A 5'-CG-3'-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues.
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.
Munnes
M
M
Institut für Genetik, Universität zu Köln, Germany.
Patrone
G
G
Schmitz
B
B
Romeo
G
G
Doerfler
W
W
D000328:Adult; D001483:Base Sequence; D001665:Binding Sites; D001921:Brain / Q000378:metabolism; D002460:Cell Line; D018899:CpG Islands; D003562:Cytidine / Q000737:chemistry*; D019175:DNA Methylation; D029721:Drosophila Proteins; D005786:Gene Expression Regulation; D018507:Gene Expression Regulation, Developmental; D015972:Gene Expression Regulation, Neoplastic; D006627:Hirschsprung Disease / Q000235:genetics; D006801:Humans; D008969:Molecular Sequence Data; D009419:Nerve Tissue Proteins / Q000096:biosynthesis / Q000235:genetics; D009447:Neuroblastoma / Q000378:metabolism; D009693:Nucleic Acid Hybridization; D009928:Organ Specificity; D016133:Polymerase Chain Reaction; D011401:Promoter Regions, Genetic; D011518:Proto-Oncogene Proteins / Q000096:biosynthesis / Q000235:genetics*; D051096:Proto-Oncogene Proteins c-ret; D011519:Proto-Oncogenes; D020794:Receptor Protein-Tyrosine Kinases / Q000096:biosynthesis / Q000235:genetics*; D011993:Recombinant Fusion Proteins / Q000096:biosynthesis; D017422:Sequence Analysis, DNA; D014157:Transcription Factors / Q000378:metabolism; D014158:Transcription, Genetic; D014407:Tumor Cells, Cultured
D029721:Drosophila Proteins; D009419:Nerve Tissue Proteins; D011518:Proto-Oncogene Proteins; D011993:Recombinant Fusion Proteins; D014157:Transcription Factors; D003562:Cytidine; D051096:Proto-Oncogene Proteins c-ret; D020794:Receptor Protein-Tyrosine Kinases; C416649:Ret protein, Drosophila