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Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation.

Ancelin K, Syx L, Borensztein M, Ranisavljevic N, Vassilev I, Briseño-Roa L, Liu T, Metzger E, Servant N, Barillot E, Chen CJ, Schüle R, Heard E - Elife (2016)

Bottom Line: Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency.The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated.At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Paris, France.

ABSTRACT
Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.

No MeSH data available.


Related in: MedlinePlus

EdU labeling and γH2AX immunofluorescence of Kdm1a mutant versus control two-cell stage embryos.(A) Two-cell stage embryos (f/wt and Δm/wt) were collected at 40–41 hr post hCG and culture for EdU pulse for 45 min. After fixation, they were analyzed by Click -It reaction to reveal the incorporated EdU (shown in green), then subjected to immunostaining for γH2AX (shown in red). DNA is counterstained with DAPI (in blue). Shown are maximal projection of confocal z series of representative embryos. f/wt control embryo n = 17 and Δm/wt mutant embryo n = 11. Scale bar, 10 μm. (B) quantification of embryo percentage according to the presence of EdU incorporation or γH2AX immunofluorescence.DOI:http://dx.doi.org/10.7554/eLife.08851.015
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fig5s2: EdU labeling and γH2AX immunofluorescence of Kdm1a mutant versus control two-cell stage embryos.(A) Two-cell stage embryos (f/wt and Δm/wt) were collected at 40–41 hr post hCG and culture for EdU pulse for 45 min. After fixation, they were analyzed by Click -It reaction to reveal the incorporated EdU (shown in green), then subjected to immunostaining for γH2AX (shown in red). DNA is counterstained with DAPI (in blue). Shown are maximal projection of confocal z series of representative embryos. f/wt control embryo n = 17 and Δm/wt mutant embryo n = 11. Scale bar, 10 μm. (B) quantification of embryo percentage according to the presence of EdU incorporation or γH2AX immunofluorescence.DOI:http://dx.doi.org/10.7554/eLife.08851.015

Mentions: To investigate whether the increase in nascent LINE-1 transcription observed might correspond to full length LINE-1 elements, we assessed by IF for the presence of ORF1, one of the two LINE-1 encoded proteins. At the two-cell stage, we found an approximately four-fold increase in the proportion of △m/wt embryos displaying a stronger IF signal, notably in the nucleus (Figure 5F). These results suggest that the LINE-1 deregulation observed at the RNA level might indeed lead to the production and nuclear import of increased levels of LINE-1 ORF1 proteins. We next investigated whether expression of such proteins from transposable elements would have any consequences. We thus performed γH2AX IF staining to assess whether increased DNA damage signalling could be seen in △m/wt compared to f/wt embryos (Figure 5G; Figure 5—figure supplement 2). Half of the mutant embryos displayed a stronger staining for γH2AX, with a significant increase compared to controls (Figure 5G). We also assessed whether this accumulation of γH2AX signals could also be related to replication delays, as reported previously in the case of maternal loss of two components of the polycomb complex PRC1 (Posfai et al., 2012). We performed EdU pulse treatment (a nucleoside analog of thymidine incorporated into DNA) in two-cell embryos, at a stage when they have normally completed S phase (40–41 hr post hCG injection). This revealed that S phase is delayed in the △m/wt embryos given the incorporation of EdU in the mutants, while none of the control embryos used in parallel were stained (Figure 5—figure supplement 2). All the mutant embryos delayed in their replication displayed concomitantly intense γH2AX signals. However, 38% of the mutant embryos did not show any EdU incorporation, indicating that they exit S phase, yet, they still show high levels of γH2AX signals. Finally, although, no significant enrichment was directly found for DNA damage pathways when running our GO analysis (Figure 4E), many genes related to DNA damage repair were upregulated (Supplementary file 2). Taken all together, these results suggest that the elevated DNA damage signalling observed could be independent from replication defaults in KDM1A maternally depleted embryos, but might be related either to changes in transcript levels for DNA damage genes or else to the observed increase in LINE-1 activity in Kdm1a mutant embryos at this stage.


Maternal LSD1/KDM1A is an essential regulator of chromatin and transcription landscapes during zygotic genome activation.

Ancelin K, Syx L, Borensztein M, Ranisavljevic N, Vassilev I, Briseño-Roa L, Liu T, Metzger E, Servant N, Barillot E, Chen CJ, Schüle R, Heard E - Elife (2016)

EdU labeling and γH2AX immunofluorescence of Kdm1a mutant versus control two-cell stage embryos.(A) Two-cell stage embryos (f/wt and Δm/wt) were collected at 40–41 hr post hCG and culture for EdU pulse for 45 min. After fixation, they were analyzed by Click -It reaction to reveal the incorporated EdU (shown in green), then subjected to immunostaining for γH2AX (shown in red). DNA is counterstained with DAPI (in blue). Shown are maximal projection of confocal z series of representative embryos. f/wt control embryo n = 17 and Δm/wt mutant embryo n = 11. Scale bar, 10 μm. (B) quantification of embryo percentage according to the presence of EdU incorporation or γH2AX immunofluorescence.DOI:http://dx.doi.org/10.7554/eLife.08851.015
© Copyright Policy
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC4829419&req=5

fig5s2: EdU labeling and γH2AX immunofluorescence of Kdm1a mutant versus control two-cell stage embryos.(A) Two-cell stage embryos (f/wt and Δm/wt) were collected at 40–41 hr post hCG and culture for EdU pulse for 45 min. After fixation, they were analyzed by Click -It reaction to reveal the incorporated EdU (shown in green), then subjected to immunostaining for γH2AX (shown in red). DNA is counterstained with DAPI (in blue). Shown are maximal projection of confocal z series of representative embryos. f/wt control embryo n = 17 and Δm/wt mutant embryo n = 11. Scale bar, 10 μm. (B) quantification of embryo percentage according to the presence of EdU incorporation or γH2AX immunofluorescence.DOI:http://dx.doi.org/10.7554/eLife.08851.015
Mentions: To investigate whether the increase in nascent LINE-1 transcription observed might correspond to full length LINE-1 elements, we assessed by IF for the presence of ORF1, one of the two LINE-1 encoded proteins. At the two-cell stage, we found an approximately four-fold increase in the proportion of △m/wt embryos displaying a stronger IF signal, notably in the nucleus (Figure 5F). These results suggest that the LINE-1 deregulation observed at the RNA level might indeed lead to the production and nuclear import of increased levels of LINE-1 ORF1 proteins. We next investigated whether expression of such proteins from transposable elements would have any consequences. We thus performed γH2AX IF staining to assess whether increased DNA damage signalling could be seen in △m/wt compared to f/wt embryos (Figure 5G; Figure 5—figure supplement 2). Half of the mutant embryos displayed a stronger staining for γH2AX, with a significant increase compared to controls (Figure 5G). We also assessed whether this accumulation of γH2AX signals could also be related to replication delays, as reported previously in the case of maternal loss of two components of the polycomb complex PRC1 (Posfai et al., 2012). We performed EdU pulse treatment (a nucleoside analog of thymidine incorporated into DNA) in two-cell embryos, at a stage when they have normally completed S phase (40–41 hr post hCG injection). This revealed that S phase is delayed in the △m/wt embryos given the incorporation of EdU in the mutants, while none of the control embryos used in parallel were stained (Figure 5—figure supplement 2). All the mutant embryos delayed in their replication displayed concomitantly intense γH2AX signals. However, 38% of the mutant embryos did not show any EdU incorporation, indicating that they exit S phase, yet, they still show high levels of γH2AX signals. Finally, although, no significant enrichment was directly found for DNA damage pathways when running our GO analysis (Figure 4E), many genes related to DNA damage repair were upregulated (Supplementary file 2). Taken all together, these results suggest that the elevated DNA damage signalling observed could be independent from replication defaults in KDM1A maternally depleted embryos, but might be related either to changes in transcript levels for DNA damage genes or else to the observed increase in LINE-1 activity in Kdm1a mutant embryos at this stage.

Bottom Line: Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency.The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated.At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced.

View Article: PubMed Central - PubMed

Affiliation: Institut Curie, Paris, France.

ABSTRACT
Upon fertilization, the highly specialised sperm and oocyte genomes are remodelled to confer totipotency. The mechanisms of the dramatic reprogramming events that occur have remained unknown, and presumed roles of histone modifying enzymes are just starting to be elucidated. Here, we explore the function of the oocyte-inherited pool of a histone H3K4 and K9 demethylase, LSD1/KDM1A during early mouse development. KDM1A deficiency results in developmental arrest by the two-cell stage, accompanied by dramatic and stepwise alterations in H3K9 and H3K4 methylation patterns. At the transcriptional level, the switch of the maternal-to-zygotic transition fails to be induced properly and LINE-1 retrotransposons are not properly silenced. We propose that KDM1A plays critical roles in establishing the correct epigenetic landscape of the zygote upon fertilization, in preserving genome integrity and in initiating new patterns of genome expression that drive early mouse development.

No MeSH data available.


Related in: MedlinePlus