Supplementary information to the paper:

DNMT3A oligomerization regulates de novo DNA methylation

Celeste Holz-Schietinger, Douglas M. Matje, Madeleine Flexer Harrison and Norbert O. Reich

Summary of Supplementary Information:

Supplemental Table 1: Conservation of residues along the DNMT3A tetramer interface Supplemental Table 2: Kinetics of M.HhaI mutants F101A and F102A Supplemental Table 3: Biophysical characterization of DNMT3A tetramer interface mutants Supplemental Table 4: Virtual alanine scan of homotetramer model Supplemental Table 5: Functional characterization of DNMT3A tetramer interface mutants Supplemental Table 6: Modelled processivity values for DNMT3A tetramers from time course data Supplemental Table 7: Biophysical characterization of DNMT3L with DNMT3A tetramer interface mutants Supplemental Table 8: Virtual alanine scan of heterotetramer model Supplemental Table 9: Functional characterization of DNMT3A tetramer interface mutants with DNMT3L Supplemental Table 9: DNMT3A mutants along the tetramer interface found in AML and MDS patients with adverse outcome Supplemental Figure 1: Structural alignment DNMT3A with homologs Supplemental Figure 2: Structural position of conserved residues between M.HhaI and DNMT3A Supplemental Figure 3: DNMT3A homotetramer model Supplemental Figure 4: Light scattering with size exclusion chromatography Supplemental Figure 5: Gel Shifts to determine oligomeric state Supplemental Figure 6: DNMT3A dimers have linear product formation with time Supplemental Figure 7: Changes in Km for oligomeric mutants Supplemental Figure 8: koff rates for DNMT3A and mutants Supplemental Figure 9: Kinetic traces of DNMT3A with DNMT3L Supplemental Figure 10: Processivity chase assay, showing dimers are non-processivity Supplemental Figure 11: DNMT3A-DNMT3L homotetramer alanine scanning results Supplemental Figure 12: All heterotetramers are processive Supplemental Figure 13: Tetramerization locks the catalytic loop associated with DNA

Supplemental Table 1: Conservation of residues along the DNMT3A tetramer interface. Comparison of residues along DNMT3A tetramer interface among bacterial C5 DNA methyltransferases that function as monomers and mammalian DNMTs. Human (h) DNMT3A F731, F732 and R736 are conserved residues in bacterial C5 monomers. When M.HhaI residues equivalent to hDNMT3A F731, F732 and R736 were mutated they show a reduction in activity. Residues mutated in this study are indicated as well as DNMT3A mutations found in AML and MDS patients.

* Same structural position as in DNMT3A

^ Bacterial homology: functional monomers: DsaV, ScrFI, HpaII, MspI, BsuFI, HaeII, NgoBI, HaeIII and HhaI Conservation between DNMT3A to bacterial homologs

** Values in Supplemental Table 2 1 Values determined by Sharma et al. 2005 2 Values determined by Estabrook et al. 2004

Supplemental Table 2: Kinetics of M.HhaI mutants F101A and F102A.

from Lindstrom et al. 20003 Supplemental Table 3: Biophysical characterization of DNMT3A tetramer interface mutants. Molecular weights where determined by light scattering. Light scattering data shows that mutants in solution are either mostly monomers or dimers, unlike wild type, which is mostly tetrameric. The form of the enzyme on DNA was determined by gel shifts showing mutants are dimers on DNA or tetrameric and even larger structures like wild type enzyme. Computational ΔΔG values were determined by Rosetta alanine scanning4 of the generated homotetramer model. Combined ΔΔG values of greater than 1.0 result in dimers on DNA. Chain A is the center DNMT3A molecule, chain D is the outer molecule, modeled over DNMT3L.

mDnmt3a (crystal structure)

hDNMT3A Mutantions this study

AML/MDS mutations

M.HhaI (monomer) hDNMT3B hDNMT3L M.HhaI ala mutation

reduction in kcat

Predicted F727 F731 F101* F790 F260 F (8), I 8 fold**3A-3A F728 F732 A F102* F791 F261 F (7), Y, H 13 fold**Tetramer Y731 Y735 A A105 Y794 H264 A (5),V,L,E

H735 H739 A R109 N798 Q268 K (5),R,E,NR767 R771 A L E134 R830 R300 K (4),Q,R,ER732 R736 A, H H R106* H795 R265 R(5),E,A,K 3 fold1

E729 E733 A D103 E792 Q262 D (3),Q,E,NS760 S764 H127 G823 E293 (m=D) H (7),Q,NE721 E725 D95 E784 ---- D (7),HD764 D768 A N131 D827 V297 (m=T) K,N (4),R(4)R725 R729 A W T99 R788 W257 K,N,T(6),QD761 D765 D128 D824 D294 D (8), R 2 fold2

D736 D740 E110 Y799 Y269 D,E,R,T,K,A,Q3A-3L only D682 D686 D60* D745 D226 D (9) ND

V683 V687 I61 V746 V687 I (9)

9 bacterial homologs^ (# used if greater then 2)

high conservationhigh conservation

conservation

high conservation

high conservation

M.HhaI Fold ! Fold ! Fold ! Fold !Wild Type 0.056 ± 0.002 1 0.19 ± 0.02 1 281 3 ± 22 1 6.6 1 ± 0.8 1F102A 0.005 ± 0.001 11.2 0.11 ± 0.02 1.727 ND ND 20 ± 4 3.0F101A 0.004 ± 0.0003 14 0.06 ± 0.02 3.167 4200 ± 200 14.95 143 ± 25 21.7

kcat (s-1) kchem (s-1) KmAdoMet (nM) Km

DNA (nM)

Enzyme Form in solution Form on DNA DNMT3L binding Computional !!GWT 127 ± 2.1 Tetramer Tetramer + yes

D768A 68.1 ± 3.1 Dimer Tetramer + yes -0.23H739A 76.2 ± 2.3 Dimer Tetramer + yes 0.29R736A 70.0 ± 2.9 Dimer Tetramer + yes 0.77Y735A Dimer no 1.29R729A 42.9 ± 1.6 Monomer/Dimer Dimer yes 1.13R771A 43.5 ± 0.7 Monomer/Dimer Dimer yes -0.38E733A 38.7 ± 1.9 Monomer/Dimer Dimer yes 0.44F732A 43.7 ± 2.7 Monomer/Dimer Dimer no 1.83

Not determined

Molecular Weight

Supplemental Table 4. Virtual alanine scan of homotetramer model. Computational ΔΔG values were determined by Rosetta alanine scanning4 of the generated homotetramer model. h= human, m= mouse. Chain A is the center DNMT3A molecule, chain D is the outer molecule, modeled over DNMT3L.

Supplemental Table 5: Functional characterization of DNMT3A tetramer interface mutants. kcat and Km values were determined by monitoring the ability of the enzyme to incorporate tritiated methyl groups transferred from cofactor AdoMet onto DNA (poly-dIdC). koff values were determined by binding excess enzyme to 5’ FAM-6 labeled GCbox30 duplex and measuring the rate of change in anisotropy upon the addition of excess unlabeled GCbox30 (100-fold labeled DNA). Change in fluorescence anisotropy was plotted against time and fit to a single exponential decay to give the koff value. Fold changes was compared to wild type enzyme. Processivity was determined by pulse-chase assays, traces shown in supplemental Fig. 11. The addition of chase DNA (pCpGL) stops product formation with a non-processive enzyme, little effect is seen for processive enzymes.

Residue # h Residue # m chain ddG ddG (partner)721 717 A 0 -0.14724 720 A 1.64 1.01725 721 A -0.11 -0.33729 725 A 0.54 -0.14732 728 A 1.2 2.25733 729 A 0.91 -0.21735 731 A 0.67 1.88736 732 A 0.17 0.51739 735 A 0.47 -0.39744 740 A 0.31 -0.48745 741 A 0.34 -0.28767 763 A -0.11 -0.24768 764 A 0.34 -1.03771 767 A 1.27 0.05772 768 A 1.25 1.2774 770 A -0.21 -0.21692 688 D -0.05 -0.19725 721 D -0.12 0.46729 725 D 0.98 0.3732 728 D 0.54 2.47733 729 D 1.31 -0.18735 731 D 1.27 1.96736 732 D 0.86 -1.5739 735 D 0.46 0.2740 736 D -0.3 -0.43744 740 D 0.49 -0.8745 741 D 0.09 -0.35768 764 D 0.09 -1.26771 767 D 0.93 -0.37772 768 D 1.4 1.41774 770 D -0.25 -0.19

Enzyme Fold ! Fold ! Fold ! Fold ! Processivity WT 3.53 ± 0.15 1.00 1.37 ± 0.09 1.00 214.0 ± 14.4 1.0 0.203 ± 0.008 1.00 yes

D768A 2.88 ± 0.10 1.23 0.83 ± 0.05 0.61 332.0 ± 30.0 1.6 0.216 ± 0.007 1.06 yesH739A 2.60 ± 0.04 1.36 0.63 ± 0.03 0.46 284.0 ± 12.7 1.3 0.209 ± 0.008 1.03 yesR736A 0.64 ± 0.03 5.52 1.85 ± 0.19 1.35 1249.0 ± 111 5.8 0.292 ± 0.010 1.44 noY735A 2.86 ± 0.29 1.23 15.5 ± 0.66 11.3 477.0 ± 13.6 2.2 0.371 ± 0.015 1.83 noR729A 4.85 ± 0.15 0.73 30.8 ± 3.06 22.5 443.0 ± 54.2 2.1 0.500 ± 0.021 2.46 noR771A 4.00 ± 0.23 0.88 44 ± 1.73 32.1 538.0 ± 23.7 2.5 0.558 ± 0.019 2.75 noE733A 3.06 ± 0.09 1.15 14.3 ± 1.33 10.4 850.0 ± 29.4 4.0 0.404 ± 0.018 1.99 noF732A 0.23 ± 0.03 15.26 4.47 ± 0.15 3.26 1140.0 ± 47.3 5.3 0.903 ± 0.092 4.45 no

Dimers on DNA

Tetramers on DNA

kcat h-1 koff min-1Km AdoMet nMKm

DNA µM bp

Supplemental Table 6: Modelled processivity values for DNMT3A tetramers from time course data. Modeling was done as shown in Holz-Schietinger et al. 20115. Kinetic modeling was originally developed for helicase processivity studies6,7 and previously used for DNMT18. The modeling is based on the shape of the curve for the non-chase experiment, which determines the methylation rate (k) and dissociation rate (koff). A processivity value (p) was determined from the k and koff rates. The processivity values (p) were used to calculate n1/2, which indicates the average numbers of catalytic turnovers before half of the enzymes are dissociated from the original substrate.

Supplemental Table 7: Biophysical characterization of DNMT3L with DNMT3A tetramer interface mutants. Computational ΔΔG values were determined by Rosetta alanine scanning4 of the interface between DNMT3A and DNMT3L revealed in the crystal structure. DNMT3L binding to DNMT3A was determined by super-shifting on gel shift. No super-shifting occurred for Y735A and F732A. Activation occurred by preincubating DNMT3L with DNMT3A and measuring the increase in activity over an hour with and without DNMT3L, at a 1:1 DNMT3A:DNMT3L ratio was needed for full activation.

p n 1/2

Wild type 0.99 26H739A 0.97 22D768A 0.97 20

Enzyme !!G Binding Activation FoldWT yes yes 5

F732A 1.83 no no ----Y735A 1.29 no no ----E733A 0.44 yes yes 11H739A 0.29 yes yes 5D768A -0.23 yes yes 5R771A -0.38 yes yes 10R736A 0.77 yes yes 5R729A 1.13 yes yes 8

Supplemental Table 8: Virtual alanine scan of heterotetramer model. Computational ΔΔG values were determined by Rosetta alanine scanning4 of the interface between DNMT3A and DNMT3L revealed in the crystal structure. h= human and m= mouse.

Supplemental Table 9: Functional characterization of DNMT3A tetramer interface mutants with DNMT3L. Values were determined as stated in table 4 but with DNMT3L preincubated with enzyme for one hour at 37°C in reaction buffer with AdoMet at a 1:1 ratio of DNMT3A to DNMT3L. Fold stimulation were determined by the fold change in methyl-group transferred in one hour comparing with and without DNMT3L.

Supplemental Table 10: DNMT3A mutants along the tetramer interface found in AML and MDS patients with adverse outcome. DNMT3A mutants found in AML patients shown in Ley et al. 20109 study, and DNMT3A mutants found in MDS patients shown in Walter et al. 201110 paper. In this study we mutated three of these residues to alanine, preliminary data shows the R736H mutant also disrupts the interface.

Residue # h Residue # m ddG Residue # h Residue # m ddG DNMT3A DNMT3L688 684 0.92 225 261 0.97692 688 -0.01 226 262 -0.64723 719 0.05 229 265 0.08724 720 2.19 257 293 1.11725 721 -0.26 258 294 0.43729 725 1.13 261 297 3732 728 1.83 262 298 0.35733 729 0.44 264 300 0.5735 731 1.29 265 301 0.92736 732 0.77 268 304 0.1739 735 0.29 269 305 0.96742 738 0.02 273 309 0.01744 740 0.02 293 329 -0.03745 741 0.1 294 330 1.33767 763 0 297 333 -0.14768 764 0.23 300 336 0.8771 767 0.38 301 337 2.1772 768 2.1 303 339 -0.13774 770 0.21

Enzyme Fold ! Fold ! Fold ! ProcessivityWT 4.67 ± 0.29 1.0 1.2 ± 0.1 1.0 0.1503 ± 0.01 1.0 yes

D768A 5.37 ± 0.37 1.1 1.2 ± 0.1 1 0.1713 ± 0.01 1.1 yesH739A 5.04 ± 0.17 1.1 1.5 ± 0.2 1.2 0.176 ± 0.01 1.2 yesR736A 5.45 ± 0.21 1.2 1.2 ± 0.1 1.0 0.1524 ± 0.01 1.0 yesY735A 1.17 ± 0.05 0.3 NAR729A 8.10 ± 0.40 1.7 1.8 ± 0.3 1.5 0.1174 ± 0.01 0.78 yesR771A 10.25 ± 0.56 2.2 2.0 ± 0.2 1.7 0.1367 ± 0.02 0.91 yesE733A 11.22 ± 0.44 2.4 2.0 ± 0.2 1.7 0.1808 ± 0.01 1.20 yesF732A 1.03 ± 0.02 0.2 NA

NA NA

Fold Stimulation

NA NA

Km DNA µM bp koff min-1

R729WR729QR736H R736A noP718LA741V

L737RR771L R771A yesS770WS714C

R729A yes

AML mutations

MDS mutations

mutanted in current study

disrupts interface

Supplemental Figure 1: Structural alignment DNMT3A with homologs. (a) Cα trace of DNMT3A (pink) structurally aligned to DNMT3L (yellow) using Pymol, which shows a root-mean-square deviation (RMSD) of 1.39 Å, DNMT3L does not have a target recognition domain (TRD). The homotetramer model was generated based off the alignment of DNMT3A to DNMT3L, in the heterotetramer crystal structure (PDB:2QRV11). (b) C-α trace of DNMT3A catalytic domain structurally aligned to M.HhaI (PDB:2HR112) using Pymol, DNMT3A in pink, M.HhaI in cyan. Shows RMSD of 2.2 Å for 220 amino acid pairs, this excludes TRD, which is larger in M.HhaI.

Supplemental Figure 2: Structural position of conserved residues between M.HhaI and DNMT3A. (a) DNMT3A F732A (PDB:2QRV11) overlay M.HhaI F102A (PDB:2HR112). DNA is from the M.HhaI structure. (b) DNMT3A R736A overlay M.HhaI R106 showing these conserved residues are also conserved in their structurally position.

Supplemental Figure 3: DNMT3A homotetramer model. (a) Model of DNMT3A homotetramer was generated by DNMT3A Chain A (PDB file 2QRV11) being kept rigid while the backbone of Chain D was aligned to the backbone of DNMT3L (Chain B) using Pymol. Chain D was then dragged into a random orientation ~10Å away from the tetramer interface of Chain A. The complex was submitted to the RosettaDock Server4 to generate the initial tetramer interface model. After visual inspection, the best scoring models were re-submitted until all iterations repeatedly converged on a common model. This model was submitted to the Rosetta computational alanine scanning13 server to evaluate the contribution of individual residues to the tetramer interface. The side chains of residues R771, E733, and R720 were manually optimized by choosing backbone dependent rotamers conformations that gave the highest scores using the computational alanine scanning protocol. The DNA was then modeled in from M.HhaI structure (PDB: 2HR1). (b) Residues at the homotetramer interface are colored based off the computationally calculated ΔΔG values for the energetic contribution of the residue to the interface. Bright yellow indicates the most important residues for the interface. ΔΔG values are shown in Supplemental Table 4. (c,d) DNMT3A residues at the heterotetramer interface, showing aromatic residues in the center of the interface and charge-charge interactions on the edge provides the greatest contribution to the interface. (e) Bar chart of calculated ΔΔG values from virtual alanine scan of DNMT3A:DNMT3A tetramer interface. A residue’s total predicted energetic contribution for the tetramer interface is for both DNMT3A molecules at the interface (pink and blue). The five residues that disrupt the interface also have the greatest calculated ΔΔG.

Supplemental Figure 4: Light scattering with size exclusion chromatography. (a) Light scattering trace of wild type DNMT3A (black) at 30 µM, with SAH saturating, M.HhaI (30 µM) with SAH and DNA (GCbox30) 30 µM (blue trace), M.HhaI with SAH (red trace). M.HhaI a monomer in solution, and binds DNA as a monomer, narrow peaks indicating single molecular state, unlike the broad peak shown for mostly tetramer forming DNMT3A catalytic domain. (b) Light scattering trace of wild type DNMT3A catalytic domain, with SAH (black), without SAH (blues) and with SAH and DNA (GCbox30), 30 µM. SAH does not change the oligomeric state, but DNA results in mostly DNMT3A aggregates. (c) Light scattering traces of all mutants, indicating WT elutes first, followed by D768A and H739A, R736A shows slightly slower elution, followed by R736H, R771A, R729A, E733A and F732A (Y735A predicated at high concentration required for light scattering, no data was obtained). The molecular weights were calculated from the light scattering data and are in Supplemental Table 3.

Supplemental Figure 5: Gel shifts to determine oligomeric state. (a) Gel shift of EcoRV size control, where DNA is constant and enzyme is varied. At enzyme concentration equal to DNA concentration two distinct bands occur, a dimer and tetramer bands. (b) Gel shift varying wild type DNMT3A (40-360 nM) with DNA at 160 nM. (c) Gel shift varying R729A, Y735A and F732A concentrations (40-360 nM), showing all these enzymes are dimers and no monomers where observed.

Supplemental Figure 6: DNMT3A dimers have linear product formation with time. Dimers and tetramers have similar rates of reaction. Time course data showing curved product formed with time for tetramers and dimers shows linear time courses. All reactions are under steady state conditions, with 30 nM active enzyme and poly-dIdC as DNA in reaction buffer. All error bars are at least three experiments given as SE.

Supplemental Figure 7: Changes in Km for oligomeric mutants. (a) Eliminating tetramer formation increases the enzymes Km for DNA, (b) Km

AdoMet increases 2-3 fold for dimer mutants, and 5-6 fold for residues conserved in M.HhaI (c) Km

AdoMet traces, where AdoMet was varied as DNA as at saturating concentrations.

Supplemental Figure 8: koff rates for wild type DNMT3A and mutants. Homodimers (blue trace) show increased product release rates compared to homotetramers (gray-wild type and red trace), DNMT3A-DNMT3L heterotetramers (black traces) show increased off rates compared to homotetramers and homodimers. koff values were determined by binding excess enzyme to 5’ 6-FAM labeled GCbox30 duplex and measuring the rate of change in anisotropy upon the addition of excess unlabeled GCbox30 (100-fold labeled DNA). Change in fluorescence anisotropy was plotted against time and fit to a single exponential decay to give the koff value. Rates shown in Supplemental Table 4.

Supplemental Figure 9: Kinetic traces of DNMT3A with DNMT3L. (a) DNMT3A homodimers were either activated by DNMT3L 2-fold more than homotetramers or no activation was seen (Y735A and F732A). DNMT3L concentration was varied (0.25-4-fold DNMT3A), reactions were run for one hour. (b) Heterotetramer formation decreases Km

DNA for dimer mutants, and no change is observed for homotetramers. All reactions were with 30 nM active enzyme and poly-dIdC as DNA in reaction buffer. Three independent experiments were run to generate error bars (standard error).

Supplemental Figure 10: Processivity chase assay, showing dimers are not processive. (a) Diagram of processivity assay (b) DNMT3A homotetramers (left side) were processive and dimers (right side) were non-processive. Processive pulse chase assays, ■ = only substrate 25 µM bp substrate; ● = substrate and then 400 µM bp pCpGL (chase) at 20 min; ▴ = substrate and chase at the start of the reaction. Indicating dimers dissociate within one turnover of chase DNA being added, tetramers have little change in product formation when chase DNA is added.

Supplemental Figure 11: DNMT3A-DNMT3L heterotetramer alanine scanning results (a) DNMT3L:DNMT3A heterotetramer as shown in the crystal structure (PDB: 2QRV11), DNA is from M.HhaI (PDB: 2HR112). (b) The tetramer interface between DNMT3L and DNMT3A. Residues at the interface are colored according to the computationally calculated ΔΔG values13. Bright yellow indicates the most important residues for the interface. ΔΔG values are shown in Supplemental Table 8. (c) ΔΔG values for mutated residues (d) DNMT3L residues at the heterotetramer interface, showing F261 in the center of the interface is predicted to make the largest energetic contribution. (e) DNMT3A residues at the heterotetramer interface, showing aromatic residues (F732, Y735 and F772) in the center of the interface provides the greatest contribution.

Supplemental Figure 12: All heterotetramers are processive. DNMT3L increases DNMT3A processivity, restoring processivity in homodimers (right site) and homotetramers (left side) by forming heterotetramers. Processive pulse chase assays, ■ = only substrate 25 µM bp substrate; ● = substrate and then 400 µM bp pCpGL (chase) at 20 min; ▴ = substrate and chase at the start of the reaction. Indicating ~ 100 turnovers are occurring before the enzyme dissociates from the DNA.

 

Supplemental Figure 13: Tetramerization locks the catalytic loop associated with DNA. Catalytic loop position in cytosine DNA methyltransferases, showing that DNMT3A tetramer formation prevents loop motion into M.HhaI corresponding open position that occurs with no DNA or non-cognate DNA (a) M.HhaI structure with cognate DNA (PDB:2HR112), showing closed loop position (cyan), aligned with M.HhaI open catalytic loop occurring with no DNA (PDB:IHMY14) (blue), DNMT3A (pink) catalytic loop (b) Loop position of all crystallized cytosine DNA methyltransferase, M.HhaI open (blue), closed (cyan), HaeIII (PDB:1DCT15) (purple), DNMT3A (PDB:2QRV11) (pink) and DNMT1 (PDB:3PT616) (yellow) (c) DNMT3A dimer overlay with M.HhaI catalytic loop open (blue), closed (cyan), indicating loop is free to move as a dimer. (d) DNMT3A homotetramer overlay with M.HhaI catalytic loop open (blue), closed (cyan), showing that DNMT3A prevents the catalytic loop in the open position (e) same as (d) but DNMT3L heterotetramer, showing DNMT3L also prevents loop motion.  Supplemental References 1. Sharma, V., Youngblood, B. & Reich, N. (2005) Journal of biomolecular structure & dynamics 22, 533-43. 2. Estabrook, R.A., Lipson, R., Hopkins, B. & Reich, N. (2004) JBCchemistry 279, 31419-28. 3. Lindstrom, W.M., Jr., Flynn, J. & Reich, N.O. (2000)JBC 275, 4912-9. 4. Lyskov, S. & Gray, J.J. (2008) Nucleic acids research 36, W233-8. 5. Holz-Schietinger, C. & Reich, N.O. (2010) JBC 285, 29091-100. 6. Ali, J. A., and Lohman, T. M. (1997) Science 275, 377–380 7. Porter, D. J., Short, S. A., Hanlon, M. H., Preugschat, F., Wilson, J. E., Willard, D. H., Jr., and Consler, T.

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