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.

NARRATIVE: In this grant we are investigating how hexavalent chromium induces small stressor protein Nupr1 and what role this may have in epigenetic reprograming and chromium-induced carcinogenesis.