Sp1/Sp3 and the myeloid zinc finger gene MZF1 regulate the human N-cadherin promoter in osteoblasts.
To determine the molecular mechanisms by which N-cadherin transcription is regulated, we cloned and sequenced a 3681-bp of the 5'-flanking region of the human N-cadherin gene. Deletion analysis of the proximal region identified a minimal 318-bp region with strong promoter activity in human osteoblasts. The cryptic promoter is characterized by high GC content and a GA-rich binding core that may bind zing finger transcription factors. Electrophoretic mobility shift assays (EMSA), competition and supershift EMSA revealed that an Sp1/Sp3 binding site acts as a basal regulatory element of the promoter in osteoblasts. Incubation of osteoblast nuclear extracts with -163/-131 wild-type probe containing the GA-rich binding core revealed another specific complex, which was not formed with a -163/-131 probe mutated in the GA repeat. EMSA identified the nuclear factor involved as myeloid zinc finger-1 (MZF1). Mutation analysis showed that Sp1/Sp3 and MZF1 binding sites contribute to basal promoter activity. Cotransfection analyses showed that Sp1 and MZF1 overexpression increases whereas Sp3 antagonizes Sp1-induced N-cadherin promoter activity in osteoblasts. RT-PCR analysis showed that human osteoblastic cells express MZF1 and that Sp1/MZF1 overexpression increased N-cadherin expression. These results indicate that Sp1/Sp3 and MZF1 are important transcription factors regulating N-cadherin promoter activity and expression in osteoblasts.
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Laboratory of Osteoblast Biology and Pathology, INSERM U606, Lariboisière Hospital, 75475 Cedex 10 Paris, France.
Fromigué
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Marie
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PJ
D024506:5' Flanking Region / Q000235:genetics; D000595:Amino Acid Sequence / Q000235:genetics; D001483:Base Sequence / Q000235:genetics; D001665:Binding Sites / Q000235:genetics; D015820:Cadherins / Q000235:genetics*; D002454:Cell Differentiation / Q000235:genetics; D002461:Cell Line, Transformed; D045744:Cell Line, Tumor; D004268:DNA-Binding Proteins / Q000235:genetics / Q000378:metabolism*; D005091:Exons / Q000235:genetics; D005786:Gene Expression Regulation / Q000235:genetics; D005809:Genes, Regulator / Q000235:genetics; D006801:Humans; D051741:Kruppel-Like Transcription Factors; D015335:Molecular Probes; D008969:Molecular Sequence Data; D010006:Osteoblasts / Q000378:metabolism*; D011401:Promoter Regions, Genetic / Q000235:genetics*; D017434:Protein Structure, Tertiary / Q000235:genetics; D016329:Sp1 Transcription Factor / Q000235:genetics / Q000378:metabolism*; D051705:Sp3 Transcription Factor; D014157:Transcription Factors / Q000235:genetics / Q000378:metabolism*; D015854:Up-Regulation / Q000235:genetics
D015820:Cadherins; D004268:DNA-Binding Proteins; D051741:Kruppel-Like Transcription Factors; C085969:MZF1 protein, human; D015335:Molecular Probes; C497121:SP3 protein, human; D016329:Sp1 Transcription Factor; D014157:Transcription Factors; D051705:Sp3 Transcription Factor