A novel nuclear protein complex controlling the expression of developmentally regulated genes in Toxoplasma gondii [CUT&TAG]

Toxoplasma gondii is a ubiquitous protozoan parasite with a complex life cycle containing multiple developmental stages. The parasites have distinct gene expression patterns at different stages to enable stage specific life activities, but the underlying regulatory mechanisms are largely unknown. In this study, we identified a nuclear complex that controls the expression of developmentally regulated genes, especially merozoite specific genes. This complex consists of the AP2 family transcription factor AP2Ⅻ-5, the epigenetic factors MORC and HDAC3, as well as a novel AP2Ⅻ-5 interacting protein 1 (AIP1) that stabilizes this complex. At the tachyzoite stage when the parasites proliferate rapidly by asexual endodyogeny, AP2Ⅻ-5 binds to the promoter regions of developmentally activated genes and recruits MORC and HDAC3 to suppress their expression. When sexual commitment and merozoite development is triggered, the abundance of AP2Ⅻ-5 decreases and its suppression on target genes is relieved. In contrast to MORC and HDAC3, which regulate Toxoplasma development but are also essential for tachyzoite growth, AP2Ⅻ-5 and AIP1 are dispensable for tachyzoite proliferation in vitro. These data suggest that while MORC and HDAC3 have broad regulatory activities, forming a complex with AP2Ⅻ-5 and AIP1 enables them to specifically regulate gene expression during development. Chromatin immunoprecipitation followed by DNA sequencing (ChIP-Seq) to determine the HDAC3 binding sites in the genomes of ME49 and ΔⅫ-5 strains using CUT&Tag.

刚地弓形虫（Toxoplasma gondii）是一种分布广泛的原生动物寄生虫，其生活周期复杂，包含多个发育阶段。该寄生虫在不同发育阶段具有独特的基因表达模式，以支撑阶段特异性的生命活动，但其背后的核心调控机制目前仍有待阐明。本研究鉴定出一种可调控发育调控基因（尤其是裂殖体特异性基因）表达的核复合物。该复合物由AP2家族转录因子AP2Ⅻ-5、表观遗传因子MORC与组蛋白脱乙酰酶3（HDAC3），以及一种新型AP2Ⅻ-5互作蛋白1（AIP1）组成，该蛋白可稳定该复合物的结构。在寄生虫通过无性内二芽殖快速增殖的速殖子期，AP2Ⅻ-5可结合至发育激活基因的启动子区域，并招募MORC与HDAC3以抑制这些靶基因的表达。当触发性分化启动与裂殖体发育进程时，AP2Ⅻ-5的蛋白丰度会下降，其对靶基因的抑制作用也随之解除。与调控弓形虫发育且对速殖子生长不可或缺的MORC和HDAC3不同，AP2Ⅻ-5与AIP1在体外对速殖子的增殖并非必需。上述实验结果表明，尽管MORC与HDAC3具有广泛的调控活性，但与AP2Ⅻ-5和AIP1形成复合物后，它们可在寄生虫发育过程中特异性调控基因的表达。本研究通过染色质免疫共沉淀测序（ChIP-Seq）结合CUT&Tag技术，对ME49株与ΔⅫ-5株基因组中的HDAC3结合位点进行了检测与分析。