A Short DNA Methyltransferase Isoform Restores Methylation In Vivo

Abstract

Two murine DNA methyltransferase isoforms (MTases) have been observed, a longer form in somatic and embryonic stem (ES) cells and a shorter form in oocytes and preimplantation embryos. While the longer MTase is associated with maintenance methyltransferase activity in replicating cells, little is known about the shorter form. We present genetic and biochemical evidence that both isoforms are expressed from the same Dnmt1 gene by using different translation initiation sites in exons 1 and 4. We further demonstrate that the shorter isoform can functionally rescue Dnmt1 null ES cells that have a hypomethylated genome. These rescued ES cells differentiate in vivo into a variety of cell types, unlike the Dnmt1 null ES cells that die upon induction of differentiation. These results show that the shorter isoform can substitute for the longer maintenance MTase in ES and differentiated cells. Our data further indicate that the shorter MTase isoform found in oocytes is fully functional in vivo and may play an active role in the regulation of DNA methylation and the establishment of imprinting patterns.

Increased Protein Stability Causes DNA Methyltransferase 1 Dysregulation in Breast Cancer

“…This differential protein accumulation was dependent on the N-terminal 120 amino acids, which seemed to function normally as a destruction domain in HMECs causing ubiquitination and proteasome-mediated degradation. Interestingly, an oocyte-specific DNMT1 isoform, DNMT1o, which is translated from a downstream start codon from an alternatively spliced transcript, thus lacking the N-terminal 118 amino acids (31), has been shown to be significantly stabilized in vivo relative to the full-length somatic DNMT1 by cycloheximide translation experiments (32). This stabilization results in an accumulation of ϳ5-fold of protein levels, similar to our findings in MCF-7 breast cancer cells relative to HMECs.…”

DNA methylation, chromatin inheritance, and cancer

“…Many other cancers have LOH of this region and it will be interesting to see if certain methylation abnormalities correlate with this loss. In addition, because additional 5' coding sequences were only recently identi®ed (Yoder et al, 1996;Gaudet et al, 1998), the DNMT1 overexpression experiments described above were conducted with a form of DNMT1 which lacks the ®rst 118 amino acid found normally in somatic cells (Wu et al, 1993;Vertino et al, 1996). This missing portion contains the DMAP1 binding region and, therefore, the exogenously expressed DNMT1 could not have interacted with DMAP1 and this fact must now be considered as contributing to these experimental results.…”

Dnmt1Overexpression Causes Genomic Hypermethylation, Loss of Imprinting, and Embryonic Lethality

“…Recipient blastocysts were BALB/c derived, while injected ES cells originated from 129/SvJae mice. Teratomas were generated by the injection of 10 7 ES cells into the flank of isogenic 129/SvJae mice and excision 3 weeks after injection (11).…”