Transcriptional regulation of the human beta3-adrenergic receptor gene

Abstract

Regulatory elements responsible for tissue-specific transcriptional regulation of the human β 3 -adrenergic receptor (β 3 -AR) were identified. A region localized between −6.50 and −6.30 kb of the proximal promoter contained three sequences that act synergistically to achieve full transcriptional activity. One segment, termed segment A, contains an Sp1 binding site. Another of the sequences, termed segment B, is a binding site for a trans-acting factor present in cells that constitutively express β 3 -AR. In a specific embodiment, the trans-acting factor is expressed in neuroblastoma (SK-N-MC) and brown adipose tissue cells, but little or not at all in CV-1, HeLa, or white adipose tissue cells. The third segment, C, is an S1 nuclease-sensitive site having CCTT repeats. Recombinant vectors under control of this transcriptional regulation region, particularly containing the B and C segments, provide a substrate for high throughput assays, such as reporter gene assays, to identify compounds that can increase the level of expression of β 3 -AR. The B segment nucleic acids also provide for isolation and cloning of the trans-acting factor. Mechanisms of transcriptional regulation and identification of other adjacent proteins involved in the regulation of the hβ 3 -AR gene expression are provided.

What is claimed is:  
     
         1 . An isolated β 3 -AR trans-activating factor polypeptide, wherein said polypeptide has the following characteristics: 
 (a) it binds specifically to a nucleic acid having the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO: 1);  
 (b) it is expressed by brown adipose tissue cells;  
 (c) it is expressed at very low levels by cells isolated from the perirenal depot;  
 (d) an AP-2 binding nucleic acid does not compete with a nucleic acid comprising a nucleotide sequence GCCTCTGGGGAG (SEQ ID NO: 1) for binding the polypeptide; and,  
 (e) when complexed to a nucleic acid comprising SEQ ID NO: 1, it is not recognized by an antibody to AP-2.  
 
     
     
         2 . A composition comprising an isolated β 3 -AR trans-activating factor polypeptide, wherein said polypeptide has the following characteristics: 
 (a) it binds specifically to a nucleic acid having the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO: 1);  
 (b) it is expressed by brown adipose tissue cells;  
 (c) it is expressed at very low levels by cells isolated from the perirenal depot;  
 (d) an AP-2 binding nucleic acid does not compete with a nucleic acid comprising a nucleotide sequence GCCTCTGGGGAG (SEQ ID NO: 1) for binding the polypeptide; and,  
 (e) when complexed to a nucleic acid comprising SEQ ID NO: 1, it is not recognized by an antibody to AP-2.  
 
     
     
         3 . The composition of  claim 2  further comprising an isolated nucleic acid having the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO: 1).