背景:微孢子虫是多种孢子形成的,与真菌相关的专性细胞内病原体感染了多种宿主。这种多样性反映在基因组水平上,大小变化了一个数量级,范围从头孢菌素物种中的不到3Mb(真核生物中已知的最小)到Edhazardia物种中的超过50Mb。作为真核生物基因组减少的范例,小的头部动物基因组引起了很多关注,研究显示基因密集,重复和内含子较差的基因组,其特征是分子功能的彻底修剪不再与其专性细胞内生活方式相关。然而,因为还没有从端粒到端粒的端粒测序,并且由于这些物种没有甲基化数据,我们对它们的整体遗传和表观遗传结构的理解是不完整的。
方法:在本研究中,我们对三个感染人的头孢菌素菌的端粒到端粒的完整基因组进行了测序。-E.使用短和长读取平台,并利用作为测序过程一部分的数据来研究这些基因组中表观遗传标记的存在。我们还使用了基于序列和结构的混合计算方法,包括蛋白质结构预测,为了帮助确定哪些头孢菌素蛋白参与端粒维持,表观遗传调控,和异染色质形成。
结果:发现头孢菌素染色体被TTAGG5-mer端粒重复序列加帽,然后是端粒相关重复元件(TAREs)侧翼的超甲基化核糖体RNA(rRNA)基因位点,其特征为5-甲基胞嘧啶(5mC)和5-半甲基胞嘧啶(5hmC),其次是较少的甲基化端粒和低甲基化的染色体核心。在端粒/亚端粒和染色体核心之间发现了强烈的核苷酸偏差,GC/AT发生了显着变化,GT/AC和GA/CT含量。存在几种编码端粒维持所必需的蛋白质的基因,表观遗传调控,异染色质的形成在头孢菌素基因组中得到了进一步证实。
结论:总而言之,我们的研究结果强烈支持端粒下作为头孢菌素基因组中异染色质形成的位点,并进一步提示这些物种可能通过在这些位点使用5mC/5hmC甲基化和兼性异染色质形成沉默rRNA基因而关闭其能量消耗核糖体机制,同时作为孢子休眠.
BACKGROUND: Microsporidia are diverse spore forming, fungal-related obligate intracellular pathogens infecting a wide range of hosts. This diversity is reflected at the genome level with sizes varying by an order of magnitude, ranging from less than 3 Mb in
Encephalitozoon species (the smallest known in eukaryotes) to more than 50 Mb in Edhazardia spp. As a paradigm of genome reduction in eukaryotes, the small
Encephalitozoon genomes have attracted much attention with investigations revealing gene dense, repeat- and intron-poor genomes characterized by a thorough pruning of molecular functions no longer relevant to their obligate intracellular lifestyle. However, because no
Encephalitozoon genome has been sequenced from telomere-to-telomere and since no methylation data is available for these species, our understanding of their overall genetic and epigenetic architectures is incomplete.
METHODS: In this study, we sequenced the complete genomes from telomere-to-telomere of three human-infecting
Encephalitozoon spp. -E. intestinalis ATCC 50506, E. hellem ATCC 50604 and E. cuniculi ATCC 50602- using short and long read platforms and leveraged the data generated as part of the sequencing process to investigate the presence of epigenetic markers in these genomes. We also used a mixture of sequence- and structure-based computational approaches, including protein structure prediction, to help identify which Encephalitozoon proteins are involved in telomere maintenance, epigenetic regulation, and heterochromatin formation.
RESULTS: The
Encephalitozoon chromosomes were found capped by TTAGG 5-mer telomeric repeats followed by telomere associated repeat elements (TAREs) flanking hypermethylated ribosomal RNA (rRNA) gene loci featuring 5-methylcytosines (5mC) and 5-hemimethylcytosines (5hmC), themselves followed by lesser methylated subtelomeres and hypomethylated chromosome cores. Strong nucleotide biases were identified between the telomeres/subtelomeres and chromosome cores with significant changes in GC/AT, GT/AC and GA/CT contents. The presence of several genes coding for proteins essential to telomere maintenance, epigenetic regulation, and heterochromatin formation was further confirmed in the Encephalitozoon genomes.
CONCLUSIONS: Altogether, our results strongly support the subtelomeres as sites of heterochromatin formation in Encephalitozoon genomes and further suggest that these species might shutdown their energy-consuming ribosomal machinery while dormant as spores by silencing of the rRNA genes using both 5mC/5hmC methylation and facultative heterochromatin formation at these loci.