Targets of the Entamoeba histolytica Transcription Factor URE3-BP

The Entamoeba histolytica transcription factor Upstream Regulatory Element 3-Binding Protein (URE3-BP) is a calcium-responsive regulator of two E. histolytica virulence genes, hgl5 and fdx1. URE3-BP was previously identified by a yeast one-hybrid screen of E. histolytica proteins capable of binding to the sequence TATTCTATT (Upstream Regulatory Element 3 (URE3)) in the promoter regions of hgl5 and fdx1. In this work, precise definition of the consensus URE3 element was performed by electrophoretic mobility shift assays (EMSA) using base-substituted oligonucleotides, and the consensus motif validated using episomal reporter constructs. Transcriptome profiling of a strain induced to produce a dominant-positive URE3-BP was then used to identify additional genes regulated by URE3-BP. Fifty modulated transcripts were identified, and of these the EMSA defined motif T[atg]T[tc][cg]T[at][tgc][tg] was found in over half of the promoters (54% p
Author Summary Most infections with Entamoeba histolytica are asymptomatic. However, in a minority of cases, they develop into invasive and even life-threatening amebiasis. We suspect, based on prior studies of invasive amebae, that changes in amebic gene expression enable the transition from asymptomatic to invasive infection. Our long-term goal is to identify the genetic program required to cause amebic colitis. Here, we studied a transcription factor named URE3-BP that controls the expression of two virulence genes, the Galactose and Galactose N- acetyl- galactosamine inhibitable lectin (Gal/GalNAc lectin) and ferredoxin. We suspected that this factor might coordinate invasiveness by co-regulating additional virulence factors. The consensus DNA motif that is recognized by URE3-BP was identified by reporter gene assays and by electromobility shift assays. We then inducibly expressed a constitutively active form of the transcription factor, and measured the changes in total amebic gene expression mediated by overexpression of this dominant-positive version of URE3-BP. This analysis allowed for a further definition of the functional URE3 motif. Inducible expression of URE3-BP led to changes in the transcript levels of several novel amebic membrane proteins. In conclusion, this genome-wide analysis of a transcription factor and its cis-acting regulatory sequence in Entamoeba histolytica has identified new transcripts regulated by URE3-BP that may play a role in trophozoite motility within a coordinated virulence-specific gene regulatory network.