Shyam Ramchandani

A mammalian protein with specific demethylase activity for mCpG DNA.

DNA-methylation patterns are important for regulating genome functions, and are determined by the enzymatic processes of methylation and demethylation. The demethylating enzyme has now been identified: a mammalian complementary DNA encodes a methyl-CpG-binding domain, bears a demethylase activity that transforms methylated cytosine bases to cytosine, and demethylates a plasmid when the cDNA is translated or transiently transfected into human embryonal kidney cells in vitro. The discovery of this DNA demethylase should provide a basis for the molecular and developmental analysis of the role of DNA methylation and demethylation.

Transcriptional regulation of the human DNA Methyltransferase (dnmt1) gene

DNA methylation is an important component of the epigenetic control of genome functions. Understanding the regulation of the DNA Methyltransferase (dnmt1) gene expression is critical for comprehending how DNA methylation is coordinated with other critical biological processes. In this paper, we investigate the transcriptional regulatory region of the human dnmt1 gene using a combination of RACE, RNase protection analysis and CAT assays. We identified one major and three minor transcription initiation sites in vivo (P1-P4), which are regulated by independent enhancers and promoter sequences. The minimal promoter elements of P1, P2 and P4 are mapped within 256 bp upstream of their respective transcription initiation sites. P1 is nested within a CG-rich area, similar to other housekeeping genes, whereas P2-P4 are found in CG-poor areas. Three c-Jun-dependent enhancers are located downstream to P1 and upstream to P2-P4, thus providing a molecular explanation for the responsiveness of dnmt1 to oncogenic signals that are mediated by the Ras-c-Jun oncogenic signaling pathway.

Antisense MBD2 gene therapy inhibits tumorigenesis.

Aberration in the pattern of DNA methylation is one of the hallmarks of cancer. We present data suggesting that dysregulation of MBD2, a recently characterized member of a novel family of methylated DNA binding proteins, is involved in tumorigenesis. Two functions were ascribed to MBD2, DNA demethylase activity and repression of methylated genes.

Identification of Initiation Sites for DNA Replication in the Human dnmt1 (DNA-methyltransferase) Locus

Vertebrates have developed multiple mechanisms to coordinate the replication of epigenetic and genetic information.Dnmt1 encodes the maintenance enzyme DNA-methyltransferase, which is responsible for propagating the DNA methylation pattern and the epigenetic information that it encodes during replication. Direct sequence analysis and bisulfite mapping of the 5′ region of DNA-methyltransferase 1 (dnmt1) have indicated the presence of many sequence elements associated with previously characterized origins of DNA replication. This study tests the hypothesis that thednmt1 region containing these elements is an origin of replication in human cells. First, we demonstrate that a vector containing this dnmt1 sequence is able to support autonomous replication when transfected into HeLa cells. Second, using a gel retardation assay, we show that it contains a site for binding of origin-rich sequences binding activity, a recently purified replication protein. Finally, using competitive polymerase chain reaction, we show that replication initiates in this region in vivo. Based on these lines of evidence, we propose that initiation sites for DNA replication are located between the first intron and exon 7 of the human dnmt1 locus.

A Conserved 3′-Untranslated Element Mediates Growth Regulation of DNA Methyltransferase 1 and Inhibits Its Transforming Activity

Ectopic expression of DNA methyltransferase 1 (DNMT1) has been proposed to play an important role in cancer.dnmt1 mRNA is undetectable in growth-arrested cells but is induced upon entrance into the S phase of the cell cycle, and until now, the mechanisms responsible for this regulation were unknown. In this report, we demonstrate that the 3′-untranslated region (3′-UTR) of the dnmt1 mRNA can confer a growth-dependent regulation on its own message as well as a heterologous β-globin mRNA. Our results indicate that a 54-nucleotide highly conserved element within the 3′-UTR is necessary and sufficient to mediate this regulation. Cell-free mRNA decay experiments demonstrate that this element increases mRNA turnover rates and does so to a greater extent in the presence of extracts prepared from arrested cells. A specific RNA-protein complex is formed with the 3′-UTR only in growth-arrested cells, and a UV cross-linking analysis revealed a 40-kDa protein (p40), the binding of which is dramatically increased in growth-arrested cells and is inversely correlated with dnmt1 mRNA levels as cells are induced into the cell cycle. Although ectopic expression of humanDNMT1 lacking the 3′-UTR can transform NIH-3T3 cells, inclusion of the 3′-UTR prevents transformation. These results support the hypothesis that deregulated expression of DNMT1with the cell cycle is important for cellular transformation.