Interpreting regulatory mechanisms of Hippo signaling through a deep learning sequence model

How specific cells respond to signaling pathways is largely encoded in the DNA sequence. However, the sequence rules result from complex interactions between signaling and cell type-specific transcription factors and are considered intractable by traditional methods. Here, we leverage interpretable deep learning on high-resolution data and extensive validation experiments to identify the sequence rules for the Hippo pathway in mouse trophoblast stem cells. We show that TEAD4 and YAP1 engage in two types of cooperativity. First, their binding is enhanced by cell type-specific transcription factors, including TFAP2C, in a distance-dependent manner. Second, a strictly-spaced Tead double motif is a canonical Hippo pathway element that mediates strong Tead4 cooperativity through transient protein-protein interactions on DNA. These mechanisms occur genome-wide and allow us to predict how small sequence changes alter the activity of enhancers in vivo. This illustrates the power of interpretable deep learning to decode canonical and cell type-specific sequence rules of signaling pathways. These super series include ChIP-nexus data for TF binding (TEAD4, YAP1, CDX2, TFAP2C, GATA3), H3K27ac Chip-seq, ATAC-seq, RNA-seq, and Nascent RNA captured through the TT-seq method in mouse trophoblast stem cells. For RNA seq: STAR count table, RSEM-TPM table, and edgeR analysis of wild-type and CRISPR mutated Tead4 single motif to TEAD4 double motif at chr17:6,827,739-6,828,394 genomic region (putative enhancer of Ezrin gene) ChIP-nexus of TEAD4 and Pol II along with H3K27ac ChIP-seq performed in wild-type and CRISPR mutated cells. The mutation was at the putative enhancer region of the Rin3 gene (chr12:102,261,820-102,262,484), where the TEAD4 single motif moved away from the TFA2PC motif from 20bp in wild-type to 60 bp in CRISPR mutated cells. Also, the ChIP-nexus of TEAD4 was performed in wild-type and CRISPR-mutated cells. The mutation was at the putative enhancer region of the Ezrin gene (chr17:6,827,739-6,828,394 ), where the Tead4 single motif was replaced with the TEAD4 double motif.