Localization of upstream elements involved in transcriptional regulation of the rat testis-specific histone H1t gene in somatic cells

The testis-specific histone H1t is synthesized exclusively in late pachytene primary spermatocytes during spermatogenesis. The mechanisms involved in transcriptional repression of the H1t gene during development before the spermatocyte stage and in later stages of germinal cell maturation and in nonexpressing somatic tissues are unknown. To assess the contribution of the upstream DNA sequence to H1t transcriptional silencing in nonexpressing cells, a set of histone Hit-promoted reporter vectors was constructed. Transient transfection of mouse C127I cells with these reporter vectors allowed us to identify a transcriptional silencer located between 948 base pairs (bp) and 780 bp upstream from the H1t transcriptional initiation site. Histone H1t-promoted luciferase activity increased 4-fold when the region between 948 bp and 875 bp upstream from the transcriptional initiation site was eliminated. Addition of a 73-bp rat H1t promoter fragment (-948 to -875, containing the 5' portion of the silencer region) to a site immediately upstream from the histone H1d proximal promoter led to significantly reduced luciferase expression upon transient transfection (56% in C127I cells and 44% in HeLa cells). Nuclear proteins were found to bind to DNA within the H1t silencer region when assayed by in vitro deoxyribonuclease (DNase) I footprinting. Thus, our data suggest that an active transcriptional silencer mechanism involving a specific and autonomous H1t promoter element (nucleotides -948/-875) may be operative to minimize expression of the H1t gene in nontesticular cells.