Quantitative analysis of transcriptomes of MKN1 control/LRRFIP2 variant 3 exon 7 knockout cell lines
Ontology highlight
ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE194309 | GEO | 2022/09/05
REPOSITORIES: GEO
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Project description:Quantitative analysis of transcriptomes of MKN1 control/LRRFIP2 variant 3 exon 7 knockout cell lines
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Project description:p130Cas (Cas, Crk-associated substrate) is an adaptor molecule composed of an N-terminal Src homology 3 (SH3) domain, a substrate domain (SD), and a C-terminal Src binding domain (SBD). The SH3 domain of Cas has been shown to associate with various signaling molecules, including focal adhesion kinase (FAK), but its role in cellular function remains unclear. To address this issue, we established and analyzed primary fibroblasts derived from mice expressing a truncated Cas lacking the exon 2 encoding the SH3 domain (Cas exon 2∆/∆). In comparison to wild-type (Cas exon 2+/+) cells, Cas exon 2∆/∆ primary fibroblasts showed delayed migration in wound healing and reduced spreading on fibronectin (FN), which would be due to reduced complex formation of Cas exon 2∆/∆ with FAK and CrkII and also to impaired localization of Cas exon 2∆/∆ to focal adhesions on FN. In addition, to analyze downstream signaling pathway regulated by Cas exon 2, we compared gene expression profiles between Cas exon 2+/+ and Cas exon 2∆/∆ cells by a microarray analysis. Interestingly, we found that Cas exon 2-deficiency upregulated expression of CXC Chemokine Receptor-4 (CXCR4), CC Chemokine Receptor-5 (CCR5), and thrombospondin 4, which are implicated in cell motility and adhesion. These results define the role of Cas SH3-encoding exon in cell motility, FAK/Cas/CrkII complex formation, recruitment of Cas to focal adhesions, and regulation of cell adhesion-associated gene expression in primary fibroblasts. Keywords: SH3-encoding exon, p130Cas
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Project description:Multiplexed Assays of Variant Effects (MAVE) of PALB2 single nucleotide variants in exon 10.
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Project description:We investigate the contribution of each SUZ12 isoform on PRC2 activity on chromatin using our different ESC lines. As an additional comparison for ∆ex4 cells (SUZ12-S), we included an ESC clone in which an unsuccessful CRISPR editing event resulted in a mutant allele with nearly constitutive splicing of exon 4 (herein, CSex4) and normal Suz12 expression levels (expressing only SUZ12-L). First, we profiled H3K27 bulk PTMs by WB and observed that, while CSex4 cells showed no major differences to control cells, ∆ex4 cells displayed lower levels of H3K27me2 and -me3, with higher levels of mono-methylation and acetylation. Similar results were obtained when comparing SUZ12-L and SUZ12-S ESC rescue lines. To validate these findings with an antibody-independent technique, and to additionally profile other histone PTMs, we analysed these cells with histone-MS. In line with the previous estimates26, WT cells displayed approximately 85% of H3K27 methylation. This proportion was largely similar in CSex4 cells, while it dropped to ~50% in ∆ex4 cells, with H3K27me2 and -me3 being the most affected modifications. Notably, H3K27me2/3 loss in ∆ex4 cells occurred at histone peptides regardless of their H3K36 methylation status. Moreover, the global H3K36 methylation rates were not affected in either CSex4 or ∆ex4 cells (Figure S4C), and no major changes in methylation or acetylation levels were observed at other residues. Importantly, reintroduction of SUZ12-L in KO cells rescued a higher degree of methylation compared to SUZ12-S. Overall, these results indicate that SUZ12-S alone is unable to maintain global physiological levels of H3K27 methylation; rather SUZ12-L is also necessary for this task.
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