E. histolytica strains HM-1:IMSS, Rahman, and 200:NIH have similar but not identical expression profiles of genes encoding histone-modifying enzymes
To account for differences in levels of histone acetylation between E. histolytica strains, we analyzed previously published data from a whole genome microarray to compare the gene expression profiles of three strains of E. histolytica (HM-1:IMSS, Rahman and 200:NIH) [14]. Overall, the expression profiles of the three E. histolytica strains were highly similar although some genes whose transcript levels were significantly different between strains (± 2-fold and p-value <0.05) were identified. Overall, 127 genes had higher expression in E. histolytica HM-1:IMSS, 261 genes had higher expression in E. histolytica 200:NIH, and 71 genes had higher expression in E. histolytica Rahman compared to the other two E. histolytica strains (Additional File 1). For our purposes, we focused on the expression levels of genes involved in the regulation of histone acetylation and chromatin structure. Surprisingly, given the absence of multiply acetylated histones in E. histolytica HM-1:IMSS, several HAT genes (2.m00560 and 67.m00100) were expressed at relatively high levels in HM-1:IMSS trophozoites (Additional File 2). Some differences in expression levels of histone modification genes between the strains were identified. One HAT (100.m00145) had significantly higher expression E. histolytica 200:NIH. Two Sir2 family HDAC genes were expressed differentially between strains: 251.m00088 was expressed at significantly higher levels in E. histolytica HM-1:IMSS and 2.m00521 was more highly expressed in E. histolytica 200:NIH. The overexpression of particular Sir2 genes in yeast leads to global histone deacetylation [6]. The increased expression of a HAT gene in E. histolytica 200:NIH trophozoites and high expression of a Sir2 HDAC gene in E. histolytica HM1:IMSS is consistent with the histone acetylation patterns in these strains. However, the actual levels histone proteins and their relative enzyme activities in these parasite strains will need to be established before conclusions can be made about the causes of the differences in levels of multiply-acetylated histones in the isolates.
Growth of E. histolytica in the presence of SCFA has minimal effects on parasite gene expression
SCFA have substantial effects on histone acetylation and development in Entamoeba [19,23]. To determine the effect of SCFA on amebic growth, we tested the ability of E. histolytica to grow in TYI-S-33 LG medium ± SCFA. In media with SCFA, there were no significant alterations in growth of E. histolytica HM-1:IMSS and 200:NIH parasites at 16 hours, the time points at which the microarray experiments were performed (data not shown). To determine if growth with SCFA altered parasite gene expression, we compared the transcriptional profile of E. histolytica 200:NIH trophozoites grown in TYI-S-33 LG medium ± SCFA. The E. histolytica 200:NIH strain was used for this and subsequent microarray analyses because it has an histone acetylation pattern similar to encystation-competent E. invadens and hence was the most likely to provide insights into developmental pathways regulated by histone acetylation [19]. For each strain and culture condition the number of arrays and the correlations between array data sets are outlined in Tables 1 and 2. Genes were considered differentially expressed if they had a ≥ 2-fold change and were significant with an FDR of < 0.05. Overall, few changes in gene expression were observed when we compared E. histolytica 200:NIH grown in TYI-S-33 or LG medium to those grown in LG medium with SCFA. Only 11 genes were differentially regulated by addition of SCFA, and there were no changes in genes associated with histone modifications (Table 3; Additional File 3). Thus, although growth of E. histolytica 200:NIH trophozoites with SCFAs results in hypoacetylation of H4 histones [19], these changes are apparently not associated with significant alterations in the gene expression profile of axenically grown trophozoites.
Growth of E. histolytica 200:NIH in the presence of TSA changes the amebic transcriptional profile
To determine the effect of TSA on amebic growth, E. histolytica strains HM-1:IMSS and 200:NIH were grown in TYI-S-33 LG medium ± TSA. E. histolytica HM-1:IMSS trophozoites died within 2–3 days in medium with 150 nM TSA, whereas E. histolytica 200:NIH survived and grew but at a much slower rate than in TYI-S-33 LG medium alone (Figure 1). As treatment with TSA has been shown to cause histone H4 hyperacetylation [19], the effects of TSA on parasite gene expression were determined by comparing the transcriptional profile of E. histolytica 200:NIH trophozoites grown in TYI-S-33 LG medium ± TSA. Four microarrays were hybridized with RNA from E. histolytica 200:NIH parasites grown in TYI-S-33 or LG medium and compared to three microarrays hybridized with RNA from E. histolytica 200:NIH parasites grown in LG medium plus TSA for 16–72 hours. In contrast to the minimal transcriptional changes seen in the SCFA treatment, TSA exposure resulted in significant changes in gene expression. Overall, 163 genes, ~2% of the genes tested, showed altered transcript abundance, with 122 genes upregulated and 41 genes downregulated by TSA exposure (Tables 4 and 5; Additional File 4). Of the 122 genes whose expression increased with TSA treatment, 46 (38%) had normalized expression < 0.2 in 200:NIH trophozoites grown in TYI-S-33 or LG, indicating that they may be silenced under normal in vitro growth conditions.
Semi-quantitative RT-PCR confirmation of array results
To confirm the expression patterns observed by microarray analysis, we performed semi-quantitative RT-PCR on 5 genes upregulated by exposure to TSA (135.m00113, 14.m00310, 337.m00049, 340.m00050 and 146.m00117), and 4 genes downregulated by exposure to TSA (1.m00712, 223.m00071, 223.m00075, and 77.m00173) (Figure 2). The gene for ssRNA, 247.m00075, 13.m00291 and 7.m00480 were used as loading controls. Serial dilutions of cDNA were performed for each sample. In all cases, RT-PCR results confirmed the array data.
A substantial number genes regulated by TSA are also developmentally regulated in E. histolytica
We compared the lists of genes regulated by TSA to a number of transcriptional profiles previously generated with E. histolytica parasites. There was no significant overlap of genes modulated by TSA with parasite genes modulated in a mouse model of colitis [27] or after exposure to 5-Azacytidine [16]. However, the profile of genes regulated by TSA was found to overlap substantially with the profiles of genes differentially expressed in the two developmental stages (trophozoites and cysts) of E. histolytica [14] (Figure 3 and Tables 4 and 5). There was significant overlap between genes upregulated by TSA treatment and cyst-specific genes, with 73 of the 122 genes upregulated by TSA also upregulated in cysts (p-value = 6 × 10-53). Genes downregulated by TSA treatment overlapped significantly with trophozoite-specific genes, with 15 of the 41 genes downregulated by TSA also downregulated in cysts (p-value = 3 × 10-7). There was no significant overlap in the opposite direction (4 genes downregulated by TSA were upregulated in cysts and 4 genes upregulated by TSA were downregulated in cysts). Genes that were upregulated in both TSA-treated trophozoites and in cysts include some of the most highly induced genes under both conditions. An example is the hypothetical protein (489.m00024), which shows a ~40-fold increase in expression in TSA treated parasites and >500-fold increase in cysts [14]. Also included in this group are several genes encoding heat shock proteins (418.m00028 and 136.m00105) and putative signaling molecules (acid sphingomyelinase, 18.m00321 and a protein kinase, 395.m00030).
Genes regulated in E. histolytica 200:NIH by exposure to Trichostatin A
Heat shock proteins
A number of heat shock proteins, including Hsp70 isoforms (64.m00148, 584.m00019, 65.m00150 and 418.m00028) were induced by TSA treatment. Whether these genes are regulated by histone acetylation, or whether their induction is due to a stress response of the parasites to growth in TSA is unclear at this point. A gene expression response to heat shock was previously reported to be linked to encystation in E. invadens [28], thus high expression of these genes indeed appears to be characteristic of the transcriptional profile of stage conversion.
Signaling molecules
Genes regulated by treatment with TSA include several that are likely to have functions in signal transduction. These include protein kinases (14.m00339 and 395.m00030) and a Rho family GTPase (110.m00118), all with increased expression in TSA-treated parasites. A protein kinase (223.m00070) and a protein phosphatase (131.m00139) are both downregulated during TSA treatment. The regulation of these putative signaling molecules by TSA may suggest a role for histone acetylation in modulating signal transduction and responses to environmental factors in E. histolytica. Also upregulated by TSA are several genes, which could play a role in transcriptional regulation such as a Myb family protein (175.m00117 and zinc finger domain containing proteins (211.m00072 and 68.m00203).
Virulence
Several genes with roles in E. histolytica virulence were downregulated by TSA treatment. This includes two genes encoding cysteine proteases: CP1 (242.m00078) and a putative CP (10.m00362), lysozyme (52.m00148) and a gene encoding the 35 kDa subunit of the amebic Gal/GalNAc lectin (17.m00351). Several of these genes have previously been identified as being trophozoite-specific, thus their down regulation is a further indication of the transcriptional activation of the encystation pathway in TSA-treated parasites [14].
Genomic regions controlled by histone acetylation
We investigated whether there were genomic regions containing multiple genes that were regulated by TSA. Such regions may be indicative of regions where gene expression is regulated by chromatin structure. We identified a cluster of three genes on scaffold 123 (123.m00113, 123.m00122, and 123.m00123) that were all upregulated by TSA. Additionally, a large cluster of genes strongly down regulated by TSA was observed on scaffold 223 (223.m00067, 223.m00068, 223.m00069, 223.m00070, 223.m00071, 223.m00074, 223.m00075, 223.m00076, 223.m00077, 223.m00078 and 223.m00079). The 223 chromosomal region had also been identified as being enriched for trophozoite-specific genes [14]. Whether expression from these genomic regions is repressed by histone acetylation, or whether the effect is indirect, needs to be determined experimentally.
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