Transcriptional regulation of cell-specific expression of the human cystathionine beta-synthase gene by differential binding of Sp1/Sp3 to the-1b promoter

Cystathionine beta -synthase (CBS) catalyzes the condensation of serine and homocysteine to form cystathionine, an intermediate step in the synthesis of cysteine. We previously characterized the CBS -1b minimal promoter (-379 2 to -3667) and found that Sp1/Sp3, nuclear factor Y, and USF-1 were involv ed in the regulation of basal promoter activity (Ge, Y., Konrad, M. A., Mat herly, L. H., Taub, J. W. (2001) Biochem. J. 357, 97-105). In this study, t he critical cis-elements and transcription factors in the CBS -1b upstream region (-4046 to -3792) were examined in HT1080 and HepG2 cells, which diff er 10-fold in levels of CBS transcripts transcribed from the CBS -1b promot er. In DNase I footprint and gel shift analyses and transient transfections of mutant CBS -1b promoter constructs into HT1080 and HepG2 cells, transcr iptionally important roles for Sp1/Sp3 binding to three GC boxes and one GT box and for binding of myeloid zinc finger 1-like proteins to two myeloid zinc finger I elements were indicated. In gel shift assays, very low levels of Sp1/Sp3 DNA-protein complexes were detected in HT1080 cells compared wi th HepG2 cells despite comparable levels of nuclear factor Y and USF-1 bind ing and similar levels of Spl and Sp3 proteins on Western blots. Mixing of HT1080 and HepG2 nuclear extracts resulted in no difference in total Sp fac tor binding in gel shift assays, thus excluding a role for an unknown activ ator or inhibitor in the disparate Sp1/Sp3 binding between the lines. Incre ased Sp1/Sp3 binding in gel shift assays was observed upon treatment of HT1 080 nuclear extracts with protein kinase A, and decreased Sp1/Sp3 binding r esulted from treatment of HepG2 nuclear extracts with calf alkaline phospha tase, suggesting a role for changes in Sp1/Sp3 phosphorylation in transcrip tion factor binding and transactivation of the CBS -1b promoter. Characteri zation of CBS promoter structure and function should clarify the molecular bases for variations in CBS gene expression in genetic diseases and the rel ationship between CBS and Down syndrome.