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

Outline of the experimental strategy.

<p>The nuclear transcriptome as well as RNAPII-associated genomic sequences of actively transcribing cells are analysed by nucRNA-Seq and RNAPII ChIP-Seq, respectively, as indicated. Top: schematic representation of transcription in the nucleus: four transcribing RNAPII complexes depicted as green shapes are associated with two chromatin fibres, DNA shown in red and blue, respectively; a third chromatin region, which is not being transcribed, is shown with DNA in black; histone complexes are yellow circles, nascent transcripts are shown as thin wavy lines, colours corresponding to chromatin. The nucRNA-Seq procedure is outlined on the left; purified nuclear RNA from the two transcribed regions is shown as wavy or straight lines colour-coded as above, DNA is depicted as thicker lines, random primers are black arrows, a putative genomic region with aligned Illumina paired-end (PE) tags signifies nucRNA-Seq data. The RNAPII ChIP-Seq procedure is outlined on the right; immunoprecipitated RNAPII-associated nucleosomes are depicted and colour-coded as above with cross-links as yellow crosses, anti-RNAPII antibodies are shown as red Y shapes, purified DNA is represented by thick lines, a putative genomic region with PE tags signifies RNAPII ChIP-Seq 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

Luciferase reporters (Fig 4E).

<p>Luciferase reporters (<a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g004" target="_blank">Fig 4E</a>).</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

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 mediates active enhancer chromatin and expression of target genes.

<p>(A) YAP1 immunofluorescence staining in SF268 cells grown at low (LD) or high density (HD). The corresponding DNA Hoechst 33342 staining is shown. Scale bar = 100μm. (B) Western blot analysis of YAP1, TEAD1 and H3K27ac from LD and HD SF268 cells. β-Actin and histone H3 served as loading controls. (C) mRNA expression of <i>YAP1</i>, <i>TEAD1</i>, <i>KISS1</i>, <i>NEXN</i>, <i>PAWR</i>, <i>S1PR1</i>, and <i>SNAPC1</i> from cells cultured at LD or HD (normalized to <i>Ubiquitin C</i> (<i>UBC</i>)). Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.(D, E, F and G) Analysis of (D) YAP1, (E) TEAD1, (F) H3K27ac, and (G) p300 occupancy at YAP1/TEAD1 peak regions from cells cultured at LD or HD by ChIP-qPCR. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR data.</p

YAP1/TEAD1 target genes.

<p>(A) Gene expression of target genes from all, proximal (≤2Kb), distal (>2Kb) or random YAP1/TEAD1 peaks. Peaks were assigned to their closest gene TSS. (B and C) Gene enrichment analysis of YAP1/TEAD1 target genes for (B) gene ontology biological processes and (C) WikiPathways. (D) Number of genes at selected expression fold change also targeted by YAP1/TEAD1 peaks. (E) Prediction of <i>YAP1</i> expression (high: purple vs. low: green) in glioblastoma and head and neck squamous cell tumor samples using the gene features extracted from 70 genes 2-fold down-regulated in YAP1 siRNA knockdowns and targeted by a YAP1/TEAD1 peak.</p

Primers used for ChIP-qPCR (Figs 5 and S7 and S9).

<p>Primers used for ChIP-qPCR (Figs <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.g005" target="_blank">5</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.s007" target="_blank">S7</a> and <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1005465#pgen.1005465.s009" target="_blank">S9</a>).</p