A DOT1B/RH2 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 Ribonuclease 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 Ribonuclease 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.

布氏锥虫（Trypanosoma brucei）会周期性改变保护性变异表面糖蛋白（variant surface glycoproteins, VSGs）的表达，以此逃避宿主免疫系统，该过程被称为抗原变异（antigenic variation）。调控VSG表达的途径主要有两种：其一为转录激活此前处于沉默状态的VSG表达位点（VSG expression site, ES）——这类位点是包含VSG基因的亚端粒区域；其二则是将大型储备库中的另一VSG通过同源重组整合至活跃的ES中。保守的组蛋白甲基转移酶DOT1B（histone methyltransferase DOT1B）参与非活跃ES的转录沉默过程，并可影响ES的转换动力学。然而，介导DOT1B发挥上述调控功能的分子机制仍有待阐明。为进一步解析DOT1B介导的调控过程，我们采用互补生化实验方法纯化了与DOT1B相关的蛋白，并鉴定出多个新型DOT1B互作蛋白。其中一个互作蛋白为核糖核酸酶H2（Ribonuclease H2）复合物，该复合物此前已被证实可解旋RNA-DNA杂交体、维持基因组完整性，并在抗原变异过程中发挥作用。我们的研究显示，敲低DOT1B会导致RNA-DNA杂交体水平升高、DNA损伤累积以及ES转换事件增多。令人意外的是，在核糖核酸酶H2突变体中也观察到了类似的VSG表达失调模式。据此我们提出，这两种蛋白可协同作用以解旋R环，从而保障基因组完整性，并参与到严格调控的抗原变异过程中。