Functional and Mechanistic Studies of XPC DNA-Repair Complex as Transcriptional Coactivator in Embryonic Stem Cells
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE64040 | GEO | 2015/04/20
SECONDARY ACCESSION(S): PRJNA269957
REPOSITORIES: GEO
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Project description:We recently identified the DNA repair complex XPC-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4 and SOX2 transcriptional activation. Here we investigate genome-wide the role of SCC in murine ESCs by mapping regions bound by its RAD23B subunit in wild type and Xpc-/- ESCs, and analyzing transcriptional profiles of SCC-depleted ESCs. RAD23B ChIP-seq in wild type murine ESCs was performed in two replicates; normal IgG were used as controls. RAD23B ChIP-seq in Xpc-/- mESCs was compared to normal IgG. Antibodies to immunoprecipitate RAD23B were raised in guinea pigs against the 108-177 peptide of murine RAD23B protein (NP_033037). Anti-sera were affinity-purified. For RNA-seq, we compared wild type JM8.N4 cells with either Rad23b-/- cells, Rad23b-/-Xpc KD1 or Rad23b-/-Xpc KD2 cells. Xpc was knocked down with two independent shRNAs (KD1 and KD2).
2015-04-20 | E-GEOD-64040 | biostudies-arrayexpress
Project description:The embryonic stem cell (ESC) state is transcriptionally controlled by OCT4, SOX2, and NANOG with cofactors, chromatin regulators, noncoding RNAs, and other effectors of signaling pathways. Uncovering components of these regulatory circuits and their interplay provides the knowledge base to deploy ESCs and induced pluripotent stem cells. We recently identified the DNA-repair complex xeroderma pigmentosum C (XPC)-RAD23B-CETN2 as a stem cell coactivator (SCC) required for OCT4/SOX2 transcriptional activation. Here we investigate the role of SCC genome-wide in murine ESCs by mapping regions bound by RAD23B and analyzing transcriptional profiles of SCC-depleted ESCs. We establish OCT4 and SOX2 as the primary transcription factors recruiting SCC to regulatory regions of pluripotency genes and identify the XPC subunit as essential for interaction with the two proteins. The present study reveals new mechanistic and functional aspects of SCC transcriptional activity, and thus underscores the diversified functions of this regulatory complex.
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Project description:The study examines the impact of XPC overexpression on DNA demethylation in human dermal fibroblasts (HDFs) and during the early phase of somatic cell reprogramming (pre-iPSCs). We profiled the 5mC status in wild type and XPC gain-of-function HDFs and pre-iPSCs on a genome-wide scale by MeDIP-seq. XPC overexpression results in a loss of DNA methylation in both HDFs and pre-iPSCs. In XPC gain-of-function cells, not only does DNA methylation occur in fewer regions compared to control WT cells, but overall methylation levels are lower than in WT cells.
2017-05-22 | GSE92445 | GEO
Project description:Cells employ global genome DNA damage repair (GGR) to eliminate a broad spectrum of DNA lesions, including those induced by UV light. The lesion-recognition factor XPC initiates repair of helix-distorting DNA lesions, but binds inefficiently to lesions that cause poor helix distortion. How such difficult-to-repair lesions are detected in chromatin is unknown. Here, we identify the poly-(ADP-ribose) polymerases PARP1 and PARP2 as constitutive interactors of XPC. The close interaction between these proteins results in the PARylation of XPC at UV lesions, and an XPC-dependent stimulation of the poly-(ADP-ribose) response, which facilitates the recruitment of the poly-(ADP-ribose)-dependent chromatin remodeler ALC1. Both ALC1 and in particular PARP2 are required for the efficient clearing of difficult-to-repair DNA lesions. Our study offers key insights into the molecular mechanisms of GGR by revealing a molecular bookmarking system, which primes chromatin containing difficult-to-repair DNA lesions for efficient repair.
2022-07-15 | PXD025226 | Pride
Project description:The transcription factor Oct3/4 is essential to maintain pluripotency in mouse embryonic stem (ES) cells. It was reported that the Xpc DNA repair complex is involved in this process. Here we examined the role of Xpc on the transcriptional activation of the target genes by Oct3/4 using the inducible knockout strategy. We found that the removal of the C-terminal region of Xpc, including the interaction sites with Rad23 and Cetn2, showed faint impact on the gene expression profile of ES cells and the functional Xpc-ΔC ES cell lines retained proper gene expression profile as well as pluripotency to contribute chimeric embryos. These data indicated that the C-terminal region of Xpc is dispensable for the transcriptional activity of Oct3/4 in mouse ES cells. An inducible knockout ES cell line of Xpc with the Cre-loxP system was generated and gene expression was measured without Xpc deletion, after 4 days of Xpc deletion or constitutive knocked out cells. Each sample was prepared in triplicate.
2015-06-09 | E-GEOD-69640 | biostudies-arrayexpress
Project description:The Xeroderma pigmentosum complementation group C (XPC) complex is a versatile factor involved in both nucleotide excision repair and transcriptional coactivation as a critical component of the NANOG, OCT4, and SOX2 pluripotency gene regulatory network. Here we present the structure of the human holo-XPC complex determined by single-particle electron microscopy to reveal a flexible, ear-shaped structure that undergoes localized loss of order upon DNA binding. We also determined the structure of the complete yeast homolog Rad4 holo-complex to find a similar overall architecture to the human complex, consistent with their shared DNA repair functions. Localized differences between these structures reflect an intriguing phylogenetic divergence in transcriptional capabilities that we present here. Having positioned the constituent subunits by tagging and deletion, we propose a model of key interaction interfaces that reveals the structural basis for this difference in functional conservation. Together, our findings establish a framework for understanding the structure-function relationships of the XPC complex in the interplay between transcription and DNA repair.
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Project description:The transcription factor Oct3/4 is essential to maintain pluripotency in mouse embryonic stem (ES) cells. It was reported that the Xpc DNA repair complex is involved in this process. Here we examined the role of Xpc on the transcriptional activation of the target genes by Oct3/4 using the inducible knockout strategy. We found that the removal of the C-terminal region of Xpc, including the interaction sites with Rad23 and Cetn2, showed faint impact on the gene expression profile of ES cells and the functional Xpc-ΔC ES cell lines retained proper gene expression profile as well as pluripotency to contribute chimeric embryos. These data indicated that the C-terminal region of Xpc is dispensable for the transcriptional activity of Oct3/4 in mouse ES cells.
2015-06-09 | GSE69640 | GEO
Project description:Oct4 is considered a master transcription factor for pluripotent cell self-renewal and embryo development. It primarily collaborates with other transcriptional factors or coregulators to maintain pluripotency. However, it is still unclear how Oct4 interacts with its partners. Here, we show that the Oct4 linker interface mediates competing and balanced Oct4 protein interactions which are crucial for maintaining pluripotency. Linker mutant ESCs maintain the key pluripotency genes expression, but show decreased expression of self-renewal genes and increased expression of differentiation genes which result in impaired ESCs self-renewal and early embryonic lethality. Linker mutation dose not affect Oct4 genomic binding and transactivation potential, but breaks the balanced Oct4 interactome. In mutant ESCs, the interaction between Oct4 and Klf5 was decreased, but interactions between Oct4 and Cbx1, Ctr9, Cdc73 were increased which disrupt the epigenetic state of ESCs. Overexpression of Klf5 or knockdown Cbx1, Cdc73 rescue the cellular phenotype of linker mutant ESCs by rebalancing Oct4 interactome indicating that different partners interact with Oct4 competitively. Thus, by showing how Oct4 interacts with different partners, we provide novel molecular insights to explain how Oct4 contributes to the maintenance of pluripotency.
2021-12-10 | PXD017456 | Pride
Project description:Protein arginine methyltransferases (PRMTs) catalyze the transfer of methyl groups from S-adenosylmethionine (SAM) to the guanidinium group of arginine residues in a number of important cell signaling proteins. PRMT1 is the founding member of this family, and its activity appears to be dysregulated in heart disease and cancer. To begin to characterize the catalytic mechanism of this isozyme, we assessed the effects of mutating a number of highly conserved active site residues (i.e., Y39, R54, E100, E144, E153, M155, and H293), which are believed to play key roles in SAM recognition, substrate binding, and catalysis. The results of these studies, as well as pH-rate studies, and the determination of solvent isotope effects (SIEs) indicate that M155 plays a critical role in both SAM binding and the processivity of the reaction but is not responsible for the regiospecific formation of asymmetrically dimethylated arginine (ADMA). Additionally, mutagenesis studies on H293, combined with pH studies and the lack of a normal SIE, do not support a role for this residue as a general base. Furthermore, the lack of a normal SIE with either the wild type or catalytically impaired mutants suggests that general acid/base catalysis is not important for promoting methyl transfer. This result, combined with the fact that the E144A/E153A double mutant retains considerably more activity then the single mutants alone, suggests that the PRMT1-catalyzed reaction is primarily driven by bringing the substrate guanidinium into the proximity of the S-methyl group of SAM and that the prior deprotonation of the substrate guanidinium is not required for methyl transfer.
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Project description:The actions of transcription factors, chromatin modifiers, and noncoding RNAs are crucial for the programming of cellular states. Although chromatin remodeling factors regulate the functional status of cells including pluripotency and differentiation, how they cross-talk with embryonic stem (ES) cell-specific transcription factors and noncoding RNAs to coordinate networks controlling of ES cell identity remain unknown. Here, we find that Pontin chromatin remodeling factor plays an essential role as a coactivator for Oct4 target genes and large intergenic noncoding RNAs (lincRNAs) in ES cells. mRNA- and ChIP-sequencing analyses reveal that Pontin and Oct4 share a substantial set of target genes involved in maintenance of ES cells. Intriguingly, Oct4-dependent coactivator function of Pontin extends to transcription of lincRNAs that are mainly involved in repression of differentiation in ES cells. Together, our findings demonstrate newly identified Oct4-Pontin-lincRNA module plays critical roles in the ES cell circuitry to orchestrate cell fate determination program.
2015-04-08 | GSE58206 | GEO