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| Status |
Public on Feb 03, 2017 |
| Title |
Distinct roles for motif affinity, chromatin state, and co-regulatory motifs in PPARγ binding and enhancer activity |
| Organisms |
Mus musculus; synthetic construct |
| Experiment type |
Expression profiling by high throughput sequencing
Genome binding/occupancy profiling by high throughput sequencing
Other
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| Summary |
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.
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| Overall design |
The study consisted of 7 MPRA experiments and 2 ChIP-seq experiments. Raw data for MPRA experiments are provided as Illumina reads of the 16 bp barcode from the RNA extracted 16 hours post transfection as well as from the plasmid library used for transfection. Raw data for ChIP-seq experiments are provided as paired-end Illumina reads for PPARg ChIP DNA fragments extracted 16 hours post transfection as well as input DNA fragments. For pools 4-7, we have provided barcode/oligo combinations as paired-end Illumina reads covering the barcode and enhancer sequence. Processed count files are counts corresponding to each barcode (Pools 1-3) or counts summed across all barcodes for each oligo (Pools 4-7).
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| Contributor(s) |
Grossman SR |
| Citation(s) |
28137873 |
| |
| Submission date |
Jul 27, 2016 |
| Last update date |
May 15, 2019 |
| Contact name |
Sharon R Grossman |
| E-mail(s) |
grossman@broadinstitute.org
|
| Organization name |
Broad Institute
|
| Street address |
415 Main Street
|
| City |
Cambridge |
| State/province |
MA |
| ZIP/Postal code |
02142 |
| Country |
USA |
| |
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| Platforms (4)
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| GPL13112 |
Illumina HiSeq 2000 (Mus musculus) |
| GPL15228 |
Illumina HiSeq 2000 (synthetic construct) |
| GPL16417 |
Illumina MiSeq (Mus musculus) |
| GPL17769 |
Illumina MiSeq (synthetic construct) |
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| Samples (21)
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| Relations |
| BioProject |
PRJNA335554 |
| SRA |
SRP079958 |
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