Molecular Mechanisms of Complex Mixture Toxicity

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PROJECT SUMMARY Environmental exposures to toxic compounds rarely result from the action of single toxicants. Often, the toxic agent is a complex mixture of chemical entities in numbers ranging from a few, such as in occupational exposures, to several thousand, as in cigarette smoke. The long-range goal of the research supported by this grant is to develop an understanding of the mechanisms responsible for the adverse health effects of exposure to mixtures of chromium (VI) and benzo[a]pyrene (BaP), focusing on the mechanisms causing gene expression deregulation. We have shown that high-dose acute chromium treatment activates MAP kinases, interferes with the assembly of transcriptional complexes, cross-links HDAC1∙DNMT1 complexes to promoter chromatin and inhibits epigenetic phosphorylation, acetylation and methylation marks established by Cr/BaP-induced gene transactivation in histones H3 and H4. These changes inhibit recruitment of RNA polymerase II to target promoters, and block inducible gene expression, increasing genomic instability, DNA damage and apoptosis while decreasing clonogenic ability. We used three different analytical approaches, namely FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements), DANPOS (Dynamic Analysis of Nucleosome Positioning and Occupancy by Sequencing), and ATAC (Assay for Transposase-Accessible Chromatin), to test whether chromium could cause epigenetic changes in chromatin organization and architecture that could explain this diversity of phenotypic effects. With high statistical significance, all three tests showed that chromium causes chromatin domains surrounding the binding sites for CTCF (CCCTC binding factor) and its analog, BORIS (Brother of the Regulator of Imprinted Sites) to switch from states of closed to open chromatin or the reverse. CTCF/BORIS binding sites are the sole determinants of chromosome boundary-insulation in the mammalian genome, playing a critical role in transcriptional regulation. In addition, CTCF is also uniquely responsible for establishing chromatin topological domains and maintaining the 3-dimensional structure of the genome. Our novel findings lead us to the hypothesis that Cr(VI) breaks the links created by CTCF connecting genome architecture and function. Specifically, we propose that Cr(VI) disrupts 3-dimensional chromatin organization and boundary formation between topologically associated domains in chromosomes, destroying the interactions between transcription regulatory sequences. Based on these findings, we propose to test this hypothesis by determining whether Cr(VI) treatment disrupts the long-range genome-wide intrachromosomal and interchromosomal interactions established by CTCF and whether it interferes with the insulator function of CTCF and disrupts transcriptional regulation in CTCF-bound domains. The knowledge derived from the research proposed here will have a major impact on the biological and medical translation of epidemiological findings of chromium exposure and, by identifying molecular targets useful to reduce disease incidence, will significantly contribute to the development of therapeutic and preventative measures.