Transcriptional Regulation of the MDR1 Gene by Histone Acetyltransferase and Deacetylase Is Mediated by NF-Y

ABSTRACT

Recent studies have shown that the histone-modifying enzymes histone acetyltransferase (HAT) and histone deacetylase (HDAC) are involved in transcriptional activation and repression, respectively. However, little is known about the endogenous genes that are regulated by these enzymes or how specificity is achieved. In the present report, we demonstrate that HAT and HDAC activities modulate transcription of the P-glycoprotein-encoding gene, MDR1. Incubation of human colon carcinoma SW620 cells in 100-ng/ml trichostatin A (TSA), a specific HDAC inhibitor, increased the steady-state level ofMDR1 mRNA 20-fold. Furthermore, TSA treatment of cells transfected with a wild-type MDR1 promoter/luciferase construct resulted in a 10- to 15-fold induction of promoter activity. Deletion and point mutation analysis determined that an inverted CCAAT box was essential for this activation. Consistent with this observation, overexpression of p300/CREB binding protein-associated factor (P/CAF), a transcriptional coactivator with intrinsic HAT activity, activated the wild-type MDR1 promoter but not a promoter containing a mutation in the CCAAT box; deletion of the P/CAF HAT domain abolished activation. Gel shift and supershift analyses identified NF-Y as the CCAAT-box binding protein in these cells, and cotransfection of a dominant negative NF-Y expression vector decreased the activation of the MDR1promoter by TSA. Moreover, NF-YA and P/CAF were shown to interact in vitro. This is the first report of a natural promoter that is modulated by HAT and HDAC activities in which the transcription factor mediating this regulation has been identified.

ACKNOWLEDGMENTS

We thank Y. Nakatani for the P/CAF wild-type and HAT deletion expression vectors, R. Mantovani and R. A. Currie for antibodies against NF-YA and NF-YB, R. Mantovani for the NF-YA29 dominant negative construct, and Victoria Richon and Steven Swendenman for helpful discussions. We also thank the members of our laboratory, particularly Tan Ince, for critical review of the manuscript, Sarah Thayer forMDR1 promoter constructs, and Yixing Lin and Kirk Pabon for gel shift studies.

This work was supported by National Cancer Institute grants P30-CA-08748 (Memorial Sloan-Kettering Cancer Center) and RO1-CA-57307 (K.W.S.).

ADDENDUM IN PROOF

While this paper was under review, R. A. Currie also described a direct physical interaction between NF-Y and both P/CAF and GCN5 (J. Biol. Chem. 273:1430–1434, 1998).