Proteomic Analysis of an Arsenic Response Locus

Arsenic exposure is a global health problem. Millions of people encounter arsenic through contaminated drinking water, consumption, and inhalation. The arsenic response locus in budding yeast is responsible for the detoxification of arsenic and its removal from the cell. This locus constitutes a conserved pathway ranging from prokaryotes to higher eukaryotes. The goal of this study was to identify how the arsenic response locus is regulated in an arsenic dependent manner. An affinity enrichment strategy called CRISPR-Chromatin Affinity Purification with Mass Spectrometry (CRISPR-ChAP-MS) was used that provides for the proteomic characterization of a given locus. CRISPR-ChAP-MS was applied to the arsenic response locus and uncovered 40 nuclear-annotated proteins showing enrichment. Functional assays, identified the histone acetyltransferase activity of SAGA and the ATPase chromatin remodeling activity of SWI/SNF to be required for activation of the locus. Furthermore, SAGA and SWI/SNF were both found to specifically organize the chromatin structure at the arsenic response locus for activation of gene transcription. This study provides the first proteomic characterization of an arsenic response locus and key insight into the mechanism of transcriptional activation that is necessary for detoxification of arsenic from the cell.

砷暴露是一项全球性的公共卫生难题。全球数以百万计的人群通过受污染的饮用水、饮食摄入以及呼吸道吸入途径接触砷。酿酒酵母中的砷响应位点负责砷的解毒过程，并将砷从细胞内清除。该位点所构成的保守通路广泛存在于原核生物至高等真核生物中。本研究的目标在于阐明砷响应位点如何以砷依赖的方式被调控。本研究采用了一种名为CRISPR-染色质亲和纯化联用质谱（CRISPR-Chromatin Affinity Purification with Mass Spectrometry, CRISPR-ChAP-MS）的亲和富集策略，该方法可实现特定基因组位点的蛋白质组学表征。将CRISPR-ChAP-MS应用于砷响应位点后，本研究共鉴定得到40个具有核定位注释的富集蛋白。功能实验证实，SAGA的组蛋白乙酰转移酶活性与SWI/SNF的ATP酶依赖型染色质重塑活性，是该位点激活所必需的。此外，研究还发现SAGA与SWI/SNF均可特异性重塑砷响应位点处的染色质结构，以驱动基因转录的激活。本研究首次完成了砷响应位点的蛋白质组学表征，并为细胞砷解毒所必需的转录激活机制提供了关键见解。