In vivo stabilization of the Dnmt1 (cytosine-5)- methyltransferase protein

Abstract

The Dnmt1o form of the Dnmt1 (cytosine-5)-methyltransferase enzyme is synthesized and stored in the cytoplasm of the oocyte and is used after fertilization to maintain methylation patterns on imprinted genes. After implantation of the blastocyst, Dnmt1o is replaced by the Dnmt1 form, which has an additional 118 aa at its amino terminus. To investigate functional differences between Dnmt1o and Dnmt1, mice were generated with a mutant allele, Dnmt1 V , which synthesized Dnmt1o instead of Dnmt1 in all somatic cells. Homozygous Dnmt1 V mice were phenotypically normal, and had normal levels of genomic methylation, indicating that Dnmt1o adopts the maintenance methyltransferase function of Dnmt1. Despite the apparent equivalence of Dnmt1o and Dnmt1 maintenance methyltransferase function in somatic cells, the Dnmt1o protein was found at high levels (with a corresponding high enzymatic activity) in Dnmt1 V mice. In heterozygous Dnmt1 V ͞؉ embryonic stem cells and early embryos, equal steadystate levels of Dnmt1o and Dnmt1 proteins were produced from the Dnmt1 V and the WT Dnmt1 alleles, respectively. However, in older embryos and adults, the Dnmt1 V allele produced five times the steady-state level of protein of the WT Dnmt1 allele. The difference in Dnmt1o and Dnmt1 levels is due to a developmentally regulated mechanism that degrades the Dnmt1 protein. The intrinsic stability of the Dnmt1o protein is the most likely reason for its use as a maternal-effect protein; stable ooplasmic stores of Dnmt1o would be available to traffick into the nuclei of the eight-cell stage embryo and maintain methylation patterns on alleles of imprinted genes during the fourth embryonic S phase.T he Dnmt1 (cytosine-5)-methyltransferase catalyzes the addition of methyl groups to cytosine bases in DNA, and it is found in most, if not all, cells of the mammalian organism (1). Based on in vitro studies, Dnmt1 has a 5-to 30-fold preference for hemimethylated DNA substrates over unmethylated substrates, indicating that the main function of Dnmt1 is to maintain methylation patterns (2). There are two isoforms of Dnmt1, which are expressed in a sequential pattern during development (3, 4). The Dnmt1o protein, which has a relative molecular mass (M r ) of 175,000, is expressed during oocyte growth and maturation, and also during preimplantation development. The M r 190,000 Dnmt1 form of the enzyme replaces Dnmt1o after implantation of the embryo (1). Dnmt1 has the same primary structure as Dnmt1o, except for the addition of a unique 118-aa domain at its amino terminus. Homozygous mutant offspring of mice that are heterozygous for Dnmt1 hypomorphic alleles have reduced levels of Dnmt1 protein and exhibit marked reductions in the level of genomic methylation, including reduction in the methylation associated with imprinted genes (5-7).Dnmt1o is a maternal-effect protein that is synthesized in the growing oocyte, stored in the ooplasm of the mature M2 oocyte, and functions after fertilization to maintain DNA methylation patterns on alleles of imprinted ...

Identification of a region of the DNMT1 methyltransferase that regulates the maintenance of genomic imprints

“…This maternal-effect DNMT1o function can be restored by oocyte-derived DNMT1s protein, but not restored by embryo-derived DNMT1o expressed from a modified endogenous Dnmt1 allele (6,14). These findings are further support for sequence-specific DNMT1 activity during preimplantation development, and suggest that the developmental source of the protein (oocyte versus embryo) determines this specificity.…”

“…In the absence of doxycycline, there was a significantly higher level of DNMT1s expression in Dnmt1 tet/tet cells, presumably due to the strong tTA transactivation. This level of increase in DNMT1s expression is not likely to influence genomic methylation (14,19). Following 3 days of exposure of Dnmt1 tet/tet cells to doxycycline, the level of DNMT1s protein fell to undetectable levels.…”

“…1 A and B). Amino acids 1-118 of DNMT1s define a region that interacts with the DMAP1 protein (21), and this region has been shown to be dispensable for viability and fertility in mice (14). The region defined by amino acids 119-410 is located just C terminal to the DMAP1-interaction domain, and is approximately the same as part A of the tripartite structure of DNMT1o proposed by Margot et al (22).…”

Identification of DNMT1 (DNA methyltransferase 1) hypomorphs in somatic knockouts suggests an essential role for DNMT1 in cell survival

“…The deletion covered most of the DMAP1 interaction domain, a region that is also deleted in the murine oocyte-specific isoform Dnmt1o. Deletion of this region produced no abnormal phenotype in mice, which expressed the shortened protein in all somatic tissues instead of full-length Dnmt1 (27). In fact, this protein could maintain normal levels of DNA methylation and appeared to be more stable than the full-length form of Dnmt1 containing the DMAP1 interaction domain (27).…”

Diverse Domains of (Cytosine-5)-DNA Methyltransferases: Structural and Functional Characterization

“…Though, in adult mice the Dnmt1o amount is 5 times higher than the Dnmt1 amount. So Dnmt1o seems to be more stable than Dnmt1 [89]. The DMAP1-bind-ing domain is likely to decrease Dnmt1 stability in vivo and thus could be involved in Dnmt1 degradation.…”