Munnes M, Patrone G, Schmitz B, Romeo G, Doerfler W
Institut für Genetik, Universität zu Köln, Germany.
Oncogene. 1998 Nov 19;17(20):2573-83. doi: 10.1038/sj.onc.1202165.
In a large proportion of familial and sporadic cases of Hirschsprung disease (HSCR) mutations in the RET (rearranged during transfection) protooncogene have been described. We have investigated the structure of the RET gene promoter and have analysed a region of approximately 1000 nucleotides in its promoter and 5'-upstream segments for the occurrence of 5-methyldeoxycytidine (5-mC) residues by using the bisulfite protocol of the genomic sequencing method. With an estimated sensitivity of about 93% of this technique, not a single 5-mC residue could be detected in the control region of a gene that seems to be silenced or exhibit low activity in many adult tissues. In these experiments, the DNAs of peripheral white blood cells (PWBC) from four healthy individuals, from seven patients with familial HSCR, as well as DNAs from different human tissues and from a human embryonic kidney (HEK) cell line have been included. In a DNA segment starting 790 nucleotides upstream of the transcriptional start site of the RET gene, a few 5-mC residues have been identified. This region possibly constitutes the transition site from an unmethylated promoter to a more extensively methylated region in the human genome. The data presented are remarkable in that a highly 5'-CG-3'-enriched segment of the human genome with 49 5'-CG-3' dinucleotide pairs in 400 bp within the putative promoter region is completely devoid of 5-mC residues, although this control region is not actively transcribed in most adult human tissues. By hybridization of a PCR-amplified RET protooncogene cDNA probe harboring exons 9-15 to a membrane (Clontech) containing poly-A selected RNAs from 50 different human tissues, weak RET protooncogene expression in many of the neural cell derived tissues has been detected. RNAs extracted from many other human tissues do not share sequence homologies to this 32P-labeled probe. Mechanisms other than DNA methylation obviously play the crucial role in the inactivation of the RET gene promoter in these tissues. We have also demonstrated by the in vitro premethylation of a RET promoter-chloramphenicol acetyltransferase (CAT) gene construct and transient transfection experiments into neuroblastoma cells that the transcriptional activity of the RET promoter is decreased by HpaII (5'-CCGG-3') methylation and abolished by SssI (5'-CG-3') methylation. Hence, the RET promoter region is sensitive to this regulatory signal. However in vivo, DNA methylation of the promoter region seems not to be the predominant regulatory mechanism for the RET protooncogene. Possibly, in adults the RET gene can be occasionally activated.
在大部分家族性和散发性先天性巨结肠病（HSCR）病例中，已发现转染期间重排（RET）原癌基因存在突变。我们研究了RET基因启动子的结构，并使用基因组测序方法的亚硫酸氢盐方案分析了其启动子和5'上游区段中约1000个核苷酸区域内5-甲基脱氧胞苷（5-mC）残基的存在情况。由于该技术的估计灵敏度约为93%，在一个在许多成人组织中似乎沉默或活性较低的基因的对照区域中未检测到单个5-mC残基。在这些实验中，纳入了来自四名健康个体、七名家族性HSCR患者的外周血白细胞（PWBC）的DNA，以及来自不同人类组织和人胚肾（HEK）细胞系的DNA。在RET基因转录起始位点上游790个核苷酸处开始的一个DNA片段中，已鉴定出一些5-mC残基。该区域可能构成了人类基因组中从未甲基化启动子到甲基化程度更高区域的过渡位点。所呈现的数据值得注意的是，在假定启动子区域内400 bp中有49个5'-CG-3'二核苷酸对的人类基因组中高度富含5'-CG-3'的片段完全没有5-mC残基，尽管该对照区域在大多数成人人类组织中不活跃转录。通过将携带外显子9 - 15的PCR扩增RET原癌基因cDNA探针与含有来自50种不同人类组织的聚腺苷酸选择RNA的膜（Clontech）杂交，已检测到许多神经细胞衍生组织中RET原癌基因的弱表达。从许多其他人类组织中提取的RNA与该32P标记探针没有序列同源性。在这些组织中，除DNA甲基化外的其他机制显然在RET基因启动子的失活中起关键作用。我们还通过RET启动子 - 氯霉素乙酰转移酶（CAT）基因构建体的体外预甲基化和向神经母细胞瘤细胞的瞬时转染实验证明，RET启动子的转录活性因HpaII（5'-CCGG-3'）甲基化而降低，并因SssI（5'-CG-3'）甲基化而被消除。因此，RET启动子区域对这种调节信号敏感。然而在体内，启动子区域的DNA甲基化似乎不是RET原癌基因的主要调节机制。可能在成年人中，RET基因偶尔会被激活。
