The sea lamprey (Petromyzon marinus) represents one of the few vertebrate species known to undergo large-scale programmatic elimination of genomic DNA over the course of its normal development. Programmed genome rearrangements (PGRs) result in the reproducible loss of ~20% of the genome from somatic cell lineages during early embryogenesis. Studies of PGR hold the potential to provide novel insights related to the maintenance of genome stability during the cell cycle and coordination between mechanisms responsible for the accurate distribution of chromosomes into daughter cells, yet little is known regarding the mechanistic basis or cellular context of PGR in this or any other vertebrate lineage. Here we identify epigenetic silencing events that are associated with the programmed elimination of DNA and describe the spatiotemporal dynamics of PGR during lamprey embryogenesis. In situ analyses reveal that the earliest DNA methylation (and to some extent H3K9 trimethylation) events are limited to specific extranuclear structures (micronuclei) containing eliminated DNA. During early embryogenesis a majority of micronuclei (~60%) show strong enrichment for repressive chromatin modifications (H3K9me3 and 5meC). These analyses also led to the discovery that eliminated DNA is packaged into chromatin that does not migrate with somatically retained chromosomes during anaphase, a condition that is superficially similar to lagging chromosomes observed in some cancer subtypes. Closer examination of “lagging” chromatin revealed distributions of repetitive elements, cytoskeletal contacts and chromatin contacts that provide new insights into the cellular mechanisms underlying the programmed loss of these segments. Our analyses provide additional perspective on the cellular and molecular context of PGR, identify new structures associated with elimination of DNA and reveal that PGR is completed over the course of several successive cell divisions.

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Notes/Citation Information

Published in PLOS Genetics, v. 12, no. 6, e1006103, p. 1-20.

© 2016 Timoshevskiy et al.

This is an open access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.

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Funding Information

Research reported in this publication was supported by the National Institute of General Medical Sciences of the National Institutes of Health (https://www.nigms.nih.gov/Pages/default.aspx) under award number R01GM104123. This work was also supported by startup funds from the University of Kentucky, Department of Biology.

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S1 Fig. H3K9me3/5MeC immunolabeling of lamprey cells.

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S2 Fig. Immunolabeling of nuclear envelope markers in rearranging embryos.

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S3 Fig. Proportion of cells in anaphase stage and interphase cells with MNi in PACT-cleared whole embryos.

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S4 Fig. Tracing lagging chromatin from late anaphase through telophase.

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S5 Fig. Progressive formation of micronuclei and delay of envelope assembly around lagging chromatin.

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S6 Fig. Fluorescence in situ hybridization Cot1 repetitive DNA (red) on lamprey chromosomes.

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S7 Fig. Detection of DNA breaks in paraffin section of 2 dpf lamprey embryos (TUNEL assay).

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S8 Fig. Examples of anaphases with lagging chromosomes and peripheral micronucleated chromatin.

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S9 Fig. Examples of cytokinetic morphology 1st dpf embryos with specific chromatin structures extending between daughter nuclei, across the cleave plane.

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S1 Movie. Three-dimensional video-image of anaphase from 1 dpf embryo after hybridization with Cot1 DNA (red) and staining with SYTO-24 (green).

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S2 Movie. Three-dimensional video-image of anaphase from a 1 dpf embryo.

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S1 Table. In situ hybridization of the germline-specific marker Germ1.

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S2 Table. Micronuclei and epigenetic modifications.

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S3 Table. Lagging chromatin in the context of early embryogenesis and PGR.