Abstract
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.
Highlights
A precise map of transcription start sites (TSSs) is indispensable for identifying promoters and other regulatory factors that mediate gene expression in an organism
The RAMPAGE protocol preserves the mRNA polarity in the resulting cDNA library by the directed introduction of Illuminasequencing adapters for Read 1 at the 5′ end via a template switching oligo (TSO), and for Read 2 toward the 3′ end of the mRNA transcript via a randomly-priming reverse-transcription (RT) primer (Figure 1A, panel 3)
We identify genes that putatively exhibit alternative TSSs, some of which appear to be differentially regulated in a stage-specific manner suggesting an additional transcript diversity that has not previously been appreciated
Summary
A precise map of transcription start sites (TSSs) is indispensable for identifying promoters and other regulatory factors that mediate gene expression in an organism. Quantitative maps of transcription initiation have revealed the complex and dynamic nature of transcription initiation landscapes in a range of model eukaryotes, including yeast, flies, worms, zebrafish, and mammals (e.g., Carninci et al, 2006; Hoskins et al, 2011; Chen et al, 2013; FANTOM Consortium and the RIKEN PMI and CLST (DGT) et al, 2014; Haberle et al, 2014; Lu and Lin, 2019) These studies have elucidated the architecture of prototypical eukaryotic promoters (Lenhard et al, 2012; Haberle and Stark, 2018). Apicomplexa include the causative agents of widespread human diseases, such as Toxoplasma gondii, Plasmodium spp., and Cryptosporidium spp These parasites have complex life cycles that typically involve several developmental stages underpinned by distinct transcriptional programs. Transcriptome analyses using microarray and RNA sequencing (RNA-seq) methods have highlighted coordinate changes in the expression levels of numerous mRNAs as Toxoplasma transitions between intraand extracellular environments (Hassan et al, 2017), converts between sexual and asexual stages (Fritz et al, 2012; Behnke et al, 2014; Farhat et al, 2020), and between acute (Tz) and chronic (Bz) stages (reviewed in Jeffers et al, 2018, Garfoot et al, 2019; Ramakrishnan et al, 2019; Waldman et al, 2020; Xue et al, 2020)
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