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Supplementary Figures S1-3, Tables S1-3. Supplementary Figure S1. Heatmaps depicting gene expression in Hic1flox/flox MEFs treated with 4- OHT when compared to MEFs treated with vehicle (ethanol) only. Genes (299 in total) displaying significantly (q < 0.05) changed expression in at least one of the indicated time points are listed. The position of Hic1 and six genes whose expression was changed more than twice (|log FC| > 1) in at least two time points is highlighted. Supplementary Figure S2. Loss of Hic1 results in increased counts of goblet and enteroendocrine cells. A,B, goblet (A) and enteroendocrine (B) cell distribution in the indicated segments of the small intestine. Specimens obtained from four Hic1flox/flox and four Hic1flox/flox Villin-Cre+ mice were stained using Periodic acid Schiff (PAS) and an anti-chromogranin A antibody to visualize goblet and enteroendocrine cells, respectively. Stained cells (indicated by black arrowheads in the histology images on the right) were in several different fields indicated by numbers on the X axis. C, no changes in enterocytes were noted in the Hic1-deficient small intestine. Left, qRT-PCR analysis of enterocytespecific markers hairy and enhancer of split-1 (Hes1) and sucrase-isomaltase (SI). The expression level of the respective gene in wt mice (upon normalization to Ubb) was arbitrarily set to 1. Right, staining of brush border enzyme alkaline phosphatase (AP) produced by differentiated enterocytes in the small intestine. Scale bar: 0.15 mm; error bars: SDs. Supplementary Figure S3. DSS-induced transcriptional response in the colon of Hic1flox/flox and Hic1flox/flox Villin-Cre+ mice six and nine days after DSS withdrawal. Epithelial lining of the colon obtained from four animals of each genotype was analyzed using qRT-PCR. The results were normalized to the Ubiquitin B (Ubb) housekeeping gene; the relative expression of another housekeeping gene, β-actin, is also shown. The expression level of the corresponding gene in mice without DSS treatment was arbitrarily set to 1. Error bars: SDs. Supplementary Table S1 Primers used for qRT-PCR analysis Supplementary Table S2. List of genes differentially expressed in Hic1flox/flox MEFs treated with 4- OHT for 48, 72, and 120 hours when compared to control MEFs treated (for the given time periods) with vehicle. Selection criterion: q < 0.05. Of note, none of the genes passed the selection criterion for the 24-hour time point. Supplementary Table S3. The most different 'Gene Ontology Biological Processes' (GO BPs) and WikiPathways categories in expression profiles of Hic1flox/flox MEFs 72 hours after addition of 4-OHT when compared to MEFs treated with vehicle only. In total, 268 gene probes passed the significance criterion (q-value < 0.05). Corresponding annotated genes (listed in Supplementary Table S2) were analyzed using the GO BP and WikiPathways 2015 Enricher datasets. The results were sorted according to the p-value; GO BPs and WikiPathways with p < 0.05 are listed.

Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses “danger” signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells.Implications: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells. Mol Cancer Res; 13(7); 1139–48. ©2015 AACR.

Hypermethylated in cancer 1 (HIC1) represents a prototypic tumor suppressor gene frequently inactivated by DNA methylation in many types of solid tumors. The gene encodes a sequence-specific transcriptional repressor controlling expression of several genes involved in cell cycle or stress control. In this study, a Hic1 allele was conditionally deleted, using a Cre/loxP system, to identify genes influenced by the loss of Hic1. One of the transcripts upregulated upon Hic1 ablation is the toll-like receptor 2 (TLR2). Tlr2 expression levels increased in Hic1-deficient mouse embryonic fibroblasts (MEF) and cultured intestinal organoids or in human cells upon HIC1 knockdown. In addition, HIC1 associated with the TLR2 gene regulatory elements, as detected by chromatin immunoprecipitation, indicating that Tlr2 indeed represents a direct Hic1 target. The Tlr2 receptor senses “danger” signals of microbial or endogenous origin to trigger multiple signaling pathways, including NF-κB signaling. Interestingly, Hic1 deficiency promoted NF-κB pathway activity not only in cells stimulated with Tlr2 ligand, but also in cells treated with NF-κB activators that stimulate different surface receptors. In the intestine, Hic1 is mainly expressed in differentiated epithelial cells and its ablation leads to increased Tlr2 production. Finally, in a chemical-induced mouse model of carcinogenesis, Hic1 absence resulted in larger Tlr2-positive colonic tumors that showed increased proportion of proliferating cells. Implications: The tumor-suppressive function of Hic1 in colon is related to its inhibitory action on proproliferative signaling mediated by the Tlr2 receptor present on tumor cells. Mol Cancer Res; 13(7); 1139–48. ©2015 AACR.