Differential activation of the SMalpha A promoter in smooth vs. skeletal muscle cells by bHLH factors

E-box/basichelix-loop-helix (bHLH)-dependent regulation of promoters for skeletalmuscle-specific genes is well established, but similar regulation ofsmooth muscle-selective promoters has not been reported. Usingtransient transfection assays of smooth muscle -actin (SMA)promoter-chloramphenicol acetyltransferase (CAT) reporter constructs inrat vascular smooth muscle cells (SMCs) and L6 skeletal myotubes, weidentified two activator elements, smE1 and smE2, with sequencescorresponding to E-box (5'-CAnnTG-3') motifs. In L6myotubes, 4-bp mutations of smE1 or smE2 E-box motif alone completelyabolished promoter activity. In contrast, mutation of smE1 and smE2 wasrequired to reduce promoter activity in SMCs. Supershift analysesidentified a myogenin-containing complex as the predominant smE1 andsmE2 binding activity in skeletal muscle, and myogenin overexpressiontransactivated the promoter. Supershift analyses with SMC extractsdemonstrated that the bHLH protein upstream stimulatory factor (USF)bound smE1, and USF overexpression transactivated the promoter in ansmE1-dependent manner. In summary, our results provide novel evidenceimplicating E-box elements in directing expression of the SMApromoter through distinct bHLH factor complexes in skeletal vs. smooth muscle.

     

Characterization of adjacent E-box and nuclear factor 1-like DNA binding sequence in the human CYP1A2 promoter

To better understand the molecular mechanisms of cytochrome P450 1A2 (CYP1A2) regulation, we have characterized a region of the promoter (+3 to -176) that contains a single E-box and an adjacent nuclear factor 1 (NF1)-like DNA binding site. The E-box was shown to specifically bind nuclear proteins that were recognized by antibodies against upstream stimulatory factor (USF) 1 and 2. Comparison of NF1 binding proteins in HepG2 cells and primary cultures of rat hepatocytes revealed different patterns of DNA-protein complexes, all of which were recognized by a general NF1 antibody. Mutations of the E-box resulted in substantial reduction of promoter activity in either primary hepatocytes or HepG2 cells regardless of the presence in the reporter constructs of other CYP1A2 regulatory elements, such as the hepatic nuclear factor 1 (HNF-1) binding site. In contrast, reporter gene activity of the promoter construct harboring the mutated NF1-like binding site was affected by upstream sequences when transfected into HepG2 cells, but not in primary hepatocytes. We conclude that both USF proteins and different isoforms of NF1 contribute to the constitutive expression of CYP1A2.