Supplementary Figure S26.

Legend – Supplementary Figure S26. The Distribution of Genomic Locations to Which the PCR products Representing PB-induced Unique Regions of Altered DNA Methylation (RAMs) in B6C3F1 Mice Aligned. PCR products representing unique B6C3F1 RAMs were assigned a genomic location relative to an annotated gene, based on the classification scheme in Figure 2.

Supplementary Figure S27.

Legend - Supplementary Figure S27. The Distribution of Chromosomes Within the Mouse Genome to Which the PCR Products Representing PB-induced Unique Regions of Altered DNA Methylation (RAMs) in B6C3F1 Mice Aligned. The chromosomes proceed from 1 to 19, including X, in a clockwise fashion. No PCR products aligned to chromosomes 20, 21 or Y.

Supplementary Figure S28.

Legend - Supplementary Figure S28. Functional Breakdown of the PCR Products that Represent Unique PB-induced Regions of Altered DNA Methylation (RAMs) in B6C3F1 Mice. The genes representing unique RAMs were broken down into 12 functional categories, 10 of which are based on Gene Ontology functional information; examples of GO functional labels are shown on the right side of the figure. For genes in the “unknown” pie slice, no information about them exists in the literature. Regions in the “uncharacterized” pie slice align to the mouse genome greater than 10 kilobases away from an annotated gene.

Nuclear receptor corepressor 2 (Ncor2) is newly methylated uniquely in the B6C3F1 at 4 weeks. Ncor2 physically interacts with many nuclear hormone receptors, inhibiting their transcriptional activation. Ncor2 forms repressive transcriptional complexes with Sin3a and HDAC1, a histone deacetylase. Ncor2 physically binds c-fos, c-jun, and NFkB1, inhibiting transactivation by NFkB. In colorectal tumors, IKK phosphorylates Ncor2, which leads Ncor2 release from and activation of Notch signaling target genes, which is thought to contribute to tumor formation. Ncor2 is a nuclear receptor corepressor. Supplementary Figure S29.

Legend - Supplementary Figure S29. Informatics Analysis of Nuclear Receptor Corepressor 2 (Ncor2/Smrt) Which is Uniquely Newly Methylated in B6C3F1 Mice at 4 Weeks of Phenobarbital (PB) Treatment. Ncor2 is a corepressor for a variety of nuclear receptors, including the constitutive active/androstane receptor (CAR).

Ral guanine nucleotide dissociation stimulator (Ralgds) is hypomethylated uniquely in the B6C3F1 at 4 weeks. A Ras effector protein with guanine- nucleotide exchange factor activity for Ral. RalGDS can induce c-fos promoter activity. H-Ras activates RalGDS. The RalGDS-Ral pathway plays a major role in the oncogenic Ras pathway. Activation of the RalGDS pathway in NIH3T3 cells results in an invasive tumor phenotype and aggressive metastasis. Supplementary Figure S30.

Legend - Supplementary Figure S30. Informatics Analysis of Ral Guanine Nucleotide Dissociation Stimulator (Ralgds), a Potential Oncogene that is Uniquely Hypomethylated in B6C3F1 Mice at 4 Weeks of Phenobarbital (PB) Treatment. Ralgds is activated by Ha-ras and plays a critical role in the oncogenic Ras pathway.

A basic helix-turn-helix transcription factor which can bind B-catenin and activate Wnt-responsive genes. MMP7 is a target of TCF-4/B-catenin and its protein product plays a role in tumor invasion and metastasis. Tcf-4 activation increases transcription of the anti-apoptotic survivin gene. In human HCC cells, Tcf-4 expression is increased and a dominant negative mutant can inhibit cell growth. Smad3 is thought to increase VEGF expression in response to TGF-beta. Transcription factor 4 (Tcf4) is hypomethylated uniquely in the B6C3F1 at 4 weeks. Constitutive activation of Tcf-4 results in accumulation of c-myc and cyclin D1 oncogenes. Supplementary Figure S31.

Legend - Supplementary Figure S31. Informatics Analysis of Transcription Factor 4 (Tcf4), a Potential Oncogene that is Uniquely Hypomethylated in B6C3F1 Mice at 4 Weeks of Phenobarbital (PB) Treatment. Tcf4 binds to beta- catenin to activate Wnt signaling-responsive genes. Tcf4 is also involved in the epithelial to mesenchymal cell transition.

Heterodimerizes with Tgfbr1 and binds Tgfb. This complex then phosphorylates proteins which regulate the transcription of a subset of genes related to cell proliferation. Loss or decreased epxression of Tgfbr2 seems to be a common event in sporadic ovarian cancer and colon cancer. Increased expression of Tgfbr2 occurs in pancreatic ductal adenocarcinoma cells, and the stromal tissue of breast cancers where expression is associated with a poorer prognosis. A Tgfbr2 DN mutant inhibited the epithelial- mesenchymal transition, invasiveness, and metastasis of epithelial carcinoma cells. Loss of Tgf beta signaling during early stages of tumorigenesis may serve a growth suppression role, whereas increased signaling in later stages may play an oncogenic role. Transforming growth factor, beta receptor II (Tgfbr2) is hypomethylated uniquely in the B6C3F1 at 4 weeks. Supplementary Figure S32.

Legend - Supplementary Figure S32. Informatics Analysis of Transforming Growth Factor Beta Receptor II (Tgfbr2), a Potential Oncogene that is Uniquely Hypomethylated in B6C3F1 Mice at 4 Weeks of Phenobarbital (PB) Treatment. Tgfbr2 might be involved in invasion and metastasis, including the epithelial- mesenchymal transition, in epithelial carcinoma cells.

Tyrosine kinase non-receptor 2 (Tnk2/Ack1) is hypomethylated uniquely in the B6C3F1 at 4 weeks. Intracellular RTK that is involved in a regulatory mechanism that sustains the GTP-bound active form of Cdc42Hs, which is directly linked to a tyrosine phosphorylation signal transduction pathway and GEF activation. Tnk2 mediates EGF signals, leading to actin cytoskeletal rearrangements. Tnk2 activates BCAR1, which contributes to cell survival, invasion and metastasis. Tnk2 phosphorylates and inactivates Wwox, a tumor suppressor gene. Primary prostate tumors show an increase of activated Tnk2 and a decrease of Wwox. Tnk2 also accelerates tumorigenesis in nude mice and increases anchorage-independent growth. A variety of human tumor types show that Tnk2 amplification correlates with poor prognosis, and overexpression in cancer cell lines increases the invasive phenotype of cells and increases mortality in a mouse model of metastasis. Supplementary Figure S33.

Legend - Supplementary Figure S33. Informatics Analysis of Tyrosine Kinase Non-receptor 2 (Tnk2), a Potential Oncogene that is Uniquely Hypomethylated in B6C3F1 Mice at 4 Weeks of Phenobarbital (PB) Treatment. An increase in Tnk2 expression can result in increased cell survival, an increased invasive and metastatic phenotype, an elevated mortality rate in a mouse model of metastasis and accelerated tumorigenesis in nude mice.

Supplementary Figure S34.

Legend - Supplementary Figure S34. Common Cellular Regulators of Genes that Represent Unique Regions of Altered DNA Methylation (RAMs) in B6C3F1 Mice at 4 Weeks of Phenobarbital (PB) Treatment. An informatics approach was utilized to uncover cellular regulators that affect 2 or more genes representing unique PB-induced B6C3F1 RAMs at 4 weeks. Red symbols are common regulators of the unique RAMs. The arrows point away from the common regulator and towards the unique RAM; positive arrows ( ) indicate that the regulator positively affects the common RAM while negative arrows ( ) denote a negative effect. Unique RAMs are hypomethylated (green), hypermethylated (orange) or newly methylated (blue). A pink center depicts a carry forward RAM from the 2 to 4 week time point. A combination of colors (i.e. green and orange) depicts a RAM with an ambiguous methylation status at the 4 week time point. The shapes of the entities represent a specific class of molecules to which the RAM or common regulator belongs: extracellular proteins or nuclear receptors ( ), ligands ( ), kinases ( ), and transcription factors ( ).

Tgfbr2 (H, 4 wk) Tcf4 (H, 4 wk) Hras (H, 4 wk) 0.05% PB for 2 or 4 weeks Identified and annotated unique RAMs in sensitive B6C3F1 mice Epithelial-mesenchymal cell transition Invasion and metstasis Efnb2 (H or Y, 4 wk) Cma1 (H, 4 wk) Trio (H or Y, 4 wk) Tnk2 (H, 4 wk) ↑ Vegf Angiogenesis Nf-kβ activation Ppp4c (H, 2 wk) Ncor2 (N, 4 wk) ↑ IL-6 ↑ IL-8 Tumor cell survival/proliferation B6C3F1 vs. C57BL/6 1 2, , , Supplementary Figure S35. Ralgds (H, 4 wk) Ralgds (H, 4 wk) 4 5,6 Hras (H, 4 wk) 4 8

Legend - Supplementary Figure S35. The Possible Functional Significance, Regarding Tumorigenesis, of Selected Genes Which Exhibited Altered Methylation Uniquely in the B6C3F1 Mouse Following Treatment with Phenobarbital (PB). The current study identified regions of altered DNA methylation (RAMs) at 2 or 4 weeks of 0.05% phenobarbital treatment and annotated 51 genes that represent these regions. Several of these genes are well- documented as playing critical roles in tumorigenesis (dark gray ovals). Unique PB- induced hypomethylation and decreased expression of Ha-ras (light gray oval) in B6C3F1 mice has been demonstrated (Bachman et al., 2006b). The appropriate time point of PB treatment is denoted (2 or 4 wk), and the methylation statuses of the RAMs are indicated by H (hypomethylation), Y (hypermethylation) or N (new methylation). RAMs with an ambiguous methylation status are indicated by the presence of 2 letters (i.e. H or Y). Interleukin 6 (IL-6) is a common 4 week regulator, and interleukin 8 (IL-8) and vascular endothelial growth factor (Vegf) are common 4 week targets, of multiple unique RAMs in the liver tumor-susceptible B6C3F1 mice (white ovals). Ralgds is a key down-stream effector of Ha-ras. The red numbers correspond to selected literature references, listed in Supplemental Table 2, for the relationships depicted in this figure.