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. 2015 Aug 21;11(8):e1005465.
doi: 10.1371/journal.pgen.1005465. eCollection 2015 Aug.

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

Claudia Stein  1 , Anaïs Flore Bardet  2 , Guglielmo Roma  1 , Sebastian Bergling  1 , Ieuan Clay  1 , Alexandra Ruchti  1 , Claudia Agarinis  1 , Tobias Schmelzle  3 , Tewis Bouwmeester  1 , Dirk Schübeler  4 , Andreas Bauer  1
Affiliations

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

Claudia Stein et al. PLoS Genet. .

Abstract

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 YAP1 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.

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Conflict of interest statement

The authors of this manuscript have read the journal's policy and have the following competing interests: CS, GR, SB, IC, AR, CA, TS, TB and AB are employees of Novartis Pharmaceuticals.

Figures

Fig 1
Fig 1. Genome-wide binding of YAP1 to chromatin in SF268 cells.
(A) Expression levels of YAP1, YAP1 target genes and non-target genes in SF268 (red) and LN229 (grey) cells measured by RNA-seq. (B) Correlation between replicates of YAP1 binding analysis by ChIP-seq. (C) Genomic views of YAP1 ChIP enrichment at gene promoters of known target genes. (D) Validation of YAP1 binding to known and novel sites, and control regions using ChIP-qPCR. Data are representative of at least three independent experiments. Error bars indicate the standard deviation of triplicate qPCR.
Fig 2
Fig 2. TEAD single and double motifs occur within most YAP1 binding sites.
(A and B) Enrichment of (A) TEAD and (B) AP-1 motifs in YAP1 peaks. Full list provided in S4 Table. (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.
Fig 3
Fig 3. YAP1 peaks are co-occupied by TEAD1.
(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.
Fig 4
Fig 4. YAP1/TEAD1 associate with active enhancers.
(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.
Fig 5
Fig 5. YAP1 mediates active enhancer chromatin and expression of target genes.
(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 YAP1, TEAD1, KISS1, NEXN, PAWR, S1PR1, and SNAPC1 from cells cultured at LD or HD (normalized to Ubiquitin C (UBC)). 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.
Fig 6
Fig 6. YAP1 binding sites largely overlap in cancer cell lines from distinct lineages.
(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.
Fig 7
Fig 7. YAP1/TEAD1 target genes.
(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 YAP1 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.
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