A novel RHH family transcription factor aCcr1 and its viral homologs dictate cell cycle progression in archaea [RNA-seq]

Cell cycle regulation is of paramount importance for all forms of life. Here we report that a conserved and essential cell cycle-specific transcription factor (designated as aCcr1) and its viral homologs control cell division in Sulfolobales. We show that the transcription level of accr1 reaches peak during active cell division (D-phase) subsequent to the expression of CdvA, an archaea-specific cell division protein. Cells over-expressing the 58-aa-long RHH (ribbon-helix-helix) family cellular transcription factor as well as the homologs encoded by large spindle-shaped viruses Acidianus two-tailed virus (ATV) and Sulfolobus monocaudavirus 3 (SMV3) display significant growth retardation and cell division failure, manifested as enlarged cells with multiple chromosomes. aCcr1 over-expression results in downregulation of 17 genes (>4-folds) including cdvA. A conserved motif, aCcr1-box, located between the TATA-binding box and the translation initiation site in the promoters of 13 out of the 17 highly repressed genes, is critical for aCcr1 binding. The aCcr1-box is present in the promoters of cdvA genes across Sulfolobales, suggesting that aCcr1-mediated cdvA repression is an evolutionarily conserved mechanism by which archaeal cells dictate cytokinesis progression, whereas their viruses take advantage of this mechanism to manipulate the host cell cycle. Strains of Sis/pSeSD and Sis/pSeSD-aCcr1 were cultured in ATV medium under the conditions as described above. For transcriptomic analysis, culture was inoculated with an initial OD600 of 0.05. The cells were pelleted at 6,000 g for 10 min after 12 h of cultivation when the OD600 reached approximately 0.2. The pellet was resuspended in 1 ml PBS buffer. The cells were pelleted again and stored at -80°C. Total RNA was extracted using the Trizol reagent (Ambion, Austin, TX, USA). Total amounts and the integrity of RNA were assessed using the RNA Nano 6000 Assay Kit of the Bioanalyzer 2100 system (Agilent Technologies, CA, USA). Transcriptomic analysis was performed by Novogene (Beijing, China). About 3 μg of high-quality RNA per sample was used for the construction of RNA-Seq libraries. The libraries are sequenced by the Illumina NovaSeq 6000. Clean reads were aligned to the reference genome sequence of S. islandicus REY15A (31). The resulting data were then analysed by Fragments Per Kilobase of transcript sequence per Million base pairs sequenced (FPKM) analysis to reveal expression levels of all genes in the S. islandicus genome. Differential genome expression analysis (over-expression of aCcr1 versus empty vector) was performed using the DEGSeq R package. The resulting P-values were adjusted using the Benjamini and Hochberg’s approach for controlling the false discovery rate padj<0.05 and |log2(foldchange)| > 0 were set as the threshold for significantly differential expression.

细胞周期调控对所有生命形式均至关重要。本研究报道了一种保守且必需的细胞周期特异性转录因子（命名为aCcr1）及其病毒同源物，可调控硫化叶菌目（Sulfolobales）的细胞分裂过程。我们发现，aCcr1的转录水平在活跃细胞分裂（D期）达到峰值，该时期晚于古菌特异性细胞分裂蛋白CdvA的表达。过表达该58个氨基酸长度的RHH（丝带-螺旋-螺旋，ribbon-helix-helix）家族细胞转录因子，以及由大型纺锤形病毒嗜酸菌双尾病毒（ATV）和单尾硫化叶菌病毒3（SMV3）编码的同源蛋白的细胞，会出现显著的生长迟缓与细胞分裂失败，表现为含有多条染色体的肿大细胞。过表达aCcr1会导致包括cdvA在内的17个基因的表达下调（下调幅度超过4倍）。在17个受高度抑制的基因中，有13个基因的启动子区域，在TATA结合盒与翻译起始位点之间存在一个保守基序aCcr1-box，该基序对aCcr1的结合至关重要。在所有硫化叶菌目的cdvA基因启动子区域中均存在aCcr1-box，这表明aCcr1介导的cdvA基因抑制是一种进化保守的机制，古菌借此调控胞质分裂进程；而其病毒则利用该机制来操纵宿主的细胞周期。Sis/pSeSD与Sis/pSeSD-aCcr1菌株在ATV培养基中按照前述条件进行培养。为进行转录组分析，以初始OD600为0.05的接种量转接培养物。培养12小时后，当OD600约为0.2时，以6000 g离心10分钟收集细胞。将沉淀重悬于1 mL磷酸盐缓冲液（PBS）中。再次离心收集细胞后，于-80℃保存。使用Trizol试剂（Ambion，美国德克萨斯州奥斯汀市）提取总RNA。使用美国加利福尼亚州安捷伦科技公司的2100生物分析仪RNA Nano 6000检测试剂盒，对总RNA的总量与完整性进行评估。转录组分析由北京诺禾致源生物信息科技有限公司（Novogene）完成。每个样本取约3 μg高质量RNA用于构建RNA测序文库。文库通过Illumina NovaSeq 6000平台进行测序。将clean reads比对至冰岛硫化叶菌REY15A（S. islandicus REY15A）的参考基因组序列（参考文献31）。随后采用FPKM（每百万测序碱基片段数对应每千碱基转录本片段数，Fragments Per Kilobase of transcript sequence per Million base pairs sequenced）分析对所得数据进行处理，以获取冰岛硫化叶菌基因组中所有基因的表达水平。采用DEGSeq R软件包进行差异基因表达分析（aCcr1过表达组 vs 空载体对照组）。所得P值采用本杰明-霍赫贝格（Benjamini and Hochberg）法进行校正以控制错误发现率，将padj<0.05且|log2(倍数变化)|>0设为显著差异表达的阈值。