Limits...
Cellular and Molecular Features of Developmentally Programmed Genome Rearrangement in a Vertebrate (Sea Lamprey: Petromyzon marinus).

Timoshevskiy VA, Herdy JR, Keinath MC, Smith JJ - PLoS Genet. (2016)

Bottom Line: Programmed genome rearrangements (PGRs) result in the reproducible loss of ~20% of the genome from somatic cell lineages during early embryogenesis.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.During early embryogenesis a majority of micronuclei (~60%) show strong enrichment for repressive chromatin modifications (H3K9me3 and 5meC).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus

Variation in the content and form of eliminated DNA indicates stepwise loss of DNA.(A-C) FISH of the Germ1 probe to anaphase chromosomes from PACT-cleared embryos. (A) A representative anaphase spread from 1 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). A majority of signals corresponding to the Germ1 repeat co-migrate with retained chromosomes, and a relatively small conglomerate of chromatin is localized to the equatorial region. (B) A representative anaphase spread from 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Lagging chromosomes are enriched with Germ1 while retained chromosomes have only a single pair of Germ1 signals. (C) Germ1-negative lagging chromatin at 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Germ1 hybridizes to a pair of signals on retained chromosomes that are associated with a relatively small conglomerate of lagging chromatin that lacks Germ1 hybridization. (D-F) Late mitotic events in cells undergoing chromosome elimination. (D) Cytokinetic cells from 1 dpf possess dense and presumably heterochromatic structures located near the cleavage furrow with filamentous extensions oriented toward the enveloped nuclei (green—SYTO-24 stained DNA). Cells with multiple micronuclei from 1 dpf (E) and 2 dpf (F) stained with SYTO-24.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4920378&req=5

pgen.1006103.g005: Variation in the content and form of eliminated DNA indicates stepwise loss of DNA.(A-C) FISH of the Germ1 probe to anaphase chromosomes from PACT-cleared embryos. (A) A representative anaphase spread from 1 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). A majority of signals corresponding to the Germ1 repeat co-migrate with retained chromosomes, and a relatively small conglomerate of chromatin is localized to the equatorial region. (B) A representative anaphase spread from 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Lagging chromosomes are enriched with Germ1 while retained chromosomes have only a single pair of Germ1 signals. (C) Germ1-negative lagging chromatin at 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Germ1 hybridizes to a pair of signals on retained chromosomes that are associated with a relatively small conglomerate of lagging chromatin that lacks Germ1 hybridization. (D-F) Late mitotic events in cells undergoing chromosome elimination. (D) Cytokinetic cells from 1 dpf possess dense and presumably heterochromatic structures located near the cleavage furrow with filamentous extensions oriented toward the enveloped nuclei (green—SYTO-24 stained DNA). Cells with multiple micronuclei from 1 dpf (E) and 2 dpf (F) stained with SYTO-24.

Mentions: In addition to these large and longitudinally stretched segments, we also observed globular (presumable acentric) conglomerates of chromatin localized to the equatorial region (Fig 2C, see also Fig 5C). The presence of these conglomerates lends support to the idea that recombinational processes (intra- or inter-chromosomal) or DNA breakage contributes to PGR [4]. It seems plausible that these acentric fragments could be driven toward the equatorial region by the same polar ejection forces that normally act to orient chromosome arms during cell division [41]. The observation that eliminated material consists of both entire chromosomes and smaller chromosomal fragments mirrors observations from hagfish and parasitic nematodes, wherein both entire chromosomes and chromosomal fragments are lost from somatic lineages [2, 19, 42]. To shed further light on patterns of DNA breakage during PGR, we performed immunolabeling with an antibody to the histone variant γ-H2AX, which binds double stranded DNA breaks and recruits repair machinery [43, 44], and employed fluorescent TDT-mediated dUTP nick-end labeling (TUNEL) labeling to more generally detect DNA breaks. Although all other histone variants yielded interpretable signals, attempts to immunolabel γ-H2AX yielded no signal in embryos at 1–5 dpf. The absence of γ-H2AX immunolabeling could reflect either a paucity of double stranded breaks or failure to react with a lamprey γ-H2AX homolog. On the other hand, TUNEL labeling yielded strong and reproducible staining that was localized exclusively to MNi (S7 Fig). Given evidence that MNi represent the last visible sites of eliminated DNA, it seems plausible that TUNEL labeling reflects the degradation of germline-specific DNA within MNi. Taken together these observations indicate that DNA elimination proceeds through an ordered series of events, wherein germline-specific sequences 1) are initially slated for elimination during early anaphase (perhaps metaphase), 2) exhibit slower poleward movement in comparison to retained chromosomes and 3) condense to form MNi where they are methylated and ultimately degraded.


Cellular and Molecular Features of Developmentally Programmed Genome Rearrangement in a Vertebrate (Sea Lamprey: Petromyzon marinus).

Timoshevskiy VA, Herdy JR, Keinath MC, Smith JJ - PLoS Genet. (2016)

Variation in the content and form of eliminated DNA indicates stepwise loss of DNA.(A-C) FISH of the Germ1 probe to anaphase chromosomes from PACT-cleared embryos. (A) A representative anaphase spread from 1 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). A majority of signals corresponding to the Germ1 repeat co-migrate with retained chromosomes, and a relatively small conglomerate of chromatin is localized to the equatorial region. (B) A representative anaphase spread from 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Lagging chromosomes are enriched with Germ1 while retained chromosomes have only a single pair of Germ1 signals. (C) Germ1-negative lagging chromatin at 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Germ1 hybridizes to a pair of signals on retained chromosomes that are associated with a relatively small conglomerate of lagging chromatin that lacks Germ1 hybridization. (D-F) Late mitotic events in cells undergoing chromosome elimination. (D) Cytokinetic cells from 1 dpf possess dense and presumably heterochromatic structures located near the cleavage furrow with filamentous extensions oriented toward the enveloped nuclei (green—SYTO-24 stained DNA). Cells with multiple micronuclei from 1 dpf (E) and 2 dpf (F) stained with SYTO-24.
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4920378&req=5

pgen.1006103.g005: Variation in the content and form of eliminated DNA indicates stepwise loss of DNA.(A-C) FISH of the Germ1 probe to anaphase chromosomes from PACT-cleared embryos. (A) A representative anaphase spread from 1 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). A majority of signals corresponding to the Germ1 repeat co-migrate with retained chromosomes, and a relatively small conglomerate of chromatin is localized to the equatorial region. (B) A representative anaphase spread from 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Lagging chromosomes are enriched with Germ1 while retained chromosomes have only a single pair of Germ1 signals. (C) Germ1-negative lagging chromatin at 2 dpf (right panel: red—Germ1, blue—DAPI; left panel: DAPI). Germ1 hybridizes to a pair of signals on retained chromosomes that are associated with a relatively small conglomerate of lagging chromatin that lacks Germ1 hybridization. (D-F) Late mitotic events in cells undergoing chromosome elimination. (D) Cytokinetic cells from 1 dpf possess dense and presumably heterochromatic structures located near the cleavage furrow with filamentous extensions oriented toward the enveloped nuclei (green—SYTO-24 stained DNA). Cells with multiple micronuclei from 1 dpf (E) and 2 dpf (F) stained with SYTO-24.
Mentions: In addition to these large and longitudinally stretched segments, we also observed globular (presumable acentric) conglomerates of chromatin localized to the equatorial region (Fig 2C, see also Fig 5C). The presence of these conglomerates lends support to the idea that recombinational processes (intra- or inter-chromosomal) or DNA breakage contributes to PGR [4]. It seems plausible that these acentric fragments could be driven toward the equatorial region by the same polar ejection forces that normally act to orient chromosome arms during cell division [41]. The observation that eliminated material consists of both entire chromosomes and smaller chromosomal fragments mirrors observations from hagfish and parasitic nematodes, wherein both entire chromosomes and chromosomal fragments are lost from somatic lineages [2, 19, 42]. To shed further light on patterns of DNA breakage during PGR, we performed immunolabeling with an antibody to the histone variant γ-H2AX, which binds double stranded DNA breaks and recruits repair machinery [43, 44], and employed fluorescent TDT-mediated dUTP nick-end labeling (TUNEL) labeling to more generally detect DNA breaks. Although all other histone variants yielded interpretable signals, attempts to immunolabel γ-H2AX yielded no signal in embryos at 1–5 dpf. The absence of γ-H2AX immunolabeling could reflect either a paucity of double stranded breaks or failure to react with a lamprey γ-H2AX homolog. On the other hand, TUNEL labeling yielded strong and reproducible staining that was localized exclusively to MNi (S7 Fig). Given evidence that MNi represent the last visible sites of eliminated DNA, it seems plausible that TUNEL labeling reflects the degradation of germline-specific DNA within MNi. Taken together these observations indicate that DNA elimination proceeds through an ordered series of events, wherein germline-specific sequences 1) are initially slated for elimination during early anaphase (perhaps metaphase), 2) exhibit slower poleward movement in comparison to retained chromosomes and 3) condense to form MNi where they are methylated and ultimately degraded.

Bottom Line: Programmed genome rearrangements (PGRs) result in the reproducible loss of ~20% of the genome from somatic cell lineages during early embryogenesis.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.During early embryogenesis a majority of micronuclei (~60%) show strong enrichment for repressive chromatin modifications (H3K9me3 and 5meC).

View Article: PubMed Central - PubMed

Affiliation: Department of Biology, University of Kentucky, Lexington, Kentucky, United States of America.

ABSTRACT
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.

No MeSH data available.


Related in: MedlinePlus