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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