Spermatid-specific promoter of the SP-10 gene functions as an insulator in somatic cells

Spermatid differentiation markers such as the acrosomal protein SP-10 display remarkable testis- and germ cell-restricted gene expression. However, little is known about the mechanisms that prevent their expression in somatic tissues. We have previously noted that the -408/+28 or the -266/+28 promoter of SP-10 directed strictly spermatid-specific transcription in transgenic mice, Biol. Reprod. 61, 1256-1266). Lack of ectopic expression in these mouse lines implied that the SP-10 promoter might have protected the transgene from the influence of neighboring enhancers. The present study tested this directly by performing enhancer-blocking assays. In transiently transfected COS cells, the -408/-92 SP-10 promoter, but not stuffer DNA, blocked the transcriptional activity of a heterologous enhancer (CMV) in a position- and orientation-dependent manner. In transgenic mice, despite integration adjacent to the pan-active CMV enhancer, the -408/+28 promoter maintained spermatid-specificity and no ectopic expression of the transgene resulted. Enhancer blocking is a characteristic feature of insulators. Our results show that the SP-10 proximal promoter, which activates transcription in spermatids, functions as an insulator in somatic cells. Insulator activity mapped to the -186/-135 region and mutation of two ACACAC motifs compromised the insulator function. In conclusion, the evolutionarily conserved SP-10 insulator is novel and is the first one shown to regulate transcription of a germ cell differentiation marker.