Tip60 Is Regulated by Circadian Transcription Factor Clock and Is Involved in Cisplatin Resistance

Hiroto Izumi,Yoshihiro Nakajima,Naoya Miyamoto,Masaki Shiota,Akihiko Kidani,Takako Noguchi,Kimitoshi Kohno,Yoshihiro Ohmiya,Akihiko Tawara

doi:10.1074/jbc.m802332200

Abstract

Histone modification is important for maintaining chromatin structure and function. Recently, histone acetylation has been shown to have a critical regulatory role in both transcription and DNA repair. We report here that expression of histone acetyltransferase (HAT) genes is associated with cisplatin resistance. We found that Tip60 is overexpressed in cisplatin-resistant cells. The expression of two other HAT genes, HAT1 and MYST1, did not differ between drug-sensitive and -resistant cells. Knockdown of Tip60 expression rendered cells sensitive to cisplatin but not to oxaliplatin, vincristine, and etoposide. Tip60 expression is significantly correlated with cisplatin sensitivity in human lung cancer cell lines. Interestingly, the promoter region of the Tip60 gene contains several E boxes, and its expression was regulated by the E-box binding circadian transcription factor Clock but not by other E-box binding transcription factors such as c-Myc, Twist, and USF1. Hyperacetylation of H3K14 and H4K16 was found in cisplatin-resistant cells. The microarray study reveals that several genes for DNA repair are down-regulated by the knockdown of Tip60 expression. Our data show that HAT gene expression is required for cisplatin resistance and suggest that Clock and Tip60 regulate not only transcription, but also DNA repair, through periodic histone acetylation.

Highlights

The development of cisplatin resistance is a major clinical limitation in cancer chemotherapy
We have previously shown that expression of activating transcription factor 4 (ATF4)2 is inducible by cisplatin treatment and is high in cisplatin-resistant cells [14]
We have found that ATF4 gene expression is regulated by the circadian transcription factor Clock, which is overexpressed in cisplatin-resistant cells [16]

Summary

Histone Acetyltransferase and Cisplatin Resistance

A, control siRNA (100 pmol) or Tip siRNA (100 pmol) were transfected into PC3 cells or P/CDP6 cells, and whole-nuclear lysates (50 ␮g) were subjected to SDS-PAGE. Fifty ␮g of whole-nuclear lysates were subjected to SDS-PAGE, and Western blotting was performed with the indicated antibodies. Whole-nuclear lysates (50 ␮g for c-Myc and USF1, and 100 ␮g for Twist) were subjected to SDS-PAGE, and Western blotting was performed with the indicated antibodies. Tip, HAT1, MYST1, Twist, c-Myc, USF1, and inverted control duplex Stealth RNA (Invitrogen) diluted in 250 ␮l of. The remaining cells were seeded in 100-mm dishes with 10 ml of culture medium and harvested after 96 h culture for Western blotting, as described above

Chromatin Immunoprecipitation

Circadian Transcription Factor

Accession number

Findings

DISCUSSION