Coincidentally, the HNF4 bindi

Coincidentally, the HNF4 binding motif is over represented within AhR enriched regions lacking a DRE core. Consistent with this proposed mechanism, several HNF4a regulated genes, including Cyp7a1 and Gck, exhibited AhR enrichment and were repressed by TCDD. Cyp7a1 is the rate limiting enzyme in the bile acid biosynthetic pathway that converts cholesterol into bile acids. Transgenic mice over expressing Cyp7a1 are protected from high fat diet induced obesity, fatty liver and insulin resistance. Moreover, a genetic defi ciency of Cyp7a1 in humans results in hyperlipidemia. Gck phosphorylates glucose in the initial step of glycolysis. Mutations in Gck that reduce kinase activity are associated with insulin resistance and maturity onset diabetes of young 2 in humans.

Furthermore, mice Inhibitors,Modulators,Libraries over expressing Gck are resistant to MODY2. The down regulation Inhibitors,Modulators,Libraries of Cyp7a1 and Gck, possibly due to AhR COUP TF interactions at HNF4a response elements, is consistent GSK-3 TCDD induced hepatic lipid accumulation in mice. Interestingly, TCDD expo sure has been linked to diabetes and metabolic syn drome in humans. Studies examining AhR COUP TF interactions and their effects on HNF4 target gene expression are being investigated further. Conclusion This study identified the genome wide locations of TCDD induced hepatic AhR enrichment in vivo and incorporates DRE distribution and differential gene expression data to further elucidate the hepatic AhR regu latory network. In addition to identifying interactions in regions associated with genes, AhR enrichment in distal non coding intergenic regions was characterized.

The functional significance of these distal interactions is unknown but intergenic binding has been Inhibitors,Modulators,Libraries reported for other TFs, and warrants further investigation. Moreover, only 50% of all AhR enriched regions involved a DRE, suggesting that indirect AhR binding to DNA plays a sig nificant role in the AhR regulatory network. Methods Animal Handling and Treatment Hepatic tissue samples from immature female ovariecto mized C57BL 6 mice obtained from a previous study were used for both ChIP assays at 2 and 24 hrs, and gene expression analyses across all time points. Briefly, mice were orally gavaged with 30 ug kg TCDD and sacrificed by cervical dislocation at 2, 4, 8, 12, 18, 24, 72 or 168 hrs postdose. Tissues were removed, weighed, and multiple samples were flash frozen in liquid nitro gen and stored at 80 C until further use.

Inhibitors,Modulators,Libraries Chromatin Immunoprecipitation and ChIP chip Experiments ChIP assays were performed as previously described with the following changes. Approximately 100 mg of mouse liver was homogenized in 1% formaldehyde and incubated for 10 min at room temperature. Tissue homogenate was centrifuged at 10,000 RPM for 3 min at 4 C. Pellet was washed in ice cold PBS, centrifuged, and resuspended in 900 uL of TSEI 1�� Protease Inhi bitor Cocktail.

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