CCAAT/enhancer binding protein beta regulates expression of the cystatin-related epididymal spermatogenic (cres) gene

The CRES protein is a member of the cystatin super-family of cysteine prote ase inhibitors with restricted expression in stage-specific germ cells, pro ximal caput epididymidis, and anterior pituitary gonadotroph cells. To eluc idate the molecular mechanisms regulating the highly restricted expression of the cres gene, we have sequenced 1.6 kilobases of mouse cres 5' flanking sequence and performed studies to examine the cres gene promoter. Two puta tive CCAAT/enhancer binding protein (C/EBP) transcription factor binding mo tifs exist within the first 135 base pairs of cres promoter. Furthermore, o ur studies demonstrate that cres mRNA levels are dramatically reduced in th e epididymides of C/EBP beta -deficient mice. These data suggest that the C /EBP family of transcription factors, in particular C/EBP beta, plays a rol e in the regulation of cres gene expression. In support of this finding, No rthern blot analysis showed that C/EBP beta is the predominant C/EBP family member expressed in the L beta T2 gonadotroph cell line and the proximal c aput epididymidis. Also, gel shift and supershift assays demonstrated that C/EBP beta protein in nuclear extracts from L beta T2 gonadotroph cells and epididymal cells bound to the two C/EBP sites in the cres promoter. Finall y, to test the in vivo function of the C/EBP sites in cres gene expression, transfection studies were performed in L beta T2 gonadotroph cells and two heterologous cell systems. These experiments showed a significant reductio n of cres transactivation when either C/EBP sites were mutated, and no tran sC/EBP activation of the cres promoter when both C/EBP sites were mutated. Taken together, these studies demonstrate that the C/EBP beta transcription factor is necessary for high levels of cres gene expression in the proxima l caput epididymidis and anterior pituitary gonadotroph cells.