Figures

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

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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. 

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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. 

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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). 

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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.