Localization of Upstream Elements Involved in Transcriptional Regulation of the Rat Testis-Specific Histone H1t Gene in Somatic Cells1

Abstract

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 H1t-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.

The first bromodomain of Brdt, a testis-specific member of the BET sub-family of double-bromodomain-containing proteins, is essential for male germ cell differentiation

“…To test this hypothesis, we performed chromatin immunoprecipitation (ChIP) experiments using affinitypurified ␣-CT antibody and primers against the H1t proximal and distal promoter region. The primers were selected based on previous studies showing that the proximal and distal regions are involved in the activation and repression of H1t expression, respectively (vanWert et al, 1998;Wolfe et al, 1999). The results revealed that immunoprecipitated full-length Brdt protein (from control testes) was found in association with the distal region of the histone H1t promoter (-806 to -1027 from the transcription initiation site), but not to the more proximal region of the promoter (Fig.…”

Specific Binding of Nuclear Proteins to a Bifunctional Promoter Element Upstream of the H1/AC Box of the Testis-Specific Histone H1t Gene1

“…Most of the H1t promoter regulatory elements are located within the proximal promoter, but some upstream elements also contribute to transcriptional regulation. For example, we identified a silencer region located between 875 and 948 bp upstream from the transcription initiation site [3]. In addition, an upstream TG-rich element has been identified as a binding site for a transcriptional activator [31].…”

“…The DNase I digestions were conducted in a total volume of 40 l as previously described [3]. Following DNase I digestion, the samples were extracted twice with phenol:chloroform (1:1) and DNA precipitated from the aqueous phase with 2.5 volumes of ethanol at Ϫ20ЊC for 1 h. The DNA was recovered by centrifugation for 20 min at 16 000 ϫ g at 4ЊC.…”

“…Following DNase I digestion, the samples were extracted twice with phenol:chloroform (1:1) and DNA precipitated from the aqueous phase with 2.5 volumes of ethanol at Ϫ20ЊC for 1 h. The DNA was recovered by centrifugation for 20 min at 16 000 ϫ g at 4ЊC. Ethanol was removed and the DNA pellet was dried partially and dissolved in 3 l of loading solution before electrophoresis [3]. Samples were heated at 90ЊC for 3 min before loading onto a denaturing polyacrylamide sequencing gel (6% polyacrylamide [19:1, acrylamide: bisacrylamide] containing 7 M urea and 1ϫ TBE [0.09 M TrisBorate, 0.002 M EDTA, pH 8.3]).…”

“…In nongerminal cells, H1t transcription is repressed with contributions from silencing elements found upstream [3] and within the H1t proximal promoter [4] as well as by methylation at specific sites within its proximal promoter [5]. Although the proximal promoter contains all of the consensus elements common to the somatic promoters, including an H1/AC box, a GC box, an H1/CCAAT box, and a TATA box, transcription of the H1t gene is not detected in cells other than testis pachytene primary spermatocytes in animal studies [6][7][8].…”