Guérin Frédéric, Arnaiz Olivier, Boggetto Nicole, Denby Wilkes Cyril, Meyer Eric, Sperling Linda, Duharcourt Sandra
Institut Jacques Monod, CNRS, UMR 7592, Université Paris Diderot, Sorbonne Paris Cité, Paris, F-75205, France.
Institute of Integrative Biology of the Cell, UMR9198 CNRS CEA Univ, Paris-Sud Université Paris-Saclay, 91198, Gif-sur-Yvette, France.
BMC Genomics. 2017 Apr 26;18(1):327. doi: 10.1186/s12864-017-3713-7.
DNA elimination is developmentally programmed in a wide variety of eukaryotes, including unicellular ciliates, and leads to the generation of distinct germline and somatic genomes. The ciliate Paramecium tetraurelia harbors two types of nuclei with different functions and genome structures. The transcriptionally inactive micronucleus contains the complete germline genome, while the somatic macronucleus contains a reduced genome streamlined for gene expression. During development of the somatic macronucleus, the germline genome undergoes massive and reproducible DNA elimination events. Availability of both the somatic and germline genomes is essential to examine the genome changes that occur during programmed DNA elimination and ultimately decipher the mechanisms underlying the specific removal of germline-limited sequences.
We developed a novel experimental approach that uses flow cell imaging and flow cytometry to sort subpopulations of nuclei to high purity. We sorted vegetative micronuclei and macronuclei during development of P. tetraurelia. We validated the method by flow cell imaging and by high throughput DNA sequencing. Our work establishes the proof of principle that developing somatic macronuclei can be sorted from a complex biological sample to high purity based on their size, shape and DNA content. This method enabled us to sequence, for the first time, the germline DNA from pure micronuclei and to identify novel transposable elements. Sequencing the germline DNA confirms that the Pgm domesticated transposase is required for the excision of all ~45,000 Internal Eliminated Sequences. Comparison of the germline DNA and unrearranged DNA obtained from PGM-silenced cells reveals that the latter does not provide a faithful representation of the germline genome.
We developed a flow cytometry-based method to purify P. tetraurelia nuclei to high purity and provided quality control with flow cell imaging and high throughput DNA sequencing. We identified 61 germline transposable elements including the first Paramecium retrotransposons. This approach paves the way to sequence the germline genomes of P. aurelia sibling species for future comparative genomic studies.
DNA消除在包括单细胞纤毛虫在内的多种真核生物中是发育程序控制的，并导致产生不同的种系和体细胞基因组。纤毛虫四膜虫含有两种具有不同功能和基因组结构的细胞核。转录不活跃的微核包含完整的种系基因组，而体细胞大核包含经过简化以利于基因表达的基因组。在体细胞大核发育过程中，种系基因组经历大量且可重复的DNA消除事件。体细胞和种系基因组的可得性对于研究程序性DNA消除过程中发生的基因组变化以及最终破译种系限制序列特异性去除的机制至关重要。
我们开发了一种新的实验方法，该方法使用流动池成像和流式细胞术将细胞核亚群分选至高纯度。我们在四膜虫发育过程中分选了营养微核和大核。我们通过流动池成像和高通量DNA测序验证了该方法。我们的工作确立了这样一个原理证明，即发育中的体细胞大核可以根据其大小、形状和DNA含量从复杂的生物样品中分选至高纯度。这种方法使我们首次能够对来自纯微核的种系DNA进行测序，并鉴定新的转座元件。对种系DNA进行测序证实，Pgm驯化的转座酶是切除所有约45,000个内部消除序列所必需的。从PGM沉默细胞获得的种系DNA和未重排DNA的比较表明，后者不能忠实地代表种系基因组。
我们开发了一种基于流式细胞术的方法来将四膜虫细胞核纯化至高纯度，并通过流动池成像和高通量DNA测序进行质量控制。我们鉴定了61种种系转座元件，包括首个四膜虫逆转座子。这种方法为未来的比较基因组研究对四膜虫姊妹种的种系基因组进行测序铺平了道路。
