Changes in the transcriptome of Entamoeba histolytica accompany the epigenetic silencing of the amoebapore gene

We previously showed that epigenetic transcriptional silencing of the amoebapore gene (Ehap-a) in E. histolytica G3 trophozoites was accompanied by the down-regulation of two other members of the saposin-like gene family (Ehap-b, and EhSaplip1). Comparison of the transcriptomes of G3 trophozoites with those of the parent HM-1:IMSS strain revealed 72 additional transcripts that were either down- or up-regulated at least 7 fold. One of the polyA+, up-regulated (>200) transcripts (459.m00030), had the potential to encode a 66 amino acid protein. Antibodies raised against a synthetic N-terminal peptide of the gene did not recognize any such protein in trophozoite lysates. Trophozoite cultures grown with the proteasome inhibitor MG-132 to prevent protein degradation, also did not have any detectable protein. Transfections with a plasmid in which the Ehactin gene promoter elements were used to flank the 459.m gene, significantly increased their level transcript but again, no protein was detected. Transfectants in which the 459.m ORF was placed downstream to the GST gene, expressed a fused protein which reacted with the antibody against the m.459 peptide. Constructs with the CAT reporter gene placed under the 459.m gene promoter element or fused to the C-terminal sequence of the 459.m ORF were prepared to determine if the higher levels of 459.m transcript found in the G3 trophozoites were due to changes in the stability of the transcript or increased transcription. No difference was observed in the very weak expressions seen in both G3 and HM-1:IMSS transfectants. Similarly, transfectants with a plasmid construct in which the 459.m gene was placed under its regulatory elements, did not induce an increase in the transcript levels. Our conclusions are that the transcript of the 459.m gene is an mRNA-like ncRNA and that modulations of transcript levels, especially of hypothetical genes, may not correlate with changes in the proteome. Samples include two replicates of the HM-1:IMSS strain and the amoebapore a silenced G3 strain (G3(A))