MYST-mediated epigenetic regulation determines the adaptive artemisinin-resistant in Plasmodium falciparum [ChIP-Seq]

To investigate the artemisinin resistance mechanism, we conducted a systematical evaluation of histone acetyltransferase expression in 45 cloned P. falciparum parasites and 30 wild-type field isolates. Remarkably, PfMYST, a member of the histone acetyltransferase MYST family, emerged as the sole candidate significantly associated with prolonged ring-survive of parasites. CHIP-seq analysis revealed PfMYST’s pivotal role in mediating histone modifications, particularly in H4K5ac and H4K8ac, within the P. falciparum genome. Through single-cell RNA sequence and conditional knockdown approaches, we identified and functionally validated PfMYST-targeted genes contributing to Plasmodium’s adaptive artemisinin resistance. We generated transgenic parasite lines tagged with HA and GFP (PfMYST-HA_GFP) in 3D7 stain using the CRISPR-Cas9 technique. To explore changes in histone acetylation upon PfMYST knockdown, we conducted a series of chromatin immunoprecipitation and high-throughput sequencing (ChIP-seq) assays targeting PfMYST, H4K5ac, H4K8ac, H4K12ac, and H4K16ac in both ring-stage and schizont-stage of PfMYST-HA_GFP and PfMYST-Ribo (PfMYST-off and wild type clones) respectively.

为探究青蒿素抗性（artemisinin resistance）的作用机制，我们对45株克隆化恶性疟原虫（Plasmodium falciparum, P. falciparum）寄生虫与30株野生型野外分离株的组蛋白乙酰转移酶（histone acetyltransferase）表达水平开展了系统性评价。值得注意的是，组蛋白乙酰转移酶MYST家族成员PfMYST成为唯一与寄生虫环期存活时间延长显著相关的候选基因。染色质免疫共沉淀测序（chromatin immunoprecipitation sequencing, ChIP-seq）分析揭示了PfMYST在介导恶性疟原虫基因组组蛋白修饰中的关键作用，尤其对H4K5ac与H4K8ac修饰的调控尤为显著。通过单细胞RNA测序与条件性敲低技术，我们筛选并功能验证了参与疟原虫适应性青蒿素抗性的PfMYST靶基因。我们利用CRISPR-Cas9技术在3D7菌株中构建了带有血凝素（hemagglutinin, HA）与绿色荧光蛋白（green fluorescent protein, GFP）标签的转基因寄生虫株PfMYST-HA_GFP。为探究PfMYST敲低后组蛋白乙酰化水平的变化，我们分别在环期与裂殖体期的PfMYST-HA_GFP与PfMYST-Ribo株（包含PfMYST敲低克隆与野生型克隆）中开展了一系列针对PfMYST、H4K5ac、H4K8ac、H4K12ac及H4K16ac的染色质免疫共沉淀高通量测序（ChIP-seq）实验。