Regulation of gene expression is a complex process controlled by sequence-specific DNA binding proteins, modulation of chromatin structure, and post-transcriptional modifications. In recent years, increased attention has been given to the role of epigenetic mechanisms, such as the modification of histone proteins, in gene regulation [1]. These modifications, including methylation, phosphorylation and acetylation, occur at specific amino acids on the N-terminal tails of histone core proteins, particularly H3 and H4, and regulate chromatin structure and gene expression [2,3]. Methylation of histones at lysine residues has typically been associated with transcriptionally silent heterochromatin [4]. In contrast, lysine acetylation is generally thought to trigger the opening of chromatin structure and transcriptional activation [5,6]. However, this is an oversimplified model and does not represent the true complexity of these processes, which can also differ between lower and higher eukaryotes [7]. Individual modifications of histones may be interdependent, with methylation of certain lysine residues blocking or enhancing the addition of acetyl groups nearby [8,9]. In addition, methylation of arginine residues may actually activate the transcription of some genes. A number of proteins have been identified which regulate these modifications, including histone acetyltransferases (HATs), histone deacetylases (HDACs), histone methyltransferases (HMT), and a recently discovered class of histone demethylases [10].
The protozoan parasite Entamoeba histolytica has two morphologically distinct life cycle forms, the infectious cyst form that transmits disease from person to person, and the trophozoite form that multiplies in the colon and eventually differentiates back into the cyst form. While in the colon, the trophozoite form causes invasive disease (colitis and liver abscess) in 50 million people per year making amebiasis a leading parasitic cause of death worldwide [11]. Despite its importance for human health, little is known about how this parasite modulates its gene expression during host invasion or conversion from one life cycle form to the other. Changes in transcript abundance in E. histolytica are associated with host invasion [12], with exposure to oxidative stress [13], and with conversion between the cyst and trophozoite forms [14], but the mechanisms regulating transcript levels are poorly understood. A number of amebic promoter elements and transcription factors have been described [15] and DNA methylation has been identified as playing a role in controlling a limited amount of amebic gene expression [16,17]. Functional histone-modifying enzymes, such as HATs of the MYST and GNAT families, and a Class I HDAC, and acetylated histones have been described in E. histolytica [18], but their activities have not yet been tied to gene expression changes.
In Entamoeba invadens, a parasite of reptiles, a role for histone modifications in the regulation of stage conversion has been proposed. Histones of in vitro cultured E. invadens trophozoites are constitutively acetylated, with the levels of acetylation increasing in the presence of Trichostatin A (TSA), but decreasing in the presence short chain fatty acids (SCFA) such as butyrate [19]. The decreased histone acetylation resulting from butyrate exposure was unexpected, as this compound induces increased histone acetylation in all other eukaryotic cells in which it has been examined [20-22]. Treatment of E. invadens trophozoites with TSA or SCFAs blocks their in vitro development to the cyst stage, suggesting a biological role for histone modification in Entamoeba development [23]. The link between cyst development and histone acetylation observed in E. invadens has not been recapitulated in E. histolytica due to lack of an in vitro system for encystation. Complicating the studies of E. histolytica is the fact that individual laboratory strains of the parasite have different baseline histone acetylation patterns [19]. For example, E. histolytica HM-1:IMSS under standard culture conditions does not have any detectable acetylated H4, whereas two other strains, E. histolytica Rahman and E. histolytica 200:NIH, have multiply-acetylated H4 populations under the same growth conditions. Additionally, both of these strains shift to a hyperacetylated H4 pattern when treated with TSA. Furthermore, when grown with SCFAs, E. histolytica Rahman and E. histolytica 200:NIH H4 histones become hypoacetylated, similar to the response of E. invadens. The unusual hypoacetylation response to butyrate of Entamoeba suggests that SCFAs regulate histone acetylation and gene expression in a unique way, one that most likely reflects parasite adaptation to growth in the presence of the large amounts of the short chain fatty acids found in the colon.
In other protozoan parasites histone modification plays important roles in life cycle progression and antigenic variation. In Toxoplasma gondii, chromatin immunoprecipitation analysis has demonstrated differential acetylation and methylation in the promoters of stage-specific genes during stage conversion [24]. In addition, treatment with drugs that affect histone acetylation or arginine methylation affected both stage-conversion and overall gene expression [24,25]. In Plasmodium falciparum histone H4 acetylation states and promoter occupation by the Sir2 transcriptional regulator have been linked to changes in the expression of var genes [26].
To gain insights into the role of histone acetylation in regulating gene expression in E. histolytica we treated E. histolytica trophozoites with SCFA or TSA and performed whole genome transcriptional profiling. The data revealed that in E. histolytica there was minimal transcriptional response to SCFAs, with ~0.1% of genes modulated ± 2-fold. In contrast, the transcriptional response to TSA was greater (~2% of genes modulated ± 2-fold), and the gene expression changes overlapped significantly with the transcriptional signature of the developmental pathway to cysts [14]. This work represents the first genome wide analysis of transcriptional changes associated with histone modifications in E. histolytica and reveals a subset of developmentally regulated genes whose expression correlates with changes in the level of histone acetylation.
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