Search

H1tis a testis-specific variant histone 1 gene transcribed in pachytene spermatocytes. As part of a program to understand its transcriptional control, we have investigated the effect of the cap-proximal, GC-rich silencer element in the context of various lengths of upstream sequence. By transient transfection of NIH 3T3 cells, we showed that a targeted mutation in the silencer has a large (>10-fold) effect on reporter gene expression, regardless of the length of upstream sequence present. No other discrete silencing activity was observed in the upstream region extending to nucleotide −1842. Similarly, when the silencer mutation was introduced into the natural gene, H1texpression was readily detected in permanently transfected cells by both RNase protection and Western blot analysis, regardless of the extent of 5′ or 3′ flanking genomic DNA. In constructs with the mutated silencer, we showed interdependence of the characteristic H1 AC and TG box regulatory elements. Promoter up-regulation occurred only when both were intact, and possibly identical binding factors were demonstrated for each by electrophoretic mobility shift assays. In view of its precisely regulated but limited expression, it is interesting that H1tretains all the promoter elements known to activate standard H1 genes, including the TG/AC unit, SP1 site, and CCAAT element. Their presence emphasizes the apparent dominance of the silencer element in most cells.

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 H1tgene is an interesting challenge as its promoter shares all of the recognized control elements of standard somatic H1 genes, yet H1tis not expressed in somatic or in early spermatogenic cells. To investigate the mechanism of this apparent repression, we exchanged three promoter subregions between H1tand 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 lacZreporter gene and assayed by transient transfection in NIH3T3 fibroblasts. In this system the wild type H1dpromoter was 20-fold stronger than the H1tpromoter. 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.