De novo DNA methyltransferase DNMT3A: Regulation of oligomeric state and mechanism of action in response to pH changes.

BACKGROUND: The oligomeric state of the human DNMT3A is functionally important and cancer cells are known to undergo changes in pH (intracellular). METHODS: Light scattering, gel filtration, and fluorescence anisotropy. Also, methylation and processivity assays. CONCLUSIONS: Physiologically relevant changes in pH result in changes in DNMT3A oligomer composition which have dramatic consequences on DNMT3A function. GENERAL SIGNIFICANCE: The pH changes which occur within cancer cells alter the oligomeric state and function of DNMT3A which could contribute to changes in genomic DNA methylation observed in vivo.

RNA modulation of the human DNA methyltransferase 3A.

DNA methyltransferase 3A (DNMT3A) is one of two human de novo DNA methyltransferases essential for transcription regulation during cellular development and differentiation. There is increasing evidence that RNA plays a role in directing DNA methylation to specific genomic locations within mammalian cells. Here, we describe two modes of RNA regulation of DNMT3A in vitro. We show a single-stranded RNA molecule that is antisense to the E-cadherin promoter binds tightly to the catalytic domain in a structurally dependent fashion causing potent inhibition of DNMT3A activity. Two other RNA molecules bind DNMT3A at an allosteric site outside the catalytic domain, causing no change in catalysis. Our observation of the potent and specific in vitro modulation of DNMT3A activity by RNA supports in vivo data that RNA interacts with DNMT3A to regulate transcription.

Mutations in DNA methyltransferase (DNMT3A) observed in acute myeloid leukemia patients disrupt processive methylation.

DNA methylation is a key regulator of gene expression and changes in DNA methylation occur early in tumorigenesis. Mutations in the de novo DNA methyltransferase gene, DNMT3A, frequently occur in adult acute myeloid leukemia patients with poor prognoses. Most of the mutations occur within the dimer or tetramer interface, including Arg-882. We have identified that the most prevalent mutation, R882H, and three additional mutants along the tetramer interface disrupt tetramerization. The processive methylation of multiple CpG sites is disrupted when tetramerization is eliminated. Our results provide a possible mechanism that accounts for how DNMT3A mutations may contribute to oncogenesis and its progression.

Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation.

DNMT3A is one of two human de novo DNA methyltransferases essential for regulating gene expression through cellular development and differentiation. Here we describe the consequences of single amino acid mutations, including those implicated in the development of acute myeloid leukemia (AML) and myelodysplastic syndromes, at the DNMT3A·DNMT3A homotetramer and DNMT3A·DNMT3L heterotetramer interfaces. A model for the DNMT3A homotetramer was developed via computational interface scanning and tested using light scattering and electrophoretic mobility shift assays. Distinct oligomeric states were functionally characterized using fluorescence anisotropy and steady-state kinetics. Replacement of residues that result in DNMT3A dimers, including those identified in AML patients, show minor changes in methylation activity but lose the capacity for processive catalysis on multisite DNA substrates, unlike the highly processive wild-type enzyme. Our results are consistent with the bimodal distribution of DNA methylation in vivo and the loss of clustered methylation in AML patients. Tetramerization with the known interacting partner DNMT3L rescues processive catalysis, demonstrating that protein binding at the DNMT3A tetramer interface can modulate methylation patterning. Our results provide a structural mechanism for the regulation of DNMT3A activity and epigenetic imprinting.

The inherent processivity of the human de novo methyltransferase 3A (DNMT3A) is enhanced by DNMT3L.

Human DNMT3A is responsible for de novo DNA cytosine methylation patterning during development. Here we show that DNMT3A methylates 5-8 CpG sites on human promoters before 50% of the initially bound enzyme dissociates from the DNA. Processive methylation is enhanced 3-fold in the presence of DNMT3L, an inactive homolog of DNMT3A, therefore providing a mechanism for the previously described DNMT3L activation of DNMT3A. DNMT3A processivity on human promoters is also regulated by DNA topology, where a 2-fold decrease in processivity was observed on supercoiled DNA in comparison with linear DNA. These results are the first observation that DNMT3A utilizes this mechanism of increasing catalytic efficiency. Processive de novo DNA methylation provides a mechanism that ensures that multiple CpG sites undergo methylation for transcriptional regulation and silencing of newly integrated viral DNA.

Identification of a second DNA binding site in human DNA methyltransferase 3A by substrate inhibition and domain deletion.

The human DNA methyltransferase 3A (DNMT3A) is essential for establishing DNA methylation patterns. Knowing the key factors involved in the regulation of mammalian DNA methylation is critical to furthering understanding of embryonic development and designing therapeutic approaches targeting epigenetic mechanisms. We observe substrate inhibition for the full length DNMT3A but not for its isolated catalytic domain, demonstrating that DNMT3A has a second binding site for DNA. Deletion of recognized domains of DNMT3A reveals that the conserved PWWP domain is necessary for substrate inhibition and forms at least part of the allosteric DNA binding site. The PWWP domain is demonstrated here to bind DNA in a cooperative manner with muM affinity. No clear sequence preference was observed, similar to previous observations with the isolated PWWP domain of Dnmt3b but with one order of magnitude weaker affinity. Potential roles for a low affinity, low specificity second DNA binding site are discussed.