A single master regulator controls asexual cell cycle division patterns in Toxoplasma gondii [RNA-Seq]

ApiAP2 are a family of conserved transcription factors (TF) that play an important role in regulating gene expression in apicomplexan parasites. ApiAP2 proteins that may control the cell cycle dependent expression program are to be discovered. To better understand how these cell-cycle dependent gene expression profiles are established, we identified ApiAP2 proteins showing a cell cycle dependent expression. Using the Auxin-degradation system, we produced several inducible knock-down (iKD) mutant for these ApiAP2 proteins. Among them, a cell-cycle regulated ApiAP2 is expressed during the late S-phase of the tachyzoite cell-cycle. The iKD mutant parasites for this gene are unable to proliferate. In presence of Auxin, the mutant parasites do not produce daughter cells and are arrested in the early phase of budding. This indicates that this TF may specifically regulate the early steps of the daughter cell formation. Using RNA-Seq, we demonstrate that the level of expression of number of transcripts is affected by the knock-down of this potential TF. Three biological replicate of the mutant strain in presence and in abscence of Auxin. mRNA profiles iKD TgAP2IX-5 parasites was generated by high throughput sequencing using HiSeq 2500 system (Illumina).

ApiAP2是一类保守的转录因子（transcription factors, TF），在顶复门寄生虫的基因表达调控中发挥关键作用。目前仍有待发现可调控细胞周期依赖性表达程序的ApiAP2蛋白。为了更好地阐明这类细胞周期依赖性基因表达谱的建立机制，我们筛选出了呈现细胞周期依赖性表达的ApiAP2蛋白。利用植物生长素降解系统（Auxin-degradation system），我们为这些ApiAP2蛋白构建了多种可诱导敲低（inducible knock-down, iKD）突变体。其中，一种受细胞周期调控的ApiAP2在速殖子（tachyzoite）细胞周期的晚S期特异性表达。该基因的iKD突变寄生虫无法正常增殖：在添加植物生长素的条件下，突变寄生虫无法产生子代细胞，并在出芽早期阶段发生增殖停滞。上述结果表明，该转录因子可能特异性调控子代细胞形成的早期步骤。通过RNA测序（RNA-Seq），我们证实该潜在转录因子的敲低会影响多种转录本的表达水平。本研究设置了添加与不添加植物生长素条件下突变株的三次生物学重复。利用Illumina公司的HiSeq 2500系统进行高通量测序，获取了iKD TgAP2IX-5寄生虫的mRNA表达谱。