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Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte.

Han L, Ge J, Zhang L, Ma R, Hou X, Li B, Moley K, Wang Q - Sci Rep (2015)

Bottom Line: We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes.Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function.Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China.

ABSTRACT
Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

No MeSH data available.


Effects of Sirt6 knockdown on the lysine acetylation of histones in mouse oocytes.Control and Sirt6-MO oocytes at GV and metaphase II stages were immunostained with an array of antibodies specifically against different acetylated histones (green), and co-stained with PI for chromosomes (red). Representative confocal images of (A) acetylated H3K9 (H3K9ac), (C) acetylated H3K56 (H3K56ac), (E) acetylated H3K14 (H3K14ac), (G) acetylated H4K12 (H4K12ac), and (I) acetylated H4K16 (H4K16ac) in control and Sirt6-MO oocytes. (B,D,F,H,J) Quantification of the data shown in panel (A,C,E,G,I), respectively. At least 35 oocytes for each group were analyzed, and the experiments were repeated 3 times. Error bars indicate ± SD. *p < 0.05 vs. controls.
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f6: Effects of Sirt6 knockdown on the lysine acetylation of histones in mouse oocytes.Control and Sirt6-MO oocytes at GV and metaphase II stages were immunostained with an array of antibodies specifically against different acetylated histones (green), and co-stained with PI for chromosomes (red). Representative confocal images of (A) acetylated H3K9 (H3K9ac), (C) acetylated H3K56 (H3K56ac), (E) acetylated H3K14 (H3K14ac), (G) acetylated H4K12 (H4K12ac), and (I) acetylated H4K16 (H4K16ac) in control and Sirt6-MO oocytes. (B,D,F,H,J) Quantification of the data shown in panel (A,C,E,G,I), respectively. At least 35 oocytes for each group were analyzed, and the experiments were repeated 3 times. Error bars indicate ± SD. *p < 0.05 vs. controls.

Mentions: The effect of Sirt6 knockdown on meiotic structures prompted us to search for the underlying mechanisms that would explain these phenotypes. Sirt6 was shown to possess NAD+-dependent histone deacetylase activity for H3K9 and H3K56. Of note, Sirt6 functions in telomere replication via deacetylating H3K9 or K56, ultimately modulating genomic stability, chromosomal end-to-end fusions, and premature cellular senescence121314. On the other hand, the dramatic changes in histone acetylation have been observed during mammalian oocyte maturation2324. On the basis of these findings, we hypothesized that Sirt6 depletion might alter the status of histone acetylation in oocyte meiosis. To test this hypothesis, we first examined the global acetylation levels of H3K9 and H3K56 in Sirt6-MO oocytes. Unexpectedly, immunostaining with antibodies against specific histone residues showed that H3K9 and H3K56 acetylation remained unchanged in Sirt6-MO oocytes at both GV and metaphase stages when compared with their controls (Fig. 6A–D). Next, we further evaluate the effects of Sirt6 on the acetylation status of several other lysines on histone H3 and H4 (H3K14, H4K12, and H4K16) in oocytes. Remarkably, a specific and drastic increase in H4K16 acetylation was detected in Sirt6-MO oocytes, particularly at metaphase stage (Fig. 6I,J). By contrast, oocytes depleted of Sirt6 had little effects on both H3K14 and H4K12 acetylation (Fig. 6E–H). It is worth noting that hypoacetylation of H4K16 is essential for the establishment of functional kinetochore in both mitotic cells and meiotic oocytes252627. Collectively, Sirt6 knockdown induced hyperacetylation of H4K16 in mouse oocytes, which may in turn perturb the chromatin conformation and kinetochore function, contributing to, at least in part, the spindle defects and chromosome misalignment during meiosis.


Sirt6 depletion causes spindle defects and chromosome misalignment during meiosis of mouse oocyte.

Han L, Ge J, Zhang L, Ma R, Hou X, Li B, Moley K, Wang Q - Sci Rep (2015)

Effects of Sirt6 knockdown on the lysine acetylation of histones in mouse oocytes.Control and Sirt6-MO oocytes at GV and metaphase II stages were immunostained with an array of antibodies specifically against different acetylated histones (green), and co-stained with PI for chromosomes (red). Representative confocal images of (A) acetylated H3K9 (H3K9ac), (C) acetylated H3K56 (H3K56ac), (E) acetylated H3K14 (H3K14ac), (G) acetylated H4K12 (H4K12ac), and (I) acetylated H4K16 (H4K16ac) in control and Sirt6-MO oocytes. (B,D,F,H,J) Quantification of the data shown in panel (A,C,E,G,I), respectively. At least 35 oocytes for each group were analyzed, and the experiments were repeated 3 times. Error bars indicate ± SD. *p < 0.05 vs. controls.
© Copyright Policy - open-access
Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4612726&req=5

f6: Effects of Sirt6 knockdown on the lysine acetylation of histones in mouse oocytes.Control and Sirt6-MO oocytes at GV and metaphase II stages were immunostained with an array of antibodies specifically against different acetylated histones (green), and co-stained with PI for chromosomes (red). Representative confocal images of (A) acetylated H3K9 (H3K9ac), (C) acetylated H3K56 (H3K56ac), (E) acetylated H3K14 (H3K14ac), (G) acetylated H4K12 (H4K12ac), and (I) acetylated H4K16 (H4K16ac) in control and Sirt6-MO oocytes. (B,D,F,H,J) Quantification of the data shown in panel (A,C,E,G,I), respectively. At least 35 oocytes for each group were analyzed, and the experiments were repeated 3 times. Error bars indicate ± SD. *p < 0.05 vs. controls.
Mentions: The effect of Sirt6 knockdown on meiotic structures prompted us to search for the underlying mechanisms that would explain these phenotypes. Sirt6 was shown to possess NAD+-dependent histone deacetylase activity for H3K9 and H3K56. Of note, Sirt6 functions in telomere replication via deacetylating H3K9 or K56, ultimately modulating genomic stability, chromosomal end-to-end fusions, and premature cellular senescence121314. On the other hand, the dramatic changes in histone acetylation have been observed during mammalian oocyte maturation2324. On the basis of these findings, we hypothesized that Sirt6 depletion might alter the status of histone acetylation in oocyte meiosis. To test this hypothesis, we first examined the global acetylation levels of H3K9 and H3K56 in Sirt6-MO oocytes. Unexpectedly, immunostaining with antibodies against specific histone residues showed that H3K9 and H3K56 acetylation remained unchanged in Sirt6-MO oocytes at both GV and metaphase stages when compared with their controls (Fig. 6A–D). Next, we further evaluate the effects of Sirt6 on the acetylation status of several other lysines on histone H3 and H4 (H3K14, H4K12, and H4K16) in oocytes. Remarkably, a specific and drastic increase in H4K16 acetylation was detected in Sirt6-MO oocytes, particularly at metaphase stage (Fig. 6I,J). By contrast, oocytes depleted of Sirt6 had little effects on both H3K14 and H4K12 acetylation (Fig. 6E–H). It is worth noting that hypoacetylation of H4K16 is essential for the establishment of functional kinetochore in both mitotic cells and meiotic oocytes252627. Collectively, Sirt6 knockdown induced hyperacetylation of H4K16 in mouse oocytes, which may in turn perturb the chromatin conformation and kinetochore function, contributing to, at least in part, the spindle defects and chromosome misalignment during meiosis.

Bottom Line: We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes.Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function.Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

View Article: PubMed Central - PubMed

Affiliation: State Key Laboratory of Reproductive Medicine, Nanjing Medical University, Nanjing 210029, China.

ABSTRACT
Sirt6, a member of the sirtuin family of NAD-dependent protein deacetylases, has been implicated in multiple biological processes. However, the roles of Sirt6 in meiosis have not been addressed. In the present study, by employing knockdown analysis in mouse oocytes, we evaluated the effects of Sirt6 on meiotic apparatus. We found that specific depletion of Sirt6 results in disruption of spindle morphology and chromosome alignment in oocytes. Consistent with this observation, incidence of aneuploidy is also markedly increased in Sirt6-depleted oocytes. Furthermore, confocal scanning showed that kinetochore-microtubule interaction, an important mechanism controlling chromosome segregation, is severely impaired in metaphase oocytes following Sirt6 knockdown. Unexpectedly, we discovered that Sirt6 modulates the acetylation status of histone H4K16 as their knockdown specifically induces the hyperacetylation of H4K16 in oocytes, which may be associated with the defective phenotypes described above via altering kinetochore function. Altogether, our data reveal a novel function of Sirt6 during oocyte meiosis and indicate a pathway regulating meiotic apparatus.

No MeSH data available.