Chromosome conformation capture on chip (4C) at the Fab7 polycomb response element in the bithorax complex of Drosophila
ABSTRACT: In Drosophila melanogaster, Hox genes are organized in an anterior and a posterior cluster, called Antennapedia complex and bithorax complex, located on the same chromosome arm and separated by 10 Mb of DNA. Both clusters are repressed by Polycomb group (PcG) proteins. Here, we show that genes of the two Hox complexes can interact within PcG bodies in the cell nucleus in tissues where they are corepressed. This colocalization increases during development and depends on PcG proteins. Hox gene contacts are part of a large gene interaction network that includes other PcG target genes. Moreover, they are conserved in the distantly related Drosophila virilis species, and mutations on one of the loci weaken silencing of genes in the other locus, resulting in the exacerbation of homeotic phenotypes in a sensitized genetic background. Thus, the three-dimensional organization of Polycomb target genes in the cell nucleus is an evolutionarily conserved phenomenon that stabilizes the maintenance of epigenetic gene silencing. Chromosome Conformation Capture on Chip (4C) was used to establish chromosomal interactions involving the Fab7 polycomb response element from the bithorax complex of Drosophila melanogaster. Larval brain and anterior discs from 3rd instar larvae were dissected to produce 2 replicate experiments with wild-type (WT-r1, WT-r2) and Fab-712 (F12-r1, F12-r2) fly lines. The 3C was performed as previously described (Hagege et al., 2007; Miele and Dekker, 2009) with the main differences being the use of DpnII (New England Biolabs), a 4 bp cutter restriction enzyme, and a fixation in 3% para-formaldehyde for 30 min, maximizing sensitivity and resolution of contact detection. The 4C DNA and DpnII digested genomic DNA (Control) samples were hybridized using a tiling array mapping the whole chromosome 3R which includes the antenapedia and bithorax hox clusters.
Project description:YBX1 is a multifunctional protein involved in the control of transcription and translation. We identified YBX1 as an target of MEK/ERK signaling in colorectal cancer cell lines. We performed a ChIP-chip analysis of HCT116 cells to identify new potential target genes of YBX1. Comparison of input DNA fragments with fragments coprecipitated with YBX1 in HCT116 cells.
Project description:The forkhead transcription factor FOXM1 is a key regulator of the cell cycle and is overexpressed in cancer. Increased levels of FOXM1 are associated with both poor prognosis and oestrogen receptor (ERalpha) status in primary breast cancer. In this study, we map FOXM1 binding genome wide in both ERalpha-positive (MCF-7) and -negative (MDA-MB-231) breast cancer cells. We identify a common set of FOXM1 binding events at cell cycle-regulating genes, but in addition, in MCF-7 cells we find a high level of concordance with ERalpha-binding regions. FOXM1 binding at these co-binding sites is dependent on ERalpha binding, as depletion of ER protein levels reduced FOXM1 binding. FOXM1 interacts directly with both ERalpha co-activator CARM1 and is required for H3 arginine methylation at the ERalpha complex. Inhibition of FOXM1 activity with the ligand thiostrepton resulted in decreased FOXM1 binding at cca. 1400 sites genome wide and reduced expression of genes correlated with poor prognosis in ERalpha-positive tumour samples. These data demonstrate a novel role for the forkhead protein FOXM1 as an ERalpha cofactor and provide insight into the role of FOXM1 in ERalpha-positive breast cancer. The FOXM1-binding sites were mapped by ChIP-Seq in MCF-7 and MDA-MB-231 cells. Cells were treated either with thiostrepton, a FOXM1 inhibitor, or with DMSO (as control). Four replicates were performed in MCF7 cells and two replicates in MDA-MB-231 cells.
Project description:This SuperSeries is composed of the following subset Series: GSE20529: Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-chip GSE20530: Promoter proximal pausing and its regulation by c-Myc in embryonic stem cells: ChIP-Seq Refer to individual Series
Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-chip data for Pol II and additional factors controlling pause release in mouse ES cells.
Project description:The somatic cell fate can be converted to tumor or pluripotent ones by ectopic expression of transcription factors in vitro and in vivo. Many oncogenic transcription factors are known to mediate both fates as they share similar proliferative and metabolic properties. Paradoxically, we found c-Jun as the first oncogene that appears to specify a somatic fate, oppose the pluripotent one and impede reprogramming. We performed a series of high through out sequencing to understand the way cJun works. To understand how c-Jun drives mESCs differentiating, we obtained c-Jun TetOn mESCs, and performed RNAseq 36h later with dox inducing or not . To understand why c-Jun blocks reprogramming while c-JunDN and Jdp2 can replace Oct4, we overexpressed these factors with KSM during reprogramming and performed RNAseq 3 Days after virus transfection. Moreover, to extend understand how these factors regulate gene expression, we also overexpressed these factors in MEF and performed RNAseq. Further more, to understand how cJun regulates cell fates and gene expression, we overexpressed c-Jun in mouse ESC and performed ChIP-seq. Also, we performed c-JunDN ChIP-seq during somatic cells reprogramming on day 3, to explore the binding sites of c-JunDN.
Project description:Recruitment of the RNA Polymerase II (Pol II) transcription initiation apparatus to promoters by specific DNA binding transcription factors is well recognized as a key regulatory step in gene expression. We describe here evidence that promoter-proximal pausing is a general feature of transcription by Pol II in embryonic stem (ES) cells, and thus an additional step where regulation of gene expression may occur. We report here that c-Myc, which occupies a third of actively transcribed genes in ES cells and is a key regulator of cellular proliferation, binds P-TEFb and contributes to release of promoter-proximal paused Pol II at these genes. ChIP-seq data for Pol II and additional factors controlling pause release in mouse ES cells.
Project description:Genomic information is encoded on a wide range of distance scales, ranging from tens of base pairs to megabases. We developed a multiscale framework to analyze and visualize the information content of genomic signals. Different types of signals, such as GC content or DNA methylation, are characterized by distinct patterns of signal enrichment or depletion across scales spanning several orders of magnitude. These patterns are associated with a variety of genomic annotations, including genes, nuclear lamina associated domains, and repeat elements. By integrating the information across all scales, as compared to using any single scale, we demonstrate improved prediction of gene expression from Polymerase II ChIP-seq measurements and we observed that gene expression differences in colorectal cancer are not most strongly related to gene body methylation, but rather to methylation patterns that extend beyond the single-gene scale. ChIP-seq data of six proteins in primary murine bone marrow macrophage cells (BMMs) under unstimulated and lipopolysaccharide (LPS) stimulated conditions. The BMMs were cultured from female C57BL/6 mice (age 8-12 weeks). Amongst these six proteins were three transcription factors (TFs), ATF340, NFκB/p50 and NFκB/p65, all of which are involved in regulating macrophage activation by microbial molecular components such as LPS. The other three ChIP-seq targets were RNA polymerase II (Pol II), and two chromatin modification marks: acetylation of histone H4 (H4ac) and tri-methylation of histone H3 lysine 27 (H3K27me3).