Expression of the human beta(3)-adrenergic receptor gene in SK-N-MC cells is under the control of a distal enhancer

Mechanisms of transcriptional regulation of the human beta (3)-adrenergic r eceptor were studied using SK-N-MC cells, a human neuroblastoma cell line t hat expresses beta (3)- and beta (1)-adrenergic receptors endogenously. Del etions spanning different portions of a 7-kb 5 ' -flanking region of the hu man beta (3)-adrenergic receptor gene were linked to a luciferase reporter and transfected in SK-N-MC, CV-1, and HeLa cells. Maximal luciferase activi ty was observed when a 200-bp region located between -6.5 and -6.3 kb from the translation start site was present. This region functioned only in SK-N -MC cells. Electrophoretic mobility shift assays of nuclear extracts from S K-N-MC, CV-1, and HeLa cells using double stranded oligonucleotides spannin g different portions of the 200-bp region as probes and transient transfect ion studies revealed the existence of three cis-acting regulatory elements: A) -6.468 kb-AGGTGGACT--6.458 kb, B) -6.448 kb-GCCTCTCTGGGGAGCAGCTTCTCC-6. 428 kb, and C) -6.405 kb-20 repeats of CCTT-6.385 kb. These elements act to gether to achieve full transcriptional activity. Mutational analysis, antib ody supershift, and electrophoretic mobility shift assay competition experi ments indicated that element A binds the transcription factor Spl, element B binds protein(s) present only in nuclear extracts from SK-N-MC cells and brown adipose tissue, and element C binds protein(s) present in both SK-N-M C and HeLa cells. In addition, element C exhibits characteristics of an S1 nuclease-hypersensitive site. These data indicate that cell-specific positi ve cis-regulatory elements located 6.5 kb upstream from the translation sta rt site may play an important role in transcriptional regulation of the hum an beta (3)-adrenergic receptor. These data also suggest that brown adipose tissue-specific transcription factor(s) may be involved in the tissue-spec ific expression of the beta (3)-adrenergic receptor gene.