Primers used for ChIP-qPCR.

<p>Primers used for ChIP-qPCR.</p

YAP1 Exerts Its Transcriptional Control via TEAD-Mediated Activation of Enhancers

<div><p>YAP1 is a major effector of the Hippo pathway and a well-established oncogene. Elevated YAP1 activity due to mutations in Hippo pathway components or <i>YAP1</i> amplification is observed in several types of human cancers. Here we investigated its genomic binding landscape in YAP1-activated cancer cells, as well as in non-transformed cells. We demonstrate that TEAD transcription factors mediate YAP1 chromatin-binding genome-wide, further explaining their dominant role as primary mediators of YAP1-transcriptional activity. Moreover, we show that YAP1 largely exerts its transcriptional control via distal enhancers that are marked by H3K27 acetylation and that YAP1 is necessary for this chromatin mark at bound enhancers and the activity of the associated genes. This work establishes YAP1-mediated transcriptional regulation at distal enhancers and provides an expanded set of target genes resulting in a fundamental source to study YAP1 function in a normal and cancer setting.</p></div

siRNAs (Fig 3A).

<p>siRNAs (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g003" target="_blank">Fig 3A</a>).</p

YAP1/TEAD1 associate with active enhancers.

<p>(A) Genomic distribution of YAP1/TEAD1 peaks. Promoter class defined as 2kb upstream of gene TSS. (B) Distance of YAP1/TEAD1 peaks and H3K27ac regions to closest gene TSS. (C) Genomic views of H3K27ac, YAP1 and TEAD1 ChIP enrichment at gene promoters of known target genes. (D) YAP1, TEAD1 and H3K27ac ChIP enrichment at all YAP1 peak regions centered on peak summit. (E) Luciferase reporter assay of six YAP1/TEAD1 distal enhancer binding sites containing single or double TEAD motifs in cells treated with YAP1 or TEADs siRNA compared to control siRNA. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.</p

Additional file 3: of MiR-210 promotes sensory hair cell formation in the organ of corti

Table of miRNA raw counts. Raw counts from sequencing of each of the small miRNA libraries. The number of sequences that correspond to each miRNA is listed. (XLSM 60 kb

TEAD single and double motifs occur within most YAP1 binding sites.

<p>(A and B) Enrichment of (A) TEAD and (B) AP-1 motifs in YAP1 peaks. Full list provided in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.s017" target="_blank">S4 Table</a>. (C) YAP1 ChIP enrichment as determined by peak score in YAP1 peaks with/without TEAD and AP-1 motifs. (D) Number of TEAD motifs in YAP1 peaks. (E) Enrichment of TEAD double motif with several spacer lengths in YAP1 peaks. (F) Sequence conservation of YAP1 peak regions. (G) Sequence conservation of TEAD single and double motifs in YAP1 peak regions. (H) YAP1 ChIP enrichment as determined by peak score in YAP1 peaks with/without single/double TEAD motifs. (I) Luciferase reporter assay for two YAP1 binding regions with either intact double motif or with single or double mutations. Relative luciferase activity represents the ratio of Firefly and Renilla luciferase activity for each sample. The red line indicates the highest mean activity of the two negative control regions. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.</p

YAP1 peaks are co-occupied by TEAD1.

<p>(A) Expression changes of a YAP1/TEAD responsive luciferase reporter upon siRNA-mediated knockdown of YAP1 or TEADs normalized to a negative control siRNA in SF268 cells. (B) Correlation between TEAD1 and YAP1 SF268 ChIP-seq samples. (C) Genomic views of YAP1 and TEAD1 ChIP enrichment at gene promoters of known target genes. (D and E) Validation of (D) TEAD1 and (E) YAP1 binding to known and novel sites and control regions following siRNA depletion of TEADs as compared to control siRNA treated cells by ChIP-qPCR. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.</p

YAP1 binding sites largely overlap in cancer cell lines from distinct lineages.

<p>(A) Correlation between SF268 and NCI-H2052 YAP1 ChIP-seq samples. (B) Genomic views of YAP1 shared, SF268-, NCI-H2052 and IMR90-specific regions. (C) Correlation between SF268 and IMR90 YAP1 ChIP-seq samples. (D) H3K27ac ChIP enrichment at YAP1 peak regions (centered on peak summit) that are shared, SF268-specific or IMR90-specific.</p

siRNAs.

<p>siRNAs.</p

Primers used for RT-qPCR.

<p>Primers used for RT-qPCR.</p