ATAC-seq Reveals Global Changes in Transcription Factor Activity Upon Perturbation of Cellular Zinc

Zinc (Zn2+) is an essential metal required in approximately 10% of the human proteome. Of this, nearly half of these proteins are involved in transcription. Little is known on how these proteins get their Zn2+, but it is becoming increasingly clear in the literature that Zn2+ may function in some capability as an intracellular messenger. Here, we sought to profile the effect that Zn2+ has on global transcription. Using ATAC-seq, we observed broad changes in chromatin accessibility when Zn2+ was both increased and depleted. These changes in chromatin accessibility correlated with changes in the enrichment of hundreds of transcription factors, many of which are Zn2+ finger transcription factors. To validate whether these changes in motif enrichment correlate with changes in transcription factor occupancy, we selected p53 as a candidate to follow up on. Using previously published p53 ChIP-seq datasets and new nascent transcription datasets, we identified high-probability p53 binding sites to validate using ChIP-qPCR. We found that the global changes in p53 motif enrichment correlated with our ChIP-qPCR results but some targets yielded local accessibility differences, a reminder that genomics datasets require experimental follow-ups before generalizations can be made. We believe that the experimental and computational workflow presented here will allow researchers to use ATAC-seq in conjunction with publicly available datasets as a relative starting point for studying transcription factor occupancy in the future. Overall design: ATAC-seq on MCF10A cells exposed to the Zn2+ chelator TPA, excess Zn in the form of ZnCl2, or an untreated control

锌离子（Zn²+）是人体约10%蛋白质组所必需的金属元素，其中近半数此类蛋白质参与转录过程。目前对于此类蛋白质如何获取锌离子的机制尚不清楚，但越来越多的文献研究表明，锌离子或许可作为细胞内信使发挥某种功能。本研究旨在分析锌离子对全局转录的影响。通过转座酶可及性测序（ATAC-seq），我们观察到，当锌离子浓度升高或被耗竭时，染色质可及性发生广泛变化。此类染色质可及性的变化与数百种转录因子的富集度变化相关，其中多数为锌指转录因子。为验证这些结合基序富集度的变化是否与转录因子结合占有率的变化相关，我们选择p53作为后续研究的候选靶点。我们利用已发表的p53染色质免疫共沉淀测序（ChIP-seq）数据集与新获得的新生转录数据集，鉴定出高可信度的p53结合位点，并计划通过染色质免疫共沉淀定量PCR（ChIP-qPCR）进行验证。研究发现，p53结合基序富集度的全局变化与我们的ChIP-qPCR结果相符，但部分靶点存在局部可及性差异，这提示我们：在得出普遍性结论前，基因组学数据集需辅以实验验证。我们认为，本文所提出的实验与计算分析流程，将为未来研究者结合公开数据集开展转录因子结合占有率研究提供可参考的起点。整体实验设计：对经锌离子螯合剂TPA处理、以氯化锌（ZnCl₂）形式施加过量锌离子的MCF10A细胞，以及未处理的对照组细胞进行ATAC-seq测序。