Molecular Mechanisms of Complex Mixture Toxicity

项目总结 环境中暴露于有毒化合物的情况很少是由单一毒物的作用引起的。通常， 毒剂是一种复杂的化学物质混合物，数量从几个不等，如在职业中 暴露到几千次，如香烟烟雾。这项研究支持的长期目标是 GRANT是为了加深对接触有害健康影响的机制的理解 到铬(VI)和苯并[a]芘(BaP)的混合物，重点研究导致基因表达的机制 放松管制。我们已经证明，大剂量急性铬治疗激活了MAP激酶，干扰了 转录复合体的组装，HDAC1DNMT1与启动子染色质的交联物和∙ 抑制Cr/BaP诱导基因的表观磷酸化、乙酰化和甲基化标记 组蛋白H3和H4的反式激活。这些变化抑制了RNA聚合酶II的靶向募集。 启动子，并阻断可诱导的基因表达，增加基因组的不稳定性，DNA损伤和细胞凋亡 同时降低克隆形成能力。我们使用了三种不同的分析方法，即FIRE (甲醛辅助分离调节元件)、DANPOS(核小体动态分析) 通过测序的定位和占有率)和ATAC(转座酶可及染色质的分析)来测试 铬是否会引起染色质组织和结构的表观遗传变化 解释这种表型效应的多样性。具有高度统计学意义，所有三个测试都显示 铬导致CTCF(CCCTC结合因子)结合部位周围的染色质结构域及其 模拟，Boris(印迹位置调节器的兄弟)，从关闭状态切换到开放染色质 或者正好相反。CTCF/Boris结合位点是染色体边界隔离的唯一决定因素 哺乳动物基因组，在转录调控中起着关键作用。此外，CTCF还具有独特的 负责建立染色质拓扑域，维护细胞的三维结构 基因组。我们的新发现引导我们假设，铬(VI)打破了CTCF连接所产生的连接 基因组结构和功能。具体地说，我们认为，铬(VI)破坏了三维染色质 染色体中拓扑相关结构域之间的组织和边界形成，破坏 转录调控序列之间的相互作用。基于这些发现，我们建议对此进行测试 通过确定铬(VI)处理是否扰乱了远程全基因组染色体内染色体的假说 以及由CTCF建立的染色体间相互作用以及它是否干扰了 并扰乱CTCF结合区的转录调控。这些知识来源于 这里提出的研究将对流行病学的生物学和医学翻译产生重大影响 铬暴露的发现，并通过识别有助于减少疾病发病率的分子靶点，将 为制定治疗和预防措施作出重大贡献。

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.