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DNA-Binding Mutant EAAE-ER alpha Genome-Wide Binding in the Uterus

ABSTRACT: Transcription factors (TFs) influence cell function by interpreting information contained within cis-regulatory elements in chromatin. Whereas ChIP-sequencing has been used to identify and map transcription factor-DNA interactions, it has been difficult to assign functionality to the binding sites identified. Thus, in this study we probed the transcriptional activity, DNA-binding competence and functional activity of select nuclear receptor (NR) mutants in cellular and animal model systems and used this information to define the sequence constraints of functional steroid nuclear receptor (sNR) cis-regulatory elements. Analysis of the architecture within sNR chromatin interacting sites revealed that only a small fraction of all sNR chromatin interacting events are associated with transcriptional output, and that this functionality is restricted to elements that vary from the consensus palindromic elements by one or two nucleotides. These findings define the transcriptional grammar necessary to predict functionality from linear genome sequences.

ORGANISM(S): Mus musculus

PROVIDER: GSE94737 | GEO | 2017/04/01

SECONDARY ACCESSION(S): PRJNA373895

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

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DNA-Binding Mutant EAAE-ER alpha Genome-Wide Binding in the Uterus

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