Mutations in the DNMT3A DNA methyltransferase in acute myeloid leukemia patients cause both loss and gain of function and differential regulation by protein partners

Abstract

Eukaryotic DNA methylation prevents genomic instability by regulating the expression of oncogenes and tumor-suppressor genes. The negative effects of dysregulated DNA methylation are highlighted by a strong correlation between mutations in the de novo DNA methyltransferase gene DNA methyltransferase 3α (DNMT3A) and poor prognoses among acute myeloid leukemia (AML) patients. We show here that clinically observed DNMT3A mutations dramatically alter enzymatic activity, including mutations that lead to 6-fold hypermethylation and 3-fold hypomethylation of the human cyclin-dependent kinase inhibitor 2B (CDKN2B or p15) gene promoter. Our results provide insights into the clinically observed heterogeneity of p15 methylation in AML. Cytogenetically normal AML (CN-AML) constitutes 40-50% of all AML cases and is the most epigenetically diverse AML subtype with pronounced changes in non-CpG DNA methylation. We identified a subset of DNMT3A mutations that enhance the enzyme's ability to perform non-CpG methylation by 2-8-fold. Many of these mutations mapped to DNMT3A regions known to interact with proteins that themselves contribute to AML, such as thymine DNA glycosylase (TDG). Using functional mapping of TDG-DNMT3A interactions, we provide evidence that TDG and DNMT3-like (DNMT3L) bind distinct regions of DNMT3A. Furthermore, DNMT3A mutations caused diverse changes in the ability of TDG and DNMT3L to affect DNMT3A function. Cell-based studies of one of these DNMT3A mutations (S714C) replicated the enzymatic studies and revealed that it causes dramatic losses of genome-wide methylation. In summary, mutations in DNMT3A lead to diverse levels of activity, interactions with epigenetic machinery components and cellular changes.

Keywords: DNA binding protein; DNA methylation; DNA methyltransferase; enzyme catalysis; enzyme kinetics; enzyme mutation; epigenetics.

Figure 1.

Mutations from AML patients in a DNMT3A homotetramer model. A model of the DNMT3A homotetramer (alternating purple and cyan monomers) bound to DNA was generated by aligning DNMT3A monomers to DNMT3L in a DNMT3A–DNMT3L heterotetramer crystal structure (PDB ID code 2QRV) followed by a subsequent alignment of a DNMT3A monomer to a M.HhaI-dsDNA co-crystal structure (PDB ID code 3EEO). Arrows in front view (A) and top view (B) indicate dimer and tetramer interfaces. Mutated residues are categorized based on location as follows: surface, orange; tetramer interface, yellow; and internal, green.

Figure 2.

A subset of mutations display little change or enhanced activity for p15-pCpG^L relative to the multiple CpG site substrate poly(dI-dC). WT DNMT3A has significantly reduced activity on p15-pCpG^L (cyan, 20 μm bp) relative to poly(dI-dC) (black, 5 μm bp) due to the limited number of CpG sites on the human promoter substrate (∼10-fold less). R736H and P904L display minimal change in activity across DNA substrates, whereas W893S, R771G, and R635G lead to enhanced activity in p15-pCpG^L. R771Q maintains significantly higher activity for both DNA substrates. Enzyme concentrations are 150 nm tetramer (27 nm active tetramer) (24) and k_cat (h⁻¹) values were determined as described under “Experimental procedures.”

Figure 3.

Substrate diagram and characteristics. Although poly(dI-dC) contains an extensive number of CpG sites with virtually no space between, p15-pCpG^L consists of a limited number of sites available for methylation that are heterogeneously spread. The pCpG^L vector, to which the p15 human promoter was inserted, is 3,872 bp in size and lacks any CpG sites for methylation.

Figure 4.

Some mutations result in enhanced activity at non-CpG sites. WT DNMT3A activity is affected by the availability of CpG sites on the DNA substrate as noted by the drastic activity loss from poly(dI-dC) (black, 5 μm bp poly(dI-dC)) to p15-pCpG^L (cyan, 20 μm bp) and limited activity on a non-CpG substrate (orange, 20 μm bp pCpG^L). In contrast to the WT enzyme, R771G, R635G, and W893S display a similar pattern of activity across substrates with enhancement on p15-pCpG^L (cyan, 20 μm bp) and comparable levels on poly(dI-dC) (black, 5 μm bp poly(dI-dC)) and non-CpG substrate (orange, 20 μm bp pCpG^L). R736H activity on the non-CpG substrate is significantly higher and virtually equal to substrates with multiple CpG sites. Enzyme concentrations are 150 nm tetramer (27 nm active tetramer) (24). Data reflect the results from at least three independent reactions.

Figure 5.

AML mutants display unique alterations to processive catalysis on the poly(dI-dC) substrate. A, WT DNMT3A; B, P904L; and C, R736H at 50 nm tetramer. Substrates were added at time 0 to start the reaction and chase assay conditions were as follows: ●, substrate only (2 μm bp poly(dI dC)); red square, substrate and then 20 min chase (40 μm bp pCpG^L); blue triangle, substrate (2 μm bp poly(dI-dC)) and chase (40 μm bp pCpG^L) at the start of the reaction. Data reflect the results from at least three independent experiments.

Figure 6.

AML mutations display loss of processive catalysis on the p15-pCpG^L human promoter substrate. A, WT DNMT3A retains the ability to perform processive catalysis when tested with the p15-pCpG^L substrate, whereas B, R736H and C, P904L resulted in a loss of processivity. All enzyme concentrations were 50 nm tetramer. Substrates were added at time 0 to start the reaction and chase assay conditions were as follows: ●, substrate only (10 μm bp p15-pCpG^L); red square, substrate and then 20 min chase (200 μm bp pCpG^L); blue triangle, substrate (10 μm bp p15-pCpG^L) and chase (200 μm bp pCpG^L) at the start of the reaction. Data reflect the results from at least three independent experiments.

Figure 7.

DNMT3L has a higher affinity for binding DNMT3A compared with TDG. In A and B, DNMT3A (10 nm) was preincubated in reaction buffer for 1 h at 37 °C with varying concentrations of TDG or DNMT3L (0–300 nm). Reactions were initiated by the addition of 5 μm bp poly(dI-dC) and run for 1 h. Fold stimulation was determined by the sum of product formed by DNMT3A with DNMT3L divided by product formed by DNMT3A without DNMT3L (A). Fold inhibition was determined by product formed by DNMT3A alone divided by product formed by DNMT3A with TDG (B). Data reflect the results from at least three independent co-incubation reactions.

Figure 8.

DNMT3L or TDG binding on DNMT3A tetramer interface are not mutually exclusive. WT DNMT3A, DNMT3L, and TDG co-incubations did not reflect the modulatory effect observed when either DNMT3L or TDG are preincubated with DNMT3A. Enzyme concentrations for all the reactions performed were 150 nm and at 1:1 for co-incubations. Prior to initiating the reaction by the addition of 5 μm bp poly(dI-dC); light grey, WT DNMT3A, DNMT3L, and TDG were preincubated for 1 h at 37 °C. Under similar conditions, the following reactions were performed as controls: black, WT DNMT3A; medium grey, WT DNMT3A and DNMT3L; dark grey, WT DNMT3A and TDG. Data reflect the results from at least three independent competitive co-incubation experiments.

Figure 9.

TDG does not compete with DNMT3L for binding to DNMT3A. The addition of TDG does not disrupt the activity of a functional DNMT3A–DNMT3L heterotetramer, but the addition of DNMT3L increases the activity of a functional DNMT3A–TDG heterotetramer. In all experiments performed, enzyme concentrations were 150 nm (1:1 for co-incubations or binding competitions) and reaction were initiated by the addition of 5 μm bp poly(dI-dC). A, green, WT DNMT3A was preincubated with DNMT3L for 1 h at 37 °C and the reaction run for 30 min prior to the addition of TDG. B, green, WT DNMT3A was preincubated with TDG for 1 h at 37 °C and the reaction run for 30 min prior to the addition of DNMT3L. The following reactions were also tested as controls: A, black, WT DNMT3A and DNMT3L; B, black, WT DNMT3A and TDG; A and B, blue, WT DNMT3A; A and B, red, WT DNMT3A, DNMT3L, and TDG preincubated at the start of the reaction (A and B). Data reflect the results from three independent experiments.

Figure 10.

S714C reduces DNA methylation in mESCs. The images show the methylation level as determined by a dot-blot assay in doxycycline-inducible DNMT3A mutant-expressing DKO mESCs after doxycycline induction for 2 weeks. The upper blot represents serial dilution of DNA derived from the indicated cells in the dot-blot, probed with an antibody against 5-mC. The lower blot represents serial dilution of standard methylated DNA as a control.