Structural and functional analysis of the Entamoeba histolytica EhrabB gene promoter.
BACKGROUND
The Entamoeba histolytica EhrabB gene encodes for a Rab GTPase involved in phagocytosis. It is located at a virulence locus where the Ehcp112 gene is in the complementary strand at 332 bp of EhrabB start codon, suggesting a finely regulated transcription of both genes. However, the transcription regulation in this parasite is poorly understood.


RESULTS
To initiate the knowledge of EhrabB gene expression regulation, here we studied the structural characteristics of its gene promoter and its control transcription elements. In silico searches of the EhrabB 5'-flanking region revealed that it contains a motif similar to the upstream regulatory element 1 (URE1) of the E. histolytica hgl5 gene. It also has sequences with homology to C/EBP and GATA1 binding sites, and heat shock elements (HSE). Primer extension experiments revealed that EhrabB has at least four transcription initiation sites. The elements at the 5'-flanking region that drive EhrabB gene expression were detected and characterized using transitory transfected trophozoites with a plasmid carrying the CAT reporter gene. EhrabB transcription is negatively regulated by a sequence located between positions -491 to -428 with respect to the first transcription initiation site. We also showed that the URE1-like motif activates EhrabB transcription. In addition, heat shock activated the EhrabB promoter in episomal constructs and lead to an increase in de novo EhrabB transcription.


CONCLUSIONS
The data suggest that EhrabB transcription is controlled negatively by an unidentified sequence, but it is activated by an URE1-like motif. Our analyses also revealed the presence of activator HSE that function under stress.
Romero-Díaz
Mónica
M
Departamento de Patología Experimental, Centro de Investigación y de Estudios Avanzados del IPN, A,P, 14-740 México, DF 07360, México. rdmoni@yahoo.com.mx
Gómez
Consuelo
C
López-Reyes
Israel
I
Martínez
Máximo B
MB
Orozco
Esther
E
Rodríguez
Mario A
MA
D000595:Amino Acid Sequence; D000818:Animals; D015500:Chloramphenicol O-Acetyltransferase / Q000235:genetics; D003001:Cloning, Molecular; D016054:DNA, Protozoan / Q000737:chemistry; D024202:Electrophoretic Mobility Shift Assay; D004748:Entamoeba histolytica / Q000201:enzymology* / Q000235:genetics*; D050982:GATA1 Transcription Factor; D005786:Gene Expression Regulation / Q000502:physiology; D015971:Gene Expression Regulation, Enzymologic / Q000235:genetics*; D017125:Genes, Protozoan / Q000502:physiology; D006160:Guanosine Triphosphate / Q000378:metabolism; D006360:Heat-Shock Proteins; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000235:genetics*; D016385:TATA Box; D024363:Transcription Initiation Site / Q000502:physiology; D015533:Transcriptional Activation; D020691:rab GTP-Binding Proteins / Q000737:chemistry / Q000235:genetics* / Q000378:metabolism*
D016054:DNA, Protozoan; D050982:GATA1 Transcription Factor; D006360:Heat-Shock Proteins; D006160:Guanosine Triphosphate; D015500:Chloramphenicol O-Acetyltransferase; D020691:rab GTP-Binding Proteins