Characterizing Pneumocystis carinii centromeres with ChIP-seq (pc_210506)

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. Overall design: 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

肺孢子菌属（Pneumocystis）是研究与宿主适应相关的着丝粒（centromere）形成机制的重要遗传系统。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。着丝粒是协调有丝分裂与减数分裂过程中染色体精准分离的基因组区域。尽管着丝粒具有核心生物学功能，但其在真核生物类群中演化速率极快。着丝粒蛋白A（CENP-A）作为组蛋白H3的变体，是多数真核生物中定义着丝粒的表观遗传标记。着丝粒常为染色体断裂的发生位点，此类断裂可引发基因组重排，并通过阻碍基因流促进物种形成。基因组重排可实现基因组重构，使病原体得以适应新宿主等全新生存环境。本研究聚焦于子囊菌门（Ascomycota）中宿主专一性哺乳动物病原体的近缘物种类群的着丝粒演化。当前，肺孢子菌属物种的长期培养仍难以实现。通过异源互补实验，我们证实肺孢子菌的CENP-A同源物功能与裂殖酵母Cnp1等效。结合短期体外培养、感染动物模型及染色质免疫共沉淀测序（ChIP-seq）技术，我们在3个分化距今约1亿年的肺孢子菌物种中鉴定得到了着丝粒区域。每个物种的17条单着丝粒染色体均对应1个独特的短区域着丝粒（长度小于10 kb）。此类着丝粒以异染色质作为侧翼区域，横跨活性基因，且无保守DNA序列基序与重复序列。上述特征提示着丝粒功能由表观遗传机制决定。实验整体设计：针对卡氏肺孢子菌（Pneumocystis carinii）中的着丝粒组蛋白CENP-A与CENP-C开展染色质免疫共沉淀测序（ChIP-seq）。实验所用卡氏肺孢子菌分离自经糖皮质激素诱导的免疫缺陷Sprague-Dawley雄性大鼠的感染肺部组织。