Mono-allelic epigenetic regulation of polycistronic transcription initiation by RNA polymerase II in <i>Trypanosoma brucei</i>

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic transcription units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated and changes in gene expression are entirely post-transcriptional. <i>Trypanosoma brucei brucei</i> is unable to infect humans because of its susceptibility to an innate immune complex, trypanosome lytic factor (TLF) in the circulation of humans. The initial step in TLF-mediated lysis of <i>T.b.brucei</i> requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here, we demonstrate that by <i>in vitro</i> selection with TLF, resistance is obtained in a stepwise process correlating with loss of HpHbR expression at an allelic level. RNA-seq, Pol II ChIP, and run-on analysis indicate HpHbR silencing is at the transcriptional level, where loss of Pol II binding at the promoter region specifically shuts down transcription of the HpHbR-containing gene cluster and the adjacent opposing gene cluster. Reversible transcriptional silencing of the divergent PTUs correlates with DNA base J modification of the shared promoter region. Base J function in establishing transcriptional silencing, rather than maintenance, is suggested by the maintenance of PTU silencing following the inhibition of J-biosynthesis and subsequent loss of the modified DNA base. Therefore, we show that epigenetic mechanisms exist to regulate gene expression via Pol II transcription initiation of gene clusters in a mono-allelic fashion. These findings suggest epigenetic chromatin-based regulation of gene expression is deeply conserved among eukaryotes, including early divergent eukaryotes that rely on polycistronic transcription.IMPORTANCEThe single-cell parasite <i>Trypanosoma brucei</i> causes lethal diseases in both humans and livestock. <i>T. brucei</i> undergoes multiple developmental changes to adapt in different environments during its digenetic life cycle. With protein-coding genes organized as polycistronic transcription and apparent absence of promoter-mediated regulation of transcription initiation, it is believed that developmental gene regulation in trypanosomes is essentially post-transcriptional. In this study, we found reversible Pol II transcriptional silencing of two adjacent polycistronic gene arrays that correlate with the novel DNA base J modification of the shared promoter region. Our findings support epigenetic regulation of Pol II transcription initiation as a viable mechanism of gene expression control in <i>T. brucei</i>. This has implications for our understanding how trypanosomes utilize polycistronic genome organization to regulate gene expression during its life cycle.