Gene expression can be transcriptionally controlled by epigenetic mechanisms including DNA methylation and histone modification. In order to define the genome-wide extent of regulation of gene expression by histone modification in Entamoeba histolytica, we performed expression profiling of E. histolytica trophozoites with short chain fatty acids and Trichostatin A (both histone deacetylase inhibitors). Our results identified that in contrast to effects seen in other eukaryotic systems, and despite inducing changes in histone acetylation, SCFA induce minimal transcriptional changes in E. histolytica trophozoites. However, the parasites do modulate gene expression significantly in response to TSA. The TSA induced transcriptional signature was distinct from changes induced by inhibition of DNA methylation but strongly overlapped with the gene expression profile of encystation in E. histolytica.
E. histolytica trophozoites normally grow and differentiate in the presence of SCFA while they reside in the lumen of the colon. SCFA are known to regulate gene expression in colonic epithelial cells which are normally exposed to SCFA [29-32]. When Entamoeba parasite isolates are initially collected from infected individuals, the trophozoites are cultured with the accompanying bacteria, which produce SCFA. Subsequently, E. histolytica isolates are selected for an ability to grow in medium that does not contain bacteria or SCFA. As only a small number of genes changed expression levels in response to SCFA, either the axenic parasites have lost nearly all of their transcriptional response to SCFA, or these compounds do not normally exert a large influence on gene expression at the transcriptional level in parasites in vivo. SCFAs do inhibit encystment, however, and based on the described results here, this may be occurring via more subtle changes in transcript levels (that did not meet the fold-change criteria applied to the data) or more likely through post-transcriptional mechanisms.
In contrast to SCFA, treatment of E. histolytica 200:NIH trophozoites with TSA demonstrated changes in gene transcript levels. This indicates that when class I/II HDAC enzymes are specifically targeted in Entamoeba and increased amounts of histone hyperacetylation occur [19], transcriptional changes follow. Like other eukaryotic cells, then, the expression of a small fraction of the genome of Entamoeba parasites appears to be sensitive to hyperacetylation of core histones. Transcriptional profiling was previously performed on E. histolytica parasites treated with 5-azacytidine (5-AzaC), an inhibitor of DNA methyltransferase, showing that ~2.1% of genes were differentially regulated by 5-AzaC exposure [16]. There was no significant overlap between the genes found here to be regulated by TSA and those regulated by 5-AzaC. Thus, epigenetic types of regulation, including both DNA methylation and histone acetylation, do play roles in gene expression mechanisms in E. histolytica, but the set of genes regulated by these processes is limited and non-overlapping. This is similar to the situation in Arabidopsis thaliana, in which genes regulated by 5-AzaC and TSA do not overlap, although a synergistic effect of treatment with both compounds has been observed [33]. In contrast, in human carcinoma cells TSA treatment results in DNA demethylation [34], one indication of the increasing levels of complexity of the mechanisms that establish histone codes in higher eukaryotes [7].
The greater significance of the gene expression changes induced by TSA was their overlap with the transcription profile of parasites undergoing differentiation. Initially these data may seem at odds with previously published data in which addition of TSA to encysting cultures of E. invadens was found to block encystment [23]. However, there are several possible explanations for this result. First, here we added TSA to vegetative E. histolytica trophozoite stage cells, whereas previous studies tested the effects of TSA on encysting E. invadens, and TSA effects on trophozoites and encysting parasites may be distinct. Second, the conclusions that TSA inhibits encystation in E. invadens were based on its ability to prevent production of a chitin-containing cyst, the end product of the differentiation pathway. The transcriptome data, in contrast, is a more revealing assessment of induction of the differentiation pathway. In fact, no genes known to encode proteins involved in cyst wall synthesis, such as chitin synthase or the glycoprotein Jacob, were regulated by TSA. Histone acetylation may therefore play an early role in cell fate determination and not regulate genes involved in the terminal stages of differentiation. Another possibility is that E. histolytica and E. invadens have opposing responses to TSA. However, this seems unlikely given the recent observation that conditions that support encystation in E. histolytica also permit spontaneous encystation in E. invadens [14], and both species respond to TSA with similar hyperacetylation responses [19].
Another model to consider is based on results from Toxoplasma gondii, in which the expression of both cyst and tachyzoite-specific genes is regulated by histone acetylation states [24]. This finding implied the existence of HATs and HDACs whose activities are developmentally regulated. If a similar situation were also the case in Entamoeba, trophozoite-specific HDAC activity would normally block activation of cyst-specific genes in trophozoites, and this block would be lifted upon TSA treatment, leading to the observed expression of a fraction of the cyst-specific genes. However, TSA treatment after induction of the encystation program would repress cyst-specific HDACs and inhibit the repression of trophozoite-specific genes, hence arresting the encystation program (Figure 4). Overall, the overlap between TSA-induced and cyst-specific genes and the overlap between TSA-repressed and trophozoite-specific genes is strong evidence that histone acetylation states are part of the mechanisms that regulate developmental pathways in E. histolytica.
Answers to all your Biology Questions
