DataSheet_3_High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii.zip

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.

刚地弓形虫（Toxoplasma gondii）是一种常见的人畜共患寄生虫，可对免疫受损个体引发致命疾病，妊娠期间感染会导致胎儿畸形，还会在部分患者中引发复发性眼部病变。该原生动物的流行与致病核心在于其适应多样环境的能力，以及区分急性与慢性感染阶段的能力。这些过程依赖于大规模的基因表达重编程，但目前对该寄生虫的转录调控机制仅部分明确。要阐明这些机制，需要获得精准且全面的转录起始位点（transcription start sites, TSSs）图谱，但迄今为止刚地弓形虫的TSSs仍未被完全界定。
为解决这一难题，我们采用5'端RNA测序技术，对刚地弓形虫急性与慢性阶段的转录起始过程开展了全基因组水平的分析。本研究深入剖析了启动子区域的转录起始特征，为7603个（占比91%）蛋白编码基因提供了实验验证的TSSs，其中仅1840个与现有基因模型吻合。通过对比急性与慢性阶段的测序数据，我们鉴定出了阶段特异性可变TSSs，这些位点理论上可产生具有不同5'端结构的mRNA同工型。通过分析TSSs上下游的核苷酸组成与核小体占据情况，我们探究了TSS选择的决定因素，并梳理了刚地弓形虫启动子的结构特征。我们还发现刚地弓形虫启动子区域存在广泛的双向转录现象，这些转录事件聚集在高度对称的阶段性核小体阵列中，凸显了染色质结构对转录起始的调控作用。与此前的研究结果一致，我们证实刚地弓形虫的5'前导序列是目前已研究的真核生物中最长的之一，中位长度约为800个核苷酸。进一步凸显精准TSS图谱的应用价值，我们还定位到了与转录起始相关的调控基序，包括慢性阶段分化主调控因子BFD1的结合位点，以及一种存在于44%刚地弓形虫启动子中、具有相似位置排布的新型基序。
本研究为刚地弓形虫的功能基因组学研究提供了关键的资源基础，也为探究该寄生虫基因组序列与转录调控因子之间的相互作用奠定了研究框架。