ChIP-seq of E4F1 in SUM159 triple negative breast cancer cell line
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ABSTRACT: E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to decipher E4F1 cellular target genes, we performed chromatin immunoprecipitation of endogenous E4F1 in SUM159 triple negative breast cancer cell line. Both input and immunoprecipitated DNA of two independant experiments were exhaustively sequenced and mapped on the human genome (hg38). Peak detection has been achieved with the HOMER suite algorithms.
Project description:E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to decipher E4F1 cellular target genes, we performed chromatin immunoprecipitation of endogenous E4F1 in primary and in p53KO, Ha-RasV12-transformed MEFs. Both input and immunoprecipitated DNA were exhaustively sequenced and mapped on the mouse genome (mm9). Peak detection has been achieved by combining two peak calling algorithms. Intersection of the two E4F1 peak lists on each cell line were considered as E4F1 chromatin bound regions. Genome-wide mapping of E4F1 binding in mouse embryonic fibroblasts.
Project description:E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to decipher E4F1 cellular target genes, we performed chromatin immunoprecipitation of endogenous E4F1 in mouse embryonic stem cells. Both input and immunoprecipitated DNA were exhaustively sequenced and mapped on the mouse genome (mm9). Peak detection has been achieved by combining two peak calling algorithms. Intersection of the two E4F1 peak lists on each cell line were considered as E4F1 chromatin bound regions.
Project description:E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to decipher E4F1 cellular target genes, we performed chromatin immunoprecipitation of endogenous E4F1 in primary and in p53KO, Ha-RasV12-transformed MEFs. Both input and immunoprecipitated DNA were exhaustively sequenced and mapped on the mouse genome (mm9). Peak detection has been achieved by combining two peak calling algorithms. Intersection of the two E4F1 peak lists on each cell line were considered as E4F1 chromatin bound regions.
Project description:E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In this study we investigated the impact of direct translational target genes on DNA damage response induced by Gemcitabine. To do so, we performed RNAseq SUM159 triple negative breast cancer cell line. SUM159 was tranfected either with shE4F1 OR shscramble treated or not with Gemcitabine for 24 hours.
Project description:E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to identify the p53-independent program controlled by E4F1, we performed microarray analyses in p53 KO and p53 KO; Ha-RasV12-transformed mouse embryonic fibroblasts (MEFs) in wild type and E4F1-inactivated cells.
Project description:E4F1 is a ubiquitously expressed zinc-finger protein of the Gli-Kruppel family that was first identified, more than 30 years ago, as a cellular target of the adenoviral oncoprotein E1A13S (Ad type V), required for transcriptional regulation of adenoviral genes. In order to identify the p53-independent program controlled by E4F1, we performed microarray analyses in p53 KO and p53 KO; Ha-RasV12-transformed mouse embryonic fibroblasts (MEFs) in wild type and E4F1-inactivated cells. To address p53-independent E4F1 transcriptome, a 12 chip array study has been realized using total RNA recovered from wild-type (E4F1+/flox, CRE infected; odd Samples) MEFs and E4F1-depleted (E4F1-/flox, CRE infected; even Samples) MEFs in p53-/- (Samples 1 to 12) and p53-/-, Ha-RasV12 background (Samples 13 to 24). p53-/- MEFs were derived from 13.5-day mouse embryos. Transformed p53-/- MEFs were generated by infection with a recombinant retrovirus encoding for Ha-RasV12. Three independent biological replicates of wild-type and knock-out MEFs for E4F1 have been used on the two genetic backgrounds.
Project description:Microarray profiling was used to determine the most abundantly expressed genes in spinophilin-silenced breast cancer cells compared to control cells in the cell line SUM159. We identified several differentially expressed genes in spinophilin-silenced cells. A total number of six samples were analyzed, each in three replicates. There are three SUM159 Control samples and three samples of the cell line SUM159 with silenced spinophilin.
Project description:Purpose: The goal of this study is to identify the role of HN1L protein as a transcription factor or co-factor in regulating TNBC cells. Methods: Due to the unavailability of a ChIP-grade HN1L antibody, we overexpressed FLAG-tagged HN1L in SUM159 cells and performed ChIP using anti-FLAG antibodies. ChIP DNA was prepared into libraries and sequenced by the Epigenomics Core of Weill Cornell Medical College using SR50 lane. Results: ChIP-Seq analysis began with mapping the sequenced reads to the genome. We utilized the Burrows-Wheeler Aligner (BWA) MEM algorithm to align the sequence reads against the human genome GRCh37/hg19 Assembly. We next used the Hypergoemetric Optimization of Motif Enrichment (HOMER) suite of tools to find and annotate peaks, and identify enriched motifs. HN1L showed 2,249 binding peaks from 10k bp upstream to 5k bp downstream of transcription start site (TSS). Conclusions: among the binding peaks 35 genes overlapped with a previously published BCSC gene signature, 10 genes overlapped with the HN1L knockdown gene signature and 8 genes are well established CSC transcription factors. When applying overlapped genes in the STRING10 pathway analysis database , a protein interaction network centered with STAT3 was obtained. STAT3 and FGFR2 peaks were validated by qPCR. Model-based Analysis of ChIP-Seq (MACS) was then used to confirm the peaks found by Hypergeometric Optimization of Motif EnRichment (HOMER) . Besides the binding peaks found in HOMER, another peak was called by MACS within LEPR , and was also validated by qPCR . These findings indicate that HN1L may act as a transcription factor through binding to enhancer regions of STAT3 and other STAT3 regulators.
Project description:We conducted RNA sequencing of SUM159 DAPK3 KO and control cells followed by Gene Set Enrichment Analysis (GSEA) to identify altered gene expression pathways by DAPK3 knockout. Epithelial-mesenchymal transition (EMT) pathway was the top significantly down-regulated pathway in SUM159 DAPK3 KO cells as compared to control cells.