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.
D000177:Acrosome; D000818:Animals; D000941:Antigens; D001483:Base Sequence; D019556:COS Cells; D003587:Cytomegalovirus / Q000235:genetics; D004742:Enhancer Elements, Genetic / Q000502:physiology*; D012739:Gonadal Steroid Hormones; D008297:Male; D008565:Membrane Proteins; D051379:Mice; D008822:Mice, Transgenic; D008969:Molecular Sequence Data; D011401:Promoter Regions, Genetic / Q000502:physiology*; D011506:Proteins / Q000235:genetics*; D013087:Spermatids / Q000378:metabolism*
C080034:Acrv1 protein, mouse; D000941:Antigens; D012739:Gonadal Steroid Hormones; D008565:Membrane Proteins; D011506:Proteins