A DOT1B/Ribonuclease H2 Protein Complex Is Involved in R-Loop Processing, Genomic Integrity, and Antigenic Variation in Trypanosoma brucei.

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.

Two flagellar BAR domain proteins in Trypanosoma brucei with stage-specific regulation.

Trypanosomes are masters of adaptation to different host environments during their complex life cycle. Large-scale proteomic approaches provide information on changes at the cellular level, and in a systematic way. However, detailed work on single components is necessary to understand the adaptation mechanisms on a molecular level. Here, we have performed a detailed characterization of a bloodstream form (BSF) stage-specific putative flagellar host adaptation factor Tb927.11.2400, identified previously in a SILAC-based comparative proteome study. Tb927.11.2400 shares 38% amino acid identity with TbFlabarin (Tb927.11.2410), a procyclic form (PCF) stage-specific flagellar BAR domain protein. We named Tb927.11.2400 TbFlabarin-like (TbFlabarinL), and demonstrate that it originates from a gene duplication event, which occurred in the African trypanosomes. TbFlabarinL is not essential for the growth of the parasites under cell culture conditions and it is dispensable for developmental differentiation from BSF to the PCF in vitro. We generated TbFlabarinL-specific antibodies, and showed that it localizes in the flagellum. Co-immunoprecipitation experiments together with a biochemical cell fractionation suggest a dual association of TbFlabarinL with the flagellar membrane and the components of the paraflagellar rod.