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.

1 - 3 . (canceled)  
     
     
         4 . A method for modulating a human Sp1 or B segment-binding β 3 -adrenergic receptor (β 3 -AR) trans-activating factor in mammalian cells, which method comprises: 
 (a) contacting mammalian cells capable of producing the Sp1 or B segment-binding β 3 -AR trans-activating factor with a test compound; and    (b) detecting an increase in a level of activity of the Sp1 or B segment-binding β 3 -AR trans-activating factor,    wherein the increase in the level of activity of the Sp1 or B segment-binding β 3 -AR trans-activating factor results in an increase in the level of β 3 -AR gene product relative to a level of expression prior to contact with the test compound.    
     
     
         5 . A method of modulating activity of a human β 3 -adrenergic receptor (β 3 -AR) trans-activating factor in mammalian cells, which method comprises: 
 (a) contacting mammalian cells capable of producing the β 3 -AR trans-activating factor with a test compound; and    (b) detecting an increase in a level of activity of the β 3 -AR trans-activating factor, wherein the increase in the level of activity of the β 3 -AR trans-activating factor is detected by detecting an increase in the level of expression of a reporter gene operatively associated with an isolated nucleic acid having a nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) relative to a level of expression prior to contact with the test compound.    
     
     
         6 . A method according to  claim 4 , wherein the increase in the level of activity of the β 3 -AR trans-activating factor is detected by detecting an increase in the amount of β 3 -AR trans-activating factor present in the cells after contacting them with the test compound relative to the amount present prior to contact with the test compound.  
     
     
         7 . A method according to  claim 4 , wherein the cells do not endogenously express β 3 -AR.  
     
     
         8 . A method according to  claim 7 , wherein the cells are selected from the group consisting of HeLa cells, CV-1 cells, and WAT cells.  
     
     
         9 . A method of modulating activity of a human Sp1 or B segment-binding β 3 -adrenergic receptor (β 3 -AR) trans-activating factor in mammalian cells, which method comprises: 
 (a) contacting mammalian cells capable of producing the Sp1 or B segment-binding β 3 -AR trans-activating factor with a test compound; and    (b) detecting a decrease in a level of activity of the Sp1 or B segment-binding β 3 -AR trans-activating factor,    wherein the decrease in the level of activity of the Sp1 or B segment-binding β 3 -AR trans-activating factor results in a decrease in the level of β 3 -AR gene product relative to a level of expression prior to contact with the test compound.    
     
     
         10 . A method of modulating activity of a human β 3 -adrenergic receptor (β 3 -AR) trans-activating factor in mammalian cells, which method comprises: 
 (a) contacting mammalian cells capable of producing the β 3 -AR trans-activating factor with a test compound; and    (b) detecting a decrease in a level of activity of the β 3 -AR trans-activating factor,    wherein the decrease in the level of activity of the β 3 -AR trans-activating factor is detected by detecting a decrease in the level of expression of a reporter gene operatively associated with an isolated nucleic acid having a nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) relative to a level of expression prior to contact with the test compound.    
     
     
         11 . A method according to  claim 9 , wherein the decrease in the level of activity of the β 3 -AR trans-activating factor is detected by detecting a decrease in the amount of β 3 -AR trans-activating factor present in the cells after contacting them with the test compound relative to the amount present prior to contact with the test compound.  
     
     
         12 . A method according to  claim 9 , wherein the cells endogenously express β 3 -AR.  
     
     
         13 . A method according to  claim 11 , wherein the cells are selected from the group consisting of neuroblastoma and BAT cells.  
     
     
         14 . A method of modulating activity of a human β 3 -adrenergic receptor (β 3 -AR) trans-activating factor in mammalian cells, which method comprises: 
 (a) contacting mammalian cells capable of producing the β 3 -AR trans-activating factor with a test compound; and    (b) detecting an increase in a level of activity of the β 3 -AR trans-activating factor, wherein the level of activity of the β 3 -AR trans-activating factor is detected by an increase in the level of expression of a reporter gene operatively associated with an isolated nucleic acid selected from the group consisting of: 
 (i) about a 7 kb genomnic DNA 5′ flanking region of a β 3 -AR transcription start site,  
 (ii) a deletion construct of a 7 kb genomic DNA located upstream of a β 3 -AR transcription start site;  
 (iii) a nucleic acid comprising a nucleotide sequence that is greater than 80% identical to the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) located 5′ to an Sp-1 binding site relative to a transcription start site; and  
 (iv) a nucleic acid comprising a heterologous coding sequence operatively associated with a promoter and operatively associated with a nucleotide sequence that is greater than 80% identical to the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) in proximity to an Sp-1 binding site, whereby expression of the heterologous protein is regulated in a tissue specific manner.  
   
     
     
         15 . A method of modulating activity of a human β 3 -adrenergic receptor (β 3 -AR) trans-activating factor in mammalian cells, which method comprises: 
 (a) contacting mammalian cells capable of producing the β 3 -AR trans-activating factor with a test compound; and    (b) detecting a decrease in a level of activity of the β 3 -AR trans-activating factor, wherein the level of activity of the β 3 -AR trans-activating factor is detected by a decrease in the level of expression of a reporter gene operatively associated with an isolated nucleic acid selected from the group consisting of: 
 (i) about a 7 kb genomic DNA 5′ flanking region of a β 3 -AR transcription start site,  
 (ii) a deletion construct of a 7 kb genomic DNA located upstream of a β 3 -AR transcription start site;  
 (iii) a nucleic acid comprising a nucleotide sequence that is greater than 80% identical to the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) located 5′ to an Sp-1 binding site relative to a transcription start site; and  
 (iv) a nucleic acid comprising a heterologous coding sequence operatively associated with a promoter and operatively associated with a nucleotide sequence that is greater than 80% identical to the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) in proximity to an Sp-1 binding site, whereby expression of the heterologous protein is regulated in a tissue specific manner.  
   
     
     
         16 . A method for isolating a purified β 3 -AR trans-activating factor comprising: 
 a) contacting a sample suspected of containing the β 3 -AR trans-activating factor with a nucleic acid comprising the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1) under conditions that permit detection of binding of the β 3 -AR trans-activating factor to the nucleic acid;    b) isolating the bound β 3 -AR trans-activating factor.    
     
     
         17 . A purified β 3 -AR trans-activating factor isolated according to the method of  claim 16 , wherein said β 3 -AR trans-activating factor has the following characteristics: 
 a) the β 3 -AR trans-activating factor binds specifically to a nucleic acid comprising the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1);    b) the β 3 -AR trans-activating factor is expressed by brown adipose tissue cells;    c) the β 3 -AR trans-activating factor is expressed at lower levels by human white adipose tissue cells isolated from the perirenal depot compared to its expression level in mouse brown adipose tissue cells;    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 β 3 -AR trans-activating factor;    e) when complexed to a nucleic acid comprising SEQ ID NO:1, the β 3 -AR trans-activating factor is not recognized by an antibody to AP-2;    f) the β 3 -AR trans-activating factor binding to SEQ ID NO:1 is demonstrated by electrophoretic mobility assay;    g) the β 3 -AR trans-activating enhances β 3 -adrenergic receptor expression; and    h) wherein said purified β 3 -AR trans-activating factor is substantially free of cellular components other than β 3 -AR trans-activating factor.    
     
     
         18 . A composition comprising an isolated nucleic acid having the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1), and an β 3 -AR trans-activating factor isolated according to the method of  claim 16 , wherein said isolated trans-activating factor is free of cellular components and has the following characteristics: 
 a) the β 3 -AR trans-activating factor binds specifically to a nucleic acid comprising the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1);    b) the β 3 -AR trans-activating factor is expressed by brown adipose tissue cells;    c) the β 3 -AR trans-activating factor is expressed at lower levels by human white adipose tissue cells isolated from the perirenal depot compared to its expression level in mouse brown adipose tissue cells;    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 trans-activating factor;    e) when complexed to a nucleic acid comprising SEQ ID NO:1, the β 3 -AR trans-activating factor is not recognized by an antibody to AP-2;    f) the β 3 -AR trans-activating factor binding to SEQ ID NO:1 is demonstrated by electrophoretic mobility assay;    g) the β 3 -AR trans-activating factor enhances β 3 -adrenergic receptor expression; and    h) wherein said purified trans-activating factor is substantially free of cellular components other than β 3 -AR trans-activating factor.    
     
     
         19 . A composition comprising a purified β 3 -AR trans-activating factor isolated according to the method of  claim 16 , and other binding factors that may interact with genomic DNA, wherein said β 3 -AR trans-activating factor has the following characteristics: 
 a) the β 3 -AR trans-activating factor binds specifically to a nucleic acid comprising the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1);    b) the β 3 -AR trans-activating factor is expressed by brown adipose tissue cells;    c) the β 3 -AR trans-activating factor is expressed at lower levels by human white adipose tissue cells isolated from the perirenal depot compared to its expression level in mouse brown adipose tissue cells;    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 trans-activating factor;    e) when complexed to a nucleic acid comprising SEQ ID NO:1, it the β 3 -AR trans-activating factor is not recognized by an antibody to AP-2;    f) the β 3 -AR trans-activating factor binding to SEQ ID NO:1 is demonstrated by electrophoretic mobility assays;    g) the β 3 -AR trans-activating factor enhances β 3  adrenergic receptor expression; and    h) wherein said purified β 3 -AR trans-activating factor is substantially free of proteins with which it can be naturally found within a cell.    
     
     
         20 . The composition of  claim 19  further comprising an isolated nucleic acid having the nucleotide sequence GCCTCTGGGGAG (SEQ ID NO:1).