High-Resolution Mapping of Transcription Initiation in the Asexual Stages of Toxoplasma gondii

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.

CRISPR-Mediated Transcriptional Repression in Toxoplasma gondii

“…sgRNAs were designed for cotargeting these loci within a window from −50 to +95 bp relative to the TSS. The SAG1 gene model was based on ORF data from the reference annotation (ToxoDB.org, GT1 v.10) and work that previously mapped the dominant TSS ( 24 , 25 ). We have since appreciated that the experimentally validated 5′ UTR extends past the first ATG to a second in-frame ATG, 51 nt further into the ORF of the reference annotation ( 37 ), which explains the truncated 5′ UTR we used for the mNG reporter.…”

“…The SAG1 upstream region represented a suitable target for evaluating transcriptional repression, as (i) its high baseline activity provides a broad dynamic range for measuring repression; (ii) transcription initiation activity at the SAG1 promoter is spatially organized within a sharp peak, with the major transcription start site (TSS) located at −146 bp relative to the ORF ( 24 ); and (iii) SAG1 is largely dispensable to parasites in vitro ( 5 ), minimizing confounding effects from selective pressure for or against repression. We therefore designed five sgRNAs to uniquely target either DNA strand of the SAG1 upstream region within −50 to +95 bp of the major TSS ( 24 , 25 ) ( Fig. 2C ).…”

“…Strikingly, the most effective sgRNA in the Spy .dCas9 system and the second most effective sgRNA in the Sth .dCas9 system (sgRNAs 1) targeted protospacers offset by only one base pair and with identical strandedness, suggesting that this target site and/or spacer/protospacer sequence is particularly effective for repression of SAG1 by CRISPRi. This target is located just downstream of the six 27-bp repeats that are required for promoter activity ( 29 ) and by inference, appears to be located within the nucleosome-depleted region ( 24 ).…”

The role of upstream open reading frames in translation regulation in the apicomplexan parasites Plasmodium falciparum and Toxoplasma gondii

“…Similar to P. falciparum , the transcripts of T. gondii also have a widespread occurrence of uORFs. At least one uORF has been predicted in 90% of transcripts with annotated 5′ leader sequences (Markus et al ., 2021 ). This number is 1.8 times higher than the reported number in human transcripts where only 49% of the transcripts contain at least one uORF in their 5′ leader sequence (Calvo et al ., 2009 ).…”

“…There is evidence for uORFs playing substantive roles in translational control in apicomplexan parasites; this evidence includes high frequencies and widespread distribution of uORFs among large numbers of transcripts (Bunnik et al ., 2013 ; Caro et al ., 2014 ; Kumar et al ., 2015 ; Srinivas et al ., 2016 ; Hassan et al ., 2017 ; Holmes et al ., 2019 ; Markus et al ., 2021 ). Additionally, ribosome profiling studies in P. falciparum and T. gondii parasites reveal footprints in the 5′ leader sequences of transcripts (Lacsina et al ., 2011 ; Bunnik et al ., 2013 ; Caro et al ., 2014 ; Hassan et al ., 2017 ; Holmes et al ., 2019 ).…”