The CYP3A4 cytochrome p450 and UGT1A1 and UGT1A6 UDP-glucuronosyl transferases are some of the most important enzymes for the metabolism of drugs, exogenous carcinogens and endogenous compounds such as steroid hormones. The activities of these enzymes vary up to 40-fold between individuals, and this can have a major effect on drug efficacy or toxicity and cancer risk. Genetic polymorphisms in the promoters of these genes can significantly upregulate or downregulate their expression. In many cases, however, there is no obvious explanation for these transcriptional effects, since the polymorphisms often do not alter known transcription factor binding sites. We hypothesize that these polymorphisms may regulate transcription factor binding indirectly, by altering the positions of promoter nucleosomes. DNA sequence can affect nucleosome positions by creating low energy, default nucleosome binding sites, and our initial computational predictions indicate that CYP3A4, UGT1A1 and UGT1A6 promoter polymorphisms can significantly alter default nucleosome positions. Our preliminary results have also revealed that DNA sequence determines the locations of nucleosomes moved and/or structurally-altered by the chromatin remodeling complex human SWI/SNF, and that these positions differ from the default positions. Despite the importance of CYP3A4, UGT1A1 and UGT1A6, there is currently no detailed information about the chromatin structures of their wild type or variant promoters. In the work proposed here, we will examine the default and hSWI/SNF-remodeled chromatin structures of these promoters in both a defined in vitro system and in vivo, in a human liver cell line (using RNAi to control hSWI/SNF levels). We will then test the hypothesis that regulatory polymorphisms alter this chromatin structure by comparing the results of wild-type and variant promoters in vitro and in vivo. This work is ideal for the PA-06-149 R21 grant mechanism because it will explore a novel hypothesis which may provide essential insights into the origins of hepatocellular variability in drug metabolizing gene expression, and because it will develop novel techniques for studying the effects of chromatin structure on transcription. Interindividual variability in CYP3A4, UGT1A1 and UGT1A6 expression and activity is of significant clinical relevance, since this variability can greatly influence the systemic bioavailability and/or toxicity of many drugs, and is linked to many forms of cancer as well as diseases arising from improper metabolism of endogenous compounds. The successful completion of these proposed studies will provide the first detailed information about the promoter chromatin structure of these genes, and may also reveal a novel mechanism by which promoter polymorphisms that do not directly alter transcription factor binding sites can nonetheless greatly affect transcription. These insights may suggest new ways by which drug metabolism rates can be controlled clinically, and may highlight a novel paradigm for the genetic control of transcriptional regulation. ? ? ?
| Denis, Gerald V; Nikolajczyk, Barbara S; Schnitzler, Gavin R (2010) An emerging role for bromodomain-containing proteins in chromatin regulation and transcriptional control of adipogenesis. FEBS Lett 584:3260-8 |
| Sims, Hillel I; Baughman, Cassandra B; Schnitzler, Gavin R (2008) Human SWI/SNF directs sequence-specific chromatin changes on promoter polynucleosomes. Nucleic Acids Res 36:6118-31 |
