Identification of a novel member of the Snail/Gfi-1 repressor family, mlt 1, which is methylated and silenced in liver tumors of SV40 T antigen transgenic mice

DNA methylation is the only known mechanism for an epigenetic genomic DNA m odification that is capable of altering gene expression. A recent study rev eals that the pattern of CpG island methylation is largely characteristic o f tumor type, suggesting that distinct sets of genes are inactivated by met hylation during development of each tumor type. We compared previously the methylation status between normal liver and liver tumors in SV40 T/t antige n transgenic mice (MT-D2 mice) using Restriction Landmark Genomic Scanning for Methylation (RLGS-M) and identified several loci/spots that appeared to be methylated frequently in liver tumors. One of these spots, B236, identi fied a locus on chromosome 12 (D12Ncvs7) syntenic with human 14q12-q21 that is frequently Lost in certain human cancers. Shotgun sequencing of a bacte rial artificial chromosome clone containing this spot/locus was performed t o identify genes within this region. The Genescan program predicted an open reading frame of a novel, intron-less gene adjacent to the B236 spot that encodes a putative 493-amino acid protein containing the SNAG repressor mot if in the NH2-terminal region and five C2H2-type zinc finger motifs in the COOH-terminal half. This putative gene, methylated in liver lumor (mlt 1), is a novel member of the SNAG transcriptional repressor family with 43% ami no acid identity to insulinoma-associated protein 1. An open reading frame encoding a protein quite similar to mouse mlt 1 (56% amino acid identity) w as located in the syntenic region of the human genome, indicating that mlt 1 is evolutionarily conserved in human. Northern blot analysis revealed tha t mlt 1 is normally expressed in brain, spleen, stomach, and liver, However , mlt 1 expression was silenced in the liver tumors of MT-D2 mice. The puta tive promoter region of mlt 1 is unmethylated in normal tissues but methyla ted in all liver tumors from 11 MT-D2 mice. We also found that mlt 1 was me thylated and not expressed in N18TG-2 cells, a mouse neuroblastoma cell lin e. Treatment of N18TG-2 tells with a demethylating agent, 5-aza-deoxycytidi ne, resulted in an expression of mlt 1, indicating that the repression of m lt 1 is attributable to methylation. Thus, mlt 1 is a novel target gene tha t is silenced by methylation during liver tumorigenesis initiated by SV40 T antigen.