Enhancers are cis-elements that activate transcription and play critical roles in tissue- and cell type-specific gene expression. During spermatogenesis, genes coding for specialized sperm structures are expressed in a developmental stage- and cell type-specific manner, but the enhancers responsible for their expression have not been identified. Using the mouse acrosomal vesicle protein (Acrv1) gene that codes for the acrosomal protein SP-10 as a model, our previous studies have shown that Acrv1 proximal promoter activates transcription in spermatids; and the goal of the present study was to separate the enhancer responsible. Transgenic mice showed that three copies of the –186/–135 fragment (50 bp enhancer) placed upstream of the Acrv1 core promoter (–91/+28) activated reporter expression in testis but not somatic tissues (n = 4). Immunohistochemistry showed that enhancer activity was restricted to the round spermatids. The Acrv1 enhancer failed to activate transcription in the context of a heterologous core promoter (n = 4), indicating a likely requirement for enhancer-core promoter compatibility. Chromatin accessibility assays showed that the Acrv1 enhancer assumes a nucleosome-free state in male germ cells (but not liver), indicating occupancy by transcription factors. Southwestern assays (SWA) identified specific binding of the enhancer to a testis nuclear protein of 47 kDa (TNP47). TNP47 was predominantly nuclear and becomes abundant during the haploid phase of spermatogenesis. Two-dimensional SWA revealed the isoelectric point of TNP47 to be 5.2. Taken together, this study delineated a 50-bp enhancer of the Acrv1 gene for round spermatid-specific transcription and identified a putative cognate factor. The 50-bp enhancer could become useful for delivery of proteins into spermatids.

Summary Sentence

Functional assays and chromatin characteristics show that the –186/–135 region of the mouse Acrv1 gene acts as a transcriptional enhancer and activates round spermatid-specific gene expression in vivo.