Leiden Malaria Research Group, Department of Parasitology, Leiden University Medical, Albinusdreef 2, 2333 ZA Leiden, the Netherlands.
Cell Microbiol. 2011 Dec;13(12):1956-74. doi: 10.1111/j.1462-5822.2011.01683.x. Epub 2011 Oct 14.
Human FACT (facilitates chromatin transcription) consists of the proteins SPT16 and SSRP1 and acts as a histone chaperone in the (dis)assembly of nucleosome (and thereby chromatin) structure during transcription and DNA replication. We identified a Plasmodium berghei protein, termed FACT-L, with homology to the SPT16 subunit of FACT. Epitope tagging of FACT-L showed nuclear localization with high expression in the nuclei of (activated) male gametocytes. The gene encoding FACT-L could not be deleted indicating an essential role during blood-stage development. Using a 'promoter-swap' approach whereby the fact-l promoter was replaced by an 'asexual blood stage-specific' promoter that is silent in gametocytes, transcription of fact-l in promoter-swap mutant gametocytes was downregulated compared with wild-type gametocytes. These mutant male gametocytes showed delayed DNA replication and gamete formation. Male gamete fertility was strongly reduced while female gamete fertility was unaffected; residual ookinetes generated oocysts that arrested early in development and failed to enter sporogony. Therefore FACT is critically involved in the formation of fertile male gametes and parasite transmission. 'Promoter swapping' is a powerful approach for the functional analysis of proteins in gametocytes (and beyond) that are essential during asexual blood-stage development.
人源 FACT（促进染色质转录）由 SPT16 和 SSRP1 蛋白组成，在转录和 DNA 复制过程中作为核小体（和染色质）结构组装和解组装的组蛋白伴侣发挥作用。我们鉴定了一种疟原虫蛋白，称为 FACT-L，其与 FACT 的 SPT16 亚基具有同源性。FACT-L 的表位标记显示其具有核定位，在（激活的）雄性配子体细胞核中高表达。FACT-L 基因不能被删除，表明其在血期发育过程中具有必需的作用。使用“启动子交换”方法，即用配子体中沉默的“无性血期特异性”启动子替换 fact-l 启动子，与野生型配子体相比，启动子交换突变体配子体中的 fact-l 转录被下调。这些突变型雄性配子体显示出 DNA 复制和配子形成延迟。雄性配子体的生育力大大降低，而雌性配子体的生育力不受影响；剩余的子孢子产生的卵囊在发育早期停滞，无法进入孢子发生。因此，FACT 对于形成有活力的雄性配子体和寄生虫传播至关重要。“启动子交换”是一种用于在有丝分裂血期发育过程中必需的配子体（及其他）蛋白的功能分析的强大方法。
