Molecular & Biochemical Parasitology 138 (2004) 205–216 Histone acetyltransferases and deacetylase in Entamoeba histolytica Girija Ramakrishnan a , Carol A. Gilchrist a , Hussein Musa a , Michael S. Torok d , Patrick A. Grant d , Barbara J. Mann a,b , William A. Petri Jr. a,b,c,∗ a Department of Medicine, University of Virginia, Charlottesville, VA 22908, USA b Department of Microbiology, University of Virginia, Charlottesville, VA 22908, USA c Department of Pathology, University of Virginia, Charlottesville, VA 22908, USA d Department of Biochemistry and Molecular Genetics, University of Virginia, Charlottesville, VA 22908, USA Received 30 March 2004; accepted 2 September 2004 Available online 3 October 2004 Abstract In our efforts to understand how transcription may be regulated in Entamoeba histolytica, we have examined if this parasite has conserved enzymatic mechanisms for targeted acetylation and deacetylation of histones. Western blotting indicated that basic nuclear proteins in the size range of 16–23kDa were acetylated in amebic trophozoites, suggesting histone acetylation. Single representatives of the GNAT and MYST family of histone acetyltransferases (HATs) were identified in the E. histolytica genome and their expression in amebic trophozoites was detected by reverse transcription of RNA followed by the polymerase chain reaction (RT-PCR). Full-length recombinant EhMYST protein demonstrated HAT activity with calf thymus histones and showed a preference for histone H4, similar to the yeast MYST protein, Esa1. However, ehMYST did not complement a yeast esa1 mutation. Histone deacetylase (HDAC) activity was detected in nuclear extracts from E. histolytica, and characteristically, was inhibited by trichostatin A (TSA). Consistent with the observation of HDAC activity, RT-PCR analysis demonstrated that an amebic hdac1 homolog (ehHDAC) is expressed and appropriately spliced in E. histolytica trophozoites. Our results suggest that mechanisms for histone acetylation and deacetylation are operational in E. histolytica. © 2004 Elsevier B.V. All rights reserved. Keywords: Entamoeba; Histone acetyltransferase; Histone deacetylase; Acetylation; Transcription 1. Introduction The intestinal protozoan parasite, Entamoeba histolytica, annually infects approximately 50 million people worldwide [1]. Invasive disease, manifesting as amebic dysentery and liver abscesses, develops in perhaps 10% of infected pa- tients. The host and parasite factors influencing development of invasive disease are not well understood. Transcription of several different amebic genes is altered in response to environmental factors such as presence of bacteria, calcium Abbreviations: HAT, histone acetyltransferase; HDAC, histone deacety- lase; ORF, open reading frame; RT-PCR, reverse transcription-polymerase chain reaction ∗ Corresponding author. Tel.: +1 434 982 0003; fax: +1 434 924 0075. E-mail address: wap3g@virginia.edu (W.A. Petri Jr.). levels, and temperature, and also in response to undefined host factors [2–5]. The E. histolytica genome encodes mul- tiple copies of genes that have been implicated in virulence functions. These include 5–6 genes for each of the heavy and the light subunits of the galactose/N-acetyl galactosamine in- hibitable adherence lectin, 3 genes encoding amebapores and 20 different cysteine proteinase genes; while all of these genes are not expressed in the trophozoite under laboratory condi- tions, they may be important for the parasitic lifestyle [6–8]. Transcriptional modulation of virulence genes is believed to be one mechanism for regulating pathogenicity of the parasite. E. histolytica is an early-branching eukaryote, and con- sequently, its transcriptional determinants are rather unique in comparison to the higher eukaryotes. Whereas the aver- age metazoan promoter is influenced by several kilobases 0166-6851/$ – see front matter © 2004 Elsevier B.V. All rights reserved. doi:10.1016/j.molbiopara.2004.09.002