Transcription Start Regions in PTU-intergenic regions drive cell cycle-dependent transcriptional activation events in Leishmania donovani

Abstract

ABSTRACT Leishmania displays an unconventional mode of transcription, with long clusters of genes being transcribed polycistronically from Transcription Start Regions (TSRs), being processed into monocistronic units prior to translation. It has long been believed that transcription is constitutive: failure to identify consensus sequences across TSRs (except a GT-rich motif supporting transcription in Trypanosoma brucei) and absence of canonical eukaryotic transcription factors led to the conclusion that regulation is primarily post-transcriptional, with epigenetics playing a role in triggering transcription initiation. This study stems from our previous findings identifying a few genes to be activated in a cell cycle-dependent manner. Using nuclear run-on assays to analyze nascent transcripts of two chromosomes, chromosomes 2 and 14, we find that while most genes are constitutively transcribed, a subset of genes gets activated at specific cell cycle stages. Reporter assays reveal that this transcriptional activation is driven by the regions immediately upstream of the genes. Sequence analyses of these TSRs lying in polycistronic intergenic regions (PIRs) uncovered a 10-mer GT-rich motif, in synchrony with earlier findings in T. brucei identifying a GT-rich motif at bidirectional TSRs. We also identify a second 25-mer motif at these TSRs, and deletion analyses find this motif to be critical for regulating gene expression. The findings of this study reveal that transcriptional events in these unicellular parasites are more complex than believed thus far: not all transcriptional events are constitutive, polycistronic transcription is not the only mode of transcription, and cis-acting sequence elements regulate at least some transcriptional events in these parasites. IMPORTANCE Endemic to 90 countries, Leishmania parasites cause a spectrum of diseases called Leishmaniases. No vaccines for human use are available to date, and the drugs currently used to treat the disease are expensive, have toxic side effects, and have complex administration regimens, with emerging drug resistance compounding problems. Researchers continue to investigate Leishmania cellular processes, with the hope of uncovering new therapeutic target sites. Gene regulation in these parasites is unusual, being modulated by various mechanisms, including epigenetic modifications, gene dosage, and post-transcriptional processing. Transcription is typically polycistronic and constitutive, initiating from Transcription Start Regions (TSRs) lying upstream of the first gene in the polycistronic transcription unit (PTU). The work presented here reveals that a subset of genes is transcribed monocistronically in a cell cycle-dependent manner from Transcription Start Regions lying in the PTU-intergenic regions (PIRs), underscoring the complexities of gene regulation in these parasites. Endemic to 90 countries, Leishmania parasites cause a spectrum of diseases called Leishmaniases. No vaccines for human use are available to date, and the drugs currently used to treat the disease are expensive, have toxic side effects, and have complex administration regimens, with emerging drug resistance compounding problems. Researchers continue to investigate Leishmania cellular processes, with the hope of uncovering new therapeutic target sites. Gene regulation in these parasites is unusual, being modulated by various mechanisms, including epigenetic modifications, gene dosage, and post-transcriptional processing. Transcription is typically polycistronic and constitutive, initiating from Transcription Start Regions (TSRs) lying upstream of the first gene in the polycistronic transcription unit (PTU). The work presented here reveals that a subset of genes is transcribed monocistronically in a cell cycle-dependent manner from Transcription Start Regions lying in the PTU-intergenic regions (PIRs), underscoring the complexities of gene regulation in these parasites.