Two CCAAT/enhancer binding protein sites are cis-activator elements of the Entamoeba histolytica EhPgp1 (mdr-like) gene expression.
Here, we show the relevance of promoter regions (-74 to +24, -167 to -75 and -259 to -168 bp) in the transcriptional activation of the multidrug resistance gene EhPgp1 in Entamoeba histolytica, using mutated plasmids and transfection assays. We also demonstrate that both CCAAT/enhancer binding protein sites (-54 to -43 bp and -198 to -186 bp) are cis-activating elements of gene expression in the drug-resistant (clone C2) and -sensitive (clone A) trophozoites. Nuclear proteins from trophozoites of both clones and C/EBP sequences of the core promoter formed specific complexes, which were abolished by anti-human C/EBPbeta antibodies. UV cross-linking and Western blot assays revealed 25 and 65 kDa bands in urea treated and untreated proteins respectively. The nuclear factors that bind to C/EBP sites were semi-purified by affinity chromatography. They were immunodetected by anti-human C/EBPbeta antibodies and formed a specific complex with the C/EBP probe. The antibodies recognized proteins in the cytoplasm, nucleus and EhkO organelles in immunofluorescence and confocal microscopy experiments. Based on our results, we propose that the C/EBP site at -54 bp stabilizes the transcription pre-initiation complex, whereas the other site at -198 bp may be involved in the formation of a multiprotein complex, which provokes DNA folding and promotes the EhPgp1 gene transcription.
Marchat
Laurence A
LA
Programa de Biomedicina Molecular, ENMYH-IPN, Guillermo Massieu Helguera 239 Fracc. La Escalera, Ticoman, CP 07320 México D. F.
Gómez
Consuelo
C
Pérez
D Guillermo
DG
Paz
Francisco
F
Mendoza
Leobardo
L
Orozco
Esther
E
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1 / Q000235:genetics* / Q000378:metabolism; D000818:Animals; D001665:Binding Sites; D022762:CCAAT-Enhancer-Binding Proteins / Q000378:metabolism*; D018432:Drug Resistance, Multiple / Q000235:genetics / Q000502:physiology; D004748:Entamoeba histolytica / Q000235:genetics*; D005786:Gene Expression Regulation; D006801:Humans; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D009687:Nuclear Proteins / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D011485:Protein Binding; D015800:Protozoan Proteins / Q000235:genetics* / Q000378:metabolism; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism; D015533:Transcriptional Activation
D020168:ATP Binding Cassette Transporter, Subfamily B, Member 1; D022762:CCAAT-Enhancer-Binding Proteins; D046911:Macromolecular Substances; D046912:Multiprotein Complexes; D009687:Nuclear Proteins; D015800:Protozoan Proteins; D014157:Transcription Factors