Project description:Genome-wide positions of Z-DNA are mapped. ChIP was performed against transiently expressing Flag-Za or Flag-Zaa using Flag antibody in HeLa cells. Za and Zaa ChIP DNA and fragmented genomic DNA were used for sequencing library construction. Each library was sequenced on Illumina GAIIx and HiSeq 2500 and sequenced reads were mapped and normalized.
Project description:In order to assess Tet1 binding, we first generated a Flag tagged Tet1 ES cells and then knocked out Dnmt3a in the [WT, Tet1-Flag] cells. By Tet1 ChIP and Flag ChIP, we showed that Tet1 binding was complementary to Dnmt3a. And Tet1 binding was not affected or slightly increased at majority of its targets.
Project description:NIPP1, an established interactor of protein phosphatase 1 (PP1), is implicated in PRC2-mediated regulation of gene expression. Here, we explore whether PP1 associated with NIPP1 is involved in NIPP1-mediated regulation of genes. Therefore, we generated Hela Tet-off (HTO) cell lines that stably and inducibly express a Flag-tagged version of wild-type NIPP1 (HTO-NIPP1wt) or mutant NIPP1 (HTO_NIPP1m). The latter mutant lacks the major PP1 binding site by two point mutations in the RVXF-motif, an established PP1 - binding motif. All cell lines were derived from the same parental HeLa Tet-Off (HTO-PT) cell line which expresses only tTA transactivator and is used as a control. The Flag fusions were only expressed in the absence of doxycyline and at levels that were up to twofold higher than that of endogenous NIPP1. We performed a gene expression profiling of the HTO cell lines, using Whole Human Genome Oligo microarrays from Agilent. A Paired SAM analysis identified 1365 genes with an altered expression (P < 0.01) between the Flag-NIPP1 and parental cell lines. Importantly, only 185 genes were differentially expressed (P<0.01) between the Flag-NIPP1m and parental cell lines. Even more strikingly, only 5% of the genes that were affected by the expression of Flag-NIPP1 also showed a significantly different expression in the Flag-NIPP1m cells. A similar small overlap was noted when the analysis was restricted to the 50 genes that were most upregulated or downregulated by the expression of Flag-NIPP1. Finally, scatter plot analysis revealed no significant correlation between the genes that were affected by the expression of Flag-NIPP1 or Flag-NIPP1m. Collectively, these data demonstrate that a moderate increase in the concentration of NIPP1 affects the expression of numerous genes by a mechanism that depends on associated PP1.
Project description:CHD (chromodomain helicase DNA binding protein) family is composed of nine members of chromatin remodeling factors that regulate chromatin structure in an ATP-dependent manner. Among them, CHD4 contributes to many basic cellular functions during development mainly through multiple proteins interacting with CHD4 including NuRD (nucleosome remodeling and deacetylase activities) complex, which contains histone deacetylase HDAC1/2. However, functions of CHD4 that are not mediated by NuRD complexes have also been found, implying the existence of unknown proteins that are associated with CHD4. In the present study, we generated CHD4 FLAG-tag knock-in cells in HeLa-S3 and HEK293T cells and sought proteins bound to CHD4 with the use of immunoprecipitation and liquid chromatography and tandem MS (LC-MS/MS) analysis. We found that LCORL (ligand dependent nuclear receptor corepressor like) and NOL4L (nucleolar protein 4 like) were reproducibly identified as a novel CHD4 interactors. RNA-sequencing analysis of HEK293T cells depleted of CHD4, LCORL, and NOL4L revealed consistent upregulation of genes related to the Notch pathway. Our results thus suggest that both NOL4L and LCORL may cooperate with CHD4 to suppress the Notch pathway in mammalian cells.
Project description:This project aimed at assessing the influence of hypoxic stress on Estrogen receptor alpha genome wide binding to chromatin, in presence or absence of E2 ligand. I also aimed at assessing if a 1-month hypoxic stress followed by 1 month of normoxia imprints an epigenetic memory, therefore altering genome wide ER binding (rescue condition).
Project description:We generated CHD4 FLAG-tag knock-in cells in HeLa-S3 and HEK293T cells and sought proteins bound to CHD4 with the use of immunoprecipitation and liquid chromatography and tandem MS (LC-MS/MS) analysis.
Project description:FOXRED2 plays important role in ER stress induced by CD3δ. To figure out how FOXRED2 effect ER stress, we set up immunoprecipitation-mass spectrometry (IP-MS) in HepG2 cells overexpressed with Flag-tagged FOXRED2 to screen for potential FOXRED2-binding proteins under ER stress.
Project description:We identified the mRNA targets of the insulin-like growth factor-2 (IGF2) mRNA-binding proteins 1, 2, and 3 (IGF2BP1/2/3) by RNA immunoprecipitation and sequencing (RIP-seq). HEK293T cells transfected with Flag-tagged IGF2BP1/2/3 plasmids were expanded and UV-crosslinked before harvest. We performed RIP of individual IGF2BP using anti-Flag antibody from nuclear extractions, and identified the associated mRNAs by next generation sequencing. More than 5000 transcripts, including protein coding and non-coding transcripts, were identified from each RIP-seq sample.