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.