HNF4α and CDX2 Regulate Intestinal YAP1 Promoter Activity.

Larsen Larsen,Pedersen Pedersen,Troelsen Troelsen,Dahlgaard Dahlgaard,Davidsen Davidsen

doi:10.3390/ijms20122981

Abstract

The Hippo pathway is important for tissue homeostasis, regulation of organ size and growth in most tissues. The co-transcription factor yes-associated protein 1 (YAP1) serves as a main downstream effector of the Hippo pathway and its dysregulation increases cancer development and blocks colonic tissue repair. Nevertheless, little is known about the transcriptional regulation of YAP1 in intestinal cells. The aim of this study to identify gene control regions in the YAP1 gene and transcription factors important for intestinal expression. Bioinformatic analysis of caudal type homeobox 2 (CDX2) and hepatocyte nuclear factor 4 alpha (HNF4α) chromatin immunoprecipitated DNA from differentiated Caco-2 cells revealed potential intragenic enhancers in the YAP1 gene. Transfection of luciferase-expressing YAP1 promoter-reporter constructs containing the potential enhancer regions validated one potent enhancer of the YAP1 promoter activity in Caco-2 and T84 cells. Two potential CDX2 and one HNF4α binding sites were identified in the enhancer by in silico transcription factor binding site analysis and protein-DNA binding was confirmed in vitro using electrophoretic mobility shift assay. It was found by chromatin immunoprecipitation experiments that CDX2 and HNF4α bind to the YAP1 enhancer in Caco-2 cells. These results reveal a previously unknown enhancer of the YAP1 promoter activity in the YAP1 gene, with importance for high expression levels in intestinal epithelial cells. Additionally, CDX2 and HNF4α binding are important for the YAP1 enhancer activity in intestinal epithelial cells.

Highlights

The Hippo pathway is a signaling pathway that is important for tissue homeostasis, regulation of organ size and tumorigenesis [1,2]
The yes-associated protein 1 (YAP1) gene was analyzed by aligning all data tracks and identifying gene-regulatory regions by comparing the signal intensity from the ChIP-seq data for transcription factors and the DNase I track which is an indicator for transcriptionally active regions (Figure 1)
Three regions with clear caudal type homeobox 2 (CDX2) ChIP-seq peaks were chosen for further analysis, one region was in the YAP1 promoter, one in intron 3 and another was in intron 4

Summary

Introduction

The Hippo pathway is a signaling pathway that is important for tissue homeostasis, regulation of organ size and tumorigenesis [1,2]. It acts as a switch between proliferation and differentiation [1,3]. A study in hepatocellular carcinoma cells showed that knockdown of the heparan sulfate proteoglycan Glypican-3 decreased YAP1 expression [8]. Both transcriptional regulator ERG (ERG) and TEAD4 can interact with the YAP1 promoter and increase H3K9/14Ac acetylation which leads to increased YAP1 expression in prostate tumors [9]. Methylation of the YAP1 promoter in polycystic ovary syndrome (PCOS) was shown to increase YAP1 protein levels [10]

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