Characterizing Pneumocystis carinii centromeres with ChIP-seq III

Pneumocystis is a relevant genetic system to study centromere formation in relation with host adaptation. How centromeres are formed and maintained in strongly host adapted fungal pathogens is poorly investigated. Centromeres are genomic regions that coordinate accurate chromosomal segregation during mitosis and meiosis. Yet, despite their essential function, centromeres evolve rapidly across eukaryotes. CENP-A, a variant of histone H3 is the epigenetic marker that define centromeres in most eukaryotes. Centromeres are often the sites of chromosomal breaks which contribute to genome shuffling and promote speciation by inhibiting gene flow. Genome shuffling allows genome reconfiguration suitable for survival in new environment such as pathogen adaptation to new hosts. Here, we study the evolution of centromeres in closely related species of mammalian specific pathogens of the fungal phylum of Ascomycota. Long term culture of Pneumocystis species is currently untenable. Using heterologous complementation, we show that Pneumocystis CENP-A ortholog is functionally equivalent to fission yeast Cnp1. Using a short-term in vitro culture, infected animal models and ChIP-seq, we identified centromeres in three Pneumocystis species that diverged ~100 Mya ago. Each species has 17 unique short regional centromeres (< 10kb) in 17 monocentric chromosomes. The centromeres are flanked by heterochromatin. They span active genes, lack conserved DNA sequence motifs, and repeats.These features suggest an epigenetic specification of centromere function. Chromatin immunoprecipitation DNA-sequencing (ChIP-seq) for centromeric histone CENP-A and CENP-C in Pneumocystis carinii. P. carinii organisms were collected from infected lungs of corticoids-induced immunocompromised Sprague-Dawley male rats. Organisms were cocultured with a suspension of A549 and LET1 cells for 7 days

肺孢子菌属（Pneumocystis）是研究着丝粒（centromere）形成与宿主适应性相关性的重要遗传系统。目前，对于强宿主适应性真菌病原体中着丝粒的形成与维持机制，相关研究仍较为匮乏。
着丝粒是一类可协调有丝分裂（mitosis）与减数分裂（meiosis）过程中染色体精准分离的基因组区域。尽管着丝粒具有不可或缺的核心功能，但在整个真核生物类群中其演化速率极快。
CENP-A（CENP-A）是组蛋白H3（histone H3）的变体，也是大多数真核生物中界定着丝粒的表观遗传标记。着丝粒往往是染色体断裂的频发位点，这些断裂可引发基因组重排，并通过阻碍基因流促进物种形成。
基因组重排能够实现基因组重构，使其适配新环境的生存需求，例如病原体对新宿主的适应性演化。
本研究聚焦于子囊菌门（Ascomycota）中宿主特异性的哺乳动物致病真菌近缘物种的着丝粒演化。
目前，肺孢子菌属物种的长期体外培养仍无法实现。
本研究通过异源互补实验证实，肺孢子菌的CENP-A同源物与裂殖酵母（fission yeast）的Cnp1功能等效。
借助短期体外培养、感染动物模型以及染色质免疫沉淀测序（ChIP-seq）技术，我们在3个约1亿年前分化的肺孢子菌物种中鉴定出了着丝粒。
每个物种的17条单着丝粒染色体中，均存在17个独特的小型区域型着丝粒（长度小于10kb）。
这些着丝粒两侧均有异染色质（heterochromatin）分布，覆盖活跃表达的基因，既无保守的DNA序列基序，也不含重复序列。
上述特征表明，着丝粒功能是通过表观遗传机制指定的。
本研究针对卡氏肺孢子菌（Pneumocystis carinii）中的着丝粒组蛋白CENP-A与CENP-C开展了染色质免疫沉淀测序实验。
实验所用的卡氏肺孢子菌样本采集自糖皮质激素诱导的免疫缺陷型斯普拉格-道利（Sprague-Dawley）雄性大鼠的感染肺部。
研究人员将该菌与A549及LET1细胞悬液共培养7天。