The imprinting of mammalian genes depends on the maintenance of DNA methylation patterns during pre- and postimplantation development. Dnmt1o is a variant form of the somatically expressed Dnmt1 cytosine methyltransferase that is synthesized and stored in the oocyte cytoplasm and trafficks to the eight-cell nucleus during preimplantation development, where it maintains DNA methylation patterns on alleles of imprinted genes. Transcripts encoding Dnmt1 are present in preimplantation embryos, suggesting that Dnmt1 protein is also expressed in the preimplantation embryo, and may account for maintenance methylation at preimplantation stages other than the eight-cell embryo. However, using an antibody that detects Dnmt1, but not Dnmt1o, no Dnmt1 protein was detected on immunoblots or by immunocytochemical staining in wildtype preimplantation embryos. Moreover, Dnmt1 protein produced in the oocyte from a modified Dnmt1 allele, Dnmt1(1s/1o), trafficked to nuclei of eight-cell embryos, but not to nuclei of other stages. The highly restricted nuclear localization patterns of oocyte-derived Dnmt1o and Dnmt1 during preimplantation development add further support to the notion that DNA methyltransferases other than Dnmt1 are required for maintaining imprints during preimplantation development.

Background: Identical DNA methylation differences between maternal and paternal alleles in gametes and adults suggest that the inheritance of genomic imprints is strictly due to the embryonic maintenance of DNA methylation. Such maintenance would occur in association with every cycle of DNA replication, including those of preimplantation embryos.

Cloning by somatic cell nuclear transfer is inefficient. This is evident in the significant attrition in the number of surviving cloned offspring at virtually all stages of embryonic and fetal development. We find that cloned preimplantation mouse embryos aberrantly express the somatic form of the Dnmt1 DNA (cytosine-5) methyltransferase, the expression of which is normally prevented by a posttranscriptional mechanism. Additionally, the maternal oocyte-derived Dnmt1o isoform undergoes little or none of its expected translocation to embryonic nuclei at the eight-cell stage. Such defects in the regulation of Dnmt1s and Dnmt1o expression and cytoplasmic-nuclear trafficking may prevent clones from completing essential early developmental events. Furthermore, aberrant Dnmt1 localization and expression may contribute to the defects in DNA methylation and the developmental abnormalities seen in cloned mammals.

Activation-induced deaminase (AID) initiates immunoglobulin somatic hypermutation (SHM). Since in vitroAID was shown to deaminate cytosines on single-stranded DNA or the nontranscribed strand, it remained a puzzle how in vivo AID targets both DNA strands equally. Here we investigate the roles of transcription and DNA sequence in cytosine deamination. Strikingly different results are found with different substrates. Depending on the target sequence, the transcribed DNA strand is targeted as well as or better than the nontranscribed strand. The preferential targeting is not related to the frequency of AID hot spots. Comparison of cytosine deamination by AID and bisulfite shows different targeting patterns suggesting that AID may locally unwind the DNA. We conclude that somatic hypermutation on both DNA strands is the natural outcome of AID action on a transcribed gene; furthermore, the DNA sequence or structure and topology play major roles in targeting AID in vitro and in vivo. On the other hand, the lack of mutations in the first ϳ100 nucleotides and beyond about 1 to 2 kb from the promoter of immunoglobulin genes during SHM must be due to special conditions of transcription and chromatin in vivo.The variable regions of immunoglobulin (Ig) genes encode the antigen binding sites of antibodies for estimated billions of different antigenic determinants. Thousands to millions of different antibody binding sites are created when the hundred or so variable, diversity, and joining genes for Ig heavy and light chains are recombined and diversified by nucleotide deletions and insertions at the V(D)J joints in developing B lymphocytes. In mature B lymphocytes, the rearranged V(D)J sequences are extensively further diversified by somatic hypermutation (SHM) during exposure to antigens that react with the specific Igs comprising the B-cell receptors.

The process of somatic hypermutation (SHM) of immunoglobulin (Ig) genes requires activation-induced cytidine deaminase (AID). Although mistargeting of AID is detrimental to genome integrity, the mechanism and the cis-elements responsible for targeting of AID are largely unknown. We show that three CAGGTG cis-elements in the context of Ig enhancers are sufficient to target SHM to a nearby transcribed gene. The CAGGTG motif binds E47 in nuclear extracts of the mutating cells. Replacing CAGGTG with AAGGTG in the construct without any other E47 binding site eliminates SHM. The CA versus AA effect requires AID. CAGGTG does not enhance transcription, chromatin acetylation, or overall target gene activity. The other cis-elements of Ig enhancers alone cannot attract the SHM machinery. Collectively with other recent findings, we postulate that AID targets all genes expressed in mutating B cells that are associated with CAGGTG motifs in the appropriate context. Ig genes are the most highly mutated genes, presumably because of multiple CAGGTG motifs within the Ig genes, high transcription activity, and the presence of other cooperating elements in Ig enhancers.