Previously we have shown that nonsyndromic cleft lip with or without cleft palate (NSCL/P)1, is strongly associated with SNPs in Interferon Regulatory Factor 6 (IRF6)2. Here, multispecies sequence comparisons identify a common SNP (rs642961, G>A) in a novel IRF6 enhancer. The A allele is significantly overtransmitted (P=1×10−11) in families with NSCL/P, in particular with cleft lip (CL) but not cleft palate. Further, there is a dosage effect of the A allele, with the relative risk for CL 1.68 for the AG genotype and 2.40 for the AA genotype. EMSA and ChIP assays demonstrate that the risk allele disrupts the binding site of transcription factor AP-2α and expression analysis in the mouse localizes the enhancer activity to craniofacial and limb structures. Our findings place IRF6 and AP-2α in the same developmental pathway and identify a high frequency variant in a regulatory element contributing substantially to a common, complex disorder.

The development of organisms requires concerted changes in gene activity. The free radical theory of development proposes that oxygen serves as a morphogen to educe development by influencing the production of metabolic oxidants such as free radicals and reactive oxygen species. One of the central tenets of this theory is that these metabolic oxidants influence development by altering the antioxidant capacity of cells by changing their production of glutathione (GSH). Here we extend on these principles by linking GSH production and oxygen sensing in the control of gene expression to establish the epigenotype of cells during development. We prescribe this novel role to GSH and oxygen during development because these metabolites influence the activity of enzymes responsible for initiating and perpetuating epigenetic control of gene expression. Increased GSH production influences epigenetic processes including DNA and histone methylation by limiting the availability of S-adenosylmethionine, the cofactor utilized during epigenetic control of gene expression by DNA and histone methyltransferases. Moreover, the recent discovery of histone demethylases that require oxygen as a cofactor directly links epigenetic processes to oxygen gradients during development.

It has been hypothesized that cancer cells increase glucose metabolism to protect against metabolic fluxes of hydroperoxides via glutathione-dependent peroxidases. 2-Deoxy-D-glucose, inhibits glucose metabolism and has been shown to cause cytotoxicity in cancer cells that is partially mediated by disruptions in thiol metabolism. In the current study, human breast cancer cells were continuously treated (24 hours) with 2-deoxy-D-glucose, and total glutathione content as well as the expression of the first enzyme in the glutathione synthetic pathway [glutamate cysteine ligase (GCL)] were found to be induced 2.0-fold. Inhibiting GCL activity during 2-deoxy-Dglucose exposure using L-buthionine-[S ,R]-sulfoximine (BSO) significantly enhanced the cytotoxic effects of 2-deoxy-Dglucose and caused increases in endpoints indicative of oxidative stress, including % oxidized glutathione and steadystate levels of pro-oxidants as assayed using an oxidationsensitive fluorescent probe. These results show that treatment of human breast cancer cells with 2-deoxy-D-glucose causes metabolic oxidative stress that is accompanied by increases in steady-state levels of GCL mRNA, GCL activity, and glutathione content. Furthermore, inhibition of 2-deoxy-D-glucosemediated induction of GCL activity with BSO increases endpoints indicative of oxidative stress and sensitizes cancer cells to 2-deoxy-D-glucose-induced cytotoxicity. These results support the hypothesis that drug combinations capable of inhibiting both glucose and hydroperoxide metabolism may provide an effective biochemical strategy for sensitizing human cancer cells to metabolic oxidative stress. (Cancer Res 2006; 66(3): 1605-10)

Thiol antioxidants, including N-acetyl-L-cysteine (NAC), are widely used as modulators of the intracellular redox state. We investigated the hypothesis that NAC-induced reactive oxygen species (ROS) signaling perturbs cellular proliferation by regulating the cell cycle regulatory protein cyclin D1 and the ROS scavenging enzyme Mn-superoxide dismutase (MnSOD). When cultured in media containing NAC, mouse fibroblasts showed G 1 arrest with decreased cyclin D1 protein levels. The absence of a NAC-induced G 1 arrest in fibroblasts overexpressing cyclin D1 (or a nondegradable mutant of cyclin D1-T286A) indicates that cyclin D1 regulates this G 1 arrest. A delayed response to NAC exposure was an increase in both MnSOD protein and activity. NAC-induced G 1 arrest is exacerbated in MnSOD heterozygous fibroblasts. Results from electron spin resonance spectroscopy and flow cytometry measurements of dihydroethidine fluorescence showed an approximately 2-fold to 3-fold increase in the steady-state levels of superoxide (O 2˙À ) in NAC-treated cells compared with control. Scavenging of O 2˙À with Tiron reversed the NAC-induced G 1 arrest. These results show that an O 2˙À signaling pathway regulates NACinduced G 1 arrest by decreasing cyclin D1 protein levels and increasing MnSOD activity. [Cancer Res 2007;67(13):6392-9]

Many breast cancer cells typically exhibit lower expression of manganese superoxide dismutase (MnSOD) compared to the normal cells from which they arise. This decrease can often be attributed to a defect in the transcription of SOD2, the gene encoding MnSOD; however, the mechanism responsible for this change remains unclear. Here, we describe how altered histone modifications and a repressive chromatin structure constitute an epigenetic process to down regulate SOD2 in human breast carcinoma cell lines. Utilizing chromatin immunoprecipitation (ChIP) we observed decreased levels of dimethyl H3K4 and acetylated H3K9 at key regulatory elements of the SOD2 gene. Consistent with these results, we show that loss of these histone modifications creates a repressive chromatin structure at SOD2. Transcription factor ChIP experiments revealed that this repressive chromatin structure influences the binding of SP-1, AP-1, and NFκB to SOD2 regulatory cis-elements in vivo. Lastly, we show that treatment with the histone deacetylase inhibitors trichostatin A and sodium butyrate can reactivate SOD2 expression in breast cancer cell lines. Taken together, these results indicate that epigenetic silencing of SOD2 could be facilitated by changes in histone modifications and represent one mechanism leading to the altered expression of MnSOD observed in many breast cancers.