Nuclear """"""""orphan"""""""" receptors are members of the thyroid/steroid hormone receptor superfamily of transcription factors whose ligand-specificity, if any, is unknown. Rev-ErbAalpha is a fascinating orphan receptor whose gene expression may regulate alternative splicing of the alpha thyroid hormone (T3) receptor and c-erbAalpha2, another orphan receptor which inhibits T3 action, whose gene overlaps that of Rev-ErbAalpha but is transcribed from the opposite strand. Interestingly, Rev-ErbAalpha gene expression is basally repressed by labile proteins and induced during adipocyte differentiation. This laboratory is interested in the regulation and regulatory role of Rev-ErbAalpha gene expression, as well as in the function of the Rev-ErbAalpha protein, which is completely unknown at present.
The specific aims of this proposal are to: (1) study the regulation of Rev-ErbAalpha expression at the gene and protein levels, (2) determine the functional significance of the Rev-ErbAalpha/c-erbAalpha2 genomic relationship in light of the demonstrated ability of Rev-ErbAalpha to interfere with splicing in vitro, and (3) characterize the DNA-binding and transcriptional properties of Rev-ErbAalpha protein. The Rev-ErbAalpha gene promoter will be isolated and characterized, and transcription factors which regulate Rev-ErbAalpha expression by binding to these sequences will be cloned. Rev-ErbAalpha antisera will be used to demonstrate Rev- ErbAalpha protein expression, correlate mRNA and protei levels, and study post-translational modification of Rev-ErbAalpha. Also, the ability of Rev-ErbAalpha transcripts to inhibit the splicing of endogenous and cotransfected c-erbAalpha2 will be evaluated to determine whether this novel regulatory mechanism functions in vivo. In addition, the function of the Rev-ErbAalpha protein will be explored by amplifying, isolating, and sequencing its preferred DNA-binding sites. The specificity of DNA-binding by Rev-ErbAalpha will be compared with that of other nuclear receptors, and the ability of wild-type and chimeric forms of Rev-ErbAalpha to regulate gene transcription from reporter genes containing Rev-ErbAalpha binding sites will be evaluated. The long term goal of this work is to understand the role of this novel orphan receptor in cell growth and differentiation. These studies will also provide insight into the biological significance of orphan receptors as well as the mechanisms of action of nuclear hormone receptor family members.
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