RESUMO
The smallest product of the Duchenne muscular dystrophy gene, dystrophin (Dp)71, is ubiquitously expressed in nonmuscle tissues. We previously showed that Dp71 expression in hepatic cells is modulated in part by stimulating factor 1 (Sp1), stimulating protein 3 (Sp3), and yin yang 1 (YY1) transcription factors, and that the polyaromatic hydrocarbon, ß-naphthoflavone (ßNF), downregulates Dp71 expression. The aim of the present study was to determine whether ßNF represses Dp71 expression by altering mRNA stability or its promoter activity. Reverse transcriptionquantitative polymerase chain reaction was used to measure halflife mRNA levels in ßNFtreated cells exposed to actinomycin D, an inhibitor of transcription, for 0, 4, 8, 12 and 16 h. Transient transfections with a plasmid carrying the Dp71 basal promoter fused to luciferase reporter gene were carried out in control and ßNFtreated cells. Electrophoretic mobility shift assays (EMSAs) were performed with labeled probes, corresponding to Dp71 promoter sequences, and nuclear extracts of control and ßNFtreated cells. To the best of our knowledge, the results demonstrated for the first time that this negative regulation takes place at the promoter level rather than the mRNA stability level. Interestingly, using EMSAs, ßNF reduced binding of YY1, Sp1, and Sp3 to the Dp71 promoter. It also suggests that ßNF may modulate the expression of other genes regulated by these transcription factors. In conclusion, ßNF represses Dp71 expression in hepatic cells by altering binding of YY1, Sp1, and Sp3 to the Dp71 promoter.
Assuntos
Distrofina/genética , Hepatócitos/efeitos dos fármacos , Hepatócitos/metabolismo , Regiões Promotoras Genéticas , Fator de Transcrição Sp1/metabolismo , Fator de Transcrição Sp3/metabolismo , Fator de Transcrição YY1/metabolismo , beta-Naftoflavona/farmacologia , Células Hep G2 , Humanos , Ligação ProteicaRESUMO
Dystrophin Dp71, the smallest product encoded by the Duchenne muscular dystrophy gene, is ubiquitously expressed in all non-muscle cells. Although Dp71 is involved in various cellular processes, the mechanisms underlying its expression have been little studied. In hepatic cells, Dp71 expression is down-regulated by the xenobiotic ß-naphthoflavone. However, the effectors of this regulation remain unknown. In the present study we aimed at identifying DNA elements and transcription factors involved in Dp71 expression in hepatic cells. Relevant DNA elements on the Dp71 promoter were identified by comparing Dp71 5'-end flanking regions between species. The functionality of these elements was demonstrated by site-directed mutagenesis. Using EMSAs and ChIP, we showed that the Sp1 (specificity protein 1), Sp3 (specificity protein 3) and YY1 (Yin and Yang 1) transcription factors bind to the Dp71 promoter region. Knockdown of Sp1, Sp3 and YY1 in hepatic cells increased endogenous Dp71 expression, but reduced Dp71 promoter activity. In summary, Dp71 expression in hepatic cells is carried out, in part, by YY1-, Sp1- and Sp3-mediated transcription from the Dp71 promoter.