Project description:Ets family transcription factor GA-binding protein (GABP) regulates gene expression in CD4 and CD8 T cells. We used microarray to examine genes differentially expressed in GABP-sufficient (WT) and GABP-deficient (KO) CD4 and CD8 T cells
Project description:A key model for understanding how large transcription complexes are targeted is the Drosophila dosage compensation system in which the Male-Specific Lethal (MSL) transcription complex specifically identifies and regulates the male X-chromosome. MSL complex is targeted to GA-containing sequences, but the most well-studied GA-binding transcription factor, GAGA Associated Factor (GAF), does not physically associate with MSL complex. Instead the Chromatin Linked Adapter for MSL Proteins (CLAMP) zinc-finger protein specifically targets MSL complex to GA-rich sequences on the X-chromosome. Here, we compare the binding relationships of CLAMP, GAF, and the MSL3 dosage compensation complex protein using ChIP-seq.
Project description:A central question in transcription factor biology is how a specific member of a transcription factor family occupies a promoter in vivo, when all family members bind the same consensus site in vitro. To uncover the mechanisms regulating DNA binding specificity within transcription factor families, we have used the techniques of chromatin immunoprecipitation coupled with genome-wide microarray analysis to query the occupancy of three members of the ETS transcription factor family in a human T-cell line. Unexpectedly, redundant occupancy was frequently detected while specific occupancy was less likely. An unbiased bioinformatics approach correlated redundant binding with consensus ETS binding sequences near transcription start sites, whereas specific binding sites diverged dramatically from the consensus, were coupled with a site for a cooperative binding partner, and were found further from transcription start sites. The specific and redundant DNA binding modes illustrate the regulation of transcription factor specificity in vivo and suggest two distinct roles for members of the ETS transcription factor family. Keywords: ChIP-chip
Project description:The non-coding genome contributes substantially to the total cellular DNA mass and is thoutht to host regulatory cis-acting element, but may also code for some regulatory transcripts. Despite ist contribution to the genomic mass, it has barely recieved scientific attention. Much emphasis has been laid on the approximaetly 2% coding Region, although its transcription, in most cases is regulated at the level of the non-coding genome. In recent years, several studies have revealed disease-associated somatic mutations in regulatory regions of the non-coding genome. One most prominent example is the hotspot mutations in the core promoter region of the TERT gene. In several cancer entities, these alterations have been associated with dysregulation of telomerase activity and tumorigenesis. Novel binding sites are created for transcription factors of the ETS family and GABP for example has been shown to bind to these novel ETS sites. The entire spectrum of ETS family members that can bind is however still lacking. In ordert o fill this knowledge gap and understand the impact of such binding on tumor biology, we have used functional protein microarrays to identify ETS members that preferentially bind the mutant promoter and by means of transcription profiling, we have identified downstream targets of the binding events. We have used siRNA to knockdown FLI-1 and hTERT in cell lines from three cancer entities (NSCLC, PDAC and MCC) with the help oft he illumina beadchip arrays and we compared the transcriptional profiles of the cell lines under different knockdown conditions. We Identify CCND1 and E2F2 as direct targets of both TERT and FLI-1, and CMTM7 as an FLI1-only target. We show, for different cancer entities, that FLI-1 binds the TERT promoter and accelerates progression through the cell cycle by enhancing E2F2 and CCND1 expression
Project description:A central question in transcription factor biology is how a specific member of a transcription factor family occupies a promoter in vivo, when all family members bind the same consensus site in vitro. To uncover the mechanisms regulating DNA binding specificity within transcription factor families, we have used the techniques of chromatin immunoprecipitation coupled with genome-wide microarray analysis to query the occupancy of three members of the ETS transcription factor family in a human T-cell line. Unexpectedly, redundant occupancy was frequently detected while specific occupancy was less likely. An unbiased bioinformatics approach correlated redundant binding with consensus ETS binding sequences near transcription start sites, whereas specific binding sites diverged dramatically from the consensus, were coupled with a site for a cooperative binding partner, and were found further from transcription start sites. The specific and redundant DNA binding modes illustrate the regulation of transcription factor specificity in vivo and suggest two distinct roles for members of the ETS transcription factor family. Keywords: ChIP-chip Chromatin IP from uninduced asynchronous Jurkat T cells, or HT29 colon cells using antibodies to three ETS transcription factors (ETS1, ELF1, and GABPa) or the transcription factors RUNX1 or E2F4. IP and whole input DNAs are amplified (WGA2 kit sigma) and labeled and compared by promoter microarrays (Agilent). Each experiment requires 2 microarrays to cover the genome. Proximal promoter arrays assay 1kb surrounding transcription start sites, extended promoter arrays assay 7kb surrounding transcription start sites. Each microarray is performed as two biological replicates with the exception of ETS1 in HT29 proximal promoter array which were done once, and ETS1 in Jurkat proximal promoter array which was done three times.
Project description:Reactivation of telomerase reverse transcriptase (TERT) expression enables cells to overcome replicative senescence and escape apoptosis, fundamental steps in the initiation of human cancer. Multiple cancer types, including up to 83% of glioblastomas (GBM), harbor highly recurrent TERT promoter mutations of unknown function but specific to two nucleotide positions. We identify the functional consequence of these mutations in GBM to be recruitment of the multimeric GABP transcription factor specifically to the mutant promoter. Allelic recruitment of GABP is consistently observed across four cancer types, highlighting a shared mechanism underlying TERT reactivation. Tandem flanking native ETS motifs critically cooperate with these mutations to activate TERT, likely by facilitating GABP heterotetramer binding. GABP thus directly links TERT promoter mutations to aberrant expression in multiple cancers.
Project description:YBX3 is a multifunctional DNA- and RNA-binding factor of the Y-box protein family. YBX3 regulates gene expression from transcription to mRNA translation and decay. To elucidate how YBX3 regulates protein expression via its RNA binding activity we identified the RNAs bound by YBX3 using eCLIP.
Project description:To elucidate how genomic sequences build transcriptional control networks we need to understand the connection between DNA sequence and transcription factor binding and function. Binding predictions based solely on consensus predictions are limited because a single factor can use degenerate sequence motifs and related transcription factors often prefer identical sequences. The ETS family transcription factor, ETS1, exemplifies these challenges. Unexpected, redundant occupancy of ETS1 and other ETS proteins is observed at promoters of housekeeping genes in T cells due to common sequence preferences and the presence of strong consensus motifs. However, ETS1 exhibits a specific function in T cell activation, thus unique transcriptional targets are predicted. To uncover the sequence motifs that mediate specific functions of ETS1, chromatin immunoprecipitation coupled with high-throughput sequencing (ChIP-seq) identified both promoter and enhancer binding events in Jurkat T cells. A comparison with DNase I sensitivity both validated the dataset and improved accuracy. Redundant occupancy of ETS1 with the ETS protein GABPA occurred primarily in promoters of housekeeping genes, whereas ETS1 specific occupancy occurred in the enhancers of T-cell specific genes. Two routes to ETS1 specificity were identified: an intrinsic preference of ETS1 for a variant of the ETS family consensus sequence and the presence of a composite sequence that can support cooperative binding with a RUNX transcription factor. Genome-wide occupancy of RUNX factors corroborated the importance of this partnership. Furthermore, genome-wide occupancy of co-activator CBP indicated tight co-localization with ETS1 at specific enhancers, but not redundant promoters. The distinct sequences associated with redundant versus specific ETS1 occupancy were predictive of promoter or enhancer location and the ontology of nearby genes. These findings demonstrate that diversity of binding motifs may enable variable transcription factor function at different genomic sites. Each ChIP sample was pooled from three independent immunoprecipitation experiments
Project description:We determine the genome-wide transcriptome, enhancer landscape and transcription factor binding profiles of FOXA1 and GATA3 in luminal and basal subtypes of bladder cancer. Furthermore, we report the first-ever mapping of genome-wide chromatin interactions by Hi-C in both bladder cancer cell lines and primary patient tumors. We show that subtype-specific transcription is accompanied by specific open chromatin and epigenomic marks, at least partially driven by distinct transcription factor binding at distal-enhancers of luminal and basal bladder cancers. Finally, we identify a novel clinically relevant transcription factor, Neuronal PAS Domain Protein 2 (NPAS2), in luminal bladder cancers that regulates other subtype-specific genes and influences cancer cell proliferation and migration.
Project description:The tumor suppressor transcription factor p53 is mutated in nearly 50% of all cancer cases, and this frequency increases with stage and resistance to therapy. Hotspot p53 mutations lose direct DNA binding activity while gaining oncogenic function. These structural p53 mutants can bind to new regions of the genome via interactions with other transcription factors. One example is the ETS transcription factor ETS2, which was previously reported to bind mutant p53. Yet, a comprehensive understanding of mutant p53 association with the ETS family needs to be understood. Here, we compare mutant p53 interaction across 26 ETS transcription factors. Mutant p53 bound ETS proteins better than wild-type p53. All ETS proteins tested bound to mutant p53 to some extent, yet relative binding differed. The ETS DNA binding domain provided a common interaction interface, but strong binding required a second interaction domain. Genome-wide mapping found that the ETS protein ERG could recruit mutant p53 to regulatory regions of genes necessary for morphogenesis, locomotion, and androgen response in prostate cancer cells. Lastly, ETS factors that interact strongly with mutant p53 tend to be upregulated in p53 mutant ovarian cancer. These results suggest that multiple ETS proteins can work with mutant p53 in cancer.