Changes to the PML body proteome in U2OS cells after 2h treatment with 1uM arsenic
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ABSTRACT: To monitor changes in PML body compinents during exposure to 1uM arsenic for 2h in U2OS cells lacking endogenous PML but expressing YFP-PML (V).
Project description:A proteomic analysis of PML bodies purified from U2OS cells expressing YFP-PMLV either treated or not with 1uM arsenic for 2 hours, in comparison with parallel purifications from wt-U2OS cells treated identically.
Project description:The distribution of histone variants H2Abbd and macroH2A in 13 regions of the HG18 assembly have been studied using a variant of the ChIP-on-Chip technique. HeLa S3 cell lines expressing tagged histones H2A, H2Abbd or macroHA were obtained using retroviral transfer. DNA fractions associated with tagged histones were isolated using a two-step purification procedure that involved affinity chromatography on a column with anti-FLAG antibodies, followed by affinity chromatography on a Ni-agarose column. The obtained genomic DNA samples were analyzed by hybridization with custom NimbleGene genomic microarrays. Two samples. Test sample 1 is HeLa S3 cells expressing epitope-tagged histone H2Abbd and test sample 2 is HeLa S3 cells expressing epitope-tagged histone macroH2A . The control for both test sample 1 and test sample 2 is HeLa S3 expressing epitope-tagged histone H2A. Two copies of each probe per array were made.
Project description:In vertebrates, the catalytic core of PRC1 comprises RING1A/B and one of six homologues of the Polycomb group RING finger (PCGF) protein. Unique characteristics of PCGF1-6 in turn define distinct subunit assemblies, broadly subdivided into canonical and non-canonical PRC1 complexes. This RNA-seq experiment aimed to test the hypothesis that Pcgf3/5 gene knockout abrogates Xist mediated gene repression. In this experiment, Pcgf3fl/flPcgf5-/- and Pcgf3-/-Pcgf5-/- mESCs bearing Xist transgene on chromosome 16 (clone C3F8) were cultured in differentiating conditions: plated on non-gelatinized TC dish in ES medium without LIF for 72 hours with (+dox) and without doxycycline. RNA was isolated from 4 biological replicates. In analysed cells chromosome 16 bears inducible Xist and the whole study investigates role of PCGF3 and PCGF5 in Xist-dependednt gene silencing, therefore both bam files containing alignments and counts per gene were uploaded to ArrayExpress only for chromosome 16. Genome-wide data can be provided upon request.
Project description:While cellular metabolism impacts the DNA damage response, a systematic understanding of the metabolic requirements that are crucial for DNA damage repair has yet to be achieved. Here, we investigate the metabolic enzymes and processes that are essential when cells are exposed to DNA damage. By integrating functional genomics with chromatin proteomics and metabolomics, we provide a detailed description of the interplay between cellular metabolism and the DNA damage response. Subsequent analysis identified Peroxiredoxin 1, PRDX1, as fundamental for DNA damage repair. During the DNA damage response, PRDX1 translocates to the nucleus where it is required to reduce DNA damage-induced nuclear reactive oxygen species levels. Moreover, PRDX1 controls aspartate availability, which is required for the DNA damage repair-induced upregulation of de novo nucleotide synthesis. Loss of PRDX1 leads to an impairment in the clearance of γΗ2ΑΧ nuclear foci, accumulation of replicative stress and cell proliferation defects, thus revealing a crucial role for PRDX1 as a DNA damage surveillance factor.
Project description:Xist represents a paradigm for long non-coding RNA function in epigenetic regulation, although how it mediates X-chromosome inactivation (XCI) remains largely unexplained. Multiple Xist-RNA binding proteins have recently been identified, including SPEN/SHARP, whose knockdown has been associated with deficient XCI at multiple loci. Here we demonstrate that SPEN is a key orchestrator of XCI in vivo and unravel its mechanism of action. We show that SPEN is essential for initiating gene silencing on the X chromosome in preimplantation mouse embryos and embryonic stem cells. On the other hand, SPEN is dispensable for maintenance of XCI in neural progenitor cells, although it significantly dampens expression of genes that escape from XCI. During initiation of XCI, we show by live-cell imaging and CUT&RUN approaches that SPEN is immediately recruited to the X chromosome upon Xist up-regulation, where it is targeted to enhancers and promoters of actively transcribed genes. SPEN rapidly disengages from chromatin once silencing is accomplished, implying a need for active transcription to tether it to chromatin. We define SPEN’s SPOC (SPEN paralog and ortholog C-terminal) domain as a major effector of SPEN’s gene silencing function, and show that artificial tethering of SPOC to Xist RNA is sufficient to mediate X-linked gene silencing. We identify SPOC’s protein partners which include NCOR/SMRT, the m6A RNA methylation machinery, the NuRD complex, RNA polymerase II and factors involved in regulation of transcription initiation and elongation. We propose that SPEN acts as a molecular integrator for initiation of XCI, bridging Xist RNA with the transcription machinery as well as nucleosome remodelers and histone deacetylases, at active enhancers and promoters.
Project description:Syncytial skeletal muscle cells contain hundreds of nuclei in a shared cytoplasm. Using single nucleus RNA-sequencing (snRNAseq) of isolated nuclei from muscle fibers, we investigated nuclear heterogeneity and transcriptional dynamics in uninjured and regenerating muscle.
Project description:RNAPII pausing/termination shortly after initiation is a hallmark of gene regulation. However, the molecular mechanisms involved are still to be uncovered. Here, we show that NELF interacts with Integrator complex subunits (INTScom) forming a stable complex with RNPII and Spt5. The interaction between NELF and INTScom subunits is RNA and DNA independent. Using both HIV-1 promoter and genome wide analyses, we demonstrate that Integrator subunits specifically control NELF-mediated RNAPII pause/release at coding genes. The strength of RNAPII pausing is determined by the nature of the NELF-associated complex. Interestingly, in addition to controlling RNAPII pause release INTS11 catalytic subunit of the INTScom is required for the synthesis of full length mRNA. Finally, INTScom-target genes are enriched in HIV-1 TAR/ NELF-binding element and in a 3’box sequence required for snRNA biogenesis. Revealing these unexpected functions of INTScom in regulating RNAPII pausing/release and completion of mRNA synthesis of NELF-target genes will contribute to our understanding of the gene expression cycle. Binding profiles of Integrator subunits in HeLa cells by ChIP-Seq (Illumina) Please note that the MACS14*.tar.gz contains MACS output bed and xls files and the 'readme.txt' contains a detailed description of each file.
Project description:The transcription factor NF-κB is considered the master regulator of the immune response but also acts broadly to regulate gene expression that influences cell survival, proliferation and differentiation. Post-translational modification of NF-κB, phosphorylation in particular, is essential for the transactivation activity of NF-κB. Emerging evidence suggests that the regulation of NF-κB in the nucleus is critical in controlling gene expression. Promyelocytic Leukemia (PML) is a nuclear protein that forms nuclear bodies (PML NBs), sub-nuclear structures that are associated with transcriptionally active genomic regions that have been implicated in multiple processes such as apoptosis, senescence and anti-viral responses. Chromosomal translocations leading to the expression of a PML-retinoic acid receptor-α (PML-RARα) fusion protein are causative for acute promyelocytic leukemia (APL) characterised by a differentiation block at the promyelocytic state of myeloid development. Here we demonstrate that PML is required for phosphorylation of NF-κB p65 and that PML is essential for NF-κB- induced transcriptional responses. Our analysis of available transcriptional profiles of all-trans retinoic acid treated acute promyelocytic leukemia (APL) cells identifies a NF-κB transcriptional programme suppressed by PML-RARα. We further demonstrate that PML-RARα inhibits NF-κB phosphorylation and transcriptional activity. Our findings demonstrate a critical role for PML in promoting NF-κB transcriptional activity which may contribute to APL initiation and maintenance. WT and PML-/- MEFs were analysed for gene expression analysis. Total of 12 samples, inlcluding triplicates were utilized. WT MEFs and PML-/- were stimulated with TNFα for three hours and analysed for gene expresison using unstimulated WT MEFs as control.
Project description:Study the impact of the most common mutation on the PML protein found in acute promyeocytic leukemia patients resistant to arsenic therapy, which induces a disorganization of PML nuclear bodies, on gene expression.
Project description:Study the impact of the most common mutation on the PML protein found in acute promyeocytic leukemia patients resistant to arsenic therapy, which induces a disorganization of PML nuclear bodies, on gene expression.