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The parasite Trypanosoma brucei periodically changes the expression of protective variant surface glycoproteins (VSGs) to evade its host’s immune system in a process known as antigenic variation. One route to change VSG expression is the transcriptional activation of a previously silent VSG expression site (ES), a subtelomeric region containing the VSG genes. Homologous recombination of a different VSG from a large reservoir into the active ES represents another route. The conserved histone methyltransferase DOT1B is involved in transcriptional silencing of inactive ES and influences ES switching kinetics. The molecular machinery that enables DOT1B to execute these regulatory functions remains elusive, however. To better understand DOT1B-mediated regulatory processes, we purified DOT1B-associated proteins using complementary biochemical approaches. We identified several novel DOT1B interactors. One of these was the RNase H2 complex, previously shown to resolve RNA-DNA hybrids, maintain genome integrity, and play a role in antigenic variation. Our study revealed that DOT1B depletion results in an increase in RNA-DNA hybrids, accumulation of DNA damage, and ES switching events. Surprisingly, a similar pattern of VSG deregulation was observed in RNase H2 mutants. We propose that both proteins act together in resolving R-loops to ensure genome integrity and contribute to the tightly regulated process of antigenic variation. IMPORTANCE Trypanosoma brucei is a unicellular parasite that causes devastating diseases like sleeping sickness in humans and the “nagana” disease in cattle in Africa. Fundamental to the establishment and prolongation of a trypanosome infection is the parasite's ability to escape the mammalian host's immune system by antigenic variation, which relies on periodic changes of a protein surface coat. The exact mechanisms, however, which mediate these changes are still elusive. In this work, we describe a novel protein complex consisting of the histone methyltransferase DOT1B and RNase H2 which is involved in antigenic variation.

Maintenance of genome integrity is critical to guarantee transfer of an intact genome from parent to offspring during cell division. DNA polymerases (Pols) provide roles in both replication of the genome and the repair of a wide range of lesions. Amongst replicative DNA Pols, translesion DNA Pols play a particular role: replication to bypass DNA damage. All cells express a range of translesion Pols, but little work has examined their function in parasites, including whether the enzymes might contribute to host-parasite interactions. Here, we describe a dual function of one putative translesion Pol in African trypanosomes, which we now name TbPolIE. Previously, we demonstrated that TbPolIE is associated with telomeric sequences and here we show that RNAi-mediated depletion of TbPolIE transcripts results in slowed growth, altered DNA content, changes in cell morphology, and increased sensitivity to DNA damaging agents. We also show that TbPolIE displays pronounced localization at the nuclear periphery, and that its depletion leads to chromosome segregation defects and increased levels of endogenous DNA damage. Finally, we demonstrate that TbPolIE depletion leads to deregulation of telomeric variant surface glycoprotein genes, linking the function of this putative translesion DNA polymerase to host immune evasion by antigenic variation.

For persistent infections of the mammalian host, African trypanosomes limit their population size by quorum sensing of the parasite-excreted stumpy induction factor (SIF), which induces development to the tsetse-infective stumpy stage. We found that besides this cell density-dependent mechanism, there exists a second path to the stumpy stage that is linked to antigenic variation, the main instrument of parasite virulence. The expression of a second variant surface glycoprotein (VSG) leads to transcriptional attenuation of the VSG expression site (ES) and immediate development to tsetse fly infective stumpy parasites. This path is independent of SIF and solely controlled by the transcriptional status of the ES. In pleomorphic trypanosomes varying degrees of ES-attenuation result in phenotypic plasticity. While full ES-attenuation causes irreversible stumpy development, milder attenuation may open a time window for rescuing an unsuccessful antigenic switch, a scenario that so far has not been considered as important for parasite survival., Author summary African trypanosomes escape the mammalian host’s immune system by antigenic variation of their variant surface glycoprotein (VSG) coat. VSGs are expressed from a specialized region in the genome, the expression site (ES), that contains essential expression site associated genes (ESAGs). So far, it was assumed that only successful antigenic switches to an intact expression site are viable. Here we show that unsuccessful VSG switches are not a dead-end, but may rather contribute to the persistence of the trypanosomes at the population level. We have simulated an unsuccessful VSG switch in pleomorphic trypanosomes by expression of a second VSG from a locus without ESAGs. The parasites responded with surprising phenotypic plasticity. All parasites immediately exchanged the surface coat and reduced the abundance of ES-derived transcripts. However, depending on the degree of ES-attenuation, the transgenic trypanosomes either resumed growth, or stopped proliferation. We show that the growth-arrested populations synchronously differentiate to the stumpy life cycle stage and become infective for the tsetse fly. This occurs at low cell densities and in the absence of the quorum sensing factor SIF. Thus, unsuccessful VSG switches are not lethal and cell density-dependent quorum sensing is not the only path to the tsetse fly competence.

Developmental differentiation is a universal biological process that allows cells to adapt to different environments to perform specific functions. African trypanosomes progress through a tightly regulated life cycle in order to survive in different host environments when they shuttle between an insect vector and a vertebrate host. Transcriptomics has been useful to gain insight into RNA changes during stage transitions; however, RNA levels are only a moderate proxy for protein abundance in trypanosomes. We quantified 4270 protein groups during stage differentiation from the mammalian-infective to the insect form and provide classification for their expression profiles during development. Our label-free quantitative proteomics study revealed previously unknown components of the differentiation machinery that are involved in essential biological processes such as signaling, posttranslational protein modifications, trafficking and nuclear transport. Furthermore, guided by our proteomic survey, we identified the cause of the previously observed differentiation impairment in the histone methyltransferase DOT1B knock-out strain as it is required for accurate karyokinesis in the first cell division during differentiation. This epigenetic regulator is likely involved in essential chromatin restructuring during developmental differentiation, which might also be important for differentiation in higher eukaryotic cells. Our proteome dataset will serve as a resource for detailed investigations of cell differentiation to shed more light on the molecular mechanisms of this process in trypanosomes and other eukaryotes., Author Summary Trypanosoma brucei is a member of a large group of flagellated protozoan parasites that threatens the lives and husbandry of millions of people worldwide. This group includes parasites that cause devastating diseases such as leishmaniasis (caused by different species of Leishmania) and Chagas disease in South America (caused by Trypanosoma cruzi). One common feature of these parasites is a sophisticated life cycle, which requires transmission between a mammalian host and an insect vector. Over the course of this life cycle, the parasites follow a sequence of distinct developmental forms, which are perfectly adapted to the different host environments. We employed quantitative mass spectrometry techniques to unravel the molecular mechanisms that drive developmental differentiation of Trypanosoma brucei, the causative agent of African sleeping sickness. We followed the changes of expression of 4270 proteins during the development of the human-infective to the insect form of trypanosomes. The insights gained should help us to better understand not only how these dangerous parasites are able to survive in different environments, but also how they evade the defense mechanisms of their hosts. Unraveling the requirements needed for adaptation to human hosts might also be useful for targeted drug development to fight the devastating neglected tropical diseases caused by these parasites.