3 KO ES cells expressing either biotin-tagged MBD2a or MBD3a and identified known NuRD complex members using label-free mass spectrometry (Supplementary Fig. 8b-c). We then calculated the intensity-based absolute quantification (iBAQ) values of the most predominant and statistically significant MBD-interacting proteins in both cell lines, which can be used to estimate the relative abundance. While we observe very similar complex composition between MBD2a-NuRD and MBD3a-NuRD, peptides shared between SALL1-4 proteins show a preferred interaction with the MBD2a-NuRD complex.
2023-06-30 | PXD042407 | Pride
Project description:Maintenance of chromatin structure is essential to eukaryotic life; dysregulation is known to be causal for aberrant development and disease. The Mi-2/nucleosome remodeling and histone deacetylase (NuRD) complex is a multiprotein machine proposed to regulate chromatin structure by nucleosome remodeling and histone deacetylation activities. We identified the localization of MBD3, a component of Mi-2/NuRD complex, in two breast cancer cell lines (MCF7 and MDA-MB-231) using ChIP-Seq. MBD3 showed cell-type specific localization with overlap across cell lines being less than 50%. MBD3 localized across gene bodies, peaking around the transcription start site (TSS). Contrary to existing models, MBD3 preferentially associated with CpG rich promoters marked by H3K4me3. These data suggest that MBD3, and by extension the Mi-2/NuRD complex, may have roles in fine tuning expression for active genes. These data represent an important first step in defining regulatory mechanisms by which Mi-2/NuRD complex controls chromatin structure and gene expression.
2013-10-31 | GSE44737 | GEO
Project description:We report here the application of magnetic cell separation in microarray experiment to understand Drosophila gliogenesis during early embryogenesis. At stage 11, neuroectodermal cells were labeled by a fusion transmembrane protein, mCD8-GFP, in the wild-type embryos as well as in the embryos where glide/gcm was overexpressed. Following the cell dissociation, neuroectodermal cells were purified by using the commercial available anti-mCD8 microbeads. Microarray experiments based on prepared neuroectodermal cells from wild-type embryos verse glide/gcm overexpressed embryos at stage 11 have revealed 76 gene differentially regulated by overexpression of glide/gcm.
2007-07-06 | E-GEOD-618 | biostudies-arrayexpress
Project description:Genome-wide association of Geminin and Zic1 during neuroectodermal cell specification
| PRJNA322060 | ENA
Project description:The heterogeneous collection of NuRD complexes can be grouped into the MBD2 or MBD3 containing complexes MBD2-NuRD and MBD3-NuRD. MBD2 is known to bind to methylated CpG sequences in vitro in contrast to MBD3. Although functional differences have been described, a direct comparison of MBD2 and MBD3 in respect to genome-wide binding and function has been lacking. Here we show when depleting cells for MBD2, the MBD2 bound genes increase their activity, whereas MBD2 plus MBD3 bound genes reduce their activity. Most strikingly, MBD3 is enriched at active promoters, whereas MBD2 is bound at methylated promoters and enriched at exon sequences of active genes. This suggests a functional connection between MBD2 binding to chromatin and splicing. 9 Total samples were analysed. Three individual replicates for HeLa cells treated with siRNA against MBD2, MBD3 or scrambled siRNA were performed. We calculated the fold change of gene expression of cells treated with MBD2- or MBD3-siRNA over cells treated with scrambled siRNA.
2013-02-08 | E-GEOD-41003 | biostudies-arrayexpress