Abstract
The archetypal TATA-box deficient G+C-rich promoter of the murine adenosine deaminase gene (Ada) requires a 48-bp minimal self-sufficient promoter element (MSPE) for function. This MSPE was used to isolate a novel full-length cDNA clone that encodes a 66-kDa murine G+C-rich promoter binding protein (mGPBP). The mGPBP mRNAs are ubiquitously expressed as either 3.0- or 3.5-kb forms differing in 3′ polyadenylation site usage. Purified recombinant mGPBP, in the absence of any other mammalian cofactors, binds specifically to both the murine Ada gene promoter's MSPE and the nonhomologous human Topo IIα gene's G+C-rich promoter. In situ binding assays, immunoprecipitation, and Western blot analyses demonstrated that mGPBP is a nuclear factor that can form complexes with TATA-binding protein, TFIIB, TFIIF, RNA polymerase II, and P300/CBP both in vitro and in intact cells. In cotransfection assays, increased mGPBP expression transactivated the murine Ada gene's promoter. Sequestering of GPBP present in HeLa cell nuclear extract by immunoabsorption completely and reversibly suppressed extract-dependent in vitro transcription from the murine Ada gene's G+C-rich promoter. However, transcription from the human Topo IIα gene's TATA box-containing G+C-rich promoter was only partially suppressed and the adenovirus major late gene's classical TATA box-dependent promoter is totally unaffected under identical assay conditions. These results implicate GPBP as a requisite G+C-rich promoter-specific transcription factor and provide a mechanistic basis for distinguishing transcription initiated at a TATA box-deficient G+C-rich promoter from that initiated at a TATA box-dependent promoter.
ACKNOWLEDGMENTS
We thank Shao-Xia Lin, Chefin Bobonis, Teresa Szal, and Sarah Bothner for excellent technical assistance. We also thank Kelly McNagny for the cDNA library and Pradip Raychaudhuri for antibody reagents and advice and discussions.
This work was supported in part by NIH grant AG11623 and MUNIN Corp. award MC99-2 to C.-Y.Y., NIH grant CA72572 to J.M.L., RO1 GM44088 to V.J.K., and NIH SBIR grants CA78044, CA91376, and CA89777 to J.A.G. L.-C.H. was supported by a UIC University fellowship. Generous support for these studies was also provided by the American Lebanese Syrian Associated Charities and NIH (Cancer Center core grant 5 PO1 CA21765) to SJCRH for J.M.L. and V.J.K.