The testis-specific histone H1t gene is strongly repressed by a G/C-rich region just downstream of the TATA box

H1t is a testis-specific histone 1 variant restricted to the male germ line and expressed only in pachytene spermatocytes. Understanding the regulation of the H1t gene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1t is not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1t and a major somatic H1 gene (H1d) by introduction of suitable restriction sites just 5' of the TATA box and 3' of the conserved H1 AC box. Hybrid promoters were joined to a lacZ reporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1d promoter was 20-fold stronger than the H1t promoter. Much of this difference in activity was traced to inhibitory sequences immediately downstream of the TATA box in H1t, although sequences upstream of the H1t AC box and within the H1t 5'-untranslated region played some role as well. A series of deletions and short oligonucleotide mutations scanned across the region between the TATA box and cap site identified two tracts of C (GC box 2) as the inhibitory sequences. While both Sp1 and Sp3 bind to this region weakly in vitro, they are unlikely to be responsible for the inhibitory effect of GC box 2, and additional binding proteins (CTB-4 and CTB-5) were identified by electrophoretic mobility shift assays as better candidates for mediating the repressive effect. When repression of the H1t promoter was relieved by mutation of GC box 2, additional mutations introduced into GC box 1 upstream of the CAAT box led to a large decrease in activity, indicating that these two G/C-rich elements have opposite effects on promoter activity.