Trichostatin A effects on gene expression in the protozoan parasite Entamoeba histolytica
© Ehrenkaufer et al; licensee BioMed Central Ltd. 2007
Received: 17 April 2007
Accepted: 05 July 2007
Published: 05 July 2007
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.
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 × 10-53) and 15/41 genes downregulated by TSA exposure were downregulated in E. histolytica cysts (p-value = 3 × 10-7).
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.
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 . 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 . 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 . 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 .
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 . 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 , with exposure to oxidative stress , and with conversion between the cyst and trophozoite forms , but the mechanisms regulating transcript levels are poorly understood. A number of amebic promoter elements and transcription factors have been described  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 , 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 . 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 . 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 . 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 . 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 .
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 . 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.
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) . 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 . 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
An overview of the microarrays generated and used in the analysis.
Number of arrays
Minimum correlation of the arrays in condition
TYI-S-33 or LG
Correlations of arrays used in analysis.
(TYI-S-33 or LG)
200:NIH (TYI-S-33 or LG)
E. histolytica genes regulated by exposure short-chain fatty acids.
ADP-ribosylation factor, putative
conserved hypothetical protein
70 kDa heat shock protein, putative
conserved hypothetical protein
protein kinase, putative
Growth of E. histolytica 200:NIH in the presence of TSA changes the amebic transcriptional profile
Subset of E. histolytica genes upregulated by exposure to Trichostatin A.
conserved hypothetical protein
70 kDa heat shock protein, putative
Rho family GTPase
protein kinase, putative
heat shock protein 70, putative
conserved hypothetical protein
zinc finger protein, putative
conserved hypothetical protein
E. histolytica genes downregulated by exposure to Trichostatin A.
leishmaniolysin-related peptidase, putative
Beige BEACH domain protein, putative
lipid phosphatase, putative
NADP-dependent alcohol dehydrogenase
conserved hypothetical protein
scavenger mRNA decapping enzyme, putative
fatty acid elongase, putative
high mobility group protein, putative
M-phase inducer phosphatase, putative
actobindin homolog, putative
cysteinyl-tRNA synthetase, putative
conserved hypothetical protein
protein kinase, putative
pseudogene, galactose-specific adhesin light subunit
conserved hypothetical protein
integral membrane protein, putative
cysteine proteinase, putative
cysteine protease 1
galactose-inhibitable lectin 35 kda subunit precursor
glucosidase II alpha subunit, putative
fatty acid elongase, putative
surface antigen ariel1-related
Semi-quantitative RT-PCR confirmation of array results
A substantial number genes regulated by TSA are also developmentally regulated in E. histolytica
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 , thus high expression of these genes indeed appears to be characteristic of the transcriptional profile of stage conversion.
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).
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 .
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 . Whether expression from these genomic regions is repressed by histone acetylation, or whether the effect is indirect, needs to be determined experimentally.
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 , 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 . 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 . In contrast, in human carcinoma cells TSA treatment results in DNA demethylation , one indication of the increasing levels of complexity of the mechanisms that establish histone codes in higher eukaryotes .
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 . 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 , and both species respond to TSA with similar hyperacetylation responses .
We have used whole-genome expression profiling to demonstrate that E. histolytica 200:NIH trophozoites have dichotomous responses to SCFA and TSA, both histone deacetylase inhibitors. Despite affecting changes in histone acetylation, and in contrast to data from other eukaryotic systems, short chain fatty acids induce minimal transcriptional changes in E. histolytica trophozoites. In contrast, TSA has both a significant effect on histone acetylation and induces transcriptional changes. Importantly the transcriptional pathway modulated by TSA overlaps significantly with the gene expression changes seen with developmental conversion from trophozoites to cysts. This work identifies for the first time a molecular signature of TSA effects on E. histolytica parasites and lays the groundwork for further dissection of the roles of histone acetylation on amebic development.
Entamoeba strains and culture methods
The strains used in this study were E. histolytica HM-1:IMSS and E. histolytica 200:NIH both of which were grown axenically in TYI-S-33 medium under standard culture conditions at 36.5°C . Additionally, both strains were grown in TYI-S-33 medium in the absence of glucose (LG medium), in TYI-S-33 medium plus SCFA (70 mM sodium acetate, 20 mM sodium propionate and 10 mM sodium butyrate) (SCFA medium) , and in TYI-S-33 medium plus TSA (150 nM or 300 nM) (TSA medium). Genotypes of the E. histolytica strains (HM-1:IMSS and 200:NIH) were confirmed by PCR and RFLP based on previously published methods [36, 37].
RNA isolation and microarray hybridization
Total RNA was isolated using Trizol reagent (Invitrogen) using the manufacturer's protocol and purified using a Qiagen RNeasy kit before being used for microarray analysis . Samples were processed for microarray hybridization by the Stanford University Protein and Nucleic Acids facility  using standard protocols. For each sample the RNA quality was checked using an Agilent BioAnalyzer QC and 4 μg subjected to the standard labeling and hybridization method . E. histolytica 200:NIH parasites were grown in SCFA for 16 hours, harvested and RNA extracted for microarray experiments. E. histolytica 200:NIH parasites were grown in TSA (150 nM or 300 nM) for 16 or 72 hours (150 nM), harvested and RNA extracted for microarray experiments.
Labeled samples were hybridized to a custom generated Affymetrix platform full genome microarray (E_his-1a520285F), which has been previously described . This array has 7,712 unique probe sets, which represent 9,435 open reading frames. Due to the highly repetitive nature of the E. histolytica genome, some of the probe sets are predicted to cross-hybridize with other sequences. Probe sets that represent a single gene and do not cross hybridize are labeled as (_at). Probe sets in which at least one probe may cross-hybridize with another gene(s) are labeled as (_x_at). In situations where all the probes for a given gene cross-hybridize with another gene(s), the probe sets is labeled as (_s_at) and additional genes that cross-hybridize with this probe set are listed in Additional File 3B. This array also contains probes for intergenic non-coding regions, however, these probe sets were excluded from all analysis. After hybridization, arrays were scanned and probe intensities calculation using Affymetrix GCOS software .
Microarray data normalization and analysis
Normalized expression values for each probe set were obtained from raw probe intensities in R 2.2.0 downloaded from the BioConductor project , using robust multi-array averaging with correction for oligo sequence (gcRMA) . To identify differentially expressed genes, we used local pooled error testing  along with Benjamini-Hochberg multiple test correction . In addition, fold-change was calculated in Genespring GX . A minimum of three arrays from each condition were used for analysis of SCFA or TSA effects. Correlation coefficients were calculated in Genespring using standard correlation. Probe sets were considered differentially expressed between two conditions if they had at least a 2-fold change and were significant with a false discovery rate (FDR) of < 0.05, and were identified as "present" in at least one array. Datasets of transcriptional profiles from E. histolytica HM-1:IMSS, E. histolytica Rahman, parasites from an in vivo model of colitis, encystation, and 5-AzaC treatment were obtained from previously published data [14, 16, 27].
Semi-quantitative reverse transcriptase polymerase chain reaction (RT-PCR)
E. histolytica 200:NIH trophozoites grown in TYI-S-33 LG medium were transferred in mid log phase (3 × 105 per ml) into TYI-S-33 LG or TYI-S-33 LG/150nM TSA and incubated for 16 hrs. Total RNA was isolated with RNAzol, and 2ug RNA was treated with DNAase I for 5 minutes at 37°C. cDNA was synthesized with oligo-dT and Superscript III reverse transcriptase (Invitrogen) at 50°C for 2 hr. Ten-fold dilutions of cDNA were used as template for 30 cycles of PCR amplification with gene-specific primers. PCR products were fractionated on 1.5% agarose gels, stained with ethidium bromide, and photographed with a GE/Amersham ImageQuant ECL recorder. Primers used in the study are:
135.m00113 Sense (5'-CCGAATCTGCATTTCCAACT-3') and
135.m00113 Antisense (5'-CAATCCCTCCTCCAAGTGAA-3');
135.m00113 Sense (5'-TCTACTTGGAGGAGGGATTC-3') and
135.m00113 Antisense (5'-AATGAATTTGCATTGCATGG-3');
14.m00310 Sense (5'-GCCAGTTTCATTCCATGGTT-3') and
14.m00310 Antisense (5'-TCAGGACCACCAACATTTGA-3');
337.m00049 Sense (5'-TCAATGAATTGGTCGTTTGC-3') and
337.m00049 Antisense (5'-TCGTTTTGGTGTGAAATGTTG-3');
146.m00117 Sense (5'-CCCCATCCAAAATTGAACAG-3') and
146.m00117 Antisense (5'-GGATGGGGATTAGAAACCAAA-3');
223.m00071 Sense (5'-CCTAAACTTCAGCAAGTTCATTCA-3') and
223.m00071 Antisense (5'-GAAAGAAGTTGAGCCCAAAGCA-3');
1.m00712 Sense (5'-AACAATTGGTCAATGCTTCTCA-3') and
1.m00712 Antisense (5'-TCCCAAATGAACGAATAGGC-3');
223.m00075 Sense (5'-TGCAAAAATTAATAACCTTCTTCG-3') and
223.m00075 Antisense (5'-TCCACCAACAAAACCTGAAA-3');
77.m00173 Sense (5'-CAACATCTATTGGAAAAAGACCA-3') and
77.m00173 Antisense (5'-TGGAGATAACTCCTTCTCCATCA-3');
340.m00050 Sense (5'-CATCGAATATGATATTACATCAAATG-3') and
340.m00050 Antisense (5'-TTTATTGGAATTGGGTCAATAGCATTC-3');
247.m00075 Sense (5'-TGCAAAGTCCATTTCCAACA-3') and
247.m00075 Antisense (5'-TTTCAGGAGAAAAAGTGGCTTC-3');
7.m00480 Sense (5'-TGATTGCAAAAGATTCAGAAACA-3') and
7.m00480 Antisense (5'-ACTTGACCCAAAGTCATCACG-3');
13.m00291 Sense (5'-TGCTCAATGGCATCAATGTT-3') and
13.m00291 Antisense (5-'GCTTCCATTTGGGACGTAGA-3');
ssRNA Sense: (5'-ACGAACGAGACTGAAACCTAT-3') and
ssRNA Antisense: (5'-TGTTACGACTTCTCCTTCCTC-3').
reverse transcriptase polymerase chain reaction
short chain fatty acids
false discovery rate.
We gratefully acknowledge all members of the Singh and Eichinger labs for helpful comments and suggestions, especially Jason Hackney for input on statistical analyses. GME was supported by NIH grant AI-068899. US was supported in part by NIH grants AI-053724 and AI-068899. DJE was supported by NIH grant AI-044893.
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