Transcriptional integration of mitogenic and mechanical signals by Myc and YAP - PubMed (original) (raw)

. 2017 Oct 15;31(20):2017-2022.

doi: 10.1101/gad.301184.117. Epub 2017 Nov 15.

Serena De Fazio 1, Francesca Biagioni 1, Elisa Donato 1, Marieta Caganova 1, Laura Curti 1, Mirko Doni 2, Silvia Sberna 1, Deborah Aldeghi 1, Chiara Biancotto 1, Alessandro Verrecchia 2, Daniela Olivero 3, Bruno Amati 1 2, Stefano Campaner 1

Affiliations

Transcriptional integration of mitogenic and mechanical signals by Myc and YAP

Ottavio Croci et al. Genes Dev. 2017.

Abstract

Mammalian cells must integrate environmental cues to determine coherent physiological responses. The transcription factors Myc and YAP-TEAD act downstream from mitogenic signals, with the latter responding also to mechanical cues. Here, we show that these factors coordinately regulate genes required for cell proliferation. Activation of Myc led to extensive association with its genomic targets, most of which were prebound by TEAD. At these loci, recruitment of YAP was Myc-dependent and led to full transcriptional activation. This cooperation was critical for cell cycle entry, organ growth, and tumorigenesis. Thus, Myc and YAP-TEAD integrate mitogenic and mechanical cues at the transcriptional level to provide multifactorial control of cell proliferation.

Keywords: Hippo signaling; Myc; TEAD; YAP; transcription.

© 2017 Croci et al.; Published by Cold Spring Harbor Laboratory Press.

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Figures

Figure 1.

Figure 1.

Myc and YAP coregulate cell cycle entry. Serum-starved subconfluent (sparse) (A) or highly confluent (confluent) 3T9MycER;YAP (B_–_H) cells were treated with OHT to activate MycER and doxycycline (dox) to trigger the expression of YAPS127A. (A) Cell cycle entry was measured by immunofluorescence analysis of EdU incorporation. DAPI was used to color nuclei. (B) Ranked heat map based on the log2 fold change of the differentially expressed genes (DEGs) identified by RNA sequencing (RNA-seq). (C) Box plot of the mRNA expression level of the Myc-dependent serum response (MDSR) genes (D) Gene ontology map based on the DEGs determined upon both MycER activation and YAP induction. (E) Cumulative bar graph of Myc, YAP, and TEAD ChIP-seq (chromatin immunoprecipitation [ChIP] combined with high-throughput sequencing) peaks, color-coded based on their overlap. (Y) YAP; (M) Myc; (T) TEAD. (F,G) Box plot of the enrichment of Myc (F) and YAP (G) ChIP-seq peaks divided into subsets as in E. (H) Expression levels of up-regulated genes cobound at their promoters by YAP, Myc, and TEAD (YMT peaks).

Figure 2.

Figure 2.

Myc-driven cell cycle entry depends on YAP activity and cytoskeletal tension. (A_–_E) Serum-starved subconfluent fibroblasts were kept in low serum and treated as indicated. (A) Immunofluorescence analysis of Myc-induced cell cycle entry of 3T9MycER measured as EdU incorporation on cells treated with the YAP inhibitor verteporfin (VP). (B) Expression analysis (clustering) of Myc up-regulated genes following VP treatment. (C) RT-qPCR expression of Myc target genes in MycER fibroblasts either wild type (YAP+/+) or knockout (Yap−/−) for Yap. (D) S-phase entry by BrdU incorporation (by FACS) in MycER fibroblasts overexpressing YAPS127A/S318A. Cells were treated with OHT to activate MycER and with the ROCK inhibitor Y276632 (Y27) as indicated. (E) Clustered heat map of normalized mRNA expression of cells shown in D. (F) Anchorage-independent growth assay of bipotential mouse embryonic liver (BMEL) cells overexpressing MycER and tet-YAPS127A, treated as indicated. Representative pictures of cell colonies are shown.

Figure 3.

Figure 3.

Cooperative binding and transcriptional activation by Myc, TEAD, and YAP. Genome-wide analyses of livers from R26-rtTA mice either wild type (wt), transgenics for Myc (tet-Myc) or YAP (tet-YAP), or double transgenics (tet-Myc/YAP). Short-term induction was achieved by feeding mice with doxycycline-containing food for 48 h. (A) Liver sections stained with an anti-Ki67 antibody. (B) Hierarchical clustering of DEGs. (C) Box plot showing a representative cluster of YAP/Myc DEGs. (D) Venn analysis of Myc, YAP, and TEAD ChIP-seq peaks. The number of peaks determined for each TF is reported in brackets; the arrows point to the number of overlapping peaks. (E) Ranked heat maps of the ChIP-seq enrichment of the indicated TFs. (Top panel) YAP peaks detected only in tet-YAP livers. (Bottom panel) Promoters bound by YAP in tet-Myc/YAP livers. (F,G) H3K27ac (F) and H3K4me3 (G) levels at promoters of DEG-up genes cobound by Myc and YAP.

Figure 4.

Figure 4.

Myc and YAP cooperate in inducing liver growth and tumorigenesis. (A) Liver weight assessed at 5 wk of induction. Data are reported as percentage relative to total body weight. (B) Kaplan-Meier disease-free survival analysis. (C) Western blotting analysis of YAP and Myc levels in LAP-tTA tet-YAP mice at the pretumoral stage (4 wk of YAP activation) and in tumors. Vinculin (vin) was used as aninternal control for equal loading. (D) Box plot of the expression level of MDSR genes up-regulated in the liver upon YAP and/or Myc induction. (Inset at the right) Ranked heat map. (E, top panel) Heat map of Myc and YAP/TAZ gene signatures based on the expression data of breast cancers (TCGA_BRCA). The heat map was clustered by breast cancer subtypes (basal-like, normal-like, and Luminal/Her2+). (Bottom panel) The statistical track shows the logarithmic plot of _P_-values for each gene. (Red bars) Genes up in basal-like; (green bars) genes up in Luminal/Her+.

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