Protein binding regions in the mouse and rat protamine-2 genes

To help define the regulatory mechanisms for tissue-specific and temporal transcription of the mouse protamine genes, the mouse protamine-2 gene was examined in vitro using protein-DNA binding assays to determine which 5' flanking regions of the gene bind proteins. DNA binding was examined in nuclear extracts prepared from expressing and nonexpressing tissues. One fragment of the gene, from -419 to -141, bound a factor present in nuclear extracts prepared from tissues in which the gene is not expressed (testis from 16-day-old animals, liver and brain), but formed very little complex in extracts from adult testis where the gene is normally expressed. This suggests that this region may be recognized by a negative regulatory factor that prevents the gene from being expressed in inappropriate tissues. Another region of the gene, from -140 to -23, formed a complex in all extracts tested, and an additional complex specific to adult testis extacts. These complexes were not formed with the rat protamine-2 gene, whose mRNA is present in vivo at approximately 5% of the level of mouse protamine-2 mRNA and is poorly transcribed in an in vitro testicular transcription system. This suggests that the factor or factors binding to this region serve as positive regulatory factors that are necessary to maintain a high level of protamine gene transcription. These studies present the first analysis of protein binding sites within the promoter of a testis-specific gene.