In order to elucidate the effects of inhibiting genomic DNA methylation in E. histolytica, we grew parasites in 23 μM 5-AzaC for up to seven days with routine passaging and assessed growth rates and in vitro virulence. We used two different E. histolytica strains HM-1:IMSS and 200:NIH, both of which are considered virulent 2829. For E. histolytica HM-1:IMSS parasites exposed to 23 μM 5-AzaC there was some reduced growth at day 2, compared to the untreated control, however, the parasite numbers in subsequent days were consistently equivalent (Figure 1A). In E. histolytica 200:NIH treated with 23 μM 5-AzaC, we did not observe any statistically significant effect on growth for up to seven days compared to the untreated control (Figure 1B). Additionally, we grew E. histolytica HM-1:IMSS for five consecutive days without routine subculturing, and did not observe any growth differences in parasites grown with or without 23 μM 5-AzaC (see Additional file 1A). We could not reliably grow parasites for >5 days without subculturing due to overcrowding of the cultures and parasite death. For the 200:NIH strain, the parasite numbers in 5-AzaC treated and untreated cultures were also equivalent at all time points tested, up to day 5 (data not shown). Under these conditions, we assessed the protein content per cell of E. histolytica HM-1:IMSS parasites at days 2, 3, 4, and 5 of growth, and found that there were no significant differences in protein content per cell between drug treated and untreated parasites or between parasites at different time points (see Additional file 1B). Similar data were observed for the 200:NIH strain (data not shown). We also grew the E. histolytica HM-1:IMSS strain (± 23 μM 5-AzaC) with routine subculturing every two days with an equal volume of culture medium and parasites passed at each time. Under these conditions, the growth of the parasites in 23 μM 5-AzaC was slightly reduced at day 2, however the rate of growth was subsequently equivalent to the untreated control at days 4 and 6 (see Additional file 1C). Overall, these data indicate that there is not a substantial and sustained effect on the growth of the E. histolytica parasites for up to 6–7 days in the presence of 23 μM 5-AzaC.

For both E. histolytica HM-1:IMSS and E. histolytica 200:NIH, 5-AzaC exposure for 3 or 7 days resulted in parasites that were reduced in virulence (52% or 63% of untreated levels respectively at day 3 and 28% and 35% of untreated levels respectively at day 7) (Figure 1C). Exposure to 5-AzaC at 23 μM did not affect CHO cell viability and addition of 23 μM 5-AzaC to the parasites at the time of the CHO cell killing assay did not affect parasite virulence (data not shown). Thus the reduction in ability of the parasites to kill CHO cells was dependent on prior exposure of the parasites to 5-AzaC. Although this was an in vitro cell killing assay and thus an indirect measure of parasite virulence, our results correlate with previously published data of in vitro and in vivo virulence attenuation in E. histolytica HM-1:IMSS grown with 23 μM of 5-AzaC for 1 week 1516. Since E. histolytica parasite strains are genomically diverse (as shown by comparative genomic hybridizations) 30 and have genome-wide expression differences 31, it was not clear if the effects of 5-AzaC could be generalized to other E. histolytica strains. We have now shown that two genetically distinct, but virulent, strains of E. histolytica, are similarly attenuated in their virulence potential by 5-AzaC treatment. Whether this virulence attenuation is due to a direct effect of the 5-AzaC on E. histolytica genomic DNA methylation, is due to some other effect on the parasite, or is simply an associative phenomenon, cannot be definitively stated at present.

In order to identify genes whose expression was modulated by 5-AzaC treatment, expression profiling of 9,435 amebic genes was performed for untreated and 5-AzaC treated E. histolytica HM-1:IMSS. We chose to perform expression profiling with the E. histolytica HM-1:IMSS strain for a number of reasons: (i) this is the strain for which the genome sequence is available and the array was designed, (ii) transcriptome data are available for this strain from an invasive mouse model of colitis and can be used for comparative purposes 27, (iii) this is the strain for which there are published data for potential roles of the retrotransposon elements (long interspersed nuclear elements (LINEs) and short interspersed nuclear elements (SINEs)) in amebic pathogenesis and gene silencing 3132, (iv) there are substantial genomic differences in coding and non-coding regions of the 200:NIH strain compared to HM-1:IMSS 30, and (v) the vast majority of relevant literature is for the HM-1:IMSS strain.

To identify genes affected by genomic DNA methylation, we chose to look at both early (3 days) and late (7 days) time points after 5-AzaC exposure. We chose the early time point to identify genes that are likely modulated by 5-AzaC as a primary effect (rather than a secondary down-stream effect of expression changes in other genes). However, in case certain genomic regions were more resistant to the effects of 5-AzaC, we also analyzed the parasite transcriptome after seven days of drug exposure. Importantly, for both these time points, there were no significant effects on parasite growth rates and both time points gave consistently attenuated virulence phenotypes in both E. histolytica strains (Figure 1 and see Additional file 1).

Genes that are endogenously silenced by genomic DNA methylation should be transcriptionally upregulated under 5-AzaC treatment. We considered genes transcriptionally regulated if they were modulated at both time points of 5-AzaC treatment compared to the untreated controls (overall 2-fold change and p-value < 0.05). To confirm the array data, a subset of genes whose expression was modulated by 5-AzaC exposure were tested by semi-quantitative RT-PCR and in all cases the array data were confirmed (Figure 2; Table 1). A total of 199 transcripts (2.1% of all amebic genes tested) were significantly modulated with 68 genes upregulated and 131 genes down regulated in 5-AzaC treated parasites compared to control, untreated parasites (Tables 2, 3, and see Additional File 4). Approximately 58% of these genes were annotated as hypothetical or of unknown function. Whether all the genes that were upregulated by 5-AzaC treatment are methylated or were secondarily controlled by other genes cannot be definitively stated. However, we required that genes be transcriptionally modulated at both early and late time points, thus hopefully minimizing the potential secondary downstream effects. Genes whose expression decreases with 5-AzaC treatment are expected to be secondary or downstream effects of regulation of another gene. Inhibition of genomic DNA methylation would not, to the best of our knowledge, be directly expected to repress gene expression 33.

Genomic regions previously known to be methylated in E. histolytica are the heat shock protein 100 (EHsp100), ribosomal RNA (rRNA), and heat shock protein 70 (HSP70), and the repetitive region EhMRS2 161834. Of these, the only gene known to be transcriptionally modulated by DNA methylation is EHsp100 16. In the study by Bernes et al., the EHsp100 gene transcript could not be detected in untreated E. histolytica HM-1:IMSS parasites but was upregulated in 5-AzaC-treated and heat-shocked parasites 16. This gene is represented by locus 192.m00086 in the E. histolytica TIGR Genome Database 35 and represented by the 64.m00187_s_at probe set in our microarray. However, in our studies this probe set showed significant hybridization under standard culture conditions in E. histolytica HM-1:IMSS parasites and was not modulated by inhibition of DNA methylation (Table 4; see Additional file 4). This probe set was also showed significant hybridization in the 200:NIH strain (data not shown). Since this probe set also represented a number of other genes (111.m00116, 181.m00064, 365.m00018, 482.m00014, 493.m00033, 511.m00026, 82.m00144, and 872.m00009), we confirmed the array results by RT-PCR using gene specific primers as designed by Bernes et al 16. The RT-PCR analysis, specific to the EHsp100 gene, confirmed our microarray data and indicated that in our E. histolytica HM-1:IMSS strain the EHsp100 gene was expressed under baseline culture conditions and was not modulated by 5-AzaC treatment (see Additional file 2). Other genes associated with the heat shock or stress response were not expressed in our parasites under standard culture conditions, indicating that the parasite cultures were not generally stressed (see Additional file 4). The difference in basal EHsp100 gene expression between our cultures and those of Bernes, et al. 16 may be due to differences in the parasite strains. It has been shown that different clones of E. histolytica HM-1:IMSS have different expression levels for certain genes, including retrotransposons 31, and have other biologically distinct properties relating to gene silencing 36.

Using bisulfite treatment followed by sequencing of the coding and promoter regions we confirmed that the EHsp100 gene in our parasite strain was methylated (Table 4). However, the extent of DNA methylation in the promoter and coding regions of the EHsp100 gene in our HM-1:IMSS parasite clone was slightly lower compared to that of Bernes, et al. 16. For example, position 45 of the promoter (according to the nucleotide positions referred to by Bernes, et al., 16) was found to be unmethylated in 6 out of 6 clones tested in our HM-1:IMSS parasite strain while 2 out of 5 clones in Bernes' HM-1:IMSS showed evidence of methylation. Similarly, for other positions fewer clones in our strain showed methylation compared to their parasites (data not shown). Whether this slight difference in DNA methylation in our strain compared to that of Bernes, et al. 16 was responsible for the baseline higher expression under standard conditions is not clear at present. While our microarray does not contain probe sets for rRNA genes, other genes previously known to contain cytosine methylation such as HSP70 (218.m00068) were not modulated by 5-AzaC treatment. In fact, it has been shown that the expression of HSP70 is not regulated by its cytosine methylation 18; thus, our data are consistent with this observation. We also identified other genes, whose transcription was marginally modulated (but which did not meet our 2-fold and p-value < 0.05 cutoff) but whose promoters were methylated (687.m00016, 3.m00674, 160.m00098, 26.m00304) (Table 4).

Thus, there appear to be at least two subsets of genes in E. histolytica. First are those that are endogenously silenced by genomic DNA methylation and for which 5-AzaC treatment induces significant transcriptional de-repression (such as 141.m00082, 115.m00143, and 97.m00140). The second category of genes are those that have genomic DNA methylation, but are not endogenously silenced (such as EHsp100, HSP70, 687.m00016, 3.m00674, 160.m00098, 26.m00304) 18. Whether the second category of genes (except for EHsp100 which is sensitive to 5-AzaC treatment) are more resistant to 5-AzaC 37 or are those in which the methylation profile is not due to methyltransferases but instead due to other factors, such as dsRNA-directed methylation 38, is not clear at present.

In order to confirm the microarray data, we isolated RNA from 5-AzaC treated and untreated E. histolytica HM-1:IMSS, and performed semi-quantitative RT-PCR on five genes: two each from significantly up (115.m00143 and 141.m00082) and down-regulated gene sets (2.m00545 and 226.m00092), respectively, and one invariant gene (147.m00095), based on Affymetrix analysis. RT-PCR agreed with the microarray data for all five transcripts tested (Figure 2).

Of the total 199 genes modulated in 5-AzaC treated E. histolytica HM-1:IMSS, 68 (0.7% of all amebic genes) were significantly up regulated by inhibition of genomic DNA methylation (Table 2). Approximately 72% of these were annotated as hypothetical proteins. Among the others were four putative protein kinases (401.m00029, 194.m00103, 141.m00082, and 159.m00103), two BspA-like leucine rich repeat protein genes (371.m00031 and 64.m00173), transcription initiation factor TFIID (115.m00143), and a DNA mismatch repair protein gene (93.m00158). There are two other putative transcription initiation factor TFIIDs annotated in the E. histolytica genome (324.m00040 and 40.m00208), both of which were expressed in detectable levels in untreated HM-1:IMSS parasites and were not regulated by 5-AzaC treatment (see Additional file 4). It is not clear whether these two TFIID genes contain genomic DNA methylation. In contrast, the 115.m00143 gene had very low basal gene expression and was up regulated in 5-AzaC treated parasites, and has genomic DNA methylation of the coding region (Table 4). To the best of our knowledge, no such basal transcription factors have been shown to be modulated by DNA methylation in other systems. In prokaryotes DNA methylation is involved in DNA mismatch repair, and it was interesting to see that the amebic DNA mismatch repair gene was also regulated by DNA methylation 39.

Overall, 53 genes went from undetectable baseline expression (microarray signal < 0.2) to a detectable expression levels after treatment by 5-AzaC; only 33 genes were up regulated by ≥ 3-fold, and only 4 up-regulated by ≥ 10-fold (Table 2). Thus, cytosine methylation apparently silences a relatively small number of amebic genes. As we have shown that 23 μM 5-AzaC treatment for seven days was adequate for inhibiting methylation of the EHsp100 gene (Table 4), we disfavor a technical reason for this observation. However, we cannot exclude that this parasite contains other types of rare DNA methylation (adenine methylation or N-4 cytosine methylation) 404142, which remained unaffected by 5-AzaC treatment or that some amebic genomic regions have 5-methylcytosine that is resistant to 23 μM of 5AzaC treatment. In Arabidopsis thaliana treatment with 5-Aza-dC resulted in up-regulation of 0.9% of all genes in the array by ≥ 3-fold 26. This suggests that DNA methylation silences a relatively small number of genes in these two systems.

It has been previously shown that the E. histolytica HM-1:IMSS EHsp100 gene contained methylation in both the promoter and coding regions 16. In order to confirm these data for our E. histolytica HM-1:IMSS strain, we performed a bisulfite reaction followed by strand-specific PCR of the EHsp100 gene. This procedure converts unmethylated cytosine residues to uracil, giving rise to thymine after amplification by PCR 43. Only methylated cytosines are refractory to the deamination and are still seen as cytosines after PCR amplification. We demonstrated that the EHsp100 gene is methylated at both the coding and promoter regions and that 5-AzaC treatment (23 μM for 7 days) significantly inhibited methylation of this gene (Table 4). The level of demethylation we observed was similar to that observed by Bernes, et al. 16. This confirmed that the 5-AzaC was working as expected under our usage conditions.

In order to determine the extent of genomic DNA methylation in genes that were transcriptionally up regulated by 5-AzaC treatment, we performed sodium bisulfite treatment, PCR and sequencing of three genes significantly upregulated by 5-AzaC treatment (141.m00082, 97.m00140, and 115.m00143). These three genes had extremely low basal gene expression (0.12 ± 0.07) (median ± standard error). We identified that two genes (141.m00082 and 97.m00140) contained cytosine methylation in both coding and corresponding promoter regions, while the third one (115.m00143) showed only limited methylation in the coding region (Table 4). Cytosine methylation in the coding region has been implicated in gene silencing in other systems 44 and appears to be responsible for the endogenous silencing of the 115.m00143 gene in E. histolytica. Overall the trend was for greater methylation in coding than promoter regions, as also seen by Bernes, et al. 16. Thus, our microarray data identified novel genes whose expression was endogenously silenced by genomic DNA methylation.

Since the number of genes that were apparently regulated by DNA methylation was limited, we investigated whether genes that were somewhat transcriptionally modulated (but that did not meet our significance fold-change or p-value criteria) may also be genomically methylated. Four such genes were subjected to bisulfite treatment and sequencing: 687.m00016, 3.m00674, 160.m00098, and 26.m00304. We picked genes that were transcriptionally upregulated early (day 3 of 5-AzaC treatment) (see Additional file 4) in order to improve our chances of finding genes partially regulated by methylation. Surprisingly, all four genes had substantial methylation in their promoter regions (Table 4). Interestingly, despite the significant extent of genomic DNA methylation, these four genes were not transcriptionally silenced under basal conditions (basal expression levels, 6.04 ± 3.25) (median ± standard error). A similar trend was previously noticed for the HSP70 gene, which has genomic methylation, but is not transcriptionally modulated by DNA methylation 18. Additionally, in our strain of E. histolytica HM-1:IMSS, the EHsp100 gene is methylated but not endogenously silenced or transcriptionally modulated by 5-AzaC treatment (Table 4).

Thus, the extent of genomic DNA methylation in E. histolytica is likely greater than that indicated by the expression profiling as not all genomic regions that are methylated appear to be either transcriptionally silenced or substantially regulated by 5-AzaC treatment. We do not feel that this represents a technical flaw (e.g. the amount of 5-AzaC used or duration of drug exposure) as 23 μM 5-AzaC for 7 days did completely abolish methylation at the Hsp100 coding region (Table 4). Additionally, higher does of 5-AzaC (50 μM and higher) are rapidly lethal to the parasite (our observations and 15). As mentioned earlier, each gene that is methylated in E. histolytica does not necessarily respond to 5-AzaC treatment 18. Similar observations have also been made in several insect systems. DNA methylation of defensin gene in the lepidopteran Mamestra brassicae is not involved in gene silencing as it is constitutively expressed 45. Likewise, highly expressed genes in Myzus persicae 4647 and Planococcus citri 4849 contain DNA methylation.

As expected, a gene whose expression levels significantly decreased after 5-AzaC treatment showed no cytosine methylation (226.m00092). Genes whose expression decreases after exposure to 5-AzaC would not be expected to be directly transcriptionally modulated by drug treatment, but likely change expression levels as a downstream effect of other transcriptional changes in the parasites.

In most systems, genomic DNA methylation occurs at CpG dinucleotides 50. The skewed AT content of the amebic genome (~75% A/T) predicts that methylation will frequently not occur at CpG dinucleotides 51. The majority of methylations we identified were located in non-CpG sites, confirming previous data for the EHsp100 gene 16. Non-CpG methylation was also detected in Entamoeba invadens 52. In order to determine if we could bioinformatically predict the location of potential DNA methylation in E. histolytica genomic DNA, we analyzed the frequencies of mono- and di-nucleotides in genes transcriptionally regulated by 5-AzaC compared to the whole genome. No significant differences in any of the mono or di-nucleotides were identified in up-regulated or down-regulated genes compared to the whole genome (Table 5). Thus, in the E. histolytica genome the frequency of the mono- or di-nucleotide occurrence was in itself not predictive of transcriptional regulation by genomic DNA methylation.

Retrotransposable elements can have substantial effects on genome structure and gene expression 53. One method by which they can modulate gene expression is by regulating the expression of adjacent genes by methylation 54. Retrotransposable elements are abundant in E. histolytica and some are maintained in hundreds of copies per genome 55. Previously we have identified that two such retrotransposable elements, LINEs and SINEs, have higher expression in virulent strains of E. histolytica HM-1:IMSS compared with nonvirulent Entamoeba 31. In the reptile parasite Entamoeba invadens the reverse transcriptase of LINE (RT LINE) contains DNA methylation and it has been suggested that both E. invadens and E. histolytica possess a mechanism for DNA methylation of LINE retrotransposons 52. It is also not clear how many fully functional LINE and SINE elements are present in E. histolytica (the majority of those that are sequenced are mutated/truncated) but we do know that at least some of the LINE and SINE elements are transcribed 31.

We were unable to detect the transcriptional profile of LINEs/SINEs as there are no probe sets representing these regions in our microarray. We wished to determine if genes adjacent to LINEs or SINEs were preferentially modulated by 5-AzaC treatment. The normalized expression values for all genes adjacent to LINEs or SINEs were 0.761 ± 0.09 (median ± std error) and 0.509 ± 0.362 (median ± std error), respectively, in untreated E. histolytica HM-1:IMSS, and 0.633 ± 0.207 (median ± std error) and 0.449 ± 0.786 (median ± std error), respectively, in 5-AzaC treated E. histolytica HM-1:IMSS. Thus, we did not identify any significant association of genes that were modulated by 5-AzaC and their genomic proximity to LINE or SINE elements compared with untreated parasites. This finding indicates that even if the LINE/SINE elements in E. histolytica contain DNA methylation they do not necessarily co-regulate adjacent genes by this mechanism.

Additionally, we identified that adjacent loci in the same scaffold did not show co-regulation by DNA methylation (data not shown). This phenomenon is also observed in Arabidopsis thaliana, in which transcriptional profiling reveals that 5-Aza-dC responsive genes are distributed randomly throughout the chromosome arms and are not clustered in any obvious pattern 26.

Virulent strains of E. histolytica HM-1:IMSS and 200:NIH grown with 23 μM of 5-AzaC for three or seven days showed no substantial growth differences but displayed severely reduced virulence phenotype compared to untreated parasites (Figure 1) 15. Importantly, others have demonstrated that the effect of 5-AzaC on parasite virulence is reversible and parasite virulence returns to baseline when drug exposure is withdrawn 15. Whether the effect of 5-AzaC on parasite virulence is simply an associative or causal finding is unclear at present. However, we did find some genes that were down regulated in 5-AzaC treated parasites, which have previously been shown to function as virulence determinants (Table 3). These included a putative cysteine proteinase (2.m00545), lysozyme (52.m00148), and a myosin Ca-binding protein (1.m00663). Cysteine proteinases (CP) are important virulence determinants in E. histolytica and serve a number of functions including degradation of colonic mucin and extracellular matrix 56. Genetic proof of their importance in virulence has been shown as parasites in which members of the CP family are down regulated are less virulent in vitro and in vivo 5758. Lysozymes are important for degrading intracellular bacteria and thus play an important role in E. histolytica virulence 59. Motility is an important aspect of amebic pathogenesis and parasites that are altered in their myosin II are less virulent 60. Likewise, Toxoplasma gondii myosin A has been implicated in powering parasite gliding and host cell invasion 61. In addition, five protein kinase genes and eight GTPase family protein genes were also down regulated significantly in 5-AzaC treated parasites. Protein kinases can affect the functions of a large number of genes and the GTPase gene family is important in vesicle trafficking, an important aspect of biology in the professionally phagocytic E. histolytica 62. Thus, some roles of these genes in amebic pathogenesis can be anticipated, although functional genetic proof will be necessary to assign a definitive role to these genes in amebic virulence. Importantly, eight of the genes down regulated under 5-AzaC treatment also had decreased expression in a non-virulent strain of E. histolytica, Rahman, compared to the virulent E. histolytica HM-1:IMSS (108.m00122, 136.m00107, 22.m00298, 297.m00061, 32.m00239, 37.m00215, 442.m00023, and 460.m00024) (Table 3) 3163. Interestingly, in E. histolytica Rahman, treatment with 23 μM 5-AzaC did not significantly change the transcriptional profile of these genes (data not shown). A number of genes that have decreased expression in avirulent Entamoeba strains and species have been shown to be virulence determinants including the Gal/GalNAc light subunit lectin 64, CP1 and CP5 565758, and amebapore 32. In addition, four genes down regulated by 5-AzaC treatment (15.m00302, 2.m00545, 67.m00091, and 71.m00153) are significantly up regulated during parasite invasion in a mouse model of amebic colitis (Table 3) 27. Since E. histolytica trophozoites passed through a mouse model become more virulent, some of the upregulated genes from the in vivo mouse model of colitis may have direct roles in virulence. Thus, data from a number of different angles indicates that genes that are down regulated by 5-AzaC treatment are good candidates on which to perform directed functional studies to elucidate their potential contributions to amebic virulence.

Trophozoites of E. histolytica HM-1:IMSS and E. histolytica 200:NIH were maintained under axenic conditions in trypticase-yeast extract iron-serum medium (TYI-S-33) supplemented with 15% adult bovine serum (Sigma), at 36.5°C as previously described 66. The identity of both strains was confirmed by strain-specific PCR and RFLP analysis 67. Trophozoites in log phase of growth were used in all experiments. To inhibit genomic DNA methylation, E. histolytica trophozoites were grown with 23 μM 5-AzaC (TRC, Canada) for up to one week with routine subculturing performed every 48 hours. For the growth curves 50,000 log-phase trophozoites were inoculated in 15 ml of TYI-S-33 media in the presence or absence of 23 μM 5-AzaC, grown under standard culture conditions as outlined above, and the parasite number recorded every 24-hours. Growth curves were performed a minimum of three times. To grow parasites without subculturing, we seeded 15,000 log-phase trophozoites in 15 ml of TYI-S33 media in the presence or absence of 23 μM 5-AzaC under standard culture conditions and monitored the parasite count every day up to day 5. In order to further assess the growth kinetics of the parasites in 23 μM 5-AzaC, 50,000 log-phase trophozoites were seeded in 15 ml of TYI-S33 media in the presence or absence of 23 μM 5-AzaC and subcultured every two days with equal inoculums of media and parasites being passed each time. Cell counts were recorded at days 2, 4, and 6 after initiation of growth in 5-AzaC.

Protein concentration was determined using the method of Bradford 68. Briefly, trophozoites were lysed on ice for 2 min using Cell Culture Lysis Reagent (Promega) supplemented with protease inhibitors (500 μM AEBSF, 1 μM leupeptin, 1 μM E-64d). Cell membranes were pelleted by centrifugation for 30 sec at 14,000 × g. A standard curve for the Bradford assay was determined using bovine serum albumin as a reference. Protein concentration of the cell lysate was determined relative to this standard curve. The protein concentration (ng/cell) was determined for each time point of interest.

CHO cells were maintained in Dulbecco's minimal essential media (DMEM) supplemented with 10% fetal bovine serum, penicillin (100 U/ml), and streptomycin (100 μg/ml). CHO cell monolayer destruction by E. histolytica trophozoites was performed according to Bracha and Mirelman 69 with some modifications. Briefly 200,000 trophozoites of E. histolytica strain HM-1:IMSS or 200:NIH grown with or without 23 μM of 5-AzaC for three or seven days were incubated with confluent CHO cell monolayers in 24-well plates in 3 mls of DMEM (containing no serum) at 36.5°C for 3 hours. The integrity of the CHO cell monolayer that remained attached to the 24-well plate was determined by staining with methylene blue. The level of monolayer destruction was calculated to be inversely proportional to the amount of intact cells that remained attached to the 24-well plate. CHO cells incubated with DMEM or DMEM with 23 μM 5-AzaC served as controls. All experiments were performed at least three times. Statistical analyses were performed using a two-tailed Student's t-test and p-values < 0.05 were considered statistically significant.

A custom E. histolytica Affymetrix array (E_his-1a520285) was designed using information from the E. histolytica genome sequencing project 27. The ORF probe sets represent 9,435 of the predicted 9,938 E. histolytica ORFs 70. Most of the highly repetitive sequences in E. histolytica such as LINEs, SINEs, rRNA genes, and tRNA genes were not included in the array as specific probe sets could not be designed. There are probe sets on the Affymetrix array representing selected intergenic regions, but these were not included in the analyses described in this paper.

Total RNA was isolated from log phase trophozoites of E. histolytica HM-1:IMSS and the same strain grown with 23 μM 5-AzaC for three or seven days using Trizol reagent (Invitrogen). RNA was cleaned using the RNAeasy kit (QIAGEN) according to the manufacturer's instructions and 4 μg processed for hybridization to the E. histolytica Affymetrix array through the Stanford University Protein and Nucleic Acid Biotechnology Facility 71 using standard Affymetrix protocols. Two arrays were generated for HM-1:IMSS parasites (not treated with drug) under mid-log conditions and two arrays were generated from HM-1:IMSS parasites treated with 5-AzaC (one each from three and seven days of treatment). Each microarray was generated from RNA harvested from parasites on different days and thus represent independent biological replicates.

The raw signal intensities were scaled by GCOS software so that the average of all of the probe sets on an array was 500. All data for intergenic regions was removed prior to any further analysis. The remaining data were analyzed using the GeneSpring® 7 software 72. A global normalization was performed per chip to the median excluding the probe sets flagged absent. All the normalized expression values were averaged between the two arrays from untreated parasites (control group), and between the two arrays from three day and seven day 5-AzaC-treated parasites (treated group). We assumed that the expression variances were equal for two arrays within a particular group. To eliminate genes that were not expressed under either of the two conditions, we filtered out probe sets that had an average normalized expression value of < 0.1 in both groups analyzed (control and 5-AzaC-treated) as we have previously determined that genes with that expression level are not routinely detectable by RT-PCR (our unpublished observations). In order to identify genes that were significantly modulated in presence of 5-AzaC we used two criteria: (1) expression fold change ≥ 2-fold, and (2) a p-value < 0.05.

Total RNA was extracted from log phase E. histolytica HM-1:IMSS parasites using TRIZOL (Invitrogen) reagent according to the manufacturer's instructions. DNA contamination in the RNA preparation was removed by DNAse treatment. Reverse transcription was performed to generate cDNA for RT-PCR using SuperScript™ II Reverse Transcriptase kit (Invitrogen) according to the manufacturer's instructions. PCR was performed with serial 10-fold dilutions of cDNA; a negative control was included with each reaction. Primers used in RT-PCR are listed in Table 1.

E. histolytica HM-1:IMSS genomic DNA was isolated using standard Phenol:Chloroform method, and RNA contamination was removed by treating with RNaseA (Invitrogen) 30. Sodium bisulfite treatment of E. histolytica genomic DNA (~2 μg) was performed according to the method described by Clark and Warnecke 43. For each sample, following bisulfite treatment, two PCR amplifications were performed, each consisting of 50 cycles. Primers used to amplify promoters and genomic regions following bisulfite treatment are listed in Table 1. PCR amplified fragments derived from sodium bisulfite treated DNA were resolved on a 1.2% agarose gel, appropriate fragments excised and purified using QIAEX® II Gel Extraction Kit (QIAGEN) according to the manufacturer's instructions. Purified DNA fragments were cloned into TOPO TA vector (Invitrogen), transformed into competent Escherichia coli (Invitrogen), plasmid DNA isolated using QIAprep Miniprep Kit (QIAGEN), and sequenced using an ABI PRISM 310 genetic analyzer (PE Applied Biosystems) (Macrogen, Korea). Six plasmid clones were sequenced for each gene fragment.

In order to determine the frequency of mono and di-nucleotides in the genomic regions affected by 5-AzaC treatment we performed the following analysis. Sequences for the promoter (500 bp upstream of the predicted start codon) and coding regions of all genes modulated by 5-AzaC treatment were downloaded from The Institute for Genomic Research (TIGR) website 35. Additionally, the sequence of the most recent assembly (12/27/05) of the genome sequence was also obtained 35. Using acustom script written in the Python programming language by JAH (see Additional file 3) we determined the mono and di-nucleotide frequency in the promoter and coding regions of genes up-regulated by 5-AzaC treatment, down-regulated by 5-AzaC treatment, and the entire genome.

To determine if the genes modulated by 5-AzaC treatment were adjacent to the retrotransposon elements (LINEs and SINEs), all occurrences of the three families of EhLINEs and EhSINEs were identified in the E. histolytica genome sequence using BLAST program 73 and the consensus sequence of the LINEs and SINEs 55 with an e-value cutoff of 10^-20. The frequency of occurrence of each LINE and SINE gene family member was similar to the results identified previously 55. We subsequently identified all genes adjacent (within 10 Kb, both upstream and downstream) to each member of EhLINE or EhSINE, considering only the gene most proximal to a given LINE or SINE. Using the hypergeometric distribution, we determined if the genes modulated by 5-AzaC treatment were more likely to be adjacent to an EhLINE or EhSINE element than by random chance (p-value < 0.05).