De novo DNA cytosine methyltransferase activities in mouse embryonic stem cells

Abstract

It has been a controversial issue as to how many DNA cytosine methyltransferase mammalian cells have and whether de novo methylation and maintenance methylation activities are encoded by a single gene or two different genes. To address these questions, we have generated a null mutation of the only known mammalian DNA methyltransferase gene through homologous recombination in mouse embryonic stem cells and found that the development of the homozygous embryos is arrested prior to the 8-somite stage. Surprisingly, the null mutant embryonic stem cells are viable and contain low but stable levels of methyl cytosine and methyltransferase activity, suggesting the existence of a second DNA methyltransferase in mammalian cells. Further studies indicate that de novo methylation activity is not impaired by the mutation as integrated provirus DNA in MoMuLV-infected homozygous embryonic stem cells become methylated at a similar rate as in wild-type cells. Differentiation of mutant cells results in further reduction of methyl cytosine levels, consistent with the de novo methylation activity being down regulated in differentiated cells. These results provide the first evidence that an independently encoded DNA methyltransferase is present in mammalian cells which is capable of de novo methylating cellular and viral DNA in vivo.

DNA Methylation Dynamics and Dysregulation Delineated by High-Throughput Profiling in the Mouse

“…We assessed the impact of reduced levels of the main maintenance DNA methyltransferase, Dnmt1 on global DNA methylation distributions using gene-targeted embryonic stem cells (ES cells) (Figure 2D). We employed a series of hypomorphic alleles, R (Eads et al, 2002), P , H (Chan et al, 2001), N (Li et al ., 1992), as well as two null alleles, S (Li et al ., 1992), and C (Lei et al, 1996). Heterozygous Dnmt1 knockout ES cells did not show an appreciable reduction in DNA methylation levels, but various homozygous or compound heterozygous allelic combinations revealed substantial hypomethylation.…”

DNMT1 regulates the timing of DNA methylation by DNMT3 in an enzymatic activity-dependent manner in mouse embryonic stem cells

“…Mouse Dnmt1 -/- (1KO) ESCs were previously derived from J1 ESCs [ 11 ]. Transgenic ESC clones co-expressing a mutant gene encoding the catalytically inactive form of DNMT1 (DNMT1 CI ) and an IRES-linked zeocin resistance gene under the CAG promoter were generated by the lipofection method using the Lipofectamine™ LTX Reagent with PLUS™ Reagent (Thermo Fisher Scientific, Waltham, MA, USA).…”

“…DNMT1, a maintenance methyltransferase, copies methylation patterns on the nascent DNA strand during the S phase and partly during the G2 phase. Its deficiency leads to embryonic lethality with abnormal expression patterns, including increased expression of transposable elements [ 11 – 13 ]. DNMT1 also interacts with many proteins required for DNA binding at replicating regions, hemimethylated regions, and heterochromatin.…”

Identification of Let-7 miRNA Activity as a Prognostic Biomarker of SHH Medulloblastoma

“…First, we used a PCA on the gene expression, and as expected, it was able to separate the MB samples into the four molecular subgroups [ 8 , 9 , 10 , 11 , 12 , 13 , 14 ] ( Figure 1 A). We further explored whether the expression of genes primarily regulated by methylation (Methods) can better cluster MB into subgroups, because DNA methylation shows a highly dynamic pattern during cellular differentiation, indicating its key function related to cell fate specification [ 71 , 72 , 73 ], and patterns of DNA methylation may provide an indirect assessment of these developmental origins. Indeed, cis-methyl genes (i.e., expression regulated by the methylation level in its promoter region; see Materials and Methods for details) can better separate MB into subgroups ( Figure 1 B) than all the genes ( Figure 1 A).…”