Characterization of RNA Polymerase II occupancy in mouse embryonic stem cells under control conditions or Phf5a knockdown
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ABSTRACT: Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed for genome-wide localization of Ser-5-phosphorylated RNA polymerase II and Ser-2-phosphorylated RNA polymerase II in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the increase of the initiating form of RNA polymerase II (Ser5-phosphorylated) and in the aberrant loss of the elongating form of RNA polymerase II (Ser2-phosphorylated) of pluripotency genes in ESCs.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed for genome-wide localization of Paf1, Leo1 and Cdc73 subunits of the Paf1 complex in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the significant decrease of Paf1 complex binding to its targets in ESCs.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed for genome-wide localization of H3K4me3, H3K79me2 and H3K36me3 in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the decrease of the elongation marks H3K79me2 and H3K36me3 from the gene bodies of pluripotency genes in ESC, wherease the promoter-associated mark H3K4me3 remains unaffected.
Project description:Phf5a regulates occupancy of Paf1 complex in mouse myotubes. In this study we assayed for genome-wide localization of Leo1 subunit of the Paf1 complex in mouse myoblasts or myotubes under conditions of shControl or shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in significant decrease of Leo1 binding to its targets in myotubes.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed for genome-wide localization of H2BK120-ub in mouse ESCs under conditions of shControl and shPhf5a knockdown. These results revealed that downregualtion of Phf5a results in the increase of the elongation mark H2BK120-ub from the gene bodies of pluripotency genes in ESC.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. We used microarrays to detail the global program of gene expression after shControl, shPhf5a, or shPaf1 knockdown in mouse ESCs following 72h knockdown and identified distinct down-regulated and up-regulated genes during this process.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed gene expression profiling using RNA-sequncing in mouse ESCs under conditions of self-renewal, Phf5a knockdown and Paf1 knockdown. These results revealed that Phf5a and Paf1 regulate similar gene expression signatures in mouse ESCs.
Project description:Phf5a regulates transcription elongation in mouse embryonic stem cells (ESCs), through regulation of the Paf1 complex. In this study we assayed nascent RNA profiling using global run-on sequncing in mouse ESCs under conditions of self-renewal, differentiation and Phf5a knockdown. These results revealed that genes positively regulated by Paf1 in ESCs exibit RNA elongation pausing on their promoters. We used conditions of self-renewal, differentiation, Phf5a knockdown and Paf1 knockdown to monitor nascent transcript expression changes during Phf5a depletion using global run-on RNA sequencing (GRO-seq). These results revealed that Phf5a through the control of Paf1 complex regulates pause release and elongation of pluripoency-associated genes in embryonic stem cells.
Project description:Protein phosphatase 1 complex (PP1C), consisting of one of the phosphatases, PP1α, β and γ, and three regulatory subunits PNUTS, TOX4 and WDR82, plays critical roles in gene regulation and is mutated in over 20% uterine corpus endometrial carcinoma cases. Here we show that knockdown of PP1γ reduced chromatin occupancy of RNA polymerase II (Pol II), but increased the cellular level and chromatin occupancy of Ser-2 phosphorylated Pol II and Ser-5 phosphorylated Pol II. 4sUDRB-seq data showed that the Pol II elongation rate decreased upon PP1γ loss. These results advanced our understanding of the roles of PP1γ in transcriptional regulation.
Project description:Dysregulated alternative splicing (AS) plays critical roles in driving cancer progression, and the underlying mechanisms remain largely unknown. Here we demonstrated that PHF5A, a component of U2 snRNPs, was frequently upregulated in colorectal cancer (CRC) samples and associated with poor prognosis. PHF5A promoted proliferation and metastasis of CRC cells in vitro and in vivo. Transcriptomic analysis identified PHF5A-regulated AS targets and pathways. Particularly, PHF5A induced TEAD2 exon 2 inclusion to activate YAP signaling, and interference of TEAD2-L partially reversed the PHF5A-mediated tumor progression. Pharmacological inhibition of PHF5A using pladienolide B had potent antitumor activity. Collectively, these data revealed the oncogenic role of PHF5A in CRC through regulating AS, and established PHF5A as potential therapeutic target.
Project description:PHD-finger protein 5A (PHF5A) is a component of U2snRNP, which plays a critical role in mRNA splicing for recognizing the branch site. However, whether it also plays other functions independent of RNA splicing remains elusive. Here, we find that expression levels of PHF5A are significantly upregulated in hepatocellular carcinoma (HCC). Down-regulation of PHF5A restrains liver cancer cell proliferation in vitro and in vivo. RNA-sequencing analysis reveals that fatty-acid biosynthetic enzymes are major downstream targets of PHF5A in HCC. Through metabolomics analysis, we find that inhibition of PHF5A reduces free fatty acid and phospholipid synthesis, further suppressing plasma membrane function. Mechanistically, PHF5A acts as a transcription co-factor of the p300 acetyltransferase for the H3K27 acetylation (Ac) and promotes H3K27Ac-dependent lipogenic genes expression. Our findings demonstrated a surprising splicing-independent role of PHF5A in transcriptional regulation of lipid metabolism for HCC progression and provided a new strategy for HCC therapy by blocking the interaction of PHF5A and p300.