Co-regulation of transcription factor binding and nucleosome occupancy through DNA features of mammalian enhancers
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ABSTRACT:
ORGANISM(S): Mus musculus
PROVIDER: GSE50762 | GEO | 2014/05/08
SECONDARY ACCESSION(S): PRJNA218857
REPOSITORIES: GEO
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Project description:In mammalian cells transcription factors (TFs) preferentially bind sites contained in regions of high nucleosomal occupancy, as determined by nucleotide-dependent computational analysis. This observation suggests that nucleosomes may act as gatekeepers of TF binding sites. We hypothesized that in mammalian genomes the information controlling nucleosome assembly may partially coincide with the information that enables TFs to recognize cognate sites in cis-regulatory elements while ignoring the myriad of non-functional, randomly occurring consensus binding sites. This way, nucleosome-mediated masking would be coupled to TF binding site functionality. The hematopoietic master regulator Pu.1 maintained nucleosome depletion at macrophage-specific enhancers that were otherwise occupied by nucleosomes in other cell types and in reconstituted chromatin. We identified a minimal set of DNA sequence and shape features that predicted Pu.1 binding with 78% accuracy. The same features predicted nucleosome occupancy in cells where Pu.1 was not expressed with higher accuracy than specifically designed models. Control of nucleosome deposition by DNA sequence and shape features that also specify TF consensus site functionality may allow maintaining the gatekeeper function of nucleosomes during evolution of cis-regulatory elements. Chromatin immuno-precipitations of the transcription factor Pu.1 followed by multiparallel sequencing performed in murine bone marrow-derived macrophages. Experiments carried out in cells infected either with a retroviral vector containing a short hairpin targeting Pu.1 or with the empty vector as control. The shPU.1 hairpin (sequence available upon request) was selected among five designed using a publicly available software (http://katahdin.mssm.edu/siRNA) and was cloned in a modified version of TtRMPVIR inducible retroviral vector (Genbank HQ456318) in which the puromycin resistance gene was inserted. The empty vector, containing an sh-Renilla sequence, was used as control. Chromatin immuno-precipitations of the transcription factor PU.1 binding to in vitro reconstituted chromatin followed by multiparallel sequencing Micrococcal nuclease digestion of chromatin extracted from bone marrow derived macrophages followed by multiparallel sequencing . Experiments were carried out in untreated cells (4 replicates) and cells infected either with a retroviral vector containing a short hairpin targeting Pu.1 or with the empty vector as control (2 replicates). The shPU.1 hairpin (sequence available upon request) was selected among five designed using a publicly available software (http://katahdin.mssm.edu/siRNA) and was cloned in a modified version of TtRMPVIR inducible retroviral vector (Genbank HQ456318) in which the puromycin resistance gene was inserted. The empty vector, containing an sh-Renilla sequence, was used as control. Micrococcal nuclease digestion of in vitro reconstituted chromatin followed by multiparallel sequencing
2014-05-08 | E-GEOD-50762 | biostudies-arrayexpress
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
2014-08-04 | E-GEOD-40300 | biostudies-arrayexpress
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. The nature of interaction between TF binding and nucleosomes and what extent it influences transcription
2014-08-04 | E-GEOD-40363 | biostudies-arrayexpress
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.
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Project description:The majority of sequence-specific transcription factors bind genomic DNA only at a fraction of their potential binding sites and the ‘rules’ for binding or not-binding are only partially understood. Here, we studied the binding properties of the myeloid and B-cell specific transcription factor PU.1 in-vivo and in-vitro to unveil basic features of occupied vs. non-occupied consensus sites. In addition to published PU.1 ChIP-seq data we mapped CTCF binding sites in monocytes and macrophages to determine chromatin domain boundaries and performed MCIp-seq in monocytes to reveal DNA methylation patterns across the genome. ChIP-seq of CTCF in human monocytes and human monocyte-derived macrophages as well as MCIp-seq in human monocytes
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2014-10-01 | E-GEOD-61596 | biostudies-arrayexpress
Project description:Nucleosomes are the primary unit of chromatin structure and inhibit key intracellular interactions which regulate how eukaryotic genetic information is read, including transcription factor (TF) binding to DNA. In theory, the sterics of nucleosome-DNA interactions could inhibit TF binding to all nucleosome-bound DNA. In reality, however, nucleosome inhibition of TF binding is variable both across a genome and even within a single nucleosome. Prior studies have demonstrated that nucleosome occupancy, nucleosome positioning, TF binding site (TFBS) affinity & TFBS location can each influence TF targeting in vivo, however, this work has been largely correlative and no study has dissected collective from individual contributions of these important variables. Therefore, to address this deficiency, we have mapped genome-wide changes in chromatin structure and TF binding following experimental nucleosome depletion. Heuristic modeling of results confirmed that chromatin specification of TF targeting is a multivariate, context-specific, process, with most informative features originating from additive interactions between cis and trans variables. Additionally, we found compelling evidence that nucleosomes serve to inhibit intra-genic TF binding, as evidenced by an increasing number of sub-optimal TFBS occupied following nucleosome depletion. The majority of these conditional loci were located within open reading frames and parallel transcriptome analysis revealed their occurrence was linked to generation of intragenic cryptic transcripts. Together results support the idea that chromatin structure has evolved to coordinate specification of regulatory factor targeting & gene expression. Findings have functional relevance to emerging datasets on genetic and epigenetic variation in human disease states.
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Project description:Nucleosomes are the primary unit of chromatin structure and inhibit key intracellular interactions which regulate how eukaryotic genetic information is read, including transcription factor (TF) binding to DNA. In theory, the sterics of nucleosome-DNA interactions could inhibit TF binding to all nucleosome-bound DNA. In reality, however, nucleosome inhibition of TF binding is variable both across a genome and even within a single nucleosome. Prior studies have demonstrated that nucleosome occupancy, nucleosome positioning, TF binding site (TFBS) affinity & TFBS location can each influence TF targeting in vivo, however, this work has been largely correlative and no study has dissected collective from individual contributions of these important variables. Therefore, to address this deficiency, we have mapped genome-wide changes in chromatin structure and TF binding following experimental nucleosome depletion. Heuristic modeling of results confirmed that chromatin specification of TF targeting is a multivariate, context-specific, process, with most informative features originating from additive interactions between cis and trans variables. Additionally, we found compelling evidence that nucleosomes serve to inhibit intra-genic TF binding, as evidenced by an increasing number of sub-optimal TFBS occupied following nucleosome depletion. The majority of these conditional loci were located within open reading frames and parallel transcriptome analysis revealed their occurrence was linked to generation of intragenic cryptic transcripts. Together results support the idea that chromatin structure has evolved to coordinate specification of regulatory factor targeting & gene expression. Findings have functional relevance to emerging datasets on genetic and epigenetic variation in human disease states.
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Project description:Nucleosomes are the primary unit of chromatin structure and inhibit key intracellular interactions which regulate how eukaryotic genetic information is read, including transcription factor (TF) binding to DNA. In theory, the sterics of nucleosome-DNA interactions could inhibit TF binding to all nucleosome-bound DNA. In reality, however, nucleosome inhibition of TF binding is variable both across a genome and even within a single nucleosome. Prior studies have demonstrated that nucleosome occupancy, nucleosome positioning, TF binding site (TFBS) affinity & TFBS location can each influence TF targeting in vivo, however, this work has been largely correlative and no study has dissected collective from individual contributions of these important variables. Therefore, to address this deficiency, we have mapped genome-wide changes in chromatin structure and TF binding following experimental nucleosome depletion. Heuristic modeling of results confirmed that chromatin specification of TF targeting is a multivariate, context-specific, process, with most informative features originating from additive interactions between cis and trans variables. Additionally, we found compelling evidence that nucleosomes serve to inhibit intra-genic TF binding, as evidenced by an increasing number of sub-optimal TFBS occupied following nucleosome depletion. The majority of these conditional loci were located within open reading frames and parallel transcriptome analysis revealed their occurrence was linked to generation of intragenic cryptic transcripts. Together results support the idea that chromatin structure has evolved to coordinate specification of regulatory factor targeting & gene expression. Findings have functional relevance to emerging datasets on genetic and epigenetic variation in human disease states.
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Project description:The majority of sequence-specific transcription factors bind genomic DNA only at a fraction of their potential binding sites and the ‘rules’ for binding or not-binding are only partially understood. Here, we studied the binding properties of the myeloid and B-cell specific transcription factor PU.1 in-vivo and in-vitro to unveil basic features of occupied vs. non-occupied consensus sites. In addition to published PU.1 ChIP-seq data we mapped CTCF binding sites in monocytes and macrophages to determine chromatin domain boundaries and performed MCIp-seq in monocytes to reveal DNA methylation patterns across the genome.
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