Project description:Examination of EBf1 binding by ChIP-seq in differentiated human adipose stromal cell (hASC) pre-adipocyte Pre-Adipocytes differentiated in-vitro were fixed in 1% formaldehyde for 15 min at room temperature and quenched for 5 min by adding glycine to a final concentration of 0.125 M. ChIP assays were then performed with custom-made EBF1 antibody or rabbit IgG . ChIP-sequencing libraries were prepared using NEBnext chip-seq library Prep master mix set from 5 ng of anti-EBF1 and anti-IgG ChIP DNA, respectively. Sequence data were generated with Illumina HiSeq 2000 single-read sequencing and aligned against the human genome (hg19, NCBI).
Project description:To investgate the role of EBF1 in human adipocyte, we performed global expression profiling in human adipocytes transfected with siRNA targeting EBF1. Human adipocytes differentiated in vitro were transfected with siRNA targeting EBF1 or Non-targeting Control. The cells were incubated for approximately 48 hours upon which Total RNA were purified. Gene expression profiling was performed using Affymetrix Human Gene 1.1 ST arrays (Affymetrix, Inc., Santa Clara, CA). Data were analyzed with packages available from Bioconductor (www.bioconductor.org). Normalization and calculation of gene expression was performed with the Robust Multichip Average expression measure using oligo package. Prior to further analysis, a non-specific filter was applied to include genes with expression signal >30 in at least 20% of all samples. A total of three independent transfection experiments were performed for the pre-adipocytes obtained from one donor. Same experiment were repeated with cells from another donor.
Project description:Opaque2 (O2) is a transcription factor that plays important roles during maize endosperm development. Mutation of the O2 gene improves the nutritional value of maize seeds, but also confers pleiotropic effects that result in reduced agronomic quality. To reveal the transcriptional regulatory framework of O2, we determined O2 DNA binding targets using chromatin immunoprecipitation coupled to high-throughput sequencing (ChIP-Seq). ChIP-Seq analysis detected 1,686 O2 DNA binding sites distributed over 1,143 genes. We identified 4 new O2 binding motifs; among them, TGACGTGG appears to be the most conserved and strongest. We confirmed that, except for the 16 kD and 18 kD zeins, O2 directly regulates expression of all other zeins. O2 directly regulates two transcription factors, genes linked to carbon and amino acid metabolism and abiotic stress resistance. Examination of 15 days after pollination(DAP) wild type maize endosperm with O2 specific antibody and IgG serves as control.
Project description:The prostate cancer cell line PC-3 was transfected with pre-miR-375 or control. After 48 hours the cells were lysed and immunoprecipitation was performed using anti-panAgo (...) or isotype anti-IgG antibodies overnight. RNA of total lysates and immunoprecipitation fractions was extracted using miRNeasy kit (Qiagen) and libraries were generated using the Stranded Total RNA Sample Prep Kit (Takara Clontech). The experiment was performed in order to identify potential targets of miR-375 in prostate cancer.
Project description:We identified genome-wide sites of occupancy for the intestine-specific transcription factor ELT-2 in L3-staged N2 worm by performing ELT-2 ChIP-seq on whole worms; we performed RNA-seq on L3-staged N2 whole worms To identify DNA regions of direct ELT-2 occupancy, we performed the following ChIP-seq assays 1) ELT-2 ChIP-seq on L3 staged N2 C. elegans worms; 2) H3K4me3 ChIP-seq on matched L3 staged N2 worms; 3) Mock IgG-only ChIP-seq negative control on L3 stage N2 worms. Input DNA was also sequenced for each replicate. In addition, we performed RNA-seq on two replicates of L3 N2 C. elegans worms.
Project description:ChIP-seq data from mouse adipocyte. Mature 3T3-L1 adipocytes were cross-linked with 1% formaldehyde 10 days after induction with MDI. Frozen cell pellets were submitted to the Broad Institute for subsequent analysis of Ebf1-bound regions using anti-Ebf1 antibody (Abnova H00001879-M01).
Project description:We compared Sox9-association at chondrocyte targets to a broad catalogue of regulatory indicators of chromatin organization and transcriptional activity to determine Sox9’s direct regulatory actions in normal developing chondrocytes. Sox9-associated regions resolve into two distinct regulatory categories. Class I regions closely associate with transcriptional start sites (TSSs). Their targets reflect general regulators of basal cell activities that Sox9 engages indirectly though a likely association with the basal transcriptional complex. In contrast, Class II regions outside of the local TSS domains highlight evolutionarily conserved, active enhancers directing expression of chondrocyte specific target genes, though DNA binding of Sox9-dimers at target sites with sub-optimal binding affinity. The level of associated chondrocyte gene expression correlates with the number of enhancer modules around the target gene and grouping into super-enhancer clusters. Comparison of Sox9 programs between neural crest and mesoderm-derived chondrocytes points to similar modes of chondrocyte specification in distinct chondrocyte lineages. These data provide the first insight into mammalian Sox family actions at the genome scale in the vivo setting. The resulting enhancer sets provide a key resource for further dissection of the regulatory programs of mammalian chondrogenesis. Incorportation of ChIP-seq data of Sox9 and histone modification marks for chromatin status together with micorarray gene expression profiling in neonatal mice chondrocytes to uncover Sox9 regulatory system. Overexpression of Sox9 with a control of EGFP in human fibroblasts to identify the direct targets of Sox9 regulatory system
Project description:Early B cell factor-1 (Ebf1), a transcription factor expressed in B cells, adipocytes and neuronal cells, has been identified as a determinant in the specification of the B cell lineage in which Ebf1 acts in a regulatory network with the transcription factors E2A and Pax5. To gain insight into the molecular basis of Ebf1 function in early stage B cells, we compared the data set of a genome-wide ChIP sequencing analysis with gain- and loss-of-function transcriptome analyses. Identification of genetic loci enriched by chromatin-immunoprecipitation using antibodies against EBF1.
Project description:Background: Although genetic or epigenetic alterations have shown to affect the three-dimensional organization of genomes, the utility of chromatin conformation in the classification of human disease has never been addressed. Results: Here, we explore whether chromatin conformation can be used to classify human leukemia. We map the conformation of the HOXA gene cluster in a panel of cell lines with 5C chromosome conformation capture technology, and use the data to train and test a support vector machine classifier named 3D-SP. We show that 3D-SP is able to accurately distinguish leukemias expressing MLL-fusion proteins from those expressing only wild-type MLL, and that it can also classify leukemia subtypes according to MLL fusion partner, based solely on 5C data. Conclusions: Our study provides the first proof-of-principle demonstration that chromatin conformation contains the information value necessary for classification of leukemia subtypes. Examination of CTCF and RAD21 binding sites in THP-1 cell.