Overexpression of CLK and wild-type CPSF6 and mutant CPSF6 in HEK293T cells to verify the effect of CLK2 phosphorylation CPSF6 on APA
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE230814 | GEO | 2023/09/07
REPOSITORIES: GEO
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Project description:Overexpression of CLK and wild-type CPSF6 and mutant CPSF6 in HEK293T cells to verify the effect of CLK2 phosphorylation CPSF6 on APA
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Project description:To identify ZNF506 genome-wide target sites, we performed ChIP-seq assay and found that ZNF506 binding sites enriched at PBS-Pro-containing ERV subfamilies (ERVPs) and further motif calling analysis showed that ZNF506 binds to PBS-Pro sequences, promoting formation of H3K9me3 modifications at binding regions. And ChIP-seq assay also indicated that the distinction of H3K9me3 signals closed to ZNF506 peak regions between ZNF506 overexpressing (OE) HEK293T cells and ZNF506 Knockout (KO) HEK293T cells was correlated with the different recruitment of corepressor KAP1. The ChIP-seq experiments using GFP antibodies and H3K9me3 antibodies on ZNF506-GFP OE HEK293T cell lines, and ZNF417-GFP OE HEK293T cell lines were used as controls for H3K9me3 ChIP. Also, the ChIP-seq experiments were performed using H3K9me3 antibodies and KAP1 antibodies on ZNF506 KO HEK293T cells and ZNF506 OE HEK293T cells.
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Project description:IntroductionUbiquinone (UQ) is a redox active lipid that transfers electrons from complex I or II to complex III in the electron transport chain (ETC). The long-lived Caenorhabditis elegans mutant clk-1 is unable to synthesize its native ubiquinone, and accumulates high amounts of its precursor, 5-demethoxyubiquinone-9 (DMQ(9)). In clk-1, complexes I-III activity is inhibited while complexes II-III activity is normal. We asked whether the complexes I-III defect in clk-1 was caused by: (1) a defect in the ETC; (2) an inhibitory effect of DMQ(9); or (3) a decreased amount of ubiquinone.MethodsWe extracted the endogenous quinones from wildtype (N2) and clk-1 mitochondria, replenished them with exogenous ubiquinones, and measured ETC activities.ResultsReplenishment of extracted mutant and wildtype mitochondria resulted in equal enzymatic activities for complexes I-III and II-III ETC assays. Blue native gels showed that supercomplex formation was indistinguishable between clk-1 and N2. The addition of a pentane extract from clk-1 mitochondria containing DMQ(9) to wildtype mitochondria specifically inhibited complexes I-III activity. UQ in clk-1 mitochondria was oxidized compared to N2.DiscussionOur results show that no measurable intrinsic ETC defect exists in clk-1 mitochondria. The data indicate that DMQ(9) specifically inhibits electron transfer from complex I to ubiquinone.
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