Changes in the DNA methylation profile of the rat H19 gene upstream region during development and transgenic hepatocarcinogenesis and its role in the imprinted transcriptional regulation of the H19 gene
Changes in the DNA methylation profile of the rat H19 gene upstream region during development and transgenic hepatocarcinogenesis and its role in the imprinted transcriptional regulation of the H19 gene
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DOI：
10.1002/mc.20036
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发表时间：
2004-09-01
影响因子：
4.6
通讯作者：
Pitot, HC
中科院分区：
文献类型：
--
作者：
Manoharan, H;Babcock, K;Pitot, HC
印迹H19和胰岛素样生长因子-2(IGF2)基因的单等位基因表达依赖于H19上游区域母系等位基因的低甲基化和父系等位基因的高甲基化。本实验室在Fischer 344(F344)和SpragueDawley Alb SV40T-Ag转基因大鼠(SD)杂交F1中的肝癌致癌研究表明，H19在肝癌中存在双等位基因表达。我们对胎儿、成人和肿瘤肝脏中H19基因上游区域的DNA甲基化状态进行了比较研究。对H19上游2950至6695bp的3.745 kb DNA片段的亚硫酸氢盐DNA测序分析显示，胎儿、成人和肿瘤肝脏的甲基化模式存在显著差异。在胎肝中，等量的高甲基化链和低甲基化链显示了亲代等位基因的不同甲基化状态，但在肿瘤性肝脏中，甲基化模式发生了明显的变化，在2984-3301BP、6033-6123BP和6518-6548BP之间的短片段发生了明显的低甲基化变化。这些结果表明，该区域所有胞嘧啶的甲基化可能与大鼠H19基因的印迹状态有关。H19启动子关键顺式调控区域的差异甲基化相关表观遗传学改变影响了肝细胞癌发生过程中向双等位基因表达的转变。与小鼠和人相似，我们发现锌指CCTCC结合因子(CTCF)结合在其上游未甲基化的CTCF结合部位，从而影响单等位基因在胎肝中的印迹表达。CTCF在胎儿肝、正常肝和肿瘤性肝中似乎没有频率限制。在CTCF结合位点的3‘端，另一个DNA区域在成人肝脏的两条DNA链上显示CpG的甲基化，在胎肝保留印迹，在肿瘤性肝脏完全去甲基化。该区域还可能是一个基本螺旋-环-螺旋亮氨酸拉链转录因子TFEB的结合位点。在成人中发现的涉及TFEB结合位点的差异CpG甲基化可能解释了H19基因在成人正常肝脏中缺乏表达的原因。此外，这些发现表明，在SD Alb SV40 T Ag转基因大鼠的肝肿瘤中，H19基因印迹的丢失与该基因两个不同区域的CpG二核苷酸的差异甲基化直接相关，并且可能是这两个区域差异甲基化的结果，这两个区域位于转录起始点4 kb 5‘以内。转基因动物在结节或肿瘤出现之前对肝细胞的细胞遗传学分析表明，这种印记的丢失在肿瘤发展的非常早期起到了作用，可能是从促进阶段过渡到进展阶段。(C)2004年Wiley-Liss公司
Monoallelic expression of the imprinted H19 and insulin-like growth factor-2 (Igf2) genes depends on the hypomethylation of the maternal allele and hypermethylation of the paternal allele of the H19 upstream region. Previous studies from our laboratory on liver carcinogenesis in the F1 hybrid of Fischer 344 (F344) and Sprague-Dawley Alb SV40 T Ag transgenic rat (SD) strains revealed the biallelic expression of H19 in hepatomas. We undertook a comparative study of the DNA methylation status of the upstream region of H19 in fetal, adult, and neoplastic liver. Bisulfite DNA sequencing analysis of a 3.745-kb DNA segment extending from 2950 to 6695 bp of the H19 upstream region revealed marked variations in the methylation patterns in fetal, adult, and neoplastic liver. In the fetal liver, equal proportions of hyper- and hypomethylated strands revealed the differentially methylated status of the parental alleles, but in neoplastic liver a pronounced change in the pattern of methylation was observed with a distinct change to hypomethylation in the short segments between 2984 and 3301 bp, 6033-6123 bp, and 6518-6548 bp. These results indicated that methylation of all cytosines in this region may contribute to the imprinting status of the rat H19 gene. This phenomenon of differential methylation-related epigenetic alteration in the key cis-regulatory domains of the H19 promoter influences switching to biallelic expression in hepatocellular carcinogenesis. Similar to mouse and human, we showed that the zinc-finger CCTCC binding factor (CTCF) binds to the unmethylated CTCF binding site in the upstream region to influence monoallelic imprinted expression in fetal liver. CTCF does not appear to be rate limiting in fetal, normal, and neoplastic liver. 3' to the CTCF binding sites, another DNA region exhibits methylation of CpG's in both DNA strands in adult liver, retention of the imprint in fetal liver, and complete demethylation in neoplastic liver. in this region is also a putative binding site for a basic helix-loop-helix leucine-zipper transcription factor, TFEB. The differential CpG methylation seen in the adult that involves the TFEB binding site may explain the lack of expression of the H19 gene in adult normal liver. Furthermore, these findings demonstrate that the loss of imprinting of the H19 gene in hepatic neoplasms of the SD Alb SV40 T Ag transgenic rat is directly correlated with and probably the result of differential methylation of CpG dinucleotides in two distinct regions of the gene that are within 4 kb 5' of the transcription start site. Cytogenetic analysis of hepatocytes in the transgenic animal prior to the appearance of nodules or neoplasms indicates a role of such loss of imprinting in the very early period of neoplastic development, possibly the transition from the stage of promotion to that of progression. (C) 2004 Wiley-Liss, Inc.