Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation - PubMed (original) (raw)

Oligomerization of DNMT3A controls the mechanism of de novo DNA methylation

Celeste Holz-Schietinger et al. J Biol Chem. 2011.

Abstract

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.

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Figures

FIGURE 1.

FIGURE 1.

DNMT3A homotetramer model. A, a homotetramer model was generated by aligning a DNMT3A monomer to DNMT3L (from PDB: 2QRV) followed by prediction of the lowest energy orientation using RosettaDock. Below is a close-up of the DNMT3A·DNMT3A tetramer interface showing the core residues and the predicted interactions. The interface shows an aromatic pocket in the center of the interface, with ionic interactions on each edge of the interface. B, depiction of the interactions at the DNMT3A·DNMT3A tetramer interface identified from the model. C, DNMT3A residues for one molecule (pink) of the tetramer interface. Residues are colored based upon their contribution, in ΔΔ_G_, to the tetramer interface compared with alanine as determined using the Rosetta interface alanine-scanning module. Bright yellow residues provide the greatest contribution to the DNMT3A·DNMT3A interface. D, multiple sequence alignment of DNMT3A tetramer interface with related DNMTs and monomeric bacterial homolog (M.HhaI). Red and green residues were mutated in this study to alanine; green and purple residues are mutated in AML or MDS patients. Shaded regions show conserved residues between DNMT3A to M.HhaI.

FIGURE 2.

FIGURE 2.

DNMT3A biophysical characterization. A, light scattering data of DNMT3A mutations along the tetramer interface shows that mutants in solution are either mostly monomer or dimers unlike wild-type tetramers. Size-exclusion chromatography results are shown for light scattering traces of tetrameric wild-type catalytic domain (black trace), representative dimeric H739A (blue trace), and representative dimeric/monomeric R771A (red trace). Molecular weights were determined from the amount of scattered light, in relation to protein concentration determined by _A_280. B, diagram of oligomeric mutants with and without DNA, showing that DNA facilitates oligomerization for DNMT3A mutants via the dimer interface. C, electrophoretic mobility assay of size markers; DNA (GCbox30) has binding sites for size standards, one site for M.HhaI (37 kDa), a known monomer and two binding sites for EcoRV (29 kDa), a known dimer, which creates a dimer and ∼ tetramer band. D, DNA facilitates oligomerization for DNMT3A mutants; five mutants were dimers on DNA. The distance the fluorescently labeled duplex DNA (GCbox30) shifted with DNMT3A determined the oligomeric state. E, electrophoretic mobility assay varying the concentration of R771A and E773A (20–400 n

m

) with DNA at 200 n

m

demonstrating these mutates are dimers even at low concentrations, unlike the monomers seen in solution at 200× the concentration.

FIGURE 3.

FIGURE 3.

DNMT3A homodimers and homotetramers are both active with different mechanisms. A, dimers and tetramers have similar rates of reaction. Left: bar chart of _k_cat values for oligomeric mutants, right: representative time course for tetramer (wild type) showing curved product formation and dimer (R771A) showing linear product formation. B, eliminating tetramer formation increases _Km_DNA. Left: bar chart of _Km_DNA values for oligomeric mutants, right: representative tetramer and dimer curves. C, DNMT3A homodimers had an increase in off-rate compared with homotetramers. Left: bar chart of _k_off values for oligomeric mutants; _k_off values were determined by the enzyme being bound to fluorescein-labeled GCbox30 DNA, then adding saturating unlabeled DNA. Right: representative tetramer and dimer curves. D, DNMT3A homotetramers are processive, and dimers are non-processive. Representative tetramer and dimer processive chase assay data; ●, only substrate, 20 μ

m

bp poly(dI-dC); ■, substrate and then 400 μ

m

bp chase (pCpGL) at 20 min; ▴ = substrate and pCpGL at the start of the reaction. No methylation was detected after addition of chaser DNA with the dimer mutant (R771A), unlike tetramer (WT), which shows less than 10% change in activity. All error bars are at least three experiments given as ±S.E., one-way analysis of variance was used to compare wild-type values to each mutant; *, p > 0.05; **, p > 0.01; and ***, p > 0.001.

FIGURE 4.

FIGURE 4.

DNMT3L heterotetramers restores processivity in DNMT3A dimer mutants. A, DNMT3L binds to DNMT3A tetramers, forming defined heterotetramer. Three dimer mutants become heterotetramers with DNMT3L resulting in the loss of dimers; Y735A and F732A do not bind DNMT3L. B, schematic of the three forms that occurs with the addition of DNMT3L, how the forms change function is below. C, DNMT3L (1:1 ratio) actives DNMT3A tetramers ∼5-fold. Homodimers that bind DNMT3L were activated ∼10-fold; Y735A and F732A saw no activation by DNMT3L. D, heterotetramer formation decreased _Km_DNA for dimer mutants, and no change for homotetramers. E, DNMT3A heterotetramers had a decrease in off-rate compared with homotetramers and homodimers. All heterotetramers have similar rates. F, all DNMT3A heterotetramers are processive as demonstrated by the processive chase assay; ●, only substrate, 20 μ

m

bp poly(dI-dC); ■, substrate and then 400 μ

m

bp chase (pCpGL) at 20 min; ▴, substrate and pCpGL at the start of the reaction. Data indicates 100 turnovers occur before the enzyme dissociates from the DNA. All error bars are at least three experiments given as ±S.E., one-way analysis of variance was used to compare wild-type enzyme to mutants; *, p > 0.05; **, p > 0.01; and ***, p > 0.001.

FIGURE 5.

FIGURE 5.

Diagram of DNMT3A oligomeric states altering methylation patterns through changes in processivity. A, homodimers (R771A, R729A, E733A, Y735A, and F732A) formed by disrupting the tetramer interface resulted in enzymes that bind DNA, methylation, fast dissociation, and rebinding a new piece of DNA (non-processive catalysis). B, homotetramers (WT, H739A, D768A, and R736A) bind DNA followed by methylation then translocation along the same piece of DNA to a new site that the enzyme methylates, thus carrying out processive catalysis multiple times before slow dissociation. C, DNMT3A·DNMT3L heterotetramers (WT, H739A, D768A, R736A, R771A, E733A, and R729A) have increased processive catalysis compared with homotetramers and restores processive in the homodimer, thus carrying out processive catalysis multiple times before slow dissociation.

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References

    1. Bird A. (2002) Genes Dev. 16, 6–21 - PubMed
    1. Reik W., Dean W., Walter J. (2001) Science 293, 1089–1093 - PubMed
    1. Wu H., Coskun V., Tao J., Xie W., Ge W., Yoshikawa K., Li E., Zhang Y., Sun Y. E. (2010) Science 329, 444–448 - PMC - PubMed
    1. Okano M., Bell D. W., Haber D. A., Li E. (1999) Cell 99, 247–257 - PubMed
    1. Chen T., Ueda Y., Dodge J. E., Wang Z., Li E. (2003) Mol. Cell Biol. 23, 5594–5605 - PMC - PubMed

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