An apicomplexan bromodomain protein, TgBDP1 associates with diverse epigenetic factors to regulate essential transcriptional processes in Toxoplasma gondii. An apicomplexan bromodomain protein, TgBDP1 associates with diverse epigenetic factors to regulate essential transcriptional processes in Toxoplasma gondii

The protozoan pathogen Toxoplasma gondii relies on tight regulation of gene expression to invade and establish infection in its host. The divergent gene regulatory mechanisms of Toxoplasma and related apicomplexan pathogens rely heavily on regulators of chromatin structure and histone modifications. The important contribution of histone acetylation for Toxoplasma in both acute and chronic infection has been demonstrated, where histone acetylation increases at active gene loci. However, the direct consequences of specific histone acetylation marks and the chromatin pathway that influences transcriptional regulation in response to the modification is unclear. As a reader of lysine acetylation, the bromodomain serves as a mediator between the acetylated histone and transcriptional regulators. Here we show that the bromodomain protein TgBDP1 which is conserved amongst Apicomplexa and within the Alveolata superphylum, is essential for Toxoplasma asexual proliferation. Using CUT&TAG we demonstrate that TgBDP1 is recruited to transcriptional start sites of a large proportion of parasite genes. Transcriptional profiling during TgBDP1 knockdown revealed that loss of TgBDP1 leads to major dysregulation of gene expression, implying multiple roles for TgBDP1 in both gene activation and repression. This is supported by interactome analysis of TgBDP1 demonstrating that TgBDP1 forms a core complex with two other bromodomain proteins and an ApiAP2 factor. This core complex appears to interact with other epigenetic factors such as nucleosome remodelling complexes. We conclude that TgBDP1 interacts with diverse epigenetic regulators to exert opposing influences on gene expression in the Toxoplasma tachyzoite. Overall design: CUT&TAG analysis was performed on Toxoplasma tachyzoites expressing an epitope-tagged bromodomain protein TGME49_263580. Included are three experimental replciates using an anti-myc antibody on extracts from the tagged parasite line. Two negative control replicated were performed using anti-myc on the untransfected parental parasite line. One positive control is included that surveyed the distribution of a known histone modification (H3K9ac) using CUT&TAG.

原生动物病原体刚地弓形虫（Toxoplasma gondii）依赖严谨的基因表达调控，以入侵宿主并建立感染。弓形虫及相关顶复门（Apicomplexa）病原体所具有的独特基因调控机制，高度依赖染色质结构调控因子与组蛋白修饰因子。已有研究证实，组蛋白乙酰化在弓形虫急、慢性感染中发挥重要作用：活跃基因位点的组蛋白乙酰化水平会出现上调。然而，特定组蛋白乙酰化修饰标记的直接效应，以及响应该修饰并影响转录调控的染色质通路，目前仍不明确。作为赖氨酸乙酰化的识别阅读器，溴结构域（bromodomain）可充当乙酰化组蛋白与转录调控因子之间的介导桥梁。本研究发现，在顶复门与囊泡虫总门（Alveolata superphylum）中保守存在的溴结构域蛋白TgBDP1，对弓形虫的无性增殖至关重要。本研究借助CUT&TAG技术证实，TgBDP1会被招募至大部分寄生虫基因的转录起始位点。对TgBDP1敲低后的转录组分析显示，TgBDP1缺失会导致基因表达出现显著失调，这表明TgBDP1在基因激活与抑制过程中均发挥多重调控作用。TgBDP1的相互作用组分析结果进一步佐证了这一结论：该蛋白可与另外两种溴结构域蛋白及一个ApiAP2因子形成核心复合物。该核心复合物似乎还可与其他表观遗传因子（如核小体重塑复合物）发生相互作用。本研究最终认为，TgBDP1可通过与多种表观遗传调控因子相互作用，对弓形虫速殖子（Toxoplasma tachyzoite）的基因表达发挥双向调控作用。实验整体设计：本研究对表达表位标记溴结构域蛋白TGME49_263580的弓形虫速殖子开展CUT&TAG分析。其中，3组生物学重复采用抗myc抗体对标记寄生虫株的提取物进行检测；2组阴性对照采用抗myc抗体对未转染的亲本寄生虫株进行检测；此外设置1组阳性对照，利用CUT&TAG技术检测已知组蛋白修饰H3K9ac的分布情况。