Characterization of the H1t Promoter: Role of Conserved Histone 1 AC and TG Elements and Dominance of the Cap-Proximal Silencer1

H1t is 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, re-gardless of the length of upstream sequence present. No other discrete silencing activity was observed in the upstream region extending to nucleotide 21842. Similarly, when the silencer mu-tation was introduced into the natural gene, H1t expression was readily detected in permanently transfected cells by both RNase protection and Western blot analysis, regardless of the extent of 59 or 39 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 oc-curred only when both were intact, and possibly identical bind-ing factors were demonstrated for each by electrophoretic mo-bility shift assays. In view of its precisely regulated but limited expression, it is interesting that H1t retains all the promoter elements known to activate standard H1 genes, including the TG/AC unit, SP1 site, and CCAAT element. Their presence em-phasizes the apparent dominance of the silencer element in most cells. gene regulation, meiosis, spermatogenesis, teste