SUPPLEMENTAL TABLES
Supplemental Table 2. Targets of Dnmt3b activity as determined by MSCC
analysis. Methylation readout of 260 genes demethylated in MYC;Dnmt3b-/- tumors
relative to MYC;Dnmt3bF/F tumors. The samples are designated as follows: normal
thymocytes (Th), MYC;Dnmt3bF/F tumors (WT), MYC;Dnmt3b-/- tumors (3b), and 24 day
pre-tumor Dnmt3b-deficient thymocytes (3b24).
Supplemental Table 3. Gene expression analysis of genes with significant change
in expression as determined by microarray. Global expression patterns of all
significant changes by microarray were obtained by comparing two normal thymocytes
(N), three MYC;Dnmt3bF/F tumors, and three MYC;Dnmt3b-/- tumors. P-values are
indicated (P<0.05).
Supplemental Table 6. Primer List. List of primers used in this study for COBRA,
genotyping, real time PCR, qRT-PCR and sequencing.
SUPPLEMENTAL MATERIALS AND METHODS
Bisulfite Pyrosequencing of SINE Elements
Pure genomic DNA was treated with sodium bisulfite using the Epitect Bisulfite Kit
(Qiagen). This process deaminates unmethylated cytosine residues to uracil leaving
methylated cytosine residues unchanged. Methylated mouse DNA acquired from NEB
(Ipswich, MA) using M. SssI (CpG) methylase, was treated with sodium bisulfite as
described before served as the positive control. PCR reactions were performed in a
total volume of 25 µl for 35 cycles using Qiagen HotStarTaq DNA Polymerase (1.0U),
MgCl2 solution (3.5 mM), dNTP’s (0.2 mM), sense primer
(TTTTGGTTGTTTTGGAATTTATT, 0.24 uM), antisense primer (5-bio-
CCTTTAATCCCAACACTTAAAAAA, 0.18 µM), with denaturation at 95 ºC for 30
seconds, annealing temperature at 60 ºC for 45 seconds, and extension at 72 ºC for 1
minute. All PCR products were electrophoresed on 0.8% agarose gel, stained with
ethidium bromide, and visualized for appropriate and pure product before proceeding
with all analyses using a Bio-Rad Laboratories Gel-Doc UV illuminator. Methylation
percentage of each CpG was determined using a Qiagen Pyromark Q24 pyrosequencer
and primers (ATTCTGTAGATTAGGTTGGT) according to manufactures
recommendations. The analysed sequence was TTYGAATTTAGAAATTYGTT.
Western Blotting
The following antibodies were used for Western blot: Dnmt3b (52A1018, Imgenex),
Dnmt1 (H-300, Santa Cruz) Dnmt3a (H-295, Santa Cruz), β-Actin (R-22, Santa Cruz)
and c- MYC (9E10, Santa Cruz). For Western blotting, protein lysates were separated in
SDS acrylamide gels and blotted into Immobilon P membranes (Millipore). Blots were
incubated in blocking buffer (5% skim milk) at a concentration of 1 mg/mL. The primary
antibody was then detected using horseradish-peroxidase-conjugated secondary
antibodies and the ECL reagent as described by the manufacturer (Pierce).
Quantitative Real-Time RT-PCR (qRT PCR)
qRT-PCR was performed as described previously (1). cDNA was synthesized from 2 µg
of total RNA using Superscript III reverse transcriptase (Invitrogen) at 50°C for 60 min.
Generated cDNA samples were mixed with gene-specific primers along with SYBR
Green Supermix (Bio-Rad) in a final volume of 20 µL. qRT-PCR was performed in the
CFX96 real time detection system (Bio-Rad). Standard curves were generated using
cDNA to determine the linear range of amplification for each primer pair. Reactions were
performed in duplicate, and relative amounts of cDNAs were normalized to the
expression of Gapdh, or β-Actin.
siRNA and shRNA knockdown experiments
SMARTpool siRNA designed against mouse Gm128 (Ment), Rpl39l, Psma8, Pnldc1 and
Syce1 along with non-silencing RNA, siGLO green transfection indicator and the
DharmaFECT 4 reagent were purchased from Dharmacon. SiRNA at a final
concentration of 100 nM was transfected into 5 x104 MYC;Dnmt3b-/- cells along with
siGLO green to monitor transfection efficiency that was found to be typically ~80%.
Cells were counted 48 h later using a hemacytometer. Non-silencing RNA served as a
negative control. Each experiment was done in duplicates and repeated three times.
Knockdown efficiency was confirmed by qRT-PCR. The same approach was used to
transfect SMARTpool siRNA against CIorf56 (MENT) into JURKAT cells. HuSH 29-mer
shRNA constructs against mouse Gm128 (Ment) were purchased from Origene.
Production of retroviruses was achieved in Phoenix-Eco packaging cell line. The virus
was then used to infect MYC;Dnmt3b-/- cells as described above. Cells were placed on
puromycine selection 36 h after infection for two days. Freshly selected cells were
plated at concentration of 0.2 x 106/mL and counted using hemacytometer. Each
counting was done in triplicates every 12 h to generate growth curves.
Combined Bisulfite Restriction Analysis (COBRA) and Bisulfite Sequencing
Bisulfite conversion of genomic DNA was carried about by sodium bisulfite treatment
using the Epitect Bisulfite Kit (Qiagen). Bisulfite primers were designed using
MethPrimer (2). A fully methylated CpG control was purchased from NEB. PCR
products were digested with restriction enzymes BstUI, TaqI (both from NEB), and Tail
(Fermentas). Digested products were then loaded on an 8% PAGE gel, separated by
electrophoresis and stained by SYBR Green Gold (Invitrogen). PCR fragments from
bisulfite treated DNA were cloned using pGem Easy T-cloning kit (Promega). Plasmid
DNA isolated from individual clones was sequenced at the UNMC Sequencing Core
Facility.
Array Comparative Genomic Hybridization (aCGH)
aCGH was performed at the Comparative Molecular Cytogenetic Core at NIH, NCI at
Frederick using 4x44K arrays from NimbleGen on DNA samples isolated from three
MYC;Dnmt3bF/F and three MYC;Dnmt3b-/- lymphomas. DNA isolated from the kidney of
EμSR-tTA;Teto-Cre;Rosa26LOXPEGFP mouse served as a control.
SUPPLEMENTAL REFERENCES
1. Opavsky R, Tsai SY, Guimond M, Arora A, Opavska J, Becknell B, Kaufmann M, Walton NA, Stephens JA, Fernandez SA, et al. Specific tumor suppressor function for E2F2 in Myc-induced T cell lymphomagenesis. Proc Natl Acad Sc. 2007;104(39):15400-15405.
2. Li LC, Dahiya R. MethPrimer: designing primers for methylation PCRs. Bioinformatics. 2002;18(11):1427-1431.
3. Rai K, Sarkar S, Broadbent TJ, Voas M, Grossmann KF, Nadauld LD, Dehghanizadeh S, Hagos FT, Li Y, Toth RK, et al. DNA demethylase activity maintains intestinal cells in an undifferentiated state following loss of APC. Cell. 2010;142(6):930-942.
Hlady_Supplemental_Table_1
Supplemental Table 1. Table of cytogenetic aberrations identified using array Comparative Genomic Hybridization. Aberrations for three MYC;Dnmt3bF/F (1‐3) and three MYC;Dnmt3b‐/‐ (4‐6) tumors. Positions of amplifications and deletions are given in both chromosomal location and cytoband. The number of probes corresponding to each amplification or deletion is shown along with a P‐value.
(#1)Event Chr Cytoband #Probes Amp/Del P-value Annotations
1 chr6:40826823-40978497 qB1 7 -0.69984 1.72E-11 599, 1700074P13Rik, 1810009J02 chr6:41184457-41451795 qB1 6 -2.25136 7.94E-49 Try4, 1810049H19Rik, Try10... 3 chr13:19196860-19197260 qA2 1 -3.61305 4.27E-144 chr14:52754373-53101965 qC2 5 -2.59613 4.75E-365 chr17:3075528-93509199 qA1 -qE5 1752 0.334409 0.00E+00 Tiam2, Tiam2, Tfb1m...
(#2)Event No Chr Cytoband #Probes Amp/Del P-value Annotations
1 chr6:41184457-41451795 qB1 6 -0.91349 2.65E-14 Try4, 1810049H19Rik, Try10... 2 chr8:108832681-108833081 qD3 1 -2.26894 4.01E-12 Slc12a4 3 chr11:114487382-121517858 qE2 329 0.414111 5.71E-218 Rpl38, Rpl38, Ttyh2... 4 chr13:19196860-19197260 qA2 1 -3.53667 2.11E-105 chr14:52754373-53101965 qC2 5 -2.88431 5.05E-286 chr17:39671705-81008799 qB1 -qE3 691 0.42206 0.00E+00 Pgk2, Crisp3, Crisp1... 7 chr17:81137009-91151345 qE3 -qE5 133 -0.64555 1.55E-141 Slc8a1, AW548124, Eml4...
(#3)Event Chr Cytoband #Probes Amp/Del P-value Annotations
1 chr14:53002908-53101965 qC2 3 -3.04096 1.37E-15
(#4)Event Chr Cytoband #Probes Amp/Del P-value Annotations
1 chr3:142814058-142814458 qH1 1 2.446012 4.10E-11 Pkn2 2 chr5:5754545-5773942 qA1 2 2.144442 2.88E-18 Steap1 3 chr5:22639484-22639884 qA3 1 4.28425 1.06E-15 Lhfpl3 4 chr6:41184457-41451795 qB1 6 -2.13734 1.96E-48 Try4, 1810049H19Rik, Try10... 5 chr6:53994195-54192108 qB3 2 2.927514 1.23E-186 chr6:60656216-60918121 qB3 8 -0.99476 1.87E-22 Snca, Snca 7 chr10:14993972-14994372 qA2 1 2.180098 2.72E-128 chr10:89618998-89619398 qC2 1 2.072192 2.43E-109 chr11:116876097-116876497 qE2 1 3.015854 3.47E-1010 chr11:120426769-120427169 qE2 1 -2.46261 2.79E-16 Pcyt2 11 chr14:53065963-53101965 qC2 2 -3.21457 1.36E-1812 chr15:3228882-103359097 qA1 -qF3 1663 0.452136 0.00E+00 Sepp1, Sepp1, Sepp1...
(#5)Event Chr Cytoband #Probes Amp/Del P-value Annotations
1 chr3:127753730-127754130 qG2 1 -4.14299 1.96E-132 chr3:142814058-142814458 qH1 1 2.788929 1.06E-15 Pkn2 3 chr5:22639484-22639884 qA3 1 3.319846 2.20E-10 Lhfpl3 4 chr6:41184457-41451795 qB1 6 -3.36505 4.85E-71 Try4, 1810049H19Rik, Try10... 5 chr6:53319496-53319896 qB3 1 -2.23691 7.14E-116 chr6:54191708-54192108 qB3 1 5.100765 1.30E-197 chr10:89618998-89619398 qC2 1 1.832661 2.72E-108 chr14:52754373-53101965 qC2 5 -3.9517 3.08E-499 chr15:54493923-54494323 qD1 1 2.839228 1.60E-1110 chr19:10273547-10273947 qA 1 2.189765 3.42E-1211 chr19:13937681-13938081 qA 1 2.913638 1.12E-1412 chrX:68840554-76033941 qA7.3 -qB 132 0.789382 3.46E-276 Gabra3, Gabrq, Magea9...
(#6)Event Chr Cytoband #Probes Amp/Del P-value Annotations
1 chr3:45382016-45626918 qB 5 0.68297 1.00E-11 Pcdh10, Pcdh10, Pcdh10... 2 chr4:136972899-136973299 qD3 1 2.023405 7.43E-12 Rap1gap 3 chr5:5754545-5773942 qA1 2 2.382502 8.41E-21 Steap1 4 chr5:22639484-22639884 qA3 1 4.330955 4.57E-17 Lhfpl3 5 chr6:41184457-41451795 qB1 6 -3.16525 2.49E-68 Try4, 1810049H19Rik, Try10... 6 chr6:53319496-53319896 qB3 1 -2.41258 3.25E-117 chr6:54191708-54192108 qB3 1 4.654747 6.80E-198 chr6:121072205-121072605 qF1 1 -3.85925 2.12E-109 chr10:14993972-14994372 qA2 1 3.046566 2.40E-1510 chr11:115235363-115235763 qE2 1 2.208779 7.62E-12 Atp5h 11 chr11:116876097-116876497 qE2 1 3.652867 1.57E-1212 chr13:19196860-19197260 qA2 1 -4.69105 2.34E-1613 chr14:52754373-53101965 qC2 5 -3.89983 5.87E-5014 chr19:5706829-5707229 qA 1 3.488686 7.69E-17 Kcnk7 15 chr19:8588764-8589164 qA 1 3.413205 7.78E-1116 chr19:10273547-10273947 qA 1 2.422973 5.19E-1317 chr19:11530152-11530552 qA 1 4.064957 2.97E-14 Ms4a4b 18 chr19:13477369-13477769 qA 1 2.285003 3.43E-11 Olfr1469 19 chr19:13937681-13938081 qA 1 3.318061 4.70E-16
Hlady_Supplemental_Table_4
-12 +12
Supplemental Table 4. Significantly deregulated genes in MYC;Dnmt3b‐/‐ tumors as determined by microarray: A heatmap displaying 40 genes that are 3‐fold or more up‐regulated in MYC;Dnmt3b‐/‐ tumors relative to MYC;Dnmt3bF/F tumors as well as 25 genes showing a 3‐fold or greater decrease in expression (P<0.01). A color bar is shown to reference fold changes with red being up‐regulated, and green being down‐regulated. An average of two normal thymocytes (N) serve as a control for pairwise comparison of three MYC;Dnmt3bF/F and three MYC;Dnmt3b‐/‐ tumors.
Gene ThRpl39l 1 ‐1.15562 ‐1.0253 ‐1.06905 7.446746 39.98144 32.09777 <0.001Pnldc1 1 1.047398 1.065331 1.297218 28.04891 32.85522 32.55415 <0.001Syce1 1 ‐1.07755 1.01264 1.017302 36.96391 23.22533 12.18671 <0.001Gm4638 1 1.175584 1.703288 1.204562 5.721351 41.34575 48.20938 <0.001S100a6 1 ‐1.09384 ‐1.23316 1.08505 7.742137 18.95699 7.942287 <0.001Tns4 1 1.522487 1.648387 1.838878 37.4808 10.6516 6.672788 <0.001Epha3 1 1.014144 1.01945 1.002131 14.78668 10.72752 4.944101 <0.001Atp1a3 1 ‐1.0253 1.571848 ‐1.10419 20.37385 6.044364 3.395995 <0.001Rbpms 1 1.411509 1.795107 1.046587 16.20619 12.80345 5.128716 <0.001Apobec2 1 ‐1.08426 1.127483 1.014133 14.40467 3.144442 4.92049 <0.001Ass1 1 ‐1.46953 1.375223 ‐2.60408 5.085414 6.943577 4.219163 <0.001Qser1 1 ‐1.19144 ‐1.67619 ‐2.34626 4.758856 2.523249 4.433712 <0.001Myadm 1 1.51187 ‐1.3466 1.439309 14.92979 5.234661 3.05054 <0.001Mt2 1 4.987487 1.920478 4.876631 56.5647 8.190547 4.812219 0.003Brdt 1 ‐1.29355 ‐1.08964 ‐1.07868 3.330702 4.349762 7.080151 <0.001
700029I01R 1 1.471148 1.223514 1.739871 10.64083 6.799094 5.20817 <0.001Pkn3 1 ‐1.17639 ‐1.24369 ‐1.22728 2.7475 3.467984 5.516675 <0.001
Gm13051 1 2.050126 1.281614 2.965273 15.40008 6.903724 7.514142 <0.001Gstm1 1 2.502135 ‐1.42614 1.449584 9.139796 9.024985 3.612881 <0.001Enpp1 1 1.702507 7.879489 2.310974 26.05383 14.14353 14.1068 <0.001
Gm13251 1 2.358147 ‐1.77805 2.475884 11.44664 5.960746 6.03484 <0.001Tex14 1 1.105546 1.042973 2.112595 1.525261 8.490341 6.571019 0.002Spry2 1 ‐1.40773 ‐1.44042 ‐1.20463 3.646293 3.353952 1.537365 <0.001Psma8 1 1.065959 1.077746 1.069802 1.153524 3.507901 7.516588 0.0020Chchd10 1 1.485018 1.540731 1.33092 11.22342 1.649978 2.840991 <0.01Myo1e 1 2.236682 3.175779 1.130977 7.28581 9.136728 7.169814 <0.001Ms4a4b 1 2.026883 ‐1.59139 2.866461 11.35861 5.778969 2.612381 0.001BC021614 1 ‐1.35158 3.557845 1.646253 14.73779 3.630382 2.872842 <0.01Cd44 1 1.279539 1.027857 2.430472 11.29092 2.139615 2.87386 <0.01
Snord82 1 ‐1.06156 1.208677 ‐1.21639 6.80872 1.834695 1.567541 <0.01LOC629446 1 1.466454 ‐2.36424 ‐1.1192 2.746599 4.608141 1.885147 <0.001
Bag3 1 1.788389 1.686495 1.866465 11.18465 3.653912 2.850029 <0.01Adi1 1 1.646629 1.58198 1.304855 10.26548 2.096143 2.607854 <0.01Klf9 1 1.596809 2.678958 1.615968 9.534676 5.571789 4.272453 <0.001
Slc22a15 1 ‐1.29721 1.123893 ‐1.139 3.324351 3.076536 2.498632 <0.001Hdc 1 8.961861 14.30646 ‐1.0547 23.50522 28.44498 25.11063 <0.001
Gm128 1 ‐1.24906 ‐1.12765 ‐1.12222 2.656525 3.475067 1.924004 <0.001Slc39a4 1 1.143677 ‐1.15965 1.337107 5.555395 2.801118 1.971126 <0.001Prr5 1 2.542029 1.147679 2.505571 13.2486 2.94192 2.868272 0.01Cebpa 1 1.845383 2.043873 1.066898 4.885597 4.91725 5.10445 <0.001Gng3 1 5.016985 1.583372 6.69806 1.23458 1.31599 1.662918 <0.001Pja2 1 4.99261 4.828205 13.55035 2.005918 1.878191 3.2741 <0.001
Serpine2 1 3.340198 6.980948 3.646376 ‐1.02829 1.583021 1.67583 <0.001Crtam 1 9.789362 11.20017 15.78151 1.176679 5.398249 4.424684 <0.001Lmo2 1 13.70378 3.339565 12.79892 1.749874 4.14701 2.813448 <0.001Eomes 1 4.791118 2.012904 6.682684 ‐1.14773 1.125798 ‐1.09074 <0.001
430407P10R 1 18.93511 3.06024 23.94598 ‐1.08239 5.29791 3.638173 <0.01Spock2 1 14.52922 3.360568 23.90337 1.973858 5.096115 1.679154 <0.001Rhob 1 14.94211 1.954319 19.02698 1.284056 4.722185 1.488758 <0.01Fbn2 1 2.981732 7.154415 33.37297 1.021369 5.393258 2.020782 <0.01Ptpn14 1 3.344837 2.098433 5.849958 ‐1.99939 ‐1.54206 1.008725 <0.001Rnf144b 1 17.8035 2.967845 14.1918 4.380415 ‐1.0305 ‐1.02363 <0.001Chst2 1 9.930992 4.888061 19.17715 1.186588 3.285927 1.622769 <0.001Gpr87 1 13.68737 2.017384 1.768283 ‐1.13284 ‐1.04068 1.054432 <0.001Grb10 1 163.6838 35.88767 213.5595 ‐1.23506 27.50859 40.20214 <0.001
Sh3bgrl2 1 16.79583 1.535852 18.72166 ‐1.01153 2.188925 2.471064 <0.01Upb1 1 13.14367 2.579203 5.151394 ‐1.2251 ‐1.06658 ‐1.06123 <0.001Etv5 1 19.97266 4.513352 21.84494 1.029946 1.783429 2.794386 <0.001Palmd 1 24.75831 1.744318 27.33398 1.224537 2.960916 1.765643 <0.001Cacna1b 1 6.22978 2.639605 35.70118 1.695525 1.417433 1.73528 <0.001Efhd1 1 18.41263 1.522964 40.70466 1.102636 4.19791 1.17829 <0.01Tnni1 1 21.5886 1.955816 42.3564 1.058616 3.57441 1.361819 <0.001Slc7a8 1 10.50429 1.597538 9.09238 ‐1.76299 ‐1.48413 ‐1.70048 <0.001Hspa1a 1 22.15759 19.72648 5.274207 1.018381 1.194695 1.227554 <0.001Wif1 1 101.9889 15.90264 112.6128 ‐1.02577 2.301956 3.115846 <0.001
MYC;Dnmt3bF/F MYC;Dnmt3b‐/‐
Hlady_Supplemental_Table_5
Supplemental Table 5. Overlapping targets of Dnmt3b and “active” demethylase
activity. Out of 5315 genes found to be targets of active DNA demethylases (3), 18
genes were found to be targets of Dnmt3b methylation activity in this study.
Epo Arhgef18 Fpgs Mobkl2c Otof Tekt2
Nat11 Cpt1b Guca1b Mtmr9 Piwil1 Trpv6
Acvrl1 Ddost Mcm7 Numbl Psma8 Utp11l
Thymus Spleen Lymph Node % % %
EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8
Mutant 9+/-8 22+/-11 3+/-2 66+/-16 Mutant 78+/-5 17+/-5 5+/-1 0+/-0 Mutant 27+/-4 53+/-6 19+/-2 2+/-0
Control 7+/-2 18+/-4 3+/-2 72+/-0 Control 71+/-6 21+/-5 8+/-2 0+/-0 Control 25+/-8 54+/-8 18+/-1 3+/-1
% % %
EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8
Mutant 14+/-9 23+/-11 3+/-2 59+/-16 Mutant 75+/-6 19+/-5 6+/-1 0+/-0 Mutant 27+/-2 53+/-3 19+/-1 1+/-0
Control 17+/-13 18+/-5 2+/-1 62+/-18 Control 67+/-1 24+/-2 8+/-1 0+/-0 Control 27+/-5 53+/-4 19+/-3 2+/-1
% % %
EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8 EGFP+ DN CD4 CD8 CD4/CD8
Mutant 13+/-5 7+/-5 27+/-7 53+/-8 Mutant 70+/-7 13+/-4 15+/-8 2+/-1 Mutant 31+/-9 46+/-3 18+/-6 5+/-4
Control 19+/-4 8+/-8 23+/-8 50+/-6 Control 71+/-8 16+/-3 11+/-8 1+/-1 Control 47+/-2 39+/-2 12+/-3 2+/-1
% % %
EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8 EGFP- DN CD4 CD8 CD4/CD8
Mutant 14+/-6 6+/-5 25+/-8 54+/-3 Mutant 58+/-14 19+/-9 21+/-12 1+/-1 Mutant 24+/-5 54+/-5 17+/-5 5+/-5
Control 21+/-5 13+/-4 24+/-8 41+/-4 Control 60+/-8 22+/-3 17+/-7 0+/-0 Control 33+/-7 46+/-2 21+/-6 1+/-1
STOP
EμSRα tTa
Teto Cre
ROSA EGFP
Dnmt3b
A B C
KO
F
0
120
90
60
30
Dnmt3b-/- F/+ F/F
% o
f C
onditio
nal A
llele Dnmt3b-/-
EµSR-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F
EGFP+, Dnmt3b-deficient EGFP-
tTA+ tTA-
EGFP+ EGFP-
EµSR-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3b+/+
(Control) (Mutant)
CD
4
CD8
tTA+ tTA-
Hlady_Supplementary_Fig_1
Thymus Spleen Lymph Node
Time (months)
% S
urv
ival
0 2 4 6 8 10 12
100
75
50
25
0
Dnmt3b-/- (n=12)
Dnmt3b+/+ (n=12)
D
E
F
G
Genotype Number %EGFP+
Thymus Cells
(x10^6)
Thymus
Weight
Spleen Cells
(x10^6) Spleen Weight
Lymph Node
Cells (x10^6)
Lymph Node
Weight
Mutant 9 49+/-10 301+/-97 0.1+/-0.02 299+/-44 0.1+/-0.01 45+/-12 0.03+/-0.01
Control 6 42+/-10 332+/-162 0.1+/-0.02 348+/-54 0.15+/-0.05 45+/-9 0.04+/-0.01
Mutant 5 55+/-11 318+/-351 0.05+/-0.02 167+/-156 0.15+/-0.01 105+/-88 0.02+/-0.02
Control 4 51+/-9 476+/-288 0.04+/-0.01 226+/-126 0.12+/-0.02 272+/-218 0.03+/-0.02
Early
Late
Ea
rly
La
te
H
Hlady_Supplementary_Fig_1
Supplemental Fig. 1. Loss of Dnmt3b has no affect on T cell
development. (A) Graphical presentation of EμSRα-tTA, Teto-Cre, and
Rosa26LOXPEGFP transgenes and the Dnmt3b conditional knock out allele
used in developmental studies. The blue color indicates that tTA regulates
transcription from Teto promoters and recombination of lox P sites (triangles).
(B) Deletion efficiency of the conditional allele of Dnmt3b as determined by
real time quantitative RT-PCR (qRT-PCR) analysis on genomic DNA isolated
from EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F (Dnmt3b-/-), EμSRα-
tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/+ (F/+) and EμSRα-
tTA;Rosa26LOXPEGFP;Dnmt3bF/F (F/F) mice (n=2 for each). Error bars
represent standard error of the mean (+/- SEM). (C) PCR based analysis of
deletion efficiency of the conditional allele of Dnmt3b (F) in DNA from FACS-
sorted EGFP+ from EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F
thymocytes. (D) Kaplan-Meier survival curves of EμSRα-tTA;Teto-
Cre;Rosa26LOXPEGFP;Dnmt3bF/F (Dnmt3b-/-, red line) and EμSRα-tTA;Teto-
Cre;Rosa26LOXPEGFP;Dnmt3b+/+ (Dnmt3b+/+, blue line) mice. Number of mice
(n) is indicated. (E) Percentage of EGFP+ cells in thymus as well as organ
weights and cellularity for thymus, spleen and lymph nodes isolated from
EμSRα-tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3bF/F (Dnmt3b-/-) and EμSRα-
tTA;Teto-Cre;Rosa26LOXPEGFP;Dnmt3b+/+ (Dnmt3bF/F) mice at age 31 days
(early) and one year (late). (F) Schematic representation of inter- and intra-
mouse comparisons. EGFP-positive (EGFP+) and EGFP negative (EGFP-)
populations within an individual mouse are shown by purple (control) and red
(mutant) arrows. In mutant mice, EGFP+ cells will be Dnmt3b-deficient, while
EGFP- cells retain Dnmt3b. Comparisons between control and mutant mice
are denoted by green (EGFP+ to EGFP+) and blue (EGFP- to EGFP-) arrows.
(G) FACS profiles of CD4 and CD8 expression in thymus, spleen, and lymph
node of control and mutant mice. Green color indicates EGFP positive cells,
blue color indicates EGFP negative cells. (H) CD4/CD8 profiles for thymi,
spleens, and lymph nodes for early and late EμSRα-tTA;Teto-
Cre;Rosa26LOXPEGFP;Dnmt3bF/F (mutant) and EμSRα-tTA;Teto-
Cre;Rosa26LOXPEGFP;Dnmt3b+/+ (control) developmental mice as determined
by FACS. Average percentages of double negative CD4-CD8- (DN), single
positive CD4+ (CD4), single positive CD8+ (CD8) and CD4+CD8+ double
positive (CD4/CD8) are shown with standard deviations.
CD4hiCD8+CD4+
2
0
8
6
4
Num
ber
of
Lym
phom
as
Dnmt3bF/F
Dnmt3b-/-
Number of mice CD4hiCD8+ CD4+ CD44hiCD25- CD44loCD25- B220-CD11b-
MYC;Dnmt3b-/- 10 8 2 5 5 10
MYC;Dnmt3bF/F 10 8 2 6 4 10
A few tumors showed a small percentage of B220- and/or CD11b- positive cells
A
B
Hlady_Supplementary_Fig_2
MYC
Supplemental Fig. 2. Loss of Dnmt3b does not alter the
immunophenotypes of MYC-induced T cell lymphomas. (A)
MYC;Dnmt3bF/F (n=10) and MYC;Dnmt3b-/- (n=10) tumors were stained with
anti-CD4 and anti-CD8 antibodies and categorized as either CD4-single
positive, or CD4hiCD8+ based on FACS. (B) Summary of immunophenotypes
as determined by FACS analysis using CD4, CD8, CD44, CD25, B220, and
CD11b expression in MYC;Dnmt3bF/F and MYC;Dnmt3b-/- tumors.
A
10
/0
9/1
8/2
7/3
6/4
5/5
4/6
3/7
2/8
1/9
0/1
0
Ratio of KO/Conditional Dnmt3b DNA
MYC;Dnmt3b-/-
KO (-)
Conditional (F)
Hlady_Supplementary_Fig_3
% C
on
ditio
na
l Alle
le
B
F/F F/+ -/-0
20
40
60
80
100
120
Supplemental Fig. 3. Deletion efficiency of Dnmt3b conditional allele in
lymphomas. (A) PCR based analysis of deletion efficiency of the conditional
allele of Dnmt3b in lymphomas (n=5) derived from MYC;Dnmt3b-/- mice
(Dnmt3b-/-). Genomic DNA with differing ratios of Dnmt3b- (ko; -) and Dnmt3bF
(conditional; F) were used as reference samples. (B) Relative levels of the
conditional allele of Dnmt3b as determined by Real time PCR analysis of
genomic DNA in three MYC;Dnmt3bF/F (F/F) , two MYC;Dnmt3bF/+ (F/+) and
three MYC;Dnmt3b-/- (-/-) mice. Averages +/- SEM is shown.
P=0.32
Dnmt3b-/-0
1
2
3
Rela
tive E
xpre
ssio
n
Dnmt3bF/F
Hlady_Supplementary_Fig_4
MYC
Supplemental Fig. 4. Loss of Dnmt3b does not affect expression of MYC
transgene. Relative expression of the MYC transgene as determined by qRT-
PCR analysis in five MYC;Dnmt3bF/F and MYC;Dnmt3b-/- lymphomas with the
averages +/- SEM shown. No statistically significant difference is seen
(P=0.32).
20
0
40
60
80
CD4+ CD4+CD8+ DN CD8+
% o
f C
ells
Dnmt3b-/-
Dnmt3bF/F
Hlady_Supplementary_Fig_5
Supplemental Fig. 5. Expression of transgenic MYC does not alter T cell
development. Expression of cell surface markers CD4 and CD8 in 24 day old
MYC;Dnmt3bF/F and MYC;Dnmt3b-/- thymocytes during early tumorigenesis.
Average percentages of double negative CD4-CD8- (DN), single positive CD4+,
single positive CD8+ and CD4+CD8+ double positive are shown with standard
deviations. Error bars represent +/- SEM and no statistically significant
difference is seen.
Spleen
Lymph Node
Time (days)
60
40
20
035 50 Final
% P
rolife
rating
C
ells
Time (days)
60
40
20
035 50 Final
% P
rolife
rating
C
ells
Time (days)
0
5
20
25
15
10
% A
po
pto
tic C
ells
35 50 Final
Time (days)
0
4
8
12
16
% A
po
pto
tic C
ells
35 50 Final
A
B
Dnmt3b-/-
Dnmt3b+/+
Dnmt3b-/-
Dnmt3b+/+
Hlady_Supplementary_Fig_6
MYC
MYC
*
*
n=
11
n=
9
n=
5
n=
6
n=
6
n=
7
n=
11
n=
9
n=
5
n=
6
n=
6
n=
7
n=
11
n=
9
n=
5
n=
6
n=
6
n=
7
n=
11
n=
9
n=
5
n=
6
n=
6
n=
7
Supplemental Fig. 6. Loss of Dnmt3b results in enhanced proliferation of
tumors in vivo. Apoptosis and BrdU incorporation assay as determined by
FACS analysis of cells isolated form spleens (A) and lymph nodes (B) of
MYC;Dnmt3b+/+ (blue bars) and MYC;Dnmt3b-/- (red bars) mice at age 35 and
50 days as well as terminally ill mice (Final). Anti-Annexin V and anti-BrdU
antibodies were used to evaluate apoptosis and BrdU incorporation,
respectively. The number of mice used for each cohort is indicated by n. Error
bars represent the SEM and significant changes are denoted by (*) P<0.05.
Assay Sine B1
Sample ID: 3b Tumor
Sequence to Analyze: TTYGAATTTAGAAATTYGTT
Assay Sine B1
Sample ID: WT Tumor
Sequence to Analyze: TTYGAATTTAGAAATTYGTT
Hlady_Supplementary_Fig_7
49% 45%
54% 49%
Supplemental Fig. 7. Methylation analysis of SINE-B1 repetitive elements
by pyrosequencing. A representative pyrogram from one MYC;Dnmt3b-/-
tumor (3b Tumor) and one MYC;Dnmt3bF/F tumor (WT Tumor) from SINE-B1
repeat elements is shown. Percentages represent the amount of
methylcytosine at the highlighted locus.
0
4
567
3
2
1
Arfip 1
0
1
2
3
4
0
2
4
6
8 Fbln2
0
2
4
6
8Mb Mtap7d1
0
1
2
3
4
5
0
3
6
9
12Stx 1
Cutl 1
0
1
2
3
4
Rbbp9
0
20
40
60
80
Hlady_Supplementary_Fig_8
Fold
induction
0
5
10
15
20
25 Ahi1
A
Angpt4
0
4
8
12
16Rxfp3
Fold
induction
0
0.4
0.8
1.2
1.6
2.0 Phlpp Sla2
0
2
3
1
4
Fold
induction
Commd7
0
1.2
1.8
0.6
Fold
induction
CCGG
CCGG
HpaII digest
Methylated
No digest by HpaII
PCR successful
Not methylated
Digested by HpaII
PCR fails
C CGG
B
C
Gene Sequence Chr Position Strand N1 N2 N3 wt1 wt2 wt3 wt4 wt5 3b1 3b2 3b3 3b4 3b5
Ahi1 AGCCTGCAGAGAGGGAGG 10 20785621 R 20 50 34 2 1 6 32 4 21 6 7 7 14
Angpt4 GTTGGCTTCTCGGTTTGC 2 151765097 R 17 8 13 17 9 11 20 10 1 1 7 7 8
Arfip1 CAGGATCTCTGCAGGTCA 3 85209887 R 12 7 11 3 2 2 13 13 13 14 25 21 23
Commd7 TGGAAAGGGGTGTGGTCA 2 153458100 R 35 7 22 16 16 4 4 1 2 1 2 1 1
Cutl1 ATGCGAGCTGCTCAGGCC 5 136879583 R 3 1 2 1 1 1 3 1 1 1 2 1 1
Fbln2 CCCAGCTGCCCCCCATCC 6 91163042 R 20 31 32 20 17 16 5 1 1 1 1 1 1
Mb GGTGTGGGGCAGCCTGAG 15 76865085 R 2 4 3 8 13 17 3 38 36 26 38 50 3
Mtap7d1 TGTAGAGGGAGAGGATGG 4 125912322 R 17 1 7 4 1 1 3 1 6 10 3 2 3
Phlpp GCTCCATGGAGAGACTAC 1 108069889 R 12 8 10 8 14 2 7 4 1 1 7 6 7
Rbbp9 GAGTTGATGACCCAGAAG 2 144382198 R 42 53 44 18 38 32 27 3 1 1 1 1 1
Rxfp3 CCGCTGTAGACCACCACA 15 10965680 R 21 36 25 18 23 1 34 10 1 4 10 14 8
Sla2 GGCTTTTCTAGCTCACAG 2 156703666 R 72 232 157 57 67 185 46 138 1 1 1 1 1
Stx1 CGCCGAGTGCGCGCCGTG 10 12661433 R 12 1 7 2 2 1 3 4 11 1 2 1 11
Hlady_Supplementary_Fig_8
Supplemental Fig. 8. Validation of MSCC data by HpaII–based real time
PCR analysis. (A) A schematic for Real-Time PCR confirmation of MSCC
data. HpaII sites that corresponded with significant MSCC data had primers
designed on either side. Genomic DNA was digested with HpaII and qRT-PCR
was performed. Higher levels of PCR product correlated with more highly
methylated samples, as only methylated HpaII sites will yield successful PCR
products. (B) MSCC counts for HpaII sites tested for Real time confirmation.
HpaII sites that did not correlate with MSCC data are marked in red. (C) Real
time PCR analysis of 13 HpaII sites in normal thymocytes (N), MYC;Dnmt3bF/F
(Dnmt3bF/F), MYC;Dnmt3b-/- (Dnmt3b-/-) and fully methylated CpG control
(CpG). An MspI digest, which digests CCGG regardless of its methylation
status, is shown as a negative control. Genes that do not confirm MSCC data
are shown in red. The average values for two replicates are shown with +/-
SEM.
Speg
Cld
n4*
TE
AD
2
N Dnmt3bF/F
CpG
Dnmt3b-/-
BC
035954
Hoxa3
Rspry
1C
lic4
Tra
f3Is
ynA
1Ig
f2bp3
A
B
U
D
U
D
U
D
U
D
U
D
U
D
U
D
U
D
U
D
U
D
Hlady_Supplementary_Fig_9
MYC
Slc
39a4
Epha3
Pkn3
Ass1
Brd
t*
U
D
U
U
U
U
D
D
D
D
Arh
gef1
8
Vasn*
Pfn
3S
ycp1
†
U
D
U
D
U
D
Pnld
c1
U
D
U
D
U
D
S100a6*
N Dnmt3bF/F
CpG
Dnmt3b-/-
MYC
N Dnmt3bF/F
CpG
Dnmt3b-/-
MYC
N Dnmt3bF/F
CpG
Dnmt3b-/-
MYC
Gene Sequence Chr Position Strand Th1 Th2 Th3 wt1 wt2 wt3 wt4 wt5 3b1 3b2 3b3 3b4 3b5
Arhgef18 TCCAAAGAGTCCGCGACC chr8 3393139 F 3 1 2 4 1 2 3 43 27 33 54 54 41
Ass1 TGGCCATTCTGCCCAAAC chr2 31320382 R 4 3 3 11 2 1 8 4 9 8 26 16 18
BC035954 CGTCCTCGGCCTCCTCCT chr4 148326478 F 3 2 2 2 1 1 3 2 11 11 6 3 1
Brdt GCTGCTGACAGCCGCCAT chr5 107760221 R 1 1 1 1 1 1 1 1 1 13 23 39 1
Cldn4 CCCATTTCCCTCTTCTGC chr5 135422153 R 1 3 1 1 1 1 1 5 7 11 5 7 13
Clic4 CCCAGCGGTTTGAAGCGG chr4 134828698 F 14 53 44 21 29 35 41 15 32 34 43 25 42
Epha3 GCTGGGATAAGGCTTACA chr16 63729522 R 6 5 4 9 1 1 18 7 4 6 26 2 1
Hoxa3 CCGAGTGCCAAGACCAGG chr6 52127507 R 1 1 1 1 1 1 2 32 25 28 34 31 52
Igf2bp3 CGTCTCCGCAGCTGCCAT chr6 49164244 F 12 32 23 17 16 13 16 11 19 21 32 17 9
IsynA1 GGGTCTGGATGAGAACGG chr8 73119104 R 2 38 14 19 16 42 4 9 24 17 55 3 9
Pfn3 CGTCTGCAGAAAGGTGTG chr13 55516361 R 7 2 1 1 3 2 2 18 17 19 35 11 5
Pkn3 CCTAGGTCTGAGAAGCAA chr2 29934592 F 4 3 5 1 1 1 13 9 5 26 59 22 17
Pnldc1 CGCTAGTCCCAGGGCGCG chr17 13102800 R 1 1 1 1 1 1 1 1 1 29 39 27 1
Rspry1 GACTCCCGCGGCTCCGCT chr8 97125226 R 21 11 16 9 11 8 6 3 16 9 8 1 1
S100a6 TCCCTCACCTAAGTTGCC chr3 90417563 R 9 14 11 1 2 5 3 7 9 25 48 5 2
Slc39a4 CCCAGCTCCAGTCTGGCC chr15 76447267 F 13 9 12 23 18 12 1 11 19 27 53 15 1
Speg GGGGGTTCCCGCTTGCGA chr1 75385447 F 8 13 11 1 1 3 3 1 16 11 9 1 1
Sycp1 GGGCCCGTGGTCGCGTGG chr3 102739739 F 1 1 1 1 1 1 1 1 11 4 13 1 1
Tead2 GGGGAGCGCGCGGGGTGG chr7 52471439 R 11 14 9 6 2 4 13 5 13 9 18 5 9
Traf3 CGTCAGCATCTCCGAGGC chr12 112405082 R 37 71 52 21 29 55 47 31 45 42 67 42 38
Vasn TCAGTCCTTCACCTCTGG chr16 4640070 F 4 5 3 3 1 4 8 14 26 24 21 8 17
Hlady_Supplementary_Fig_9
Supplemental Fig. 9. Validation of MSCC counts by Combined bisulfite
restriction analysis. (A) COBRA analysis of normal thymocytes (N),
MYC;Dnmt3bF/F and MYC;Dnmt3b-/- tumors in loci surrounding 21 HpaII sites.
PCR fragments were digested with a restriction enzyme (BstUI, TaqI*, or TaiI†)
and loaded onto a PAGE gel. CpG represents PCR fragment derived from
100% methylated human genomic DNA control. Undigested (U) and digested
(D) fragments correspond to unmethylated and methylated DNA, respectively.
Non-confirming COBRAs are colored in red. (B) MSCC data correlating to
counts assessed by COBRA in (A). Higher counts inversely correlate with the
methylation status of the HpaII site.
Hlady_Supplementary_Fig_10
Th -/-F/F
MYC;Dnmt3b
Supplemental Fig. 10. Supervised hierarchical clustering of MSCC
samples based on count data. Supervised clustering was performed using
the most highly changed counts from MYC;Dnmt3bF/F and MYC;Dnmt3b-/-.
Spearman rank correlation was chosen for the similarity metric and average
linkage clustering of three normal thymus, and five MYC;Dnmt3bF/F and
MYC;Dnmt3b-/- tumors was conducted. Michael Eisen’s Gene Cluster program
was used for the clustering and Treeview to view the clusters.
Hlady_Supplementary_Fig_11
A
Thymocytes
MYC;Dnmt3bF/F
MYC;Dnmt3b-/-
Thymocytes
MYC;Dnmt3bF/F
MYC;Dnmt3b-/-
Pnldc1 Promoter CpGs (4 clones)
Arhgef18 Promoter CpGs (6 clones)B
Supplemental Fig. 11. Methylation analysis of Pnldc1 and Arhgef18
promoters. (A) Bisulfite sequencing of the Pnldc1 promoter using genomic
DNA isolated from normal thymocytes, two MYC;Dnmt3bF/F and three
MYC;Dnmt3b-/- tumors. Each pie represents a CpG dinucleotide and each
wedge of pie represents the sequence of an individual allele. Black wedges
correspond to methylated CpG dinucleotides and white wedges represent
unmethylated CpGs. (B) Bisulfite sequencing of the Arhgef18 promoter using
DNA isolated from normal thymocytes, MYC;Dnmt3bF/F and MYC;Dnmt3b-/-
tumors. (C) qRT-PCR analysis of Pnldc1 in two averaged normal thymi (Th),
three MYC;Dnmt3bF/F and three MYC;Dnmt3b-/- tumors. (D) qRT-PCR analysis
of Arhgef18 in two averaged normal thymi (Th), two MYC;Dnmt3bF/F and three
MYC;Dnmt3b-/- tumors.
0
0.5
1
1.5
C
D
0
500
1000
1500
2000
2500 Pnldc1
Arhgef18
MYC;Dnmt3b-/-Th MYC;Dnmt3bF/F
MYC;Dnmt3b-/-Th MYC;Dnmt3bF/F
Re
lative
Exp
ressio
nR
ela
tive
Exp
ressio
n
0
200
300
100
5
10
15
20
Dnmt3bF/FN Dnmt3b-/-
Re
lative
exp
ressio
n
0
500
1000
1500
2000
0Dnmt3bF/FN Dnmt3b-/-
Pnldc1
0
4000
6000
2000
Rpl39l
Dnmt3bF/FN Dnmt3b-/-
25
50
75
100
0Re
lative
exp
ressio
n
Ass1
125
250
375
500
0
Lmo2
Dnmt3bF/FN Dnmt3b-/- Dnmt3bF/FN Dnmt3b-/- Dnmt3bF/FN Dnmt3b-/-
Cfb
0
4000
6000
2000
Re
lative
exp
ressio
n
Dnmt3bF/FN Dnmt3b-/-
0
1200
1800
600
Dnmt3bF/FN Dnmt3b-/-
Crb10 Wif1
Hlady_Supplementary_Fig_12R
ela
tive
exp
ressio
n
5
10
15
20
Dnmt3bF/FN Dnmt3b-/-0
S100a6
1
2
3
4
Dnmt3bF/FN Dnmt3b-/-0
Psma8
0
100
200
300
400
500
Dnmt3bF/FN Dnmt3b-/-
Syce1
Pkn3
Supplemental Fig. 12. Validation of microarray by qRT-PCR. qRT-PCR
shows the transcript levels of 11 genes in three MYC;Dnmt3b-/- (Dnmt3b-/-)
versus three MYC;Dnmt3bF/F (Dnmt3bF/F) tumors. An average of two normal
thymocytes (N) serves as a control. Averages +/- SEM is shown, P<0.05 for all
genes.
Supplemental Fig. 13. siRNA-mediated knockdown in MYC;Dnmt3b-/- cell
lines. (A) Dnmt3b-deficient cell lines were infected with non-targeting siRNA
controls (NT) or siRNA against Ment, Psma8, Rpl39l, Syce1, and Pnldc1. qRT-
PCR was performed to measure levels of transcript after two days of in vitro
incubation. Averages +/- SEM is shown, P<0.05. (B) An additional
MYC;Dnmt3b-/- cell line was treated with Ment siRNA to confirm the ability of
Ment to decrease cell growth.
Hlady_Supplementary_Fig_13
0
20
40
60
80
100
120
Ment
Control
Rela
tive
Exp
ressio
n
A
B
Norm
alize
d C
ell C
ou
nts
0
20
40
60
80
100
120
140
Ment Psma8 Rpl39l Pnldc1
NT
siRNA
0
2
4
6
8
10
Locus A
U
D
TestesKidney
HeartLung
Thymus
Locus A 26 CpGs (7 clones)
Hlady_Supplementary_Fig_14
A
B
C
2x105
Rela
tive
Exp
ressio
n
Supplemental Fig. 14. Methylation and expression patterns of Ment in
normal tissues. (A) Expression of Ment transcript levels in 14 normal mouse
organs based on qRT-PCR data. (B) Methylation readout of the Ment promoter
in normal mouse tissues by COBRA. Undigested (U) and digested (D)
fragments correspond to unmethylated and methylated DNA, respectively. (C)
In depth methylation analysis of five normal mouse tissues – thymus, testes,
kidney, heart, and lung by bisulfite sequencing. Each pie represents a CpG
dinucleotide and each wedge of pie represents the sequence of an individual
allele. Black wedges correspond to methylated CpG dinucleotides and white
wedges represent unmethylated CpGs.
Jeko-1
Jurkat
RAJI
REH
Akata
JY
JVM-2
Daudi
0
1
2
3
4
5
JVM-2 JY Jeko1 RAJI Jurkat Akata REH Daudi
P<0.05
JVM-2 JY
JeKo-1
RAJI
Jurkat
Akata
REH
Daudi
CpG
Hlady_Supplementary_Fig_15
MENT
DNMT3B
Supplemental Fig. 15. Ment is a target of Dnmt3b-mediated methylation
in human lymphomas. (A) qRT-PCR analysis of MENT and DNMT3B
expression in human lymphoma cell lines. Statistically significant inverse
correlations were found using Spearman rank correlation (rs=-0.8 P=0.03). (B)
COBRA of MENT Locus II in human cell lines. (C) Bisulfite sequencing of
MENT Locus II in indicated cell lines.
A
B
C
D
U
Rela
tive E
xpre
ssio
n
Locus II
Locus II
Mantle Cell Lymphoma
1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 CpG
D
U
Hlady_Supplementary_Fig_16
Burkitt’s Lymphoma Diffuse Large B Cell Lymphoma
Supplemental Fig. 16. Methylation analysis of the MENT promoter in
primary human lymphomas. Methylation analysis of the MENT promoter by
COBRA in 26 human primary tumor samples from Burkitt’s Lymphoma
(samples 1-10), Diffuse Large B Cell Lymphoma (samples 11-20), and Mantle
Cell Lymphoma (samples 21-26). PCR fragments were digested with
restriction enzyme BstUI and loaded onto a PAGE gel. CpG represents a PCR
fragment derived from 100% methylated JURKAT genomic DNA control.
Undigested (U) and digested (D) fragments correspond to unmethylated and
methylated DNA, respectively.
0
1
2
3
4
5
6
7Dnmt1
0
1
2
3
4
5
6Dnmt3a
F/F F/F 3d+OHT 7d+OHT
Cre-ER
F/F F/F 3d+OHT 7d+OHT
Cre-ER
Hlady_Supplementary_Fig_17
Supplemental Fig. 17. Acute loss of Dnmt3b in MYC;Dnmt3bF/F cells
leads to upregulation of Dnmt1 and Dnmt3a. MYC;Dnmt3bF/F cell lines
were infected with MSCV-EGFP-CreER constructs. Application of 4-
hydroxytamoxifen (OHT) allows Cre to enter the nucleus and conditionally
delete Dnmt3b. qRT-PCR was performed to measure levels of transcript of
Dnmt1 and Dnmt3a after three and seven days of incubation with OHT.
Averages +/- SEM is shown, P<0.05.
Rela
tive E
xpre
ssio
nR
ela
tive E
xpre
ssio
n
1.2
1.0
0.8
0.6
0.4
0.2
0Control shRNA-1 shRNA-2
Rela
tive E
xpre
ssio
n
P<0.05
Hlady_Supplementary_Fig_18
Supplemental Fig. 18. Knockdown efficiency of Ment transcript levels by
qRT-PCR. Dnmt3b-deficient cell lines were infected with non-targeting shRNA
vector controls or one of two shRNA constructs to form stable cell lines. Cells
containing the shRNA vectors were selected for by addition of puromycin into
culture medium. qRT-PCR was performed to measure levels of transcript of
Ment after two days of in vitro selection. Averages +/- SEM is shown, P<0.05.
25 24
25 49
Contr
ol
Ment
EGFP
0 days 10 days
Hlady_Supplementary_Fig_19
Supplemental Fig. 19. Up-regulation of Ment contributes to accelerated
cell growth in vitro. EGFP expression in unselected MYC;Dnmt3bF/F cells
infected with the MSCV-IRES-EGFP (control) or MSCV-myc-Ment-IRES-EGFP
(Ment) at plating (day 0) and after 10 days of in vitro growth. The percentage
of positive cells in the FACS profile is shown in the top right corner.
0
20
40
60
80
100
120
MENT
Control
Jurkat
Hlady_Supplementary_Fig_20
Supplemental Fig. 20. Knockdown efficiency of MENT transcript levels
by qRT-PCR. A Jurkat cell line was infected with non-targeting siRNA (Control)
or siRNA designed against MENT. qRTPCR was performed to measure levels
of transcript of MENT after 48 days of in vitro incubation with siRNA. Averages
+/- SEM is shown, P<0.05.
Rela
tive
Exp
ressio
n
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