HOXC9-induced neuronal differentiation in human neuroblastoma BE(2)-C cells [ChIP-seq analysis]
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ABSTRACT: Cell differentiation is an essential process of normal development by which a stem cell or progenitor cell becomes a post-mitotic, specialized cell with unique morphology and function. Also, it has long been recognized that differentiation is associated with a marked reduction in DNA damage response at the global level. The molecular basis for the coordination between cell cycle exit, acquirement of specialized structure and function, and attenuation of DNA damage response during differentiation is not well understood. We have conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program in human neuroblastoma cells. Gene expression profiling reveals that HOXC9-induced differentiation is associated with transcriptional regulation of 2,395 genes, which is characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping demonstrates that HOXC9 occupies 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and DNA damage response. These findings suggest that HOXC9 directly activates and represses the transcription of distinct sets of genes to coordinate the cellular events characteristic of neuronal differentiation. Two independent preparations of BE(2)-C/Tet-Off/Myc-HOXC9 cells cultured in the absence of doxycycline for 6 days were used for chromatin immunoprecipitation (ChIP) against Myc-tagged HOXC9 and massively parallel sequencing by Illumina Genome Analyzer IIx.
Project description:Expression of the forkhead transcription factor FOXP1 is essential for early B cell development, whereas downregulation of FOXP1 at the germinal center (GC) stage is required for GC B cell function. Aberrantly high FOXP1 expression is frequently observed in diffuse large B cell lymphoma (DLBCL) and mucosa-associated lymphoid tissue (MALT) lymphoma, being associated with poor prognosis. Here, by gene expression microarray [GSE51382] and quantitative RT-PCR analysis upon ectopic overexpression of FOXP1 in primary human memory B cells (MBCs) and B-cell lines, combined with chromatin immunoprecipitation-sequencing (ChIP-seq), we established that FOXP1 directly represses expression of PRDM1, IRF4, and XBP1, transcriptional master regulators of plasma cell (PC) differentiation. In accordance, FOXP1 is prominently expressed in primary human naive and MBCs but expression strongly decreases during plasma PC differentiation. Moreover, as compared to IgM+ MBCs, IgG+ MBCs combine lower expression of FOXP1 with an enhanced intrinsic PC differentiation propensity, and constitutive (over)expression of FOXP1 in B cell lines and primary human MBCs represses their ability to differentiate into PCs. Taken together, our data indicate that proper control of FOXP1 expression plays a critical role in PC differentiation, whereas aberrant overexpression of FOXP1 might contribute to lymphomagenesis by blocking terminal B cell differentiation. OXP1 ChIP-seq profile in primary human memory B cells (MBCs) and B-cell lines
Project description:Cell differentiation is an essential process of normal development by which a stem cell or progenitor cell becomes a post-mitotic, specialized cell with unique morphology and function. Also, it has long been recognized that differentiation is associated with a marked reduction in DNA damage response at the global level. The molecular basis for the coordination between cell cycle exit, acquirement of specialized structure and function, and attenuation of DNA damage response during differentiation is not well understood. We have conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program in human neuroblastoma cells. Gene expression profiling reveals that HOXC9-induced differentiation is associated with transcriptional regulation of 2,395 genes, which is characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping demonstrates that HOXC9 occupies 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and DNA damage response. These findings suggest that HOXC9 directly activates and represses the transcription of distinct sets of genes to coordinate the cellular events characteristic of neuronal differentiation. Affymetrix microarray assays were performed according to the manufacturer's directions on total RNA isolated from three independent samples of BE(2)-C/Tet-Off/Myc-HOXC9 cells cultured in the presence or absence of doxycycline for 6 days.
Project description:The expression of v5-tagged Hoxc9 is induced and ChIP-seq is used to profile genome-wide occupancy in differentiating motor neurons The differentiation of ventral motor neurons is induced by treating embryonic stem cell cultures with retinoic acid and hedgehog signaling. Here, ChIP-seq is used to profile the genome-wide occupancy of Hoxc9 after five days of differentiation.
Project description:Cell differentiation is an essential process of normal development by which a stem cell or progenitor cell becomes a post-mitotic, specialized cell with unique morphology and function. Also, it has long been recognized that differentiation is associated with a marked reduction in DNA damage response at the global level. The molecular basis for the coordination between cell cycle exit, acquirement of specialized structure and function, and attenuation of DNA damage response during differentiation is not well understood. We have conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program in human neuroblastoma cells. Gene expression profiling reveals that HOXC9-induced differentiation is associated with transcriptional regulation of 2,395 genes, which is characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping demonstrates that HOXC9 occupies 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and DNA damage response. These findings suggest that HOXC9 directly activates and represses the transcription of distinct sets of genes to coordinate the cellular events characteristic of neuronal differentiation.
Project description:Cell differentiation is an essential process of normal development by which a stem cell or progenitor cell becomes a post-mitotic, specialized cell with unique morphology and function. Also, it has long been recognized that differentiation is associated with a marked reduction in DNA damage response at the global level. The molecular basis for the coordination between cell cycle exit, acquirement of specialized structure and function, and attenuation of DNA damage response during differentiation is not well understood. We have conducted a genome-wide analysis of the HOXC9-induced neuronal differentiation program in human neuroblastoma cells. Gene expression profiling reveals that HOXC9-induced differentiation is associated with transcriptional regulation of 2,395 genes, which is characterized by global upregulation of neuronal genes and downregulation of cell cycle and DNA repair genes. Remarkably, genome-wide mapping demonstrates that HOXC9 occupies 40% of these genes, including a large number of genes involved in neuronal differentiation, cell cycle progression and DNA damage response. These findings suggest that HOXC9 directly activates and represses the transcription of distinct sets of genes to coordinate the cellular events characteristic of neuronal differentiation.
Project description:FOXO transcription factors are key players in diverse cellular processes affecting tumorigenesis, stem cell maintenance and lifespan. To gain insight into mechanisms of FOXO regulated target gene expression, we studied genome-wide effects of FOXO3 activation. Profiling RNA polymerase II changes shows FOXO3 regulates gene expression through transcription initiation. Correlative analysis of FOXO3 and RNA polymerase II ChIP-seq profiles demonstrates FOXO3 to act as a transcriptional activator. Furthermore, this analysis reveals a significant part of FOXO3 gene regulation proceeds through enhancer regions. FOXO3 binds to pre-existing enhancers and further activates these enhancers as shown by changes in histone acetylation and RNA polymerase II recruitment. In addition, FOXO3-mediated enhancer activation correlates with regulation of adjacent genes and pre-existence of chromatin loops between FOXO3 bound enhancers and target genes. Combined, our data elucidate how FOXOs regulate gene transcription and provide insight into mechanisms by which FOXOs can induce different gene expression programs depending on chromatin architecture. Paper with published gene expression: PMID 22139133. Examination of FOXO3 binding and changes in RNAPII occupancy (0, 4, 24 hours after induction) in colorectal cell line
Project description:HIF1 is essential for regulation of the transcriptional response to hypoxia. Recently we showed that the transcriptional repressors E2F7 and E2F8 interact and transcriptionally cooperate with HIF1. Here we further explored this cooperation by performing genome-wide analysis, screening for novel HIF1-E2F7 targets. We show that specifically E2F7 is induced in hypoxia by HIF1. Furthermore, chip-sequencing for E2F7 and HIF1 revealed a large number of common targets of which a subset was also regulated by the complex as examined by microarray analysis. Our data show that the HIF1-E2F7 complex can function both as a repressor or activator. Notably, we identify neuropilin 1 (NRP1) as a novel HIF1-E2F7 target, which is repressed by HIF1-E2F7 in vitro and during zebrafish development, depending on E2F-binding sites present in the NRP1 promoter. In addition we show that regulation of NRP1 by the HIF1-E2F7 complex is required for normal axon guidance of spinal motorneurons in vivo. ChIP-seq analysis of HIF1a and E2F7 binding
Project description:CTCF and BORIS (CTCFL), two paralogous mammalian proteins sharing nearly identical DNA binding domains, are thought to function in mutually exclusive manners in DNA binding and transcriptional regulation. Here we show that these two proteins co-occupy a specific subset of regulatory elements consisting of clustered CTCF binding motifs (termed 2xCTSes). BORIS occupancy at 2xCTSes is largely invariant in BORIS-positive cancer cells, with the genomic pattern recapitulating the germline-specific BORIS binding to chromatin. In contrast to the single-motif CTCF target sites (1xCTSes), the 2xCTS elements are preferentially found at active promoters and enhancers, both in cancer and germ cells. 2xCTSes are also enriched in genomic regions that escape histone to protamine replacement in human and mouse sperm. Depletion of BORIS gene leads to altered transcription of a large number of genes and the differentiation of K562 cells, while the ectopic expression of this CTCF paralog leads to specific changes in transcription in MCF7 cells. In summary, we discover two functionally and structurally different classes of CTCF binding regions, 2xCTSes and 1xCTSes, revealed by their predisposition to bind BORIS. We propose that 2xCTSes play key roles in the transcriptional program of cancer and germ cells Genome-wide mapping of CTCF and BORIS occupancies in both germ and cancer cells. ChIP-seq and expression profiling by high throughput sequencing
Project description:In this study, we combined a genome-wide analysis of the Polycomb protein Bmi1 and an in vivo RNAi screening to identify critical targets whose repression in neural progenitor and Malignant Glioma cells enables normal and aberrant self-renewal. Bmi1 ChIP-sequencing generated in 2 primary adult mouse Neural Progenitor cell lines, 1 mouse astrocytic cell line, 1 mouse Glioma initiating cell line, and 1 mouse Glioma cell line. In human cell lines, Bmi1 ChIP-seq was performed for foetal Neural Progenitor Cells, and 2 Glioblastoma Stem-like cells. ChIP-seq for AP-1 in one Glioblastoma stem-like cell line. ChIP-sequencing were generated using either Illumina GxII or HiSeq. We have further performed RNA-seq of adult Ink4a/Arf -/-;BMI1or GFP dox-inducible shRNA mNPC with a doxycycline inducible shRNA targeting BMI1 or GFP (as control). These cells were either treated with doxyclyine for 48h to ablate BMI1 and GFP expression or further subjected to short-term differentiation using either BMP4 (10ng/ml or 50ng/ml for 3 or 6hrs), or FBS (1 or 10% for 3 or 6hrs). Additional mouse RNA-seq were performed in two biological replica of adult mouse brains (littermate) with either wild-type FVB background or BMI1 -/-. Finally, we performed RNA-seq in human Glioblastoma stem-like cells (most likely belonging to the mesenchymal GBM subtype) treated with BMP7, Arvanil and doxycycline or EtOH treated as ctrl.
Project description:We report the application of single-molecule-based sequencing technology for high-throughput profiling of SNF5 binding in human pluripotent embryonic carcinoma NCCIT and SNF5 overexpressed NCCIT cells. We generated genome-wide cSNF5 maps of NCCIT and SNF5 overexpressed NCCIT cells from chromatin immunoprecipitated DNA. SNF5 and OCT4 seem not to share their binding in OCT4 centered binding plot in control, while SNF5 overexpression directs SNF5 to OCT4 target genes. Examination of the relationship between SNF5 and OCT4 binding in control and SNF5 overexpressed cells.