Context-dependent roles of the Entamoeba histolytica core promoter element GAAC in transcriptional activation and protein complex assembly

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Abstract

Transcriptional control of the hgl5 gene of Entamoeba histolytica is mediated through an unusual core promoter composed of TATA, GAAC and Initiator elements. In the hgl5 promoter the GAAC element (AATGAACT) determines the site and rate of transcription initiation. Here we tested the role of the GAAC element in transcription activation from upstream regulatory elements (UREs) in the hgl5 promoter. We also examined the function of the GAAC element in the ferredoxin (fdx) promoter and characterized the protein binding to the GAAC element. Electrophoretic mobility shift assays (EMSA) demonstrated that the GAAC region is necessary for higher-order nuclear protein complex assembly. The function of the GAAC element in transcription activation mediated by UREs revealed that mutation of the GAAC element did not affect transcription activation mediated by the hgl5 URE4 but abrogated activation by the hgl5 URE3. We compared the role of the GAAC elements in the hgl5 and fdx promoters. Competitive gel shift assays were consistent with the same nuclear protein binding to the GAAC elements in both genes. Mutation of the GAAC element in the fdx gene decreased reporter gene expression, however, in contrast to hgl5 gene, had no effect on the site of transcription initiation. These results support a role for the GAAC element in assembly of nuclear proteins at the core promoter and in transcription activation mediated by URE3. The differing effect on transcription initiation in the hgl5 and fdx genes upon mutation of the GAAC element suggests a context-dependence of the GAAC-binding protein in gene expression.

Introduction

Entamoeba histolytica, an enteric protozoan parasite, is the etiologic agent of amebic colitis and liver abscesses. During its life cycle E. histolytica undergoes developmental changes including transformation from cyst to trophozoite and adaptation from an anaerobic to aerobic environment upon invasion. Coordination of these events in E. histolytica is not understood, although regulation of transcription is likely to be an important mechanism of this control. Transcriptional control of the emetine drug resistance gene in E. histolytica [1], [2] has been described and demonstrates a possible relationship between transcriptional regulation and pathogenesis. E. histolytica has many unusual features at a transcriptional level including an α-amanitin resistant RNA pol II [3], a divergent TATA binding protein (TBP) [4], short 5′ and 3′ mRNA untranslated regions [5], and a tripartite core promoter architecture [6], [7], [8]. The core promoter of the hgl5 gene of E. histolytica consists of three conserved elements that control the endogenous transcription start site: a TATA box at −30 (GTATTTAAAC), an Initiator region (Inr) overlying the site of transcription initiation (GAAAGACAA), and a third sequence with variable location between the TATA box and Inr, the GAAC region (AATGAACT) [7], [8]. We have demonstrated previously that in the hgl5 gene the GAAC region controls the rate of gene expression and the endogenous transcription start site, and is able to direct a new transcription start site independent of the TATA or Inr regions [7], [8].

Here we examine the GAAC elements of the hgl5 and fdx genes, both of which have been identified as having roles in amebic pathogenesis. The hgl5 gene is a member of a gene family encoding the galactose- and N-acetyl-d-galactosamine-inhibitable lectin (Gal/GalNAc inhibitable lectin) which has been shown to have central roles in amebic attachment, invasion, and killing of the target cells [9], [10], [11], [12]. In a related parasite, Entamoeba invadens, a role for the Gal/GalNAc lectin has been identified in stage transformation and development from the trophozoite to the cyst form [13]. The fdx gene is important for E. histolytica's survival in oxygen-rich environments of the host during amebic invasion [14] and in drug resistance to metronidazole [15].

The promoter of the hgl5 gene contains five major upstream regulatory elements (UREs) [6] in addition to the core promoter described above [7], [8]. The URE3 functions as a positive regulatory element in the hgl5 gene promoter [6] but a negative regulatory element in the fdx gene promoter [14]. Recent work has identified the URE3-binding protein and shown it to contain EF-hand calcium binding motifs [16]. The URE4, composed of two 9 bp repeats, functions as an enhancer in the hgl5 gene [17] and recently two URE4 enhancer-binding proteins that contain RNA-recognition motifs (RRM) have been identified [18].

To further clarify the role of the GAAC core promoter element in transcriptional control in E. histolytica, we have tested its roles in (i) activation pathways via upstream regulatory regions, (ii) nuclear protein binding to the core promoter, and (iii) in transcription control of the fdx gene promoter.

Section snippets

Cultivation of Entamoeba histolytica and stable transfection

E. histolytica strain HM-1:IMSS trophozoites were cultured in TYI-S-33 medium containing penicillin (100 U ml−1) (GIBCO/BRL) and streptomycin (100 μg ml−1) (GIBCO/BRL) [19]. All analyses were performed on stably transfected parasites maintained at 24 μg ml−1 of G418 [20].

Plasmid construction

Constructs in Fig. 1 were generated in the following manner. The minimal promoter (MP) plasmid (Fig. 1A) contains the hgl5 TATA-GAAC-INR with the relative orientation and spacing of the three elements identical to that in the

Activation of gene expression in the hgl5 gene of Entamoeba histolytica by the URE3 element occurred via the core promoter in a GAAC-dependent manner

The GAAC region of the hgl5 gene has been shown to control the rate and endogenous transcription start site [8] and is able to direct a new transcription start site independent of a TATA or Inr regions [7]. We questioned whether this region would also be able to mediate transcriptional activation by upstream regulatory regions. To test this hypothesis we made constructs (Fig. 1) in which a MP (TATA-GAAC-Inr), and a MP-mut GAAC (TATA-mutated GAAC-Inr), were upstream of the reporter gene luc (A

Discussion

New roles are emerging for the core promoter element GAAC in transcriptional control of E. histolytica genes. Previous data have shown that this region controls the rate and site of transcription initiation [7], [8]. We have now identified preliminary evidence that the GAAC element (i) mediates transcriptional activation by some upstream regulatory regions, (ii) is involved in protein complex assembly at the core promoter, and (iii) may function in a context-dependent manner in amebic genes.

Acknowledgements

We thank all Petri and Mann lab members for scientific discussions and helpful suggestions. This work was supported by National Institutes of Health Grants R01-AI 37941 to WAP and K08-AI 01453 to US. WAP is a Burroughs Welcome Scholar in Molecular Parasitology and US is a recipient of a Burroughs Welcome Career Award in the Biomedical Sciences.

References (37)

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