(A) Schematic representation of transgenes in MYC-induced T cell lymphoma model used in this study.
(B) Examples of RLGS profiles obtained from normal EμSR-tTA thymocytes (normal) and EμSR-tTA;Teto-MYC tumors (tumor). Loss or decreased intensity of single-copy NotI fragments in the tumors, relative to several neighboring unaltered fragments, was detected by visual inspection of overlaid autoradiographs. The arrows indicate the position of RLGS fragments 4E41, 2E46, 4B22, and 4D09, which are lost or reduced in intensity in tumor samples, indicative of specific methylation events.
(C) Summary of the frequency of methylation events observed in eight tumor samples and two normal thymic controls. Filled boxes indicate methylation at that site and open boxes indicate no methylation. Only data for RLGS fragments that have been methylated in more than 50% of tumors are shown. Methylation data from the analysis of three sets of EGFP-positive and EGFP-negative thymocytes isolated from 21-d-old EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP^EGFP mice are also shown (see text and Figure 2).
(D) Southern blot analysis of control and tumor cells using the 2C51 NotI-EcoRV DNA fragment as a probe. DNA isolated from thymocytes of EμSR-tTA mice was digested either with EcoRV alone (lane 1) or with EcoRV and NotI (lanes 2 and 3). DNA isolated from eight different T cell lymphomas (tumors; lanes 1–8) was digested with restriction enzymes EcoRV and NotI. The bands represent methylated (M) or unmethylated (U) NotI sites.
(E) Schematic representation of a CpG island located near the RLGS fragment 3F56. Nucleotide position of the NotI site (black triangle), exon 1, and the region amplified for bisulfite sequencing is indicated relative to the transcription initiation site. Bisulfite sequencing was performed on DNA isolated from two normal and seven tumor samples. Each circle represents a CpG dinucleotide and each row of circles indicates the sequence of an individual allele. Filled black circles correspond to methylated CpG dinucleotides and open circles represent unmethylated CpG dinucleotides.
(F) Expression of genes near the RLGS fragments 2C51 and 3F56 was quantified in thymocytes isolated from three EμSR-tTA mice (open bars) and eight EμSR-tTA;Teto-MYC tumors (black bars) using real-time RT-PCR. The y-axis represents fold decrease in gene expression relative to the levels of gene expression detected in the first control.

(A) Schematic representation of experimental design used for evaluation of in vitro and in vivo DNA methylation in MEFs. Early passage of primary MEFs was either harvested (C0) or infected with retroviral vectors expressing MYC and an activated form of Ras (Ras^61L). A portion of cells was harvested immediately after transformation had occurred, as determined by morphological changes (C1). At this point, the rest of the cells was either injected subcutaneously into nude mice or kept in culture. By 18 d, injected cells formed tumors that were harvested along with C1 cells kept in culture in vitro (T2 tumor and C2 culture, respectively).
(B) Methylation status of 1,635 NotI restriction sites was evaluated by RLGS in DNA from C0, C1, and C2 cultures along with five T2 tumors derived from nude mice. The Venn diagram shows the number of NotI fragments that became methylated in T2 culture and T2 tumors. Sections of RLGS profiles are shown for fragments 3B29 and 4F01.
(C) Summary of DNA methylation changes observed in C0, C1, and C2 culture and five T2 tumors as determined by RLGS. Open squares represent unmethylated sequences, black squares represent methylated events.

(A) Comparison of tumor-specific DNA methylation changes observed in EμSR-tTA;Teto-MYC;p53^−/− (left panel), EμSR-tTA;Teto-MYC;E2f2^−/− (middle panel), and EμSR-tTA;Teto-MYC; Teto-Cre;Pten^LoxP/LoxP (right panel) mice relative to EμSR-tTA;Teto-MYC mice (indicated as p53^+/+, E2f2^+/+, and Pten^+/+ in each of the panels). Data is shown for RLGS fragments methylated in >33% of tumors. Filled boxes indicate methylation and open boxes indicate a loss of methylation at those sites. Class I, Class II, and Class III RLGS DNA fragments are outlined in green, red, and blue, respectively). Data from analysis of thymocytes isolated from two age-matched normal control EμSR-tTA;p53^−/− (−/−; left panel), EμSR-tTA;E2f2^−/− (−/−; middle panel), and EμSR-tTA;Teto-Cre;Pten^LoxP/LoxP (−/−; right panel) mice are also shown. Fisher's exact test was applied and p-values are shown.
(B) Dendrogram of a two-dimensional unsupervised hierarchical cluster analysis for 28 tumors analyzed using methylation status of 1,481 NotI fragments. Control (EμSR-tTA;Teto-MYC), p53 (EμSR-tTA;Teto-MYC; p53^−/−), E2f2 (EμSR-tTA;Teto-MYC; E2f2^−/−), or Pten (EμSR-tTA;Teto-MYC; Teto-Cre;PTEN^LoxP/LoxP).

(A) Schematic representation of experimental design used in this study.
(B) EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP^EGFP mice were harvested at 21 d of age, and EGFP-positive and EGFP-negative thymocytes were isolated using high-speed FACS. Flow diagrams for nontransgenic controls (EGFP-control), as well as for EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP^EGFP thymocytes prior to sorting (unsorted), and after sorting (EGFP-negative [−] and EGFP-positive [+] cells) are shown. The purity of EGFP-negative and EGFP-positive populations sorted from either side of the threshold, which is indicated with dotted lines, is shown within each panel.
(C) Real-time RT-PCR analysis of MYC and nucleolin (nuc) expression in unsorted (gray bars), EGFP-positive (black bars) and EGFP-negative (white bars) cell populations sorted from thymocytes of EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP^EGFP mouse as described in Figure 2B. Expression data are presented as fold increase relative to the EGFP-negative population. Representative example from three independent experiments is shown.
(D) Representative examples of RLGS profile sections derived from cells isolated as described in Figure 2B. The arrows indicate the position of RLGS fragments that appear methylated in tumors.
(E) No differences in CpG methylation profiles between EGFP+ and EGFP− cell populations were detected for any of the three pairs of samples analyzed as summarized in Figure 2E where open boxes indicate no methylation at that site. Frequency of methylation events in final tumors for each RLGS fragment is shown as a percentage in corresponding boxes.
(F) COBRA analysis of bisulfite-treated DNA isolated from EGFP-negative [−] and EGFP-positive [+] thymocytes of three quadruple transgenic mice at age 21 d. PCR products for RLGS fragment 3F70 were digested with restriction enzyme BstUI and loaded onto a PAGE gel along with 100% methylated (SssI-treated DNA) control (100%). DNA from normal thymocytes (con) and two tumors is also shown. Undigested and digested fragments correspond to unmethylated and methylated DNA, respectively.
(G) Expression of genes near the RLGS fragments as determined by real-time RT-PCR analysis of EGFP-negative (white bars) and EGFP-positive (black bars) cell populations sorted from thymocytes of EμSR-tTA;Teto-MYC;Teto-Cre;Rosa26LOXP^EGFP. Expression of each gene in EGFP-positive population is presented as fold decrease relative to the expression of the same gene in EGFP-negative population. Representative examples from three independent experiments are shown.

(A) Graphical representation of the time-frame of tumor onset in EμSR-tTA;Teto-MYC (wild type), EμSR-tTA;Teto-MYC;p53^−/− (p53^−/−), EμSR-tTA;Teto-MYC;E2f2^−/− (E2f2^−/−), or EμSR-tTA;Teto-MYC;Teto-Cre;Pten^LoxP/LoxP (Pten^−/−) mice.
(B) Examples of FACS profiles of normal thymocytes (normal) and tumor cells (tumors) derived from the different genetic backgrounds as indicated. The percentage of cells that are in each quadrant of the FACS profile is indicated.
(C) Representative examples of RLGS profile sections of normal thymocytes derived from EμSR-tTA (normal p53^+/+) and EμSR-tTA; p53^−/− (normal p53^−/−) mice and tumors derived from EμSR-tTA;Teto-MYC (tumor p53^+/+) and EμSR-tTA;Teto-MYC; p53^−/− (tumor p53^−/−) mice. The position of DNA fragment 2D17 is indicated with arrows. Note the increase in the intensity of 2D17 in tumors deleted for the p53 tumor suppressor (compare p53^+/+ and p53^−/− tumor samples in the third and fourth panels).
(D) Real time RT-PCR analysis of expression of 6D20 in normal thymic controls (white bars), EμSR-tTA;Teto-MYC tumors (black bars), or EμSR-tTA;Teto-MYC tumors deleted either for p53, E2f2, or Pten (grey bars) as indicated.
(E) Summary of expression data from tumors derived in different genetic backgrounds obtained on analysis of eight RLGS fragments as indicated. Red boxes represent tumors with reduction in expression relative to normal thymic controls; yellow boxes indicate tumors with no change in expression. Repression thresholds for individual genes were chosen as described in Materials and Methods.