Flow cytometry sorting of nuclei enables the first global characterization of Paramecium germline DNA and transposable elements

Linda Sperling,Cyril Denby Wilkes,Nicole Boggetto,Frédéric Guérin,Eric Meyer,Olivier Arnaiz,Sandra Duharcourt

doi:10.1186/s12864-017-3713-7

Abstract

BackgroundDNA 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.ResultsWe 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.ConclusionsWe 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.

Highlights

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
We report the development of an efficient flow cytometry-based method to sort nuclei in P. tetraurelia
This method represents a major breakthrough over previously published methods [3, 7], in that it provides (i) improved reliability; (ii) high purity; and (iii) quality control evaluated by flow cell imaging and high throughput sequencing

Summary

Introduction

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. In diverse organisms, programmed DNA elimination leads to the removal of specific-germline DNA sequences during development of somatic cells and generates germline and somatic genomes with distinct architectures. This process has been described in a wide variety of animals and in ciliates, suggesting that it has likely arisen independently in different lineages [1]. Meiosisspecific small RNA and chromatin modification pathways, similar to those found in plants and animals for the formation of heterochromatin and silencing of repeated sequences, are involved in the epigenetic programming of DNA elimination [4, 5]

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