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The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis.

Wuelling M, Pasdziernik M, Moll CN, Thiesen AM, Schneider S, Johannes C, Vortkamp A - Cell Cycle (2013)

Bottom Line: Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation.Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase.Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Medical Biotechnology, Department of Developmental Biology, University Duisburg-Essen, Essen, Germany.

ABSTRACT
TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Here, we have identified a new function of Trps1 in controlling mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.

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Figure 7. Overexpression of Hdac4 rescues the G2/M-phase delay in Trps1-/- chondrocytes. (A and B) The level of H3K9ac in FACS sorted G2/M-phase cells after staining with DyeCycle green was quantified by western blot analysis (A). Acetylation is increased in unsorted (total cell extract) and isolated, G2/M-phase Trps1-/- chondrocytes. α-pH3 detection confirms enrichment of G2/M-phase cells. (B) The level of H3K9 acetylation in relation to total H3 is increased in sorted G2/M-phase cells (n =3; p* < 0.05). (C and D) Wild-type and Trps1-/- primary chondrocytes were transfected with a Flag-tagged Hdac4 and the overexpression was confirmed by western blotting (C). Cell cycle progression was analyzed by flow cytometry after BrdU and 7-AAD labeling. Hdac4 overexpression decreases H3K9 acetylation in Trps1-/- chondrocytes similar to wild-type levels, while H3 levels were not altered. (D) Hdac4 overexpression rescued the increased proportion of G2/M-phase cells in Trps1-/- mutants, but had little effect on wild-type chondrocytes (n =3; p* < 0.05). (E) Interaction of Trps1 with Hdac1 and Hdac4, or a complex of both, increases the histone deacetylase activity on histone H3. Loss of Trps1 reduces Hdac activity, thereby leading to hyperacetylated chromatin and disturbed chromatin condensation. Subsequently, chromosome segregation is impaired.
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Figure 7: Figure 7. Overexpression of Hdac4 rescues the G2/M-phase delay in Trps1-/- chondrocytes. (A and B) The level of H3K9ac in FACS sorted G2/M-phase cells after staining with DyeCycle green was quantified by western blot analysis (A). Acetylation is increased in unsorted (total cell extract) and isolated, G2/M-phase Trps1-/- chondrocytes. α-pH3 detection confirms enrichment of G2/M-phase cells. (B) The level of H3K9 acetylation in relation to total H3 is increased in sorted G2/M-phase cells (n =3; p* < 0.05). (C and D) Wild-type and Trps1-/- primary chondrocytes were transfected with a Flag-tagged Hdac4 and the overexpression was confirmed by western blotting (C). Cell cycle progression was analyzed by flow cytometry after BrdU and 7-AAD labeling. Hdac4 overexpression decreases H3K9 acetylation in Trps1-/- chondrocytes similar to wild-type levels, while H3 levels were not altered. (D) Hdac4 overexpression rescued the increased proportion of G2/M-phase cells in Trps1-/- mutants, but had little effect on wild-type chondrocytes (n =3; p* < 0.05). (E) Interaction of Trps1 with Hdac1 and Hdac4, or a complex of both, increases the histone deacetylase activity on histone H3. Loss of Trps1 reduces Hdac activity, thereby leading to hyperacetylated chromatin and disturbed chromatin condensation. Subsequently, chromosome segregation is impaired.

Mentions: To further enrich the population of chondrocytes in G2/M-phase, we used Vybrant DyeCycleGreen, which incorporates into DNA in living cells, thereby allowing the subsequent isolation of mitotic chondrocytes according to their DNA content by FACS (Fig. S3B and C). We isolated cells in G2/M-phase from three Trps1-/- and wild-type mice per experiment. Flow cytometric re-analysis of a subset of isolated G2/M-phase cells confirmed an enrichment to about 90% in both genotypes (Fig. S3D and E). We next quantified acetylated H3K9 in relation to total H3 protein by western blotting (Fig. 7A). We found the ratio of acetylated H3K9 to total H3 to be significantly increased from 0.6 in wild-type cells to 0.83 in Trps1-/- chondrocytes (Fig. 7B), demonstrating that the hyperacetylated chromatin is maintained during mitosis.


The multi zinc-finger protein Trps1 acts as a regulator of histone deacetylation during mitosis.

Wuelling M, Pasdziernik M, Moll CN, Thiesen AM, Schneider S, Johannes C, Vortkamp A - Cell Cycle (2013)

Figure 7. Overexpression of Hdac4 rescues the G2/M-phase delay in Trps1-/- chondrocytes. (A and B) The level of H3K9ac in FACS sorted G2/M-phase cells after staining with DyeCycle green was quantified by western blot analysis (A). Acetylation is increased in unsorted (total cell extract) and isolated, G2/M-phase Trps1-/- chondrocytes. α-pH3 detection confirms enrichment of G2/M-phase cells. (B) The level of H3K9 acetylation in relation to total H3 is increased in sorted G2/M-phase cells (n =3; p* < 0.05). (C and D) Wild-type and Trps1-/- primary chondrocytes were transfected with a Flag-tagged Hdac4 and the overexpression was confirmed by western blotting (C). Cell cycle progression was analyzed by flow cytometry after BrdU and 7-AAD labeling. Hdac4 overexpression decreases H3K9 acetylation in Trps1-/- chondrocytes similar to wild-type levels, while H3 levels were not altered. (D) Hdac4 overexpression rescued the increased proportion of G2/M-phase cells in Trps1-/- mutants, but had little effect on wild-type chondrocytes (n =3; p* < 0.05). (E) Interaction of Trps1 with Hdac1 and Hdac4, or a complex of both, increases the histone deacetylase activity on histone H3. Loss of Trps1 reduces Hdac activity, thereby leading to hyperacetylated chromatin and disturbed chromatin condensation. Subsequently, chromosome segregation is impaired.
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Figure 7: Figure 7. Overexpression of Hdac4 rescues the G2/M-phase delay in Trps1-/- chondrocytes. (A and B) The level of H3K9ac in FACS sorted G2/M-phase cells after staining with DyeCycle green was quantified by western blot analysis (A). Acetylation is increased in unsorted (total cell extract) and isolated, G2/M-phase Trps1-/- chondrocytes. α-pH3 detection confirms enrichment of G2/M-phase cells. (B) The level of H3K9 acetylation in relation to total H3 is increased in sorted G2/M-phase cells (n =3; p* < 0.05). (C and D) Wild-type and Trps1-/- primary chondrocytes were transfected with a Flag-tagged Hdac4 and the overexpression was confirmed by western blotting (C). Cell cycle progression was analyzed by flow cytometry after BrdU and 7-AAD labeling. Hdac4 overexpression decreases H3K9 acetylation in Trps1-/- chondrocytes similar to wild-type levels, while H3 levels were not altered. (D) Hdac4 overexpression rescued the increased proportion of G2/M-phase cells in Trps1-/- mutants, but had little effect on wild-type chondrocytes (n =3; p* < 0.05). (E) Interaction of Trps1 with Hdac1 and Hdac4, or a complex of both, increases the histone deacetylase activity on histone H3. Loss of Trps1 reduces Hdac activity, thereby leading to hyperacetylated chromatin and disturbed chromatin condensation. Subsequently, chromosome segregation is impaired.
Mentions: To further enrich the population of chondrocytes in G2/M-phase, we used Vybrant DyeCycleGreen, which incorporates into DNA in living cells, thereby allowing the subsequent isolation of mitotic chondrocytes according to their DNA content by FACS (Fig. S3B and C). We isolated cells in G2/M-phase from three Trps1-/- and wild-type mice per experiment. Flow cytometric re-analysis of a subset of isolated G2/M-phase cells confirmed an enrichment to about 90% in both genotypes (Fig. S3D and E). We next quantified acetylated H3K9 in relation to total H3 protein by western blotting (Fig. 7A). We found the ratio of acetylated H3K9 to total H3 to be significantly increased from 0.6 in wild-type cells to 0.83 in Trps1-/- chondrocytes (Fig. 7B), demonstrating that the hyperacetylated chromatin is maintained during mitosis.

Bottom Line: Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation.Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase.Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.

View Article: PubMed Central - PubMed

Affiliation: Center for Medical Biotechnology, Department of Developmental Biology, University Duisburg-Essen, Essen, Germany.

ABSTRACT
TRPS1, the gene mutated in human "Tricho-Rhino-Phalangeal syndrome," encodes a multi zinc-finger nuclear regulator of chondrocyte proliferation and differentiation. Here, we have identified a new function of Trps1 in controlling mitotic progression in chondrocytes. Loss of Trps1 in mice leads to an increased proportion of cells arrested in mitosis and, subsequently, to chromosome segregation defects. Searching for the molecular basis of the defect, we found that Trps1 acts as regulator of histone deacetylation. Trps1 interacts with two histone deacetylases, Hdac1 and Hdac4, thereby increasing their activity. Loss of Trps1 results in histone H3 hyperacetylation, which is maintained during mitosis. Consequently, chromatin condensation and binding of HP1 is impaired, and Trps1-deficient chondrocytes accumulate in prometaphase. Overexpression of Hdac4 rescues the mitotic defect of Trps1-deficient chondrocytes, identifying Trps1 as an important regulator of chromatin deacetylation during mitosis in chondrocytes. Our data provide the first evidence that the control of mitosis can be linked to the regulation of chondrocyte differentiation by epigenetic consequences of altered Hdac activity.

Show MeSH
Related in: MedlinePlus