Growth of the protozoan parasite Entamoeba histolytica in 5-azacytidine has limited effects on parasite gene expression

Table 4 Compilation of bisulfite treatment and sequencing results.

Probe set	Baseline expression value	Fold change	p-value	Annotation	Promoter region		Coding region
					Total bp analyzed	# methylated cytosines/# total cytosines	Total bp analyzed	# methylated cytosines/# total cytosines
Control genes
64.m00187	14.05	0.90	0.731	Hsp100	455	10/62	397	48/48
64.m00187**	14.05	0.90	0.731	Hsp100**	ND	ND	397	0/48
Significantly upregulated
141.m00082	0.32	2.98	0.010	protein kinase, putative	308	36/36	73	13/13
115.m00143	0.12	3.64	0.001	transcription initiation factor TFIID, putative	266	0/27	254	5/40
97.m00140_at	0.08	3.10	0.006	hypothetical protein	248	59/59	110	23/23
Marginally upregulated
687.m00016_at	12.16	18.68	0.091	hypothetical protein	328	18/18	ND	ND
3.m00674_at	0.62	2.17	0.089	hypothetical protein	143	16/16	ND	ND
160.m00098_at	11.46	1.72	0.112	hypothetical protein	244	16/16	ND	ND
26.m00304_at	0.54	1.64	0.086	hypothetical protein	115	19/19	ND	ND
Significantly downregulated
226.m00092_at	0.343	0.34	0.008	Rab family GTPase	347	0/27	41	0/6

64.m00187_s_at also represents 111.m00116, 181.m00064, 192.m00086, 365.m00018, 482.m00014, 493.m00033, 511.m00026, 82.m00144, and 872.m00009.
The probe set, baseline expression value in E. histolytica HM-1:IMSS trophozoites, fold change, p-value, and gene annotation are shown. For each gene the number of base pairs analyzed, the number of methylated cytosines, the number of total cytosines analyzed, and the location (promoter or coding region) are indicated. Genomic DNA from E. histolytica HM-1:IMSS parasites was treated with 10 M sodium bisulfite and PCR amplified. Six independent clones were analyzed for each region of interest to determine the extent of cytosine methylation; for a particular cytosine position to be indicated as methylated 4 out of 6 clones had to be methylated. ** represents genomic DNA from E. histolytica HM-1:IMSS parasites treated with 23 μM 5-AzaC for 7 days. ND = not done. Primers used in PCR analysis of the Hsp100 gene were specific to 192.m00086 and were identical to those used by Bernes et al. [16].

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