A 5′-CG-3′-rich region in the promoter of the transcriptionally frequently silenced RET protooncogene lacks methylated cytidine residues

Abstract

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.

Familial form of Hirschsprung disease: Nucleotide sequence studies reveal point mutations in the RET proto-oncogene in two of six families but not in other candidate genes

“…3). In a region of about 900 base pairs, 95 unmethylated 5Ј-CG-3Ј dinucleotides are located of which the majority lies within the putative promoter segment [Munnes et al, 1998]. During embryogenesis, RET proto-oncogene expression is regulated in a temporally and spatially defined pattern [Avantaggio et al, 1994;Schuchardt et al, 1995].…”

“…1, 3). sively in the substantia nigra, the adrenal medulla, cerebellum and other parts of the brain [Takaya et al, 1996;Munnes et al, 1998]. Tumor tissues like pheochromocytomas, medullary thyroid carcinomas (MTC) and papillary thyroid carcinomas (PTC) also harbor RET transcripts.…”

The gastrointestinal tract as the portal of entry for foreign macromolecules: fate of DNA and proteins

“…Details of these methods as performed in this laboratory were described previously: Southern hybridization (Southern 1975;Koetsier et al 1993), PCR (Munnes et al 1998), fluorescent in situ hybridization (Heller et al 1995).…”

De Novo Methylation, Long-Term Promoter Silencing, Methylation Patterns in the Human Genome, and Consequences of Foreign DNA Insertion

“…We investigated the level of DNA methylation in a DNA segment of about 1,000 bp in the promoter and 5 -upstream region of this gene (Munnes et al 1998). By again apply-ing the bisulfite genomic sequencing technique, DNA from peripheral white blood cells (PWBCs) from healthy individuals or from Hirschsprung disease patients was used as well as DNA from different human tissues and from a human embryonic kidney cell line.…”

“…In in vitro experiments in which the transcriptional activity of the RET gene promoter was assessed in linkage to an indicator gene after transfection into human cells, the activity of this promoter was decreased by HpaII (5 -CCGG-3 ) methylation and abolished by SssI (5 -CG-3 ) methylation. Hence the RET protooncogene promoter is sensitive to DNA methylation at least in transfection and transient transcription experiments (Munnes et al 1998). …”