Localization of upstream elements involved in transcriptional regulation of the rat testis-specific histone H1t gene in somatic cells.
Localization of upstream elements involved in transcriptional regulation of the rat testis-specific histone H1t gene in somatic cells.
复制标题
体细胞中参与大鼠睾丸特异性组蛋白 H1t 基因转录调控的上游元件的定位。
DOI：
10.1095/biolreprod61.4.1005
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发表时间：
1999
影响因子：
3.6
通讯作者：
Grimes,SR
中科院分区：
文献类型：
--
作者：
Wolfe,SA;Mottram,PJ;vanWert,JM;Grimes,SR
睾丸特异性组蛋白 H1t 在精子发生过程中仅在粗线期晚期初级精母细胞中合成。在精母细胞阶段之前的发育过程中、在生殖细胞成熟的后期阶段以及在不表达的体细胞组织中涉及H1t基因转录抑制的机制尚不清楚。为了评估上游 DNA 序列对非表达细胞中 H1t 转录沉默的贡献，构建了一组组蛋白 H1t 促进的报告载体。用这些报告载体瞬时转染小鼠 C127I 细胞，使我们能够鉴定位于 H1t 转录起始位点上游 948 bp 和 780 bp 之间的转录沉默子。当转录起始位点上游 948 bp 至 875 bp 之间的区域被消除时，组蛋白 H1t 促进的荧光素酶活性增加 4 倍。将 73 bp 的ratH1t启动子片段（-948至-875，包含沉默子区域的5'部分）添加到紧邻组蛋白H1d近端启动子上游的位点，导致瞬时转染后荧光素酶表达显着降低（C127I细胞中为56％，HeLa细胞中为44％）。通过体外脱氧核糖核酸酶 (DNase) I 足迹分析，发现核蛋白与 H1t 沉默子区域内的 DNA 结合。因此，我们的数据表明，涉及特定且自主的H1t启动子元件（核苷酸-948/-875）的主动转录沉默机制可能可有效减少非睾丸细胞中H1t基因的表达。
The testis-specific histone H1t is synthesized exclusively in late pachytene primary spermatocytes during spermatogenesis. The mechanisms involved in transcriptional repression of theH1tgene 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 toH1ttranscriptional silencing in nonexpressing cells, a set of histoneH1t-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 theH1ttranscriptional initiation site. HistoneH1t-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 ratH1tpromoter fragment (−948 to −875, containing the 5′ portion of the silencer region) to a site immediately upstream from the histoneH1dproximal 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 theH1tsilencer region when assayed by in vitro deoxyribonuclease (DNase) I footprinting. Thus, our data suggest that an active transcriptional silencer mechanism involving a specific and autonomousH1tpromoter element (nucleotides −948/−875) may be operative to minimize expression of theH1tgene in nontesticular cells.