Submission Number: KSH-4-11-13

Abstract Number: 311

TESTIS-SPECIFIC 47kDa NUCLEAR PROTEIN, A PUTATIVE TRANSCRIPTION FACTOR, BINDS TO SINGLE AND DOUBLE STRANDED SP-10 PROMOTER ELEMENTS.

Kshitish K Acharya*, John C Herr* and Prabhakara P Reddi*

Department of Cell Biology, University of Virginia, Charlottesville, VA 22908, USA ¹

Abstract:
We are using the testis-specific gene SP-10 as a model to study transcriptional regulation during acrosomal biogenesis. Promoter analysis using transgenic mice showed that the -266 to -91bp region of the SP-10 promoter is critical for spermatid-specific expression of SP-10. Southwestern analysis (SWA) revealed binding of several fragments of the -266 to -91bp SP-10 promoter to a 47kD testis nuclear protein (TNP47). In the present study, attempts were made to further characterize TNP47 and identify its cognate binding site on the promoter. In SWA, TNP47 appeared to bind with highest affinity to the -184 to -146bp region (38mer) of the SP-10 promoter. Tissue specificity examination using nuclear proteins from brain, heart, spleen, kidney, liver and lung indicated that TNP47 is testis-specific. Furthermore, in developing testis, TNP47 was more abundant on day 24 (round spermatids present) than 17 (spermatids absent) suggesting a temporal expression pattern of TNP47 that corresponds to SP-10 promoter activity. Sequence comparison of three SP-10 promoter fragments which bind to TNP47 revealed a common 5-acacac motif that was repeated within the 38mer. Mutation of either 5-acacac motif reduced TNP47 binding of 38mer whereas mutation of both the motifs abolished the binding. TNP47 also displayed single stranded DNA binding. The anti-sense strand of the 38mer bound to TNP47 with much higher affinity than the sense strand or the double stranded 38mer. Two-dimensional (2-D) electrophoresis of testis nuclear proteins (TNPs) followed by SWA analysis demonstrated that a 47 ( 3) kD protein with a pI of 5.2 ( 0.3) binds to the anti-sense strand as well as double stranded 38mer. The 2-D characterization is expected to help in micro sequencing and cloning of TNP47. In summary, a 47kD testis-specific nuclear protein and its putative cis-element 5-acacac have been identified. The relevance of these trans- and cis-elements in SP-10 gene regulation is being investigated. Supported by NIH HD 36239, HD 29099, Fogarty International Center, Andrew W Mellon Foundation and Schering A.G.

Keywords: Transcriptional regulation, SP-10, acrosomal biogenesis, testis, round spermatid, spematogenesis

This abstract is being presented at: 8:00 AM in session:
Gene Regulation and Function I