Identification of determinants of differential chromatin accessibility through a massively parallel genome-integrated reporter assay

Here we introduce the Multiplexed Integrated Accessibility Assay (MIAA), a multiplexed parallel reporter assay which measures changes to genome accessibility as a result of the integration of synthetic oligonucleotide phrase libraries into a controlled, natively inaccessible genomic context. We apply MIAA to measure the effects of sequence motifs on cell type-specific DNA accessibility between mouse embryonic stem cells and embryonic stem cell-derived definitive endoderm cells, screening a total of 7,905 distinct phrases. MIAA is able to recapitulate differential accessibility patterns of 100-nt sequences derived from natively differential genomic regions, identifying the presence of E-box motifs common to epithelial-mesenchymal transition driver transcription factors in stem cell-specific accessible regions that become repressed during differentiation to endoderm. We further present causal evidence that a single binding motif for a key regulatory transcription factor is sufficient to open chromatin, and classify sets of stem cell-specific, endoderm-specific, and shared pioneer factor motifs. We also demonstrate that over-expression of two definitive endoderm transcription factors, Brachyury and FoxA2, results in changes to accessibility in phrases containing their respective DNA-binding motifs. Finally, we use MIAA results to explore the order of motif interactions and identify preferential motif ordering arrangements that appear to have an effect on accessibility. SLOTv3 (de novo motif) Library - 2 replicates uCd8 locus in endoderm and stem cell; 4 replicates (stem cell) and 2 replicates (endoderm) in main explerimental RAR locus; 2 replicates FoxA2 and Brachyury over-expression SLOTv4 (pioneer) Library - 2 replicates of endoderm and stem cell