Histone variant 3 regulates RNA polymerase II transcription termination and dual strand transcription of siRNA loci in Trypanosoma brucei

Histone variant 3 (H3V) is a kinetoplastid specific histone variant that is enriched at RNA polymerase II termination sites and telomeres. We previously found that the DNA modification base J, which co-localizes with H3V, functions to prevent readthrough transcription within gene clusters in T. brucei. We now demonstrate a similar role for H3V, where the loss of this histone variant results in increased expression of downstream genes. The effect is larger when both H3V and J are lost. Additionally, removal of H3V results in increased siRNAs from dual strand transcribed loci as well as increased expression of silent VSGs, indicating a role of H3V in the regulation of antigenic variation The role of H3V in regulating transcription termination was investigated using WT and H3V KO T. brucei cell lines. The role of J was also studied by treating each of these cell lines with DMOG, an inhibitor of J synthesis. We used small RNA seq libraries to identify transcriptional changes following the loss of H3V and J.

组蛋白变体3（Histone variant 3, H3V）是一类动质体（kinetoplastid）特异性组蛋白变体，富集于RNA聚合酶II（RNA polymerase II）终止位点与端粒（telomeres）区域。我们此前发现，与H3V共定位的DNA修饰碱基J（DNA modification base J），可在布氏锥虫（T. brucei）的基因簇内阻断通读转录（readthrough transcription）。本研究证实H3V具有类似功能：该组蛋白变体的缺失会导致下游基因（downstream genes）表达上调，且当H3V与碱基J同时缺失时，该调控效应更为显著。此外，敲除H3V会使双链转录位点（dual strand transcribed loci）产生的小干扰RNA（small interfering RNA, siRNAs）水平升高，同时上调沉默的变异表面糖蛋白（variant surface glycoprotein, VSG）的表达，提示H3V参与抗原变异（antigenic variation）的调控。本研究通过构建布氏锥虫野生型（wild type, WT）与H3V基因敲除（knockout, KO）细胞系，探究了H3V在转录终止调控中的作用；同时通过碱基J合成抑制剂二甲基乙二酰基甘氨酸（Dimethyloxalylglycine, DMOG）处理两类细胞系，进一步解析了碱基J的相关功能。我们构建了小RNA测序（small RNA sequencing, sRNA-seq）文库，以鉴定H3V与碱基J缺失后引发的转录组变化。