NOVEL TESTIS-SPECIFIC PROTEIN-DNA INTERACTIONS ACTIVATE TRANSCRIPTIONOF THE MOUSE PROTAMINE-2 GENE DURING SPERMATOGENESIS

The mouse protamines ape expressed exclusively in postmeiotic male ger m cells and are crucial for the compaction of chromatin during the lat e stages of spermatogenesis, The temporal expression of the two mouse protamines is transcriptionally regulated in the testis. Recent studie s have demonstrated that ubiquitous and testis-specific proteins bind to the promoter of the mouse protamine 2 (mP2) gene, We have performed in vitro transcription and mobility shift assays to characterize the functional significance of the protein-DNA interactions within 180 bas e pairs upstream of the mP2 transcription start site. Deletion and mut ational analyses reveal two positive regulatory sequences for mP2 tran scription at positions -59/-47 and -83/-72 of the mP2 promoter, The pr oximal element at -59/-47 binds to a novel testis-specific protein we name protamine-activating factor 1 (PAF-1). PAF-1 reaches high levels in round spermatids at the time of mP2 transcription. Deletion of the -59/-47 sequence results in about a 3-fold reduction of mP2 transcript ion in vitro, Although the PAF-1 binding site (PAF-responsive element, PAF-RE), contains the sequence GTCA present in the cAMP-responsive el ement and is very similar to the estrogen-responsive element, mobility shift assays revealed that neither the cAMP-responsive element modula tor nor the estrogen receptor is the protein(s) binding to PAF-RE, Com petition mobility shift assays reveal that the second positive regulat ory element at -88/-72 binds a Y-box-binding protein, Using in vitro t ranscription assays, a 5-fold decrease in mP2 transcription is seen wh en both the PAF-RE and this Y-box are deleted, These data suggest that the testis-specific PAF-1 and a Y-box-binding protein are needed to a ctivate mP2 transcription in postmeiotic male germ cells.