YAP Drives Growth by Controlling Transcriptional Pause Release from Dynamic Enhancers

Abstract

The Hippo/YAP signaling pathway is a crucial regulator of tissue growth, stem cell activity, and tumorigenesis. However, the mechanism by which YAP controls transcription remains to be fully elucidated. Here, we utilize global chromatin occupancy analyses to demonstrate that robust YAP binding is restricted to a relatively small number of distal regulatory elements in the genome. YAP occupancy defines a subset of enhancers and superenhancers with the highest transcriptional outputs. YAP modulates transcription from these elements predominantly by regulating promoter-proximal polymerase II (Pol II) pause release. Mechanistically, YAP interacts and recruits the Mediator complex to enhancers, allowing the recruitment of the CDK9 elongating kinase. Genetic and chemical perturbation experiments demonstrate the requirement for Mediator and CDK9 in YAP-driven phenotypes of overgrowth and tumorigenesis. Our results here uncover the molecular mechanisms employed by YAP to exert its growth and oncogenic functions, and suggest strategies for intervention.

Figure 1. YAP binds to potent enhancers

A) Venn diagram of the common TEAD1/4 and YAP peaks in HuCCT1 cells. B) Scatter plot of the distance distribution of YAP peaks from the closest TSS in the indicated cell lines. C) Heatmap for the ChIP-seq signal of the indicated antibodies +/−2Kb from the center of YAP peaks. Clustering results from K-means method. D) Box plots of the normalized counts of H3K27ac and H3K4me1 signal at YAP+ enhancer peaks or YAP− enhancers. *** p<0.0001. E) Boxplot indicating expression levels of genes associated to YAP+ or YAP− enhancers following RNAseq in HUCCT1 cells. F) Histogram indicating the fraction of YAP− or YAP+ enhancers catalogued as regular o super- enhancers in HuCCT1 cells. G) Line plots depicting H3K27Ac and H3K3me1 average signal in YAP+ enhancers upon siYT treatment. H) Log2 changes in gene expression between siControl / siYT treated cells for genes associated with different genomic elements 48h post transfection. Error bars represent 95% CI. I) Left panel, ChIP-qPCR for H3K27ac in selected YREs in cells at low and high density. Error bars = SD. Right panel, immunofluorescence for YAP in these conditions. See also Figure S1.

Figure 2. YAP controls RNA Polymerase II promoter pause-release

A–B) Line graph representing the pausing index (PI) for YAP positive genes upon YAP/TAZ silencing (siYT) (A); and YAP+ enhancer genes vs. YAP− (B). C) Box plot representing the fold change of RNA PolII pS5 at the promoter (pink shades) or pS2 in the gene body (green shades) in YAP+ and YAP− enhancer genes upon siYT treatment. D) ChIP-seq tracks representing signal for RNA PolII pS5 or pS2 around a representative YAP− gene and a YAP+ gene. E) Metagene profile of RNAPolII pS5 (upper) and pS2 (lower) for YAP+ and YAP− super-enhancer genes in cells treated with siC and siYT. F) Box plot showing the occupancy of PolII pS5 at the promoter (pink shades) or pS2 in the gene body (green shades) in YAP+ and YAP− enhancer genes. See also Figure S2.

Figure 3. YAP recruits Mediator to regulate transcription

A) Validation co-IP between YAP-TEAD1 and MED12. B) ChIP-seq tracks of a YAP+ and YAP− locus for YAP, MED1 and SMC1 in HuCCT1 cells treated with siC or siYT. C) Boxplot representing normalized MED1 (left) or SMC1 (right) coverage in YAP+ or YAP− enhancers in siC- or siYT-treated cells. D) Scatter plot correlating YAP occupancy to loss of MED1 signal upon YAP/TAZ silencing. On the X-axis MED1 peaks are ranked according to YAP signal. Y-axis in the top panel represents MED1 signal changes between siC and siYT cells. Y-axis in the lower panel represents the counts of YAP signal in each MED1 peak. E) ChIP-qPCR for MED1 and SMC1 at selected YREs in H69 cells at low and high density. Error bars = SD. F) Tumor volume of xenograft HuCCT1 bearing TetOYAP and/or shMED1 constructs relative to volume measured before the administration of Doxycycline (n= 5 mice, error bars = SEM). See also Figure S3.

Figure 4. YAP-Mediator regulates transcriptional elongation via CDK9 recruitment

A) Normalized CDK9 coverage around the promoter of YAP negative- and YRE-associated genes. Error bars = 95% CI B) Fold change of CDK9 occupancy upon siYT treatment around the promoter of YAP negative- and YRE-associated genes. Error bars = 95% CI C) Log2 (FC) of expression of YAP− vs. YAP+ genes in HuCCT1 cells treated with CDK9 inhibitors Flavopiridol and NVP-2. Error bars = 95% CI. D) Liver/Body weight ratio of mice overexpressing YAP for 1 week treated with vehicle or Flavopiridol. E) Model for YAP/Mediator-driven transcriptional elongation in active Hippo signaling (left) or inactive Hippo signaling (right). See also Figure S4.