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
2001
DNA methylation is the only known mechanism for an epigenetic genomic
DNA modification that is capable of altering gene expression. A recent
study reveals that the pattern of CpG island methylation is largely
characteristic of tumor type, suggesting that distinct sets of genes
are inactivated by methylation during development of each tumor type.
We compared previously the methylation status between normal liver and
liver tumors in SV40 T/t antigen 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 , identified a
locus on chromosome 12 ( D12Ncvs7 ) syntenic with human
14q12–q21 that is frequently lost in certain human cancers. Shotgun
sequencing of a bacterial artificial chromosome clone containing this
spot/locus was performed to 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 motif
in the NH 2 -terminal region and five
C 2 H 2 -type zinc finger motifs in the
COOH-terminal half. This putative gene, methylated in liver
tumor ( mlt 1 ) , is a novel member
of the SNAG transcriptional repressor family with 43% amino acid
identity to insulinoma-associated protein 1. An open reading
frame encoding a protein quite similar to mouse mlt 1
(56% amino acid identity) was located in the syntenic region of the
human genome, indicating that mlt 1 is evolutionarily
conserved in human. Northern blot analysis revealed that 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 putative promoter region of mlt
1 is unmethylated in normal tissues but methylated in all liver
tumors from 11 MT-D2 mice. We also found that mlt 1 was
methylated and not expressed in N18TG-2 cells, a mouse neuroblastoma
cell line. Treatment of N18TG-2 cells with a demethylating agent,
5-aza-deoxycytidine, resulted in an expression of mlt 1,
indicating that the repression of mlt 1 is attributable
to methylation. Thus, mlt 1 is a novel target gene that
is silenced by methylation during liver tumorigenesis initiated by SV40
T antigen.
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