Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica

Abstract

Background: Histone modification regulates chromatin structure and influences gene expression associated with diverse biological functions including cellular differentiation, cancer, maintenance of genome architecture, and pathogen virulence. In Entamoeba, a deep-branching eukaryote, short chain fatty acids (SCFA) affect histone acetylation and parasite development. Additionally, a number of active histone modifying enzymes have been identified in the parasite genome. However, the overall extent of gene regulation tied to histone acetylation is not known.

Results: In order to identify the genome-wide effects of histone acetylation in regulating E. histolytica gene expression, we used whole-genome expression profiling of parasites treated with SCFA and Trichostatin A (TSA). Despite significant changes in histone acetylation patterns, exposure of parasites to SCFA resulted in minimal transcriptional changes (11 out of 9,435 genes transcriptionally regulated). In contrast, exposure to TSA, a more specific inhibitor of histone deacetylases, significantly affected transcription of 163 genes (122 genes upregulated and 41 genes downregulated). Genes modulated by TSA were not regulated by treatment with 5-Azacytidine, an inhibitor of DNA-methyltransferase, indicating that in E. histolytica the crosstalk between DNA methylation and histone modification is not substantial. However, the set of genes regulated by TSA overlapped substantially with genes regulated during parasite development: 73/122 genes upregulated by TSA exposure were upregulated in E. histolytica cysts (p-value = 6 x 10(-53)) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 x 10(-7)).

Conclusion: This work represents the first genome-wide analysis of histone acetylation and its effects on gene expression in E. histolytica. The data indicate that SCFAs, despite their ability to influence histone acetylation, have minimal effects on gene transcription in cultured parasites. In contrast, the effect of TSA on E. histolytica gene expression is more substantial and includes genes involved in the encystation pathway. These observations will allow further dissection of the effects of histone acetylation and the genetic pathways regulating stage conversion in this pathogenic parasite.

Figure 1

Growth rates of E. histolytica HM-1:IMSS and 200:NIH in medium with Trichostatin A. Log-phase trophozoites (7,500 cells) were seeded into 14 ml tubes containing fresh media (LG or LG+TSA). Aliquots were counted every 24 hours. (A) E. histolytica HM1:IMSS parasites stopped proliferating immediately upon transfer to LG+TSA containing media. (B) E. histolytica 200:NIH can grow in LG+TSA, although at a reduced rate compared to growth in LG medium. Experiments were performed a minimum of two times and standard deviation is shown.

Figure 2

RT-PCR confirms microarray data for genes regulated by TSA. Genes found to be differentially expressed based on the array data were tested by semi-quantitative RT-PCR. RNA from log phase E. histolytica 200:NIH trophozoites exposed to 150 nM TSA for 16 hours was used to generate cDNA for the analysis. (A) Genes identified by the array analysis as upregulated in TSA treated parasites (135.m00113, 14.m00410, 337.m00049, 340.m00050 and 146.m00117) are shown. (B) Genes identified as downregulated in TSA parasites (1.m00712, 223.m00071, 223.m00075, and 77.m00173) are shown. (C) Genes identified as being unchanged in TSA treated parasites (247.m00075 and 7.m00480, 13.m00291) and small subunit ribosomal RNA (X61116) were used as a loading control. For all genes, the trends indicated by the array data were recapitulated by the RT-PCR analysis. For all samples, a control reaction without reverse transcriptase control was performed, and was negative.

Figure 3

Venn diagram of genes regulated by TSA and during stage conversion. Overlap of genes regulated by TSA with developmentally regulated genes is shown. Of 122 genes upregulated by TSA treatment, 73 also show increased expression in cysts. Of the 41 genes downregulated by TSA treatment, 15 have increased expression in trophozoites. Both of these overlaps are statistically significant (p = 6 × 10^-53 and p = 3 × 10^-7 respectively).

Figure 4

A proposed model for the role of histone acetylation in Entamoeba stage conversion. Under axenic growth conditions, trophozoite-specific HATs and HDACs induce the expression of trophozoite genes while suppressing the expression of cyst genes. When TSA is added to these cultures, the repression of HDAC activity allows expression of cyst genes. In contrast, during encystation cyst-specific HATs and HDACs become active, turning off trophozoite-specific genes and inducing cyst genes. TSA treatment of cells induced to encyst may repress the activity of cyst-specific HDACs, allowing continued expression of trophozoite genes and blocking completion of the encystation pathway. Steps sensitive to TSA treatment are highlighted in red.