Kinetoplastid-specific histone variant functions are conserved in Leishmania major

Protein-coding genes in kinetoplastid protists are transcribed from polycistronic arrays, yielding RNA precursors which are processed to form mature transcripts bearing a 5’ spliced leader (SL) and 3’ poly(A) tract. Regions of transcription initiation and termination lack known eukaryotic promoter and terminator elements, and current data suggest that transcription is instead regulated predominantly through epigenetic mechanisms. Several epigenetic marks, including histone modifications, histone variants, and an atypical DNA modification known as base J have been localized to regions of transcription initiation or termination in Trypanosoma brucei, Trypanosoma cruzi, and/or Leishmania major. Despite this conservation, the phenotypes of base J mutants vary significantly across trypanosomatids, suggesting that the specific epigenetic networks governing transcription initiation and termination have diverged significantly during evolution. In this light, we sought to characterize and compare the roles of the histone variants H2A.Z, H2B.V, and H3.V in L. major. As in T. brucei, the histone variants H2A.Z and H2B.V were shown to be essential in L. major using a powerful quantitative plasmid segregation-based test. In contrast and again similar to T. brucei, H3.V is not essential in Leishmania as H3.V-null lines grew normally, resembled WT, and remained infectious. Using SL-primed RNA-seq, we found that H3.V-null parasites have steady-state transcript levels comparable to WT parasites and display no defects in the efficiency of transcription termination at convergent strand switch regions (SSRs). Our results show a conservation of histone variant phenotypes between L. major and T. brucei, in contrast to the phenotypes associated with the epigenetic DNA base J modification. Total RNA from Four LmjF samples were analyzed using RNA-Seq. One of them is wildtype parasites, one is single knockout for H3V gene and two independent double knockouts for H3V gene.

动质体原生生物（kinetoplastid protists）的蛋白质编码基因由多顺反子阵列（polycistronic arrays）转录生成，产生的RNA前体经加工后形成带有5’端剪接前导序列（spliced leader, SL）和3’端poly(A)尾（poly(A) tract）的成熟转录本。转录起始与终止区域缺乏已知的真核生物启动子和终止子元件，现有数据表明，该类生物的转录主要通过表观遗传机制（epigenetic mechanisms）进行调控。已有研究在布氏锥虫（Trypanosoma brucei）、克氏锥虫（Trypanosoma cruzi）以及硕大利什曼原虫（Leishmania major）中，将包括组蛋白修饰（histone modifications）、组蛋白变体（histone variants）以及一种被称为碱基J（base J）的非典型DNA修饰在内的多种表观遗传标记定位到转录起始或终止区域。尽管存在这种保守性，但碱基J突变体的表型在锥虫体（trypanosomatids）中差异显著，这表明调控转录起始与终止的特异性表观遗传网络在进化过程中发生了显著分化。鉴于此，我们旨在表征并比较组蛋白变体H2A.Z、H2B.V以及H3.V在硕大利什曼原虫中的功能。与布氏锥虫的研究结果一致，基于质粒分离的定量检测法证实，H2A.Z与H2B.V在硕大利什曼原虫中是必需的。与之相反，且同样与布氏锥虫的研究结果相似，H3.V在利什曼原虫中并非必需：H3.V基因敲除株的生长状态正常，与野生型（wildtype）菌株无差异，且仍保持感染性。我们采用剪接前导序列引物介导的RNA测序（SL-primed RNA-seq）分析发现，H3.V敲除寄生虫的稳态转录本水平与野生型寄生虫相当，且在反向链转换区域（convergent strand switch regions, SSRs）的转录终止效率上未出现缺陷。本研究结果表明，硕大利什曼原虫与布氏锥虫之间的组蛋白变体表型具有保守性，这与表观遗传DNA碱基J修饰相关的表型形成鲜明对比。本研究对4份硕大利什曼原虫（LmjF）样本的总RNA进行了RNA测序（RNA-Seq）分析，其中1份为野生型寄生虫样本，1份为H3V基因单敲除样本，另外2份为独立获得的H3V基因双敲除样本。