Protein phosphatase PP1 regulation of RNA polymerase II transcription termination and allelic exclusion of VSG genes in trypanosomes.

The genomes of Leishmania and trypanosomes are organized into polycistronic transcription units flanked by a modified DNA base J involved in promoting RNA polymerase II (Pol II) termination. We recently characterized a Leishmania complex containing a J-binding protein, PP1 protein phosphatase 1, and PP1 regulatory protein (PNUTS) that controls transcription termination potentially via dephosphorylation of Pol II by PP1. While T. brucei contains eight PP1 isoforms, none purified with the PNUTS complex, complicating the analysis of PP1 function in termination. We now demonstrate that the PP1-binding motif of TbPNUTS is required for function in termination in vivo and that TbPP1-1 modulates Pol II termination in T. brucei and dephosphorylation of the large subunit of Pol II. PP1-1 knock-down results in increased cellular levels of phosphorylated RPB1 accompanied by readthrough transcription and aberrant transcription of the chromosome by Pol II, including Pol I transcribed loci that are typically silent, such as telomeric VSG expression sites involved in antigenic variation. These results provide important insights into the mechanism underlying Pol II transcription termination in primitive eukaryotes that rely on polycistronic transcription and maintain allelic exclusion of VSG genes.

Epigenetic regulation of TERRA transcription and metacyclogenesis by base J in Leishmania major.

The hyper-modified DNA base J helps control termination of Pol II transcription at polycistronic transcription units (PTUs) in T. brucei and L. major , allowing epigenetic control of gene expression. The Telomere Repeat-containing RNA (TERRA) is synthesized in T. brucei by Pol I readthrough transcription of a telomeric PTU. While little is understood regarding TERRA synthesis and function, the hyper-modified DNA base J is highly enriched at telomeres in L. major promastigotes. We now show that TERRA is synthesized by Pol II in L. major and loss of base J leads to increased TERRA. For at least one site, the increased TERRA is by Pol II readthrough transcription from an adjacent PTU. Furthermore, Pol II readthrough defects and increased TERRA correlate with increased differentiation of promastigotes to the infectious metacyclic life stage and decreased cell viability. These results help explain the essential nature of base J in Leishmania and provide insight regarding epigenetic control of coding and non-coding RNA expression and parasite development during the life cycle of L. major .

Mono-allelic epigenetic regulation of bi-directional polycistronic transcription initiation by RNA Polymerase II in Trypanosoma brucei.

Unique for a eukaryote, protein-coding genes in trypanosomes are arranged in polycistronic units (PTUs). This genome arrangement has led to a model where Pol II transcription of PTUs is unregulated. The initial step in trypanosome lytic factor (TLF) mediated lysis of Trypanosoma brucei requires high affinity haptoglobin/hemoglobin receptor (HpHbR) binding. Here we demonstrate that by in vitro 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. Therefore, 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 primitive eukaryotes that rely on polycistronic transcription.