Polyisoprenylated Benzophenone, Garcinol, a Natural Histone Acetyltransferase Inhibitor, Represses Chromatin Transcription and Alters Global Gene Expression

Karanam Balasubramanyam,M Altaf,Radhika A Varier,V Swaminathan,Aarti Ravindran,Parag P Sadhale,Tapas K Kundu

doi:10.1074/jbc.m402839200

Abstract

Histone acetylation is a diagnostic feature of transcriptionally active genes. The proper recruitment and function of histone acetyltransferases (HATs) and deacetylases (HDACs) are key regulatory steps for gene expression and cell cycle. Functional defects of either of these enzymes may lead to several diseases, including cancer. HATs and HDACs thus are potential therapeutic targets. Here we report that garcinol, a polyisoprenylated benzophenone derivative from Garcinia indica fruit rind, is a potent inhibitor of histone acetyltransferases p300 (IC50 approximately 7 microm) and PCAF (IC50 approximately 5 microm) both in vitro and in vivo. The kinetic analysis shows that it is a mixed type of inhibitor with an increased affinity for PCAF compared with p300. HAT activity-dependent chromatin transcription was strongly inhibited by garcinol, whereas transcription from DNA template was not affected. Furthermore, it was found to be a potent inducer of apoptosis, and it alters (predominantly down-regulates) the global gene expression in HeLa cells.

Highlights

Histone acetylation is a diagnostic feature of transcriptionally active genes
Substantial progress has been made in the study of histone deacetylases (HDACs) inhibitors, very little is known about histone acetyltransferases (HATs) inhibitors
A polyisoprenylated benzophenone from G. indica fruit rind has been found to be a potent inhibitor of histone acetyltransferases

Summary

EXPERIMENTAL PROCEDURES

Purification of Human Core Histones and Recombinant Proteins— Human core histones were purified from HeLa nuclear pellet as described previously [32]. Indicated amounts of proteins (see figure legends) were incubated in HAT assay buffer containing 50 mM Tris-HCl, pH 8.0, 10% (v/v) glycerol, 1 mM dithiothreitol, 1 mM phenylmethylsulfonyl fluoride, 0.1 mM EDTA pH 8.0, 10 mM sodium butyrate at 30 °C for 10 min in the presence and absence of garcinol followed by addition of 1 ␮l of 4.7 Ci/mmol [3H]acetyl-CoA and were further incubated for another 10 min. HDAC Assay—The deacetylation assay was performed as described previously [31], Briefly 2.4 ␮g of core histones were incubated in HAT buffer without NaBu, with 20 ng of p300 and 1 ␮l of 4.7 Ci/mmol [3H]acetyl-CoA for 30 min at 30 °C. Harvested cells were washed with PBS and lysed with lysis buffer containing 0.5% Triton X-100, 20 mM Tris, and 15 mM EDTA at room temperature for 15 min. Guidelines set by MIAME were followed, and the raw microarray data will be deposited in the GEO data base (www.ncbi.nlm.nih.gov/geo/)

RESULTS AND DISCUSSION

TABLE I Inhibition kinetic parameters

Transcription factors

Transcription factor