Project description:We were interested to explain why p53 binds some high affinity sites in contrast to other high affinity sites that are not bound by p53. p53 binding was measured using p53 ChIP-CHIP and in parallel nucleosome occupancy was measured on these same sites Comparison between p53 binding and nucleosome occupancy at p53 predicted binding sites
Project description:We were interested to explain why p53 binds some high affinity sites in contrast to other high affinity sites that are not bound by p53. p53 binding was measured using p53 ChIP-CHIP and in parallel nucleosome occupancy was measured on these same sites Comparison between p53 binding and nucleosome occupancy at p53 predicted binding sites ChIP-CHIP of p53 from MCF7EcoR under Basal conditions and MCF7EcoR treated with NCS (Activated) and Mononucleosomal extraction from MCF7sip53, MCF7EcoR under Basal conditions and MCF7EcoR treated with NCS (Activated) Expression analysis of MCF7sip53 and MCF7EcoR treated with NCS (Activated)
Project description:It is widely believed that reorganization of nucleosomes result in availability of transcription factor (TF) binding sites for eukaryotic gene regulation. Recent findings also show TFs induced during physiological perturbations can alter nucleosome occupancy to facilitate DNA binding. Although, these suggest a close relationship between TF binding and nucleosomes, the nature of this interaction, or to what extent it influences transcription is not clear. Moreover, since physiological perturbations induced multiple TFs, relatively direct effect of any TF on nucleosome occupancy remains poorly addressed. With these in mind, we used a single TF to induce physiological changes and following characterization of the two states (before and after induction of the TF) we determined: (a) genome wide binding sites of the TF, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. We find only ~20% of TF binding results from nucleosome repositioning - interestingly, almost all corresponding genes were transcriptionally altered. Whereas, when TF-occupancy was independent of nucleosome repositioning only a small fraction of corresponding genes were expressed/repressed. These observations suggest a model where TF occupancy leads to transcriptional change only when coupled with nucleosome repositioning in close proximity. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-sequencing) typically overlaps with only a small fraction of genes that change expression. The nature of interaction between TF binding and nucleosomes and what extent it influences transcription
Project description:We report the genome-wide map of nucleosome positions in the mouse liver, with emphasis on transcriptional start sites, CpG islands, Foxa2 binding sites, and their correlation with gene expression. Despite the heterogeneity of liver tissue, we could clearly discern the nucleosome pattern of the predominant liver cell, the hepatocyte. By analyzing nucleosome occupancy and the distributions of heterochromatin protein 1 (Hp1), CBP, and p300 in Foxa1/2-deficient livers we find, surprisingly, that the maintenance of nucleosome position and chromatin structure surrounding Foxa2 binding sites is independent of Foxa1/2. Examination of nucleosome map and Foxa2 binding in the mouse liver.
Project description:Previous studies have analyzed patterns of transcription, transcription factor (TF) binding or mapped nucleosome occupancy across the genome. These suggest that the three aspects are genetically connected but the cause and effect relationships are still unknown. For example, physiologic TF binding studies involve many TFs, consequently, it is difficult to assign nucleosome reorganization to the binding site occupancy of any particular TF. Therefore, several aspects remain unclear: does TF binding influence nucleosome (re)organizations locally (in close vicinity of their binding sites) or impact the chromatin landscape at a more global level; are all or only a fraction of TF binding a result of reorganization in nucleosome occupancy; finally, do all TF binding and associated nucleosome occupancy changes result in altered gene expression? With these in mind, we sought to study a single TF that induces physiological changes, and following characterization of the two states (before and after induction of the TF) we determined: (a) genomic binding sites of the TF, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. Results demonstrate that TF binding influences expression of the target gene only when it is coupled to nucleosome repositioning at or close to its binding site, and not when transcription factor binding occurs without local associated nucleosome reorganization.
Project description:The binding sequence for any transcription factor can be found millions of times within a genome, yet only a small fraction of these sequences encode functional transcription factor binding sites. To study how the competition between nucleosomes and transcription factors helps determine a functional transcription factor site from a predicted transcription factor site, we compared experimentally-generated in vitro nucleosome occupancy with in vivo nucleosome occupancy and transcription factor binding in murine embryonic stem cells. Using a solution hybridization enrichment technique, we generated a high-resolution nucleosome map from targeted regions of the genome containing predicted sites and functional sites of Oct4/Sox2 regulation. We found that at Pax6 and Nes, which are bivalently poised in stem cells, functional Oct4 and Sox2 sites show high amounts of in vivo nucleosome displacement compared to in vitro. Oct4 and Sox2, which are active, show no significant displacement of in vivo nucleosomes at functional sites, similar to nonfunctional Oct4/Sox2 binding. This study highlights the ability of Oct4 and Sox2 transcription factors to affect nucleosome occupancy in different ways, which may reflect distinct patterns of stem cell gene regulation.
Project description:It is widely believed that reorganization of nucleosomes result in availability of transcription factor (TF) binding sites for eukaryotic gene regulation. Recent findings also show TFs induced during physiological perturbations can alter nucleosome occupancy to facilitate DNA binding. Although, these suggest a close relationship between TF binding and nucleosomes, the nature of this interaction, or to what extent it influences transcription is not clear. Moreover, since physiological perturbations induced multiple TFs, relatively direct effect of any TF on nucleosome occupancy remains poorly addressed. With these in mind, we used a single TF to induce physiological changes and following characterization of the two states (before and after induction of the TF) we determined: (a) genome wide binding sites of the TF, (b) promoter nucleosome occupancy and (c) transcriptome profiles, independently in both conditions. We find only ~20% of TF binding results from nucleosome repositioning - interestingly, almost all corresponding genes were transcriptionally altered. Whereas, when TF-occupancy was independent of nucleosome repositioning only a small fraction of corresponding genes were expressed/repressed. These observations suggest a model where TF occupancy leads to transcriptional change only when coupled with nucleosome repositioning in close proximity. This, to our knowledge, for the first time also helps explain why genome wide TF occupancy (e.g., from ChIP-sequencing) typically overlaps with only a small fraction of genes that change expression.