Epigenetic Stress and Chromate Carcinogenesis

项目摘要 六价铬（Cr（VI））已被证明在人类吸入时会导致肺癌， 已知的表观遗传机制负责铬（VI）诱导的致癌作用。Nupr 1（核 蛋白质1）是小的、高碱性的、未折叠的蛋白质，分子量为8，800道尔顿， 由各种压力源引起的。我们的初步数据表明，Nupr 1的水平显着增加， 在暴露于Cr（VI）后的人支气管上皮BEAS 2B细胞中。Cr（VI）诱导的Nupr 1激活 可能受表观遗传调节因子和AP 1转录因子控制，因为（i）Nupr 1转录是 通过用组蛋白去乙酰化酶或DNA甲基转移酶的抑制剂处理细胞而增加;（ii）Cr（VI） FAIRE（甲醛辅助分离调控元件）发现， AP 1结合位点和ChIP-seq结果（ENCODE）显示启动子周围的AP 1因子的结合 Nupr 1的区域。我们的初步结果表明，Nupr 1的过表达增加了 转录活性标记组蛋白H3 K4三甲基化（H3 K4 me 3），但降低H4 K16的水平 乙酰化（H4 K16 ac）-癌症的标志，表明Nupr 1的诱导可归因于Cr（VI）- 介导H3 K4 me 3的获得和H4 K16 ac的丧失。Nupr 1在Cr（VI）诱导的细胞内变化中的重要性 H3 K4 me 3和H4 K16 ac进一步得到以下事实的支持，即通过siRNA敲低Nupr 1极大地抑制了H3 K4 me 3和H4 K16 ac。 损害了Cr（VI）暴露后H3 K4 me 3的增加和H4 K16 ac的损失。重要的是， Nupr 1过表达诱导BEAS 2B细胞转化，而Nupr 1敲低抑制BEAS 2B细胞转化。 Cr（VI）诱导的细胞转化。我们假设Cr（VI）通过改变表观遗传学来诱导Nupr 1。 Nupr 1基因启动子中的状态和AP 1转录因子结合，并快速干扰染色质 通过改变H4 K16 ac和/或H3 K4 me 3的结构和基因表达，从而有助于Cr（VI）诱导的 致癌作用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Cr（VI）介导的Nupr 1诱导的机制。在目标2中，我们将确定Nupr 1- 染色质结构和基因表达的依赖性Cr（VI）响应性变化。我们将进一步描述 通过比较，通过诱导Nupr 1，通过Cr（VI）暴露失调的转化相关基因 通过过表达Nupr 1或Cr（VI）处理诱导的转化BEAS 2B细胞的转录谱。 在目标3中，我们将研究敲除Nupr 1表达是否阻止细胞转化和肿瘤形成。 在Cr（VI）诱导的裸鼠中。我们还将研究慢性Cr（VI）暴露在野生动物中的致癌性- 型小鼠和Nupr 1敲除小鼠中。

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.