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Distinct roles for motif affinity, chromatin state, and co-regulatory motifs in PPARγ binding and enhancer activity

ABSTRACT: Sequence-specific transcription factors (TFs) regulate gene expression by binding to cognate motifs in promoters and enhancers. However, predicting genomic TF binding events and their quantitative contribution to expression remains a major challenge. In principle, the binding and enhancer activity of specific sites in vivo might depend on: (i) latent properties of the motif instance, (ii) cooperative interactions with other TFs that bind in the immediate vicinity, and (iii) the chromatin state of the sites in the genome. Here, we used massively parallel reporter assays (MPRA) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo assays, to systematically dissect the contributions of motif affinity, cooperative interactions, and chromatin accessibility to the binding and regulatory activity of genomic sequences that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that PPARγ binding and enhancer activity are governed by distinct features. Genomic PPARγ binding to motif sites is largely governed by on larger-scale features, such as chromatin accessibility, whereas the degree to which a PPARγ motif site enhances transcriptional activity depends on the sequence immediately surround the motif. We detect and functionally validate a network of TFs comprised of multiple functional classes that collaborate with PPARγ to drive transcription. We extensively perturb this network, revealing functional cooperativity among classes of TFs that does not depend on precise positioning. Together, these results present a clear picture of how chromatin and TFs from distinct functional classes interact with PPARγ to determine binding and enhancer activity, and provide a paradigm for studying any TF.

ORGANISM(S): synthetic construct Mus musculus

PROVIDER: GSE84888 | GEO | 2017/02/03

SECONDARY ACCESSION(S): PRJNA335554

REPOSITORIES: GEO

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Project description:Contemporary high throughput technologies permit the rapid identification of transcription factor (TF) target genes on a genome-wide scale, yet the functional significance of TFs requires knowledge of target gene expression patterns, cooperating TFs and cis-regulatory element (CRE) structures. Here we investigated the myogenic regulatory network downstream of the Drosophila zinc finger TF Lame duck (Lmd) by combining both previously published and newly performed genomic data sets, including chromatin immunoprecipitation sequencing (ChIP-seq), genome-wide mRNA profiling, cell-specific expression patterns of putative transcriptional targets, analysis of histone mark signatures, studies of TF co-occupancy by additional mesodermal regulators, TF binding site determination using protein binding microarrays (PBMs), and machine learning of candidate CRE motif compositions. Our findings suggest that Lmd orchestrates an extensive myogenic regulatory network, a conclusion supported by the identification of Lmd-dependent genes, histone signatures of Lmd-bound genomic regions, and the relationship of these features to cell-specific gene expression patterns. The heterogeneous co-occupancy of Lmd-bound regions with additional mesodermal regulators revealed that different transcriptional inputs are used to mediate similar myogenic gene expression patterns. Machine learning further demonstrated diverse combinatorial motif patterns within tissue-specific Lmd-bound regions. PBM analysis established the complete spectrum of Lmd DNA binding specificities, and site-directed mutagenesis of Lmd and additional, newly discovered motifs in known enhancers demonstrated the critical role of these TF binding sites in supporting full enhancer activity. Collectively, these findings provide new insights into the transcriptional codes regulating muscle gene expression, and offer a generalizable approach for similar studies in other systems. Examination of Lmd occupancy to genomic DNA from sorted mesodermal cells

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Distinct roles for motif affinity, chromatin state, and co-regulatory motifs in PPARγ binding and enhancer activity

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