Toxoplasma gondii is a common parasite of humans and animals, causing life-threatening disease in the immunocompromized, fetal abnormalities when contracted during gestation, and recurrent ocular lesions in some patients. Central to the prevalence and pathogenicity of this protozoan is its ability to adapt to a broad range of environments, and to differentiate between acute and chronic stages. These processes are underpinned by a major rewiring of gene expression, yet the mechanisms that regulate transcription in this parasite are only partially characterized. Deciphering these mechanisms requires a precise and comprehensive map of transcription start sites (TSSs); however, Toxoplasma TSSs have remained incompletely defined. To address this challenge, we used 5′-end RNA sequencing to genomically assess transcription initiation in both acute and chronic stages of Toxoplasma. Here, we report an in-depth analysis of transcription initiation at promoters, and provide empirically-defined TSSs for 7603 (91%) protein-coding genes, of which only 1840 concur with existing gene models. Comparing data from acute and chronic stages, we identified instances of stage-specific alternative TSSs that putatively generate mRNA isoforms with distinct 5′ termini. Analysis of the nucleotide content and nucleosome occupancy around TSSs allowed us to examine the determinants of TSS choice, and outline features of Toxoplasma promoter architecture. We also found pervasive divergent transcription at Toxoplasma promoters, clustered within the nucleosomes of highly-symmetrical phased arrays, underscoring chromatin contributions to transcription initiation. Corroborating previous observations, we asserted that Toxoplasma 5′ leaders are among the longest of any eukaryote studied thus far, displaying a median length of approximately 800 nucleotides. Further highlighting the utility of a precise TSS map, we pinpointed motifs associated with transcription initiation, including the binding sites of the master regulator of chronic-stage differentiation, BFD1, and a novel motif with a similar positional arrangement present at 44% of Toxoplasma promoters. This work provides a critical resource for functional genomics in Toxoplasma, and lays down a foundation to study the interactions between genomic sequences and the regulatory factors that control transcription in this parasite.

弓形虫是人类和动物的常见寄生虫，在免疫功能低下者中引起危及生命的疾病，在妊娠期间感染时导致胎儿畸形，并在某些患者中引起复发性眼部病变。这种原生动物的流行和致病性的核心在于其适应广泛环境以及区分急性和慢性阶段的能力。这些过程的基础是基因表达的重大重新布线，但这种寄生虫中调节转录的机制仅得到部分表征。破译这些机制需要精确而全面的转录起始位点（TSS）图谱；然而，弓形虫TSS 的定义仍然不完全。为了应对这一挑战，我们使用 5' 端 RNA 测序来对急性和慢性阶段的转录起始进行基因组评估。弓形虫。在这里，我们报告了对启动子处转录起始的深入分析，并为 7603 个（91%）蛋白质编码基因提供了根据经验定义的 TSS，其中只有 1840 个与现有基因模型一致。比较急性和慢性阶段的数据，我们确定了阶段特异性替代 TSS 的实例，这些 TSS 可能会产生具有不同 5' 末端的 mRNA 亚型。对 TSS 周围的核苷酸含量和核小体占用率的分析使我们能够检查 TSS 选择的决定因素，并概述 TSS 的特征弓形虫启动子架构。我们还发现普遍存在的分歧转录弓形虫启动子聚集在高度对称相控阵的核小体内，强调染色质对转录起始的贡献。 证实了之前的观察，我们断言弓形虫5' 前导序列是迄今为止研究的所有真核生物中最长的前导序列之一，中位长度约为 800 个核苷酸。进一步强调了精确 TSS 图谱的实用性，我们查明了与转录起始相关的基序，包括慢性阶段分化的主调节因子 BFD1 的结合位点，以及一个具有相似位置排列的新基序，其出现在 44% 的转录起始中。弓形虫发起人。这项工作为功能基因组学提供了重要资源弓形虫，并为研究基因组序列与控制这种寄生虫转录的调节因子之间的相互作用奠定了基础。