A stem cell marker KLF5 regulates CCAT1 via three-dimensional genome structure in colorectal cancer cells

Takashi Takeda,Shogo Kobayashi,Toshitsugu Fujita,Yuhki Yokoyama,Daisuke Okuzaki,Mamoru Uemura,Hidekazu Takahashi,Kaho Asai,Norikatsu Miyoshi,Hidetoshi Eguchi,Yuichiro Doki,Hirofumi Yamamoto,Hiroaki Itakura,Hiroo Ueno,Hodaka Fujii,Masaki Mori

doi:10.1038/s41416-021-01579-4

Abstract

BackgroundKLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene. In this study, we aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC).MethodsIn vitro engineered DNA-binding molecule-mediated chromatin immunoprecipitation (enChIP)-seq method was used to identify the regions that bind to the KLF5 promoter.ResultsWe revealed that the KLF5 promoter region interacted with the KLF5 enhancer region as well as the transcription start site (TSS) region of the Colon Cancer Associated Transcript 1 (CCAT1) gene. Notably, the heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. In vitro analysis indicated that KLF5 regulated CCAT1 expression and we found that CCAT1 expression was highly correlated with KLF5 expression in CRC clinical samples.ConclusionsOur data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with co-factors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC.

Highlights

Krüppel-like factor 5 (KLF5) plays a crucial role in stem cells of colorectum in cooperation with Lgr5 gene
The binding DNA sequence was analysed by NGS using two guide RNAs designed 400–600 bp the chemoresistance, sphere-formation ability and expression level of Cancer stem cells (CSCs)-related genes in heterodeletion mutants of the KLF5 upstream of the transcription start site in the KLF5 promoter enhancer
A single KLF5 and Cancer Associated Transcript 1 (CCAT1) expression are correlated in colorectal cancer (CRC) clinical knockdown of BRD4, MED1 and RAD21 and triple knockdown samples of these genes led to a modest decrease in KLF5 expression We examined the expression of the KLF5 protein and CCAT1 (Fig. 2c and Supplementary Fig. S3D)

Summary

Introduction

KLF5 plays a crucial role in stem cells of colorectum in cooperation with Lgr gene. We aimed to explicate a regulatory mechanism of the KLF5 gene product from a view of three-dimensional genome structure in colorectal cancer (CRC). The heterodeletion mutants of KLF5 enhancer impaired the cancer stem-like properties of CRC cells. The KLF5 protein participated in the core-regulatory circuitry together with co-factors (BRD4, MED1, and RAD21), which constructs the three-dimensional genome structures consisting of KLF5 promoter, enhancer and CCAT1 TSS region. CONCLUSIONS: Our data propose the mechanistic insight that the KLF5 protein constructs the core-regulatory circuitry with cofactors in the three-dimensional genome structure and coordinately regulates KLF5 and CCAT1 expression in CRC. KLF5 is associated with CSC-like properties because KLF5 knockdown suppressed sphere-formation activity in colorectal cancer (CRC) cell lines [10]. KLF5 deletion prevented the tumourigenesis of Lgr5+ intestinal stem cells induced by the mutated β-catenin gene [12]

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Conclusion