1

Online Supplemental Material for Van Emburgh and Robertson,

“Modulation of Dnmt3b function in vitro by interactions with Dnmt3L,

Dnmt3a, and Dnmt3b splice variants”

Supplemental Materials and Methods

Plasmid construction, protein expression and purification

The pSL301 human satellite 2 (SAT2)-containing plasmid, described previously (1), was used to

derive the electrophoretic mobility shift assay (EMSA) DNA probe and as a substrate for DNA

methyltransferase activity assays. Recombinant hexahistidine-tagged (6XHis) DNMTs were

generated using the Bac-to-Bac Baculovirus Expression System (Invitrogen). DNMT cDNAs,

generated by subcloning or PCR-based cloning, were inserted into pFastBacHT vectors (see

Table S1 for all primer sequences), which were subsequently used to derive recombinant

baculovirus. Dnmt3b splice variants (Dnmt3b1, Dnmt3b2, and Dnmt3b3 in pBluescript SKII

provided by Dr. En Li), ∆434-531, and C657A baculovirus constructs were created by PCR and

cloned into pFastbac HT-C at the NotI site. The ∆584-859 and ∆1-140 deletion constructs were

excised from previously described expression vectors (2) with EcoRI+HindIII and cloned into

pFastbac HT-A at the EcoRI and HindIII sites. Murine Dnmt3b1 ICF syndrome mutant

constructs and wild-type human DNMT3B1 cDNAs were kindly provided by Dr. Guo-Liang Xu

(3). Murine Dnmt3b1 cDNAs were PCR amplified using the original vectors as templates and

cloned into pFastbac HT-C at the NotI site. The human DNMT3B1 cDNA was PCR amplified

using the original vector as template and cloned into pFastbac HT-C at the SalI site. The

Dnmt3b1 deletions ∆671-859, ∆225-249, and ∆227-429 in pEGFP-C1 were kindly provided by

Dr. Taiping Chen (4). The ∆225-249 and ∆227-429 cDNAs were amplified using the Dnmt3b

2

Not1 primers, ∆671-859 was amplified using the SG77 NotI primer and the Dnmt3b1 670R NotI

primer and cloned into pFastbac HT-C at the NotI site. All primer sequences are provided in

Table S1. The human Dnmt3L cDNA in pGEX-5X-1 was provided by Dr. Guo-Liang Xu. The

Dnmt3b1 ∆173-859, ∆434-859, and ∆1-560 pGEX-5X constructs have been described

previously (2). The Dnmt3b3-CD construct was made by PCR using pFastbac-Dnmt3b3 as

template and cloning into the NotI site of pGEX-5X-3. Fluorescent tagged fusion protein

expression vectors were generated using available plasmids. Full-length Dnmt3b2, Dnmt3b3,

Dnmt3b1 C657A, and Dnmt3b1 V612A cDNAs were cloned into the EcoRI and BamHI sites of

the pEGFP-C2 (Clontech) mammalian expression vector. Full-length DNMT3B3 cDNA was

cloned into the EcoRI and BamHI sites of pDsRed-C1 (Clontech). The Dnmt3b1 cDNA was

cloned into EcoRI and BamHI sites of pEGFP-C1. Using EGFP-Dnmt3b1 as template, the open

reading frame was PCR amplified using the m3b (F) EcoRI and m3b (R) BamHI primers and

cloned into these sites of pDsRed-Monomer-C1 (Clontech). Using the pGEX-Dnmt3L plasmid

as template, the Dnmt3L cDNA was PCR amplified using the Dnmt3L (F) SalI and Dnmt3L (R)

SalI primers and the resulting product cloned into this site of pDsRed-Monomer-C1. All

constructs were confirmed by DNA sequencing.

To generate 6XHis-tagged recombinant proteins, high titer (>109) baculovirus stock was

used to infect Sf9 cells cultured in SF900II media containing 5% fetal bovine serum (Invitrogen).

Recombinant proteins were purified from whole cell extract using Ni-NTA His-Bind resin

(Novagen) as described previously (5). Proteins were eluted from beads with a low salt elution

(LSE) buffer (20 mM NaH2PO4, 10 mM NaCl, 500 mM imidazole, 1% NP-40, 10% glycerol)

and stored at -70°C. Recombinant GST-tagged DNMTs were generated using the pGEX-5X

bacterial expression system (GE Healthcare). GST-DNMT fusion constructs were expressed in

3

the BL21 E. coli strain. Briefly, the culture was induced with 0.1 mM IPTG for 4 hours at 37°C.

Cells were lysed with 1X phosphate buffered saline, 20% glycerol, 0.5% triton X-100, and

sonicated 5X 1 minute at 50% duty on output setting 4 (Branson Sonifier 450). Recombinant

proteins were purified from cell extracts using glutathione sepharose 4 fast flow resin (GE

Healthcare). Cell extract was incubated with resin for 45 minutes then non-specifically bound

proteins were removed by washing 5X with cell lysis buffer. Proteins were eluted from the resin

with 50 mM Tris-HCl (pH 8.0) and 10 mM reduced glutathione. Eluted proteins were dialyzed

against LSE for buffer exchange and stored at -70°C. Bound resin was stored as a 50% slurry in

cell lysis buffer at -70°C for use in GST pull downs. Purified proteins and resin were analyzed

on SDS-PAGE gels followed by staining with coomassie blue. Eluted proteins were quantitated

using the Bradford Assay (Bio-Rad).

Supplemental Tables

Table S1: Sequences of primers used in this study.

Table S2: Amino acid conversion table showing homologous positions in the murine and human

Dnmt3b sequence where ICF-syndrome patient-associated mutations are located.

Supplemental Figure Legends

Fig. S1: Effects of Dnmt3b splice variants on the methylation of plasmid and SAT2 sequences.

(A) Methylation of individual CpG sites by Dnmt3s with and without Dnmt3L determined by

pyrosequencing on the plasmid backbone region (see also Fig. 1). Error bars indicate the

standard deviation. (B) Graphic representation of the linear correlation of methylation of

4

individual CpGs between (left panel) Dnmt3b1 with Dnmt3L and other Dnmt3s with Dnmt3L

using plasmid pyrosequencing data. Dnmt3b2 + Dnmt3L R: 0.99, R2: 0.99, Dnmt3a + Dnmt3L

R: 0.76, R2: 0.57, DNMT3B1 + Dnmt3L R: 0.99, R2: 0.98. Middle panel: Dnmt3s alone and with

Dnmt3L using SAT2 pyrosequencing data. Dnmt3a vs Dnmt3a + Dnmt3L R: 0.88, R2: 0.78,

Dnmt3b1 vs Dnmt3b1 + Dnmt3L R: 0.88, R2: 0.78. Right panel: Dnmt3s alone and with Dnmt3L

using plasmid sequencing data. Dnmt3a vs Dnmt3a + Dnmt3L R: 0.91, R2: 0.83. Dnmt3b1 vs

Dnmt3b1 + Dnmt3L R: 0.47, R2: 0.22. (C) Plasmid BGS results for the indicated Dnmt3s with

and without Dnmt3L. Overall % methylation is indicated in parentheses. White circles:

unmethylated CpGs, black circles: methylated CpGs. Each row represents one clone. (D) BGS

data for plasmid (left) and SAT2 (right) regions for the Dnmt3b2 splice variant with and without

Dnmt3L. (E) Processivity of the indicated Dnmt3 constructs with and without Dnmt3L.

Processivity data was generated using the BGS data in Figs. 2, S1C, and S1D. Processivity

indices are presented as a box plot using SigmaPlot. Black circles represent outliers of <10th or

>90th percentiles. *P<0.05, **P<0.01 (F) Non-CpG methylation activity of Dnmt3b1,

DNMT3B1, and Dnmt3a in vitro. BGS reactions were analyzed for the presence of non-CpG

methylation. The background level of non-conversion by the sodium bisulfite reaction is very

low (0.22%) as shown by BGS analysis of the Dnmt3b1 C657A mutant, which is catalytically

dead (Fig. S2). Non-CpG methylation was than stratified according to occurrence in CT, CA, and

CC dinucleotides. CG methylation frequency is also shown as a reference.

Fig. S2: Impact of Dnmt3b1 deletions on DNA binding and enzymatic activity. (A) Left panel:

representative EMSA gel showing binding of the Dnmt3b1 ∆434-859 construct to the SAT2

DNA probe (0-200 nM range) relative to full-length Dnmt3b1. Right panel: quantification of

5

DNA binding using the Hill equation. (B) Graphical summary of pyrosequencing data for the

indicated Dnmt3b deletion or mutation constructs at the plasmid region in the presence and

absence of Dnmt3L. Data is presented as the average methylation over all CpG sites (Fig. 1). (C)

BGS DNA methylation analysis of the indicated Dnmt3b deletions and the C657A mutant, with

and without Dnmt3L, for the SAT2 region. (D) BGS analysis for the plasmid sequence with the

same Dnmt3b constructs as in part C. For both C and D, the overall percent methylation is

indicated in parentheses. White circles: unmethylated CpGs, black circles: methylated CpGs.

Each row represents one clone.

Fig. S3: Pyrosequencing and BGS results for Dnmt3b1 ICF syndrome-associated mutations. (A)

Graphical summary of pyrosequencing data for the indicated Dnmt3b mutants at the plasmid

region in the presence and absence of Dnmt3L. (B) BGS DNA methylation analysis of the

indicated ICF syndrome-associated Dnmt3b mutants that demonstrated detectable enzymatic

activity in HpaII digestions, in the presence and absence of Dnmt3L for the SAT2 sequence. (C)

BGS data for the plasmid region. In B and C the overall percent methylation for the entire region

is indicated in parentheses.

Fig. S4: Impact of ICF syndrome-associated mutations in the context of murine Dnmt3b1 with

Dnmt3L on individual CpG site methylation within the SAT2 (A) and plasmid region (B) as

determined by bisulfite pyrosequencing. Error bars indicate the standard deviation from the

mean.

6

Fig. S5: Percent methylation, by individual CpG site, in the SAT2 and plasmid pyrosequencing

regions of Dnmt3a-Dnmt3L bimolecular reactions relative to Dnmt3a-Dnmt3L-Dnmt3b

trimolecular reactions. Trimolecular reactions are in the presence of the indicated Dnmt3b splice

variants (A - SAT2, D - plasmid), deletion constructs and the C657A point mutant (B - SAT2, E

- plasmid), or ICF syndrome-associated Dnmt3b1 mutants (C - SAT2, F - plasmid) as indicated

in each panel.

Fig. S6: Area plot of CpG site methylation within the SAT2 region based on the pyrosequencing

data derived from the current study (Dnmt3b vs Dnmt3a, each in the presence of Dnmt3L)

compared to our previous DNA methylation analysis of chromosome 1 SAT2 DNA methylation

patterns in HCT116 colorectal carcinoma cells using bisulfite genomic-sequencing (1,6). Note

the similar contours of the plots for Dnmt3b1+Dnmt3L and in vivo methylation patterns of SAT2

in HCT116 cells in the right panel even though absolute levels of methylation differ.

References

1. Gopalakrishnan, S., Van Emburgh, B.O., Shan, J., Su, Z., Fields, C.R., Vieweg, J.,

Hamazaki, T., Schwartz, P.H., Terada, N. and Robertson, K.D. (2009) A novel DNMT3B

splice variant expressed in tumor and pluripotent cells modulates genomic DNA

methylation patterns and displays altered DNA binding. Mol. Canc. Res., 7, 1622-1634.

2. Geiman, T.M., Sankpal, U.T., Robertson, A.K., Zhao, Y., Zhao, Y. and Robertson, K.D.

(2004) DNMT3B interacts with hSNF2H chromatin remodeling enzyme, HDACs 1 and

2, and components of the histone methylation system. Biochem. Biophys. Res. Commun.,

318, 544-555.

7

3. Xie, Z.-H., Huang, Y.-N., Chen, Z.-X., Riggs, A.D., Ding, J.-P., Gowher, H., Jeltsch, A.,

Sasaki, H., Hata, K. and Xu, G.-L. (2006) Mutations in DNA methyltransferase

DNMT3B in ICF syndrome affect its regulation by DNMT3L. Hum. Mol. Genet., 15,

1375-1385.

4. Chen, T., Tsujimoto, N. and Li, E. (2004) The PWWP domain of Dnmt3a and Dnmt3b is

required for directing DNA methylation to the major satellite repeats at pericentric

heterochromatin. Mol. Cell. Biol., 24, 9048-9058.

5. Yokochi, T. and Robertson, K.D. (2002) Preferential methylation of unmethylated DNA

by mammalian de novo DNA methyltransferase Dnmt3a. J. Biol. Chem., 277, 11735-

11745.

6. Gopalakrishnan, S., Sullivan, B.A., Trazzi, S., Della Valle, G. and Robertson, K.D.

(2009) DNMT3B interacts with constitutive centromere protein CENP-C to modulate

DNA methylation and the histone code at centromeric regions. Hum. Mol. Genet., 18,

3178-3193.

Table S1. PCR and pyrosequencing primers used in this study.

Dnmt3b PrimersSG 77 (F) NotI 5’-GGCACCGCGGCCGCATGAAGGGAGACAGCAGACAT-3’SG 78 (R) NotI 5’-GCTACTGCGGCCGCCTATTCACAGGCAAAGTAGTC-3’m3b 670R (NotI) 5’-GCT ACT GCG GCC GCT TGC GGG CAG GAT TGA CG-3’m3b (F) EcoRI 5’-GGCACCGAATTCTATGAAGGGAGACAGCAGACAT-3’m3b (R) BamHI 5’-GCTACTGGATCCCTATTCACAGGCAAAGTAGTC-3’Dnmt3b3-CD Primers161F m3b C-term 5'-GGCACCGCGGCCGCATGCTGGAAGAATTTGAGCCACCC-3'162R m3b C-term 5'-GCTACTGCGGCCGCCTATTCACAGGCAAAGTAGTCC-3'DNMT3B PrimersSG75 (F) SalI 5’-GGCTGTCGACATGAAGGGAGACACCAGGCAT-3’SG76 (R) SalI 5’-GCCTGTCGACCTATTCACATGCAAAGTAGTCCTTCAGAGG-3’Dnmt3L PrimersDnmt3L (F)SalI 5'-GGCACCGTCGACATGGCGGCCATCCCAGCC-3'Dnmt3L (R)SalI 5'-GCTACTGTCGACTTATAAAGAGGAAGTGAGTTCTGT -3'BGS PrimersBGS Plasmid F 5'-GTAGTGTTGTTATAATTATGAGTG-3'BGS Plasmid R 5’-CTTAATCAATAAAACACCTATCTC-3’BGS Sat2F 5’-GTAATGATTATTATGATAGATTTG-3’BGS Sat2R 5’-ACATCCTAAACCTATTAATATTC-3’Pyrosequencing PrimersPyro Plasmid R 5’-biot-TTTAATATAACTTCATTCAACTCC-3’Plasmid Sequencing F 5’-AATTATGAGTGATAATATTG-3’Pyro Sat2 R 5’-biot-AATAATATCAACACCAAAC-3’Sat2 Sequencing F 5’- ATTTTATTTTATTAGATG-3’

Table S2. Homologous amino acid positions in murine Dnmt3a/Dnmt3b1 compared to human DNMT3B1 relevant to this study.

Dnmt3a Dnmt3b1 DNMT3B1Dnmt3b-3L D682 D633 D627Dnmt3b-3L F636 F587 F581Dnmt3b-3L F728 F679 F673Dnmt3b-3L F768 F719 F713Dnmt3b-3b R881 R832 R826

ICF V661 V612A V606AICF G718 G669S G663SICF L719 L670T L664TICF V781 V732G V726GICF A821 A772P A766PICF H869 H820R H814RICF R878 R829G R823GICF R895 R846Q R840Q

CpG Number (Plasmid)1 2 3 4 5

% M

ethy

latio

n

0

20

40

60

80

100

B

Van Emburgh Fig. S1

Plasmid CpG Methylation

Dnmt3b1 + 3L0 10 20 30

DN

MT3

+ 3

L

0

20

40

60

80

100

120

Dnmt3b2 + 3L Dnmt3a + 3L DNMT3B1 + 3L

SAT2 CpG Methylation

Dnmt3 Alone0 20 40 60 80

Dnm

t3 +

3L

0

20

40

60

80

100

120

Dnmt3a vs Dnmt3a + 3L Dnmt3b1 vs Dnmt3b1 + 3L

Plasmid CpG Methylation

Dnmt3 Alone0 20 40 60 80 100

Dnm

t3 +

3L

0

20

40

60

80

100

120

140

Dnmt3a vs Dnmt3a 3L Dnmt3b1 vs Dnmt3b1 3L

Dnmt3aDnmt3a + Dnmt3LDnmt3b1Dnmt3b1 + Dnmt3LDnmt3b2Dnmt3b2 + Dnmt3LDNMT3B1DNMT3B1 + Dnmt3L

A

++

Dnmt3b1

3b1 +

3L

Dnmt3b2

3b2 +

3L

DNMT3B1

3B1 +

3L

Dnmt3a

3a +

3L

Proc

essi

vity

Inde

x

0.0

0.2

0.4

0.6

0.8

1.0

1.2E

* ** **

Van Emburgh Fig. S1 cont.

% Methylation of C by CpN

Dnmt3a DNMT3B1 Dnmt3b1

% o

f Met

hyla

ted

C

0

5

10

1580

CpA CpT CpC CpG

F

Dnmt3b1 (0.9%)

3b1 + 3L (16.8%)

DNMT3B1 (7.3%)

3B1 + 3L (32%)

Dnmt3a (22.9%)

3a + 3L (62.7%)

C

Dnmt3b2 (1.1%) Dnmt3b2 (0.3%)

3b2 + 3L (9%) 3b2 + 3L (23.1%)

D

Plasmid Plasmid SAT2

Plasmid

% M

ethy

latio

n

0

5

10

15

20

25

30Dnmt3b1Dnmt3b1 + 3L

∆1-140 (3.6%)

∆1-140 + 3L (6.1%)

∆1-140 (0.6%)

∆1-140 + 3L (6.4%)∆434-531+ 3L (7.8%)

∆434-531+ 3L (12.3%)

A

B

Dnmt3b1 C657A (0.3%)

Dnmt3b1 C657A (0.6%)

Van Emburgh Fig. S2

nM Dnmt3b0 200 400 600

% S

hift

0

20

40

60

80

100

120

Dnmt3b1Δ434-859

C

D

KD= 45 nM

KD= 183 nM

SAT2

Plasmid

∆434-531

Dnmt3b1 C657A + 3L

Dnmt3b1 C657A + 3L

∆434-531

Not Done

Not Done

Not Done

Not Done

G669S + 3L (1.7%)

G669S + 3L (2.5%)

R829G (0.3%)

R829G + 3L (6.1%)

R829G (1.6%)

R829G + 3L (8.2%)

R846Q (0%)

R846Q + 3L (10.3%)

R846Q (0.9%)

R846Q + 3L (5.4%)

A

B

Van Emburgh Fig. S3

Plasmid%

Met

hyla

tion

0

2

4

10

20Dnmt3b1Dnmt3b1 + 3L

C

SAT2

Plasmid

G669S

Not Done

G669S

Not Done

CpG Number (SAT2)1 2 3 4 5 6 7

% M

ethy

latio

n

0

10

20

30

40

50

CpG Number (Plasmid)1 2 3 4 5

% M

ethy

latio

n

0

10

20

30

40

50

Dnmt3b1 +3LV612A + 3LG669S + 3LL670T + 3LV732G + 3LA772P + 3LH820R + 3LR829G + 3LR846Q + 3L

Dnmt3b1 + 3LV612A + 3LG669S + 3LL670T + 3LV732G + 3LA772P + 3LH820R + 3LR829G + 3LR846Q + 3L

A

B

Van Emburgh Fig. S4

Deletions

CpG Number (SAT2)1 2 3 4 5 6 7

Rel

ativ

e %

Met

hyla

tion

80

100

120

140

Splice Variants

CpG Number (SAT2)1 2 3 4 5 6 7

Rel

ativ

e %

Met

hyla

tion

80

100

120

140

V612AG669SL670TV732G A772PH820RR829GR846Q

A

B

ICF Mutations

CpG Number (SAT2)1 2 3 4 5 6 7

Rel

ativ

e %

Met

hyla

tion

80

100

120

140

Van Emburgh Fig. S5

C

Δ584-859Δ671-859Δ1-140Δ225-249Δ227-429Δ434-531Δ1-560C657A

Dnmt3b1Dnmt3b2Dnmt3b3DNMT3B1

Deletions

CpG Number (Plasmid)1 2 3 4 5

Rel

ativ

e %

Met

hyla

tion

80

100

120

140

160

180

ICF Mutations

CpG Number (Plasmid)1 2 3 4 5

Rel

ativ

e %

Met

hyla

tion

80

90

100

110

120

130

140

150

V612AG669SL670TV732GA772PH820RR829GR846Q

Van Emburgh Fig. S5 cont. 

Splice Variants

CpG Number (Plasmid)1 2 3 4 5

Rel

ativ

e %

Met

hyla

tion

80

100

120

140

160

180

D

E

F

Δ584-859Δ671-859Δ1-140Δ225-249Δ227-429Δ434-531Δ1-560C657A

Dnmt3b1Dnmt3b2Dnmt3b3DNMT3B1

Overlay of in vivo and in vitro SAT2 methylation

CpG Number9 10 11 12 13 14 15

% M

ethy

latio

n

50

60

70

80

90

100

HCT116 in vivoDnmt3a + 3L in vitro

Overlay of in vivo and in vitro SAT2 methylation

CpG Number9 10 11 12 13 14 15

% M

ethy

latio

n

0

20

40

60

80

100

HCT116 in vivoDnmt3b1 + 3L in vitro

Van Emburgh Fig. S6