Sharon R. Grossman, Xiaolan Zhang, Li Wang, Jesse Engreitz, Alexandre Melnikov et al.
Enhancers regulate gene expression through the binding of sequence-specific transcription factors (TFs) to cognate motifs. Various features influence TF binding and enhancer function—including the chromatin state of the genomic locus, the affinities of the binding site, the activity of the bound TFs, and interactions among TFs. However, the precise nature and relative contributions of these features remain unclear. Here, we used massively parallel reporter assays (MPRAs) involving 32,115 natural and synthetic enhancers, together with high-throughput in vivo binding assays, to systematically dissect the contribution of each of these features to the binding and activity of genomic regulatory elements that contain motifs for PPARγ, a TF that serves as a key regulator of adipogenesis. We show that distinct sets of features govern PPARγ binding vs. enhancer activity. PPARγ binding is largely governed by the affinity of the specific motif site and higher-order features of the larger genomic locus, such as chromatin accessibility. In contrast, the enhancer activity of PPARγ binding sites depends on varying contributions from dozens of TFs in the immediate vicinity, including interactions between combinations of these TFs. Different pairs of motifs follow different interaction rules, including subadditive, additive, and superadditive interactions among specific classes of TFs, with both spatially constrained and flexible grammars. Our results provide a paradigm for the systematic characterization of the genomic features underlying regulatory elements, applicable to the design of synthetic regulatory elements or the interpretation of human genetic variation.
Sharon R. Grossman, Xiaolan Zhang, Li Wang, Jesse Engreitz, Alexandre Melnikov, Peter Rogov, Ryan Tewhey, Alina Isakova, Bart Deplancke, Bradley E. Bernstein, Tarjei S. Mikkelsen, Eric S. Lander. Systematic dissection of genomic features determining transcription factor binding and enhancer function. 2017-02-14. https://doi.org/10.1073/pnas.1621150114
This pool tests mutations disrupting occurrences of TF motifs correlated with activity across bound mouse genomic enhancer backgrounds. The GEO release provides oligo-level RNA and plasmid counts summed across assigned barcodes, which are packaged here with the motif and position tokens encoded in each name.
This pool tests a matrix of PPARγ core motifs exchanged across 25 genomic flanking sequences, with additional control constructs. The released matrix was assayed in differentiated 3T3-L1 adipocytes and provides matched plasmid-DNA and RNA barcode counts.
This pool contains 750 mouse genomic PPARγ ChIP-seq-bound sites, 750 matched unbound sites, and native/motif-disrupted versions of each. The constructs were tested in differentiated 3T3-L1 adipocytes with matched plasmid-DNA and RNA barcode counts.
This pool tests synthetic enhancer constructs carrying individual or paired sites from 15 positively correlated TF motifs in multiple configurations on low-activity templates. The GEO release supplies oligo-level RNA and plasmid counts summed across assigned barcodes.
This pool substitutes correlated TF motifs into existing motif sites within active PPARγ-bound genomic enhancer backgrounds. The GEO release supplies oligo-level RNA and plasmid counts summed across assigned barcodes, with target and inserted motif tokens parsed from each construct name.
This pool is an independent collection of mouse genomic PPARγ motif-containing sequences used to test the motif-composition model, together with intact and motif-disrupted construct classes. The released counts were measured in differentiated 3T3-L1 adipocytes using matched RNA and plasmid-DNA barcode channels.
This pool systematically perturbs 25 bound genomic enhancer contexts with tiled 10-bp mutations and 20-bp sequence swaps, retaining the central PPARγ motif. The released constructs were assayed in differentiated 3T3-L1 adipocytes with matched RNA and plasmid-DNA barcode counts.
Original supplemental and deposited inputs retained for this study. Download files individually or together as a ZIP; nested folders are preserved. Source reuse terms apply, and sequencing reads may be omitted.
Download all 18 files (ZIP)article_source.pdfGSE84888_family.soft.gzGSE84888_filelist.txtGSE84888_Pool5_MPRA.txt.gzGSE84888_Pool6_MPRA.txt.gzGSE84888_Pool7_MPRA.txt.gzGSM2253151_Pool1_RNA.counts.txt.gzGSM2253152_Pool1_Plasmid.counts.txt.gzGSM2253153_Pool2_RNA.counts.txt.gzGSM2253154_Pool2_Plasmid.counts.txt.gzGSM2253155_Pool3_RNA.counts.txt.gzGSM2253156_Pool3_Plasmid.counts.txt.gzGSM2253157_Pool4_RNA.counts.txt.gzGSM2253158_Pool4_Plasmid.counts.txt.gzGSM2253165_Pool5.barcodes.txt.gzGSM2253166_Pool6.barcodes.txt.gzGSM2253167_Pool7.barcodes.txt.gzREADME.txt