Novel leader exons of the cyclic adenosine 3',5'-monophosphate response element modulator (CREM) gene, transcribed from promoters P3 and P4, are highly testis-specific in primates.

Testicular expression of CREM is essential for spermatogenesis in the mouse. From a monkey testis cDNA library we isolated a CREM transcript isoform with a novel 5' exon theta2 which provides at its 3'-end an in-frame ATG to the downstream reading frame. 5'-RACE on human testis cDNA indicated that exon theta2 is > or = 113 bp in size. Moreover, a second novel leader exon, theta1, of > or = 289 bp was identified and encodes a putative open reading frame of 26 amino acids. In-vitro translation and cellular expression of CREM-theta1 and CREM-theta2 splice variants cloned from human testis yielded not only full length proteins but also shorter repressor products resulting from downstream translation initiation. Upon co-transfection, products of CREM-theta2 cDNA repressed protein kinase A-induced activation of a CRE-driven reporter construct. RT-PCR analysis of primate tissues for CREM-theta2 transcripts showed abundant expression in the testis and very low levels or absence from all other tissues tested. CREM-theta1 mRNA was exclusively expressed in the testis. Promoters P3 and P4, flanking exons theta1 and theta2, were cloned and found to be non-responsive to protein kinase A in transfection assays. Furthermore, we show differential activation of P1, P3 and P4 during mouse postnatal testicular development, suggesting cell- and stage-specific regulatory mechanisms for these CREM promoters.