Identification and characterization of methylation-dependent/independent DNA regulatory elements in the human SLC9B1 gene.
Identification and characterization of methylation-dependent/independent DNA regulatory elements in the human SLC9B1 gene.
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DOI：
10.1016/j.gene.2015.02.050
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发表时间：
2015-05-01
期刊：
影响因子：
3.5
通讯作者：
James PF
中科院分区：
文献类型：
--
作者：
Kumar PL;James PF
人类NHEDC 1（hNHEDC 1）蛋白被认为是精子运动和生育力所必需的，但调节其基因表达的机制在很大程度上是未知的。本研究在hNHEDC 1（SLC 9 B1）编码基因的5′端鉴定了多个DNA调控元件，并探讨了这些元件的DNA甲基化在其表达调控中的作用。我们首先表明，全长hNHEDC 1蛋白是睾丸特异性的组织，我们测试，它定位于曲细精管的细胞。SLC 9 B1基因位点的计算机分析鉴定了两个推定的启动子（P1和P2）和位于基因5′端的两个CpG岛-- CpGI（与P1重叠）和CpGII（基因内）。通过P1的缺失分析，我们发现相对于转录起始位点（TSS）的-23 bp到+200 bp的区域足以在生殖细胞系中获得最佳启动子活性。此外，P1（相对于TSS的-500 bp至+200 bp区域）的体外甲基化消除了其在生殖细胞和体细胞中的活性，这强烈表明该启动子的DNA甲基化可以调节SLC 9 B1的表达。此外，P1/CpGI的亚硫酸氢盐测序分析发现睾丸与肺中的甲基化降低，而CpGII在这两种组织之间的甲基化没有差异。此外，用5-氮杂2-脱氧胞苷处理HEK 293细胞导致NHEDCl转录物的上调和启动子CpGI中甲基化的降低。最后，我们发现了基因内SLC 9 B1 CpGII的增强子和沉默子功能。总之，我们的数据表明，SLC 9 B1基因的表达可以通过这些多个DNA调控元件介导的DNA甲基化依赖性和独立机制的协同作用来调节。
The human NHEDC1 (hNHEDC1) protein is thought to be essential for sperm motility and fertility however the mechanisms regulating its gene expression are largely unknown. In this study we have identified multiple DNA regulatory elements in the 5′ end of the gene encoding hNHEDC1 (SLC9B1) and have explored the role that DNA methylation at these elements plays in the regulation of its expression. We first show that the full-length hNHEDC1 protein is testis-specific for the tissues that we tested and that it localizes to the cells of the seminiferous tubules. In silico analysis of the SLC9B1 gene locus identified two putative promoters (P1 and P2) and two CpG islands - CpGI (overlapping with P1) and CpGII (intragenic) - at the 5′ end of the gene. By deletion analysis of P1, we show that the region from −23bp to +200bp relative to the transcription start site (TSS) is sufficient for optimal promoter activity in a germ cell line. Additionally, in vitro methylation of the P1 (the −500bp to +200bp region relative to the TSS) abolishes its activity in germ cells and somatic cells strongly suggesting that DNA methylation at this promoter could regulate SLC9B1 expression. Furthermore, bisulfite-sequencing analysis of the P1/CpGI uncovered reduced methylation in the testis vs. lung whereas CpGII displayed no differences in methylation between these two tissues. Additionally, treatment of HEK 293 cells with 5-Aza2-Deoxycytidine led to upregulation of NHEDC1 transcript and reduced methylation in the promoter CpGI. Finally, we have uncovered both enhancer and silencer functions of the intragenic SLC9B1 CpGII. In all, our data suggests that SLC9B1 gene expression could be regulated via a concerted action of DNA methylation-dependent and independent mechanisms mediated by these multiple DNA regulatory elements.
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影响因子：
46.9
作者：
通讯作者：
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影响因子：
16.8
作者：
Lorincz, MC;Dickerson, DR;Groudine, M
通讯作者：
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DOI：
10.2741/e115
发表时间：
2010-01-01
期刊：
Frontiers in bioscience (Elite edition)
影响因子：
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