Epigenetic Stress and Chromate Carcinogenesis

项目总结 六价铬(Cr(VI))被证明在吸入时会导致人类肺癌， 了解铬(VI)诱发致癌的表观遗传学机制。1月1日(核 蛋白质1)是一种小的、高度碱性的、未折叠的蛋白质，分子量为8800道尔顿， 由各种应激源引起的。我们的初步数据表明，Nupr1的水平显著增加 铬(VI)对人支气管上皮BEAS2B细胞的影响铬(VI)诱导的Nupr1活化 可能受表观遗传调控因子和AP1转录因子控制，因为(I)Nupr1转录是 通过用组蛋白脱乙酰酶或DNA甲基转移酶的抑制剂处理细胞而增加；(Ii) 通过FIRE(甲醛辅助分离调控元件)发现开放周围的染色质结构域 Ap1结合位点和芯片序列结果(Encode)显示ap1因子与启动子周围的结合。 1月1日地区。我们的初步结果表明，Nupr1的过度表达增加了 转录活性标志组蛋白H3K4三甲基化(H3K4me3)，但降低H4K16水平 乙酰化(H4K16ac)-癌症的标志，表明Nupr1的诱导可归因于铬(VI)- H3K4me3的介导性获得和H4K16ac的损失。Nupr1在铬(VI)诱导的细胞周期变化中的作用 H3K4me3和H4K16ac进一步得到以下事实的支持：siRNA极大地击倒了Nupr1 抑制铬(VI)暴露后H3K4me3的增加和H4K16ac的丢失。重要的是 Nupr1过表达诱导BEAS2B细胞转化，而Nupr1基因敲除则抑制其转化 铬(VI)诱导的细胞转化。我们推测，铬(VI)通过表观遗传学的改变诱导Nupr1 Nupr1基因启动子与AP1转录因子结合状态及对染色质的快速干扰 通过改变H4K16ac和/或H3K4me3的结构和基因表达，从而促进铬(VI)诱导 致癌。我们将通过三个具体目标来探讨这些假设。在目标1中，我们将确定 调节铬(VI)介导的Nupr1诱导的机制。在目标2中，我们将确定Nupr1- 依赖于铬(VI)的染色质结构和基因表达的响应变化。我们将进一步描述 比较铬(VI)暴露诱导Nupr1对转化相关基因表达的影响 Nupr1或Cr(VI)过表达诱导转化的BEAS2B细胞的转录谱 在目标3中，我们将研究Nupr1表达敲除是否阻止细胞转化和肿瘤形成 在铬(VI)诱发的裸鼠体内。我们还将调查在野外长期接触铬(VI)的致癌性。 类型小鼠和Nupr1基因敲除小鼠。

PROJECT SUMMARY Hexavalent Chromium (Cr(VI)) has been shown to cause lung cancer in humans when inhaled, with little known about the epigenetic mechanisms responsible for Cr(VI)-induced carcinogenesis. Nupr1 (nuclear protein 1) is a small, highly basic, and unfolded protein with molecular weight of 8,800 daltons, which is induced by a variety of stressors. Our preliminary data indicate that the level of Nupr1 is significantly increased in human bronchial epithelial BEAS2B cells following exposure to Cr(VI). Cr(VI)-induced activation of Nupr1 might be controlled by epigenetic regulators and AP1 transcription factors, since (i) Nupr1 transcription is increased by treating cells with inhibitors of histone deacetylases or DNA methyltransferase; (ii) Cr(VI) was found by FAIRE (Formaldehyde-Assisted Isolation of Regulatory Elements) to open chromatin domains around AP1 binding sites and ChIP-seq results (ENCODE) show the binding of AP1 factors around the promoter region of Nupr1. Our preliminary results demonstrate that overexpression of Nupr1 increases the levels of transcriptional active mark histone H3K4 trimethylation (H3K4me3) but decreases the levels of H4K16 acetylation (H4K16ac) – a hallmark of cancers, indicating that induction of Nupr1 is attributable to Cr(VI)- mediated gain of H3K4me3 and loss of H4K16ac. The importance of Nupr1 in the Cr(VI)-induced changes in H3K4me3 and H4K16ac was further supported by the fact that knockdown of Nupr1 by siRNA greatly compromised the increase of H3K4me3 and the loss of H4K16ac following Cr(VI) exposure. Importantly, overexpression of Nupr1 induced transformation of BEAS2B cells, while knockdown of Nupr1 inhibited Cr(VI)-induced cell transformation. We hypothesize that Cr(VI) induces Nupr1 via changes in epigenetic status and AP1 transcription factor binding in the promoter of Nupr1 gene and rapidly perturbs chromatin structure and gene expression by altering H4K16ac and/or H3K4me3, thereby contributing to Cr(VI)-induced carcinogenesis. We will explore these hypotheses through three specific aims. In Aim 1, we will determine mechanisms that regulate Cr(VI)-mediated induction of Nupr1. In Aim 2, We will determine the Nupr1- dependent Cr(VI)-responsive changes in chromatin structure and gene expression. We will further characterize transformation-related genes dysregulated by Cr(VI) exposure through induction of Nupr1 by comparing transcriptional profiles of transformed BEAS2B cells induced by overexpression of Nupr1 or Cr(VI) treatment. In Aim 3, we will study whether knockout of Nupr1 expression prevents cell transformation and tumor formation in nude mice induced by Cr(VI). We will also investigate the carcinogenicity of chronic Cr(VI) exposure in wild- type mice and in Nupr1-knockout mice.