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J Biol Chem, Vol. 273, Issue 21, 13208-13216, May 22, 1998

Role of Sp Proteins and ROR in Transcription Regulation of Murine Prosaposin

From the Division of Human Genetics, Children's Hospital Research Foundation, Children's Hospital Medical Center, Cincinnati, Ohio 45229-3039

Prosaposin is the precursor of four low molecular weight sphingolipid-activating proteins (SAPs) or saposins. These four proteins function as intracellular activators of several lysosomal enzymes involved in the degradation of glycosphingolipids, and prosaposin itself has neurite outgrowth effects. Expression of prosaposin is regulated in a temporal and spatial manner with expression in specific brain neurons and visceral cell types. Here a major regulatory fragment was characterized within 310 bp 5' to the transcription start site. Using electrophoretic mobility shift assay (EMSA) and DNA footprinting, members of the Sp family (Sp1, Sp3, and Sp4), the orphan nuclear receptor (ROR), and an unknown transcription factor (U; TGGGGGAG) were shown to bind to this region. To evaluate the role of such transcription factor binding sites for this locus, a series of mutant constructs was generated within this region, and their function was evaluated in cultured NS20Y neuroblastoma cells. A 3' Sp1 site, a 5' Sp1/U cluster and the ROR binding sites were functional. The data are consistent with a model in which the factors that bind to the Sp1/U cluster and RORE site interact negatively to diminish promoter activity to a background level that is determined primarily by the 3' Sp1 site. These interactions depend on the tissue-specific repertoire of transcription factors leading to differential expression of this locus.

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