ABSTRACT: This SuperSeries is composed of the following subset Series: GSE28141: Genome-wide analysis of REST knockdown responsive gene expression in mouse ES cells GSE28233: Genome-wide maps of REST and its cofactors in mouse E14 cells Refer to individual Series
Project description:We report the application of single-molecule-based sequencing technology for REST and its cofactors genome wide binding sites in E14 cells.We then combine these binding sirtes with REST regulating gene profiling, to understand REST binding and regulation in E14 cells. Examination of REST and 5 cofactors(RCOR1, RCOR2,RCOR3,SIN3A,SIN3B) in E14 cells, REST and SIN3A endogenous antibody were used for ChIP experiment. The stable E14 cells expressing low level exogenous RCOR1, RCOR2, RCOR3,and SIN3B with V5 tag were used for ChIP experiment with V5 antibody to obtain individual ChIP DNA.
Project description:After characterizing super-enhancer-associated chromatin dynamics accompanied by malignant progression of skin stem cells, we show that ETS family members auto-regulate themselves and a cohort of cancer-associated super-enhancer transcription factors which together are essential for tumor maintenance. Mouse skin squamouse cell carcinoma (SCC) tumor-initiating stem cells (SCs) were FACS-purified for ChIP-sequencing.
Project description:Estrogen Receptor (ER) is a hormonal transcription factor that plays important roles in breast cancer. It functions primarily through binding to the regulatory regions of target genes containing the consensus ERE motifs. In order to identify ER target genes and re-define the ERE motifs we performed ChIP-Seq analysis of ER in MCF7 breast cancer cell line. Applying a novel computational algorithm named Hybrid Motif Sampler (HMS), specifically designed for TFBS motif discovery in ChIP-Seq data, we were able to detect an improved ERE motif and reveal intra-motif dependency especially in neighboring base pairs. MCF7 cells were grown in starving medium (RPMI with 5% FCS) for 3 days prior to the treatment with 10 nM β-estradiol or vehicle control for 45 minutes. ChIP was done using an anti-ER antibody in both the ethl-treated and the E2-treated cells. ChIP-Seq sample prep and sequencing were done following the manufacture's protocol using the Genome Analyzer (Illumina). The read files were analyzed using ethl-treated as control for E2-treated, leading to one final peak file.
Project description:We immunoprecipitated formaldehyde crosslinked chromatin from DNMT triple knockout cells using an antibody against methylated lysine 27 on histone H3. Subsequent high-throughput sequencing allowed us to study the genome-wide distribution of this histone mark in the absence of DNA methylation. Examination of a histone modification in mESCs lacking DNA methylation.
Project description:The progression from stem cell to differentiated neuron is associated with extensive chromatin remodeling that controls gene expression, but the mechanisms that connect chromatin to gene expression are not well defined. Here we show that mutation of ZNF335 causes severe human microcephaly ("small brain"), small somatic size, and neonatal death. Germline Znf335 null mutations are embryonically lethal in mice, whereas RNA-interference studies and postmortem human studies show that Znf335 is essential for neural progenitor self-renewal, neurogenesis, and neuronal differentiation. Znf335 is a component of a vertebrate-specific, trithorax H3K4 methylation complex, while global ChIP-seq and mRNA expression studies show that Znf335 is a previously unsuspected, direct regulator of REST/NRSF, a master regulator of neural gene expression and neural cell fate, as well as other essential neural-specific genes. Our results reveal ZNF335 as an essential link between H3K4 complexes and REST/NRSF, and provide the first direct evidence that this pathway regulates human neurogenesis and neuronal differentiation. Examination of Znf335-bound genes with two separate antisera
Project description:A current model for the genomic recruitment of Kap1 is via its interaction with KRAB domain-containing zinc finger transcription factors. We have performed ChIP-seq for various mutant KAP1 proteins and shown that this recruitment mechanism mediates binding of KAP1 only to the 3M-CM-"M-BM-^@M-BM-^Y ends of zinc finger genes and that other factors are involved in recruiting KAP1 to promoter regions. 17 total ChIP-seq datasets; three different FLAG-KAP1 mutants, one FLAG-KAP1 wild type, and four different Input datasets from 4 different stable cell lines derived from HEK293 cells: 1 FLAG-KAP1 wild type dataset and 1 Input dataset done from HEK293 stable cells; 1 FLAG-KAP1 HP1BDmut dataset and 1 Input dataset done from HEK293 stable cells, 1 FLAG-KAP1 N-ter RBCC{delta}mut dataset and 1 Input dataset done from HEK293 stable cells, 1 FLAG-KAP1 C-ter PB{delta}mut dataset and 1 Input dataset done from HEK293 stable cells. One FLAG-KAP1 N/C-ter (RBCC+PB){delta}mut dataset done from T-REx HEK293 stable cells. One endogenous KAP1 dataset done from HEK293 cells. Two independent ELK4 datasets done from duplicate HEK293 cells. One endogenous Kap1 dataset and one Input dataset from a stable cell line derived from U2OS cells.
Project description:The orphan nuclear receptor TR4 (human testicular receptor 4 or NR2C2) plays a pivotal role in a variety of biological and metabolic processes. With no known ligand and few known target genes, the mode of TR4 function was unclear. We report the first genome-wide identification and characterization of TR4 in vivo binding. Using chromatin immunoprecipitation followed by high throughput sequencing (ChIP-seq), we identified TR4 binding sites in 4 different human cell types and found that the majority of target genes were shared among different cells. TR4 target genes are involved in fundamental biological processes such as RNA metabolism and protein translation. In addition, we found that a subset of TR4 target genes exerts cell-type specific functions. Analysis of the TR4 binding sites revealed that less than 30% of the peaks from any of the cell types contained the DR1 motif previously derived from in vitro studies, suggesting that TR4 may be recruited to the genome via interaction with other proteins. A bioinformatics analysis of the TR4 binding sites predicted a cis regulatory module involving TR4 and ETS transcription factors. To test this prediction, we performed ChIP-seq for the ETS factor ELK4 and found that 30% of TR4 binding sites were also bound by ELK4. Motif analysis of the sites bound by both factors revealed a lack of the DR1 element, suggesting that TR4 binding at a subset of sites is facilitated through the ETS transcription factor ELK4. Further studies will be required to investigate the functional interdependence of these two factors. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf 8 total ChIP-seq datasets; four TR4 and datasets done in duplicate from 4 different cell lines; ELK4 (Sap1a) duplicate dataset done from HeLa cells; 1 ELK1 replicate in HeLa cells, 2 individual replicates for histone mod datasets from K562 cells
Project description:Only a small percentage of human transcription factors (e.g. those associated with a specific differentiation program) are expressed in a given cell type. Thus, cell fate is mainly determined by cell type-specific silencing of transcription factors that drive different cellular lineages. Several histone modifications have been associated with gene silencing, including H3K27me3 and H3K9me3. We have previously shown that the two largest classes of mammalian transcription factors are marked by distinct histone modifications; homeobox genes are marked by H3K27me3 and zinc finger genes are marked by H3K9me3. Several histone methyltransferases (e.g. G9a and SETDB1) may be involved in mediating the H3K9me3 silencing mark. We have used ChIP-chip (GSE24480) and ChIP-seq to demonstrate that SETDB1, but not G9a, is associated with regions of the genome enriched for H3K9me3. A current model is that SETDB1 is recruited to specific genomic locations via interaction with the corepressor TRIM28 (KAP1), which is in turn recruited to the genome via interaction with zinc finger transcription factors that contain a Kruppel-associated box (KRAB) domain. However, specific KRAB-ZNFs that recruit TRIM28 (KAP1) and SETDB1 to the genome have not been identified. We now show that ZNF274 (a KRAB-ZNF that contains 5 C2H2 zinc finger domains), can interact with KAP1 in vitro and, using ChIP-seq, we show that ZNF274 binding sites co-localize with SETDB1, KAP1, and H3K9me3 at the 3’ ends of zinc finger genes. Knockdown of ZNF274 with siRNAs reduced the levels of KAP1 and SETDB1 recruitment to the binding sites. These studies provide the first identification of a KRAB domain-containing ZNFs that is involved in recruitment of the KAP1 and SETDB1 to the human genome. For data usage terms and conditions, please refer to http://www.genome.gov/27528022 and http://www.genome.gov/Pages/Research/ENCODE/ENCODEDataReleasePolicyFinal2008.pdf 7 total ChIP-seq datasets; 4 ZNF274 datasets done in duplicate from 4 different cell lines; 1 KAP1 duplicate dataset done in duplicate from K562 cells; 1 SetDB1 duplicate dataset from K562 cells; 1 H3K9me3 duplicate dataset from K562 cells
Project description:Hepatocyte nuclear factor-4α (HNF4α, NR2A1) is a nuclear receptor which has a critical role in hepatocyte differentiation and the maintenance of homeostasis in the adult liver. However, a detailed understanding of native HNF4α in the steady state remains to be elucidated. Here we report the native HNF4α isoforms, phosphorylation status and complexes in the steady state, as shown by shotgun proteomics in HepG2 hepatocarcinoma cells. Shotgun proteomic analysis revealed the complexity of native HNF4α, including multiple phosphorylation sites and inter-isoform heterodimerization. The associating complexes identified by label-free semi-quantitative proteomic analysis include the following: the DNA-dependent protein kinase catalytic subunit, histone acetyltransferase complexes, mRNA splicing complex, other nuclear receptor coactivator complexes, the chromatin remodeling complex, and the nucleosome remodeling and histone deacetylation complex. Among the associating proteins, GRB10 interacting GYF protein 2 (GIGYF2, PERQ2) is a new candidate cofactor in metabolic regulation. Moreover, an unexpected heterodimerization of HNF4α and Hepatocyte nuclear factor-4γ was found. A biochemical and genome-wide analysis of transcriptional regulation showed that this heterodimerization activates gene transcription. The genes thus transcribed include the cell death-inducing DEF45-like effector b (CIDEB) gene, which is an important regulator of lipid metabolism in the liver. This suggests that the analysis of the distinctive stoichiometric balance of native HNF4α and its cofactor complexes described here is important for an accurate understanding of transcriptional regulation. Examination of HNF4alpha binding sites with domain-specific antibodies and HNF4gamma binding sites in HepG2 cell.
Project description:Efficient in vitro generation of hematopoietic stem cells (HSCs) from embryonic stem cells (ESCs) holds great promise for cell-based therapies of hematological diseases. To date, HoxB4 remains to be the most effective transcription factor (TF) whose over-expression in ESCs confers long-term repopulating ability to ESC-derived HSCs. Despite its importance, the components and dynamics of the HoxB4 transcriptional regulatory network is poorly understood, hindering efforts to develop a more efficient protocol for in vitro derivation of HSCs. Towards this goal, we performed global gene expression profiling and chromatin immunoprecipitation coupled with deep sequencing (ChIP-Seq) at four stages of the HoxB4-mediated HSC development. Joint analyses of ChIP-Seq and gene expression profiles unveil a number of global features of the HoxB4 regulatory network. Cells from three time points of the developmental processes were collected, including days 6, 16, and 26. Deep sequencing were done for both IP and input DNA from cells from each time point.