Afar R, Silverman R, Aguanno A, Albert V R
Roche Institute of Molecular Biology, Nutley, NJ 07110, USA.
Brain Res Mol Brain Res. 1996 Feb;36(1):79-92. doi: 10.1016/0169-328x(95)00247-p.
Dopamine beta-hydroxylase catalyzes the final step in noradrenaline synthesis and is expressed exclusively in noradrenergic and adrenergic cells. In order to identify elements within the dopamine beta-hydroxylase (DBH) gene which contribute to the regulation of tissue-specific expression, we have analyzed the expression of the rat DBH promoter by transient transfection in both DBH-expressing and non-expressing cell lines. We have found that 1 kilobase of the DBH promoter can direct expression of the luciferase reporter gene in the DBH-expressing PC12, CATH.a, and SK-N-SH cell lines, but not in the non-DBH-expressing C6 glioma or CA77 cell lines. This activity was localized to a region between -133 and -173 upstream of the transcription start site. This element, however, also directed expression in non-DBH-expressing cell lines, but was inhibited when sequences between -212 and -388 were included. This inhibitory region contains sequences homologous to a silencer element recently identified in the human DBH gene, and shares homology with other previously identified silencer elements. Gel retardation experiments demonstrate that the rat DBH inhibitory region and the silencer elements found in the rat sodium type II channel and SCG10 genes bind a similar factor. The region between -133 and -173, which contains a consensus cyclic AMP response element (CRE), was also found to be responsive to cAMP in both DBH-expressing and non-expressing cells. Inclusion of sequences between -173 and -190 diminished the cAMP induction in PC12 cells, and nearly abolished the induction in C6 and CA77 cells, suggesting the presence of an additional negative element which inhibits cAMP induction in non-DBH expressing cells. DNA binding assays using antibodies to CRE binding protein-related transcription factors identified ATF-1 binding to the rat DBH-CRE, and further suggest that inhibition of cAMP regulation may be due to inhibition of ATF-1 binding by an additional factor, which binds to the DBH promoter immediately upstream of the CRE. These results demonstrate the importance of both positive and negative regulatory elements in the regulation of tissue-specific expression of the rat DBH gene.
多巴胺β-羟化酶催化去甲肾上腺素合成的最后一步，且仅在去甲肾上腺素能和肾上腺素能细胞中表达。为了鉴定多巴胺β-羟化酶（DBH）基因中有助于调节组织特异性表达的元件，我们通过在表达DBH和不表达DBH的细胞系中进行瞬时转染，分析了大鼠DBH启动子的表达情况。我们发现，DBH启动子的1千碱基可在表达DBH的PC12、CATH.a和SK-N-SH细胞系中指导荧光素酶报告基因的表达，但在不表达DBH的C6胶质瘤或CA77细胞系中则不能。这种活性定位于转录起始位点上游-133至-173之间的区域。然而，该元件也可在不表达DBH的细胞系中指导表达，但当包含-212至-388之间的序列时则受到抑制。这个抑制区域包含与最近在人DBH基因中鉴定出的沉默子元件同源的序列，并且与其他先前鉴定的沉默子元件具有同源性。凝胶阻滞实验表明，大鼠DBH抑制区域以及在大鼠II型钠通道和SCG10基因中发现的沉默子元件结合相似的因子。在-133至-173之间的区域，包含一个共有环磷酸腺苷反应元件（CRE），在表达DBH和不表达DBH的细胞中也被发现对环磷酸腺苷有反应。包含-173至-190之间的序列会减弱PC12细胞中环磷酸腺苷的诱导作用，并几乎消除C6和CA77细胞中的诱导作用，这表明存在一个额外的负性元件，它在不表达DBH的细胞中抑制环磷酸腺苷的诱导。使用针对CRE结合蛋白相关转录因子的抗体进行的DNA结合分析确定了ATF-1与大鼠DBH-CRE的结合，并进一步表明环磷酸腺苷调节的抑制可能是由于一个额外的因子抑制了ATF-1的结合，该因子与CRE上游紧邻的DBH启动子结合。这些结果证明了正负调控元件在大鼠DBH基因组织特异性表达调控中的重要性。
