ChIP-DSL H20K promoter array from human gastric cancer cells MKN28 with Sox2 overexpression
ABSTRACT: The transcription factor Sox2 inhibits human gastric cancer growth and activates Sox2-related tumor surpressive genes in human gastric cancer cells. Conditional Sox2-overexpression in cells with a low Sox2 level demonstrated that the Sox2-regulated tumor surpressive genes demand on an enhanced Sox2 activity for better expression to work in human gastric cancer. Chromatin immunoprecipitation (ChIP) of Sox2 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Sox2 directly activates the chromatin at promoters or putative enhancers of Sox2 target genes. Transcription factor Sox2 promoter array in MKN28 cells with Sox2 overexpression.
Project description:The Lhx2 transcription factor plays essential roles in morphogenesis and patterning of ectodermal derivatives, as well as in controlling stem cell activity. Lhx2 is expressed in the hair follicle (HF) buds, while in postnatal telogen HFs Lhx2+ cells reside in the stem cell-enriched epithelial compartments (bulge, secondary hair germ) and co-express selected stem cell markers (Sox9, Tcf4 and Lgr5). Lhx2+ cells represent the vast majority of cells in the bulge and secondary hair germ that proliferate in response to skin injury. This is functionally important, since the wound re-epithelialization is significantly retarded in heterozygous Lhx2 knockout (+/-) mice, while anagen onset in the HFs located closely to the wound is accelerated compared to wild-type mice. Cell proliferation in the bulge and the number of Sox9+ and Tcf4+ cells in the HFs closely adjacent to the wound in Lhx2+/- mice are decreased in comparison to wild-type controls, while expression of Lgr5 and cell proliferation in the secondary hair germ are increased. Furthermore, acceleration of wound-induced anagen development in Lhx2+/- mice is inhibited by administration of Lgr5 siRNA. In addition, Chip-on-chip/ChIP-qPCR and reporter assay analyses reveal Sox9, Tcf4 and Lgr5 as direct Lhx2 targets in keratinocytes. These data strongly suggest that Lhx2 positively regulates Sox9 and Tcf4 in the bulge cells and promotes wound re-epithelization, while it simultaneously negatively regulates Lgr5 in the secondary hair germ and inhibits HF cycling. Thus, Lhx2 operates as a regulator of epithelial stem cell activity during skin response to injury. Chromatin form primary mouse keratinocytes (PMK) was subjected to ChIP analysis with Lhx2 antibody; input and ChIP DNA were labelled with Cy3 and Cy5 respectivly and used form Nimblegen MM8 Mouse Promoter Array
Project description:Hematopoietic Stem Cells (HSC) are originated during embryonic development from endothelial-like cells located in the ventral side of the dorsal aorta around day E10-12 of murine development. This region is called AGM for Aorta/Gonad/Mesonephros refering to the tissues around the hemogenic aorta. Hematopoiesis depends on the Notch pathway and the identification of Notch-targets is important for the understanding of blood origin. Hematopoietic Stem Cells (HSCs) specification occurs in the embryonic aorta and requires Notch activation, however which are the elements regulated by Notch that control this process are mainly unknown. Here, we took a genome-wide approach to identify putative direct Notch targets by precipitating the chromatin that binds to the Notch partner RBPj in the Aorta-Gonad-Mesonephros (AGM) tissue from E11.5 mouse embryos. This assay revealed 701 gene promoter regions as candidates to be regulated by Notch in the AGM. Chromatin was obtained from a pool of 40 dissected AGMs at E11.5. Chromatin immunoprecipitation (ChIP) was performed as previously described (Aguilera et al, PNAS 2004) with minor modifications. In brief, cross-linked chromatin was sonicated for 10 minutes, medium-power, 0.5-interval; with a Bioruptor (Diagenode) and precipitated with anti-RBPJ (Chu and Bresnick, 2004). After crosslinkage reversal, DNA was used as a template for PCR or for array hybridization. Mouse promoter chip on chip microarray SET (Agilent) was used to identify RBPj targets. It covers 70,000 best identified gene regions with a-5.5 kb to + 2.5 kb range, and has on average 25 probes per gene with an average probe to probe distance of 200 bp. The ChIP-on-chip was performed with dye swaps and one IgG control was brought along. Enrichment analysis was done by comparing the precipitation normalized dye swap signal with input control signal.
Project description:This SuperSeries is composed of the following subset Series: GSE41026: Expression analysis of HepG2, HepG2-slug and HepG2-slug on Matrigel GSE41027: Chip-chip from HepG2 cells and HepG2 cells with slug overexpression Refer to individual Series
Project description:The transcription factor slug represses genes with E-box and then activates cancer stem cell related pathway: Wnt, Notch and Hedgehog pathway. Chromatin immunoprecipitation (ChIP) of slug by ChIP-on-chip analysis demonstrated that slug indirectly activates cancer stem cell related pathway and thus promotes vasculogenic mimicry formation. Comparison of HepG2-control in regular culture, HepG2-slug in regular culture and HepG2-slug on Matrigel.
Project description:ERG is a transcription factor that is involved in leukomogenesis and its mRNA overexpression has been associated with poor prognosis in a subset of patients with T-cell acute lymphoblastic leukemia (T-ALL) and acute myeloid leukemia (AML). Herein, a genome-wide screen of ERG target genes was conducted by chromatin immunoprecipitation-on-chip (ChIP-chip) in Jurkat cells. 342 significant annotated regions were derived from ChIP-chip experiments. Seventeen candidate promoter regions resulted in at least two-fold enrichment by quantitative PCR. Notably, ERG potential targets included WNT signaling genes: WNT2, WNT9A, WNT11, CCND1, and FZD7. Functionally, expression of WNT11 was downregulated with siRNA ERG knockdown and substantially upregulated in a tet-on ERG-inducible assay in K562 cells. To investigate a role for ERG in WNT signaling, a WNT agonist was used to inhibit glycogen synthase kinase (GSK-3). This treatment resulted in an ERG-dependent proliferative growth advantage in the tet-on ERG-inducible system. Lastly, chromatin immunoprecipitation assays of primary leukemia blasts confirmed WNT11 promoter enrichment dependent on ERG mRNA expression. In conclusion, ERG transcriptional networks in leukemia are revealed. Specifically, WNT11 emerged as a target of ERG. We propose that overexpression of ERG in acute leukemia may lead to a proliferative advantage upon activation of WNT signals. ChIP-chip with ERG antibody C20 and combined C17/20 and nonspecificic IgG in Jurkat
Project description:In humans, there are four Ago proteins (Ago1–4) and AGO1- and 2 were previously implicated in TGS induced by exogenous siRNAs and microRNAs (miRs) directed against gene promoter transcripts via promotion of changes in histone covalent modifications and DNA methylation. Not-with-standing, many mechanistic details of this process remain poorly defined in human cells, and very little is known about the identity of possible endogenous signals, which may drive this process in human cells. Given the evolutionary conserved role of siRNAs and AGO proteins in TGS and heterochromatin formation, we set out to analyse their possible involvement in senesence-associated repression of E2F target genes. To determine, in an unbiased manner, which genes might be under the control of AGO proteins we perfomed genome-wide promoter profiling in senescent and presenescent control WI38 primary fibroblasts applying “ChIP-on-chip” technology using 4 an anti-pan-AGO antibody. DNA from matched genomic inputs and chromatin immunoprecipitation (ChIP) samples were purified, amplified by PCR, and labeled either with Cy3 or Cy5 fluorophores. The labeled DNA from input and ChIP fractions were then combined and hybridised to human promoter arrays containing ~59,000 promoters. determination of AGO binding sites in WI-38 pre-senescent human fibroblast and in senescent human fibroblast
Project description:The chromatin remodeller p400 has oncogenic properties that promote early steps of colon cancer. Chromatin immunoprecipitation (ChIP) of p400 together with chromatin profiling by ChIP-on-chip analysis demonstrated that p400 directly binds the chromatin at promoters of p400 target genes. Study of p400 binding on promoters of HCT116 cells
Project description:Pax5 is a critical regulator of B cell commitment. Here we identified direct Pax5 target genes by streptavidin-mediated ChIP-chip analysis of pro-B cells expressing in vivo biotinylated Pax5. By binding to promoters and enhancers, Pax5 directly regulates the expression of multiple transcription factor, cell surface receptor and signal transducer genes. One of the newly identified enhancers was shown by transgenic analysis to confer Pax5-dependent B-cell-specific activity to the Nedd9 gene controlling B cell trafficking. Profiling of histone modifications in Pax5-deficient and committed wild-type pro-B cells demonstrated that Pax5 induces active chromatin at activated target genes, while eliminating active chromatin at repressed genes in committed pro-B cells. Pax5 rapidly induces these chromatin and transcription changes by recruiting chromatin-remodeling, histone-modifying and basal transcription factor complexes to its target genes. These data provide novel insight into the regulatory network and epigenetic regulation, by which Pax5 controls B cell commitment. Analysis of chromatin and TF binding in rag2-/- and pax5-/- rag2-/- pro-B cells. Chip-Chip with 1-3 experiments for each antibody and celltype combination.
Project description:The transcription factor Pax5 represses B-lineage-inappropriate genes and activates B-cell-specific genes in B-lymphocytes. Here we have identified 170 novel Pax5-activated genes. Conditional mutagenesis demonstrated that the Pax5-regulated genes require continuous Pax5 activity for normal expression in pro-B and mature B cells. Expression of half of the Pax5-activated genes is either absent or significantly reduced upon Pax5 loss in plasma cells. Direct Pax5 target genes were identified based on their protein synthesis-independent activation by a Pax5-estrogen receptor fusion protein. Chromatin immunoprecipitation (ChIP) of Pax5 together with chromatin profiling by ChIP-on-chip analysis demonstrated that Pax5 directly activates the chromatin at promoters or putative enhancers of Pax5 target genes. The novel Pax5-activated genes code for key regulatory and structural proteins involved in B cell signaling, adhesion, migration, antigen presentation and germinal center B cell formation, thus revealing a complex regulatory network, which is activated by Pax5 to control B cell development and function. Keywords: Chip-chip, cell type comparison comparison of Pax5-/-Rag2-/- vs Rag2-/- pro-B cells