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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 6. Acetylation of H3K9 is increased in mitotic, Trps1-/- cells. (A–F and M) Immunofluorescent labeling of primary chondrocyte from wild-type (A–C) and Trps1-/- (D–F) mice using α-H3K9ac and α-y-Tubulin antibodies and DAPI counterstaining revealed increased H3K9 acetylation in metaphases of Trps1-/- mutants (D–F). (M) Quantification of the mean fluorescence intensity of H3K9 acetylation in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (G–L) Immunofluorescent staining of wild-type (G–I) and Trps1-/- chondrocytes (J–L) with an α-H3K9acS10p antibody revealed an increased level of acetylated pH3 in mitotic, Trps1-/- cells. Scale bar: 10 µm. (M–R) Immunofluorescent labeling with an α-HP1β antibody as marker for pericentric heterochromatin showed reduced staining in Trps1-/- cells (P–R) compared with wild-type chondrocytes (M–O). (A) Quantification of the mean fluorescence intensity of H3K9 acetylation in mitotic cells in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (B) Quantification of the mean fluorescence intensity of Hβ1 staining in mitotic cells in relation to DAPI (n =4; 30 mitoses per animal; p* = 0.00004)
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Figure 6: Figure 6. Acetylation of H3K9 is increased in mitotic, Trps1-/- cells. (A–F and M) Immunofluorescent labeling of primary chondrocyte from wild-type (A–C) and Trps1-/- (D–F) mice using α-H3K9ac and α-y-Tubulin antibodies and DAPI counterstaining revealed increased H3K9 acetylation in metaphases of Trps1-/- mutants (D–F). (M) Quantification of the mean fluorescence intensity of H3K9 acetylation in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (G–L) Immunofluorescent staining of wild-type (G–I) and Trps1-/- chondrocytes (J–L) with an α-H3K9acS10p antibody revealed an increased level of acetylated pH3 in mitotic, Trps1-/- cells. Scale bar: 10 µm. (M–R) Immunofluorescent labeling with an α-HP1β antibody as marker for pericentric heterochromatin showed reduced staining in Trps1-/- cells (P–R) compared with wild-type chondrocytes (M–O). (A) Quantification of the mean fluorescence intensity of H3K9 acetylation in mitotic cells in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (B) Quantification of the mean fluorescence intensity of Hβ1 staining in mitotic cells in relation to DAPI (n =4; 30 mitoses per animal; p* = 0.00004)

Mentions: We next asked if the increased H3 acetylation levels were maintained during mitosis and investigated H3K9 acetylation at distinct mitotic stages in primary chondrocyte cultures after immunofluorescent labeling with an α-H3K9ac antibody. As described for other cell types,6 H3K9 acetylation decreases during prophase and prometaphase, leading to reduced acetylation levels in metaphases of wild-type chondrocytes (Fig. 6A–C). In contrast, in Trps1-/- metaphases, H3K9 acetylation appeared to be increased compared with that in wild-type cells (Fig. 6D–F). To quantify the differences in H3K9 acetylation in mitotic cells, we measured the mean fluorescent intensity of acetylated H3K9 after immunofluorescent labeling in relation to DAPI-stained DNA in 100 mitotic cells per culture of 5 embryos per genotype. In mitotic wild-type chondrocytes, the ratio of acetylated H3K9 to DAPI was 3.55, while it was significantly increased to 4.53 in Trps1-/- cells (Fig. 6S).


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 6. Acetylation of H3K9 is increased in mitotic, Trps1-/- cells. (A–F and M) Immunofluorescent labeling of primary chondrocyte from wild-type (A–C) and Trps1-/- (D–F) mice using α-H3K9ac and α-y-Tubulin antibodies and DAPI counterstaining revealed increased H3K9 acetylation in metaphases of Trps1-/- mutants (D–F). (M) Quantification of the mean fluorescence intensity of H3K9 acetylation in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (G–L) Immunofluorescent staining of wild-type (G–I) and Trps1-/- chondrocytes (J–L) with an α-H3K9acS10p antibody revealed an increased level of acetylated pH3 in mitotic, Trps1-/- cells. Scale bar: 10 µm. (M–R) Immunofluorescent labeling with an α-HP1β antibody as marker for pericentric heterochromatin showed reduced staining in Trps1-/- cells (P–R) compared with wild-type chondrocytes (M–O). (A) Quantification of the mean fluorescence intensity of H3K9 acetylation in mitotic cells in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (B) Quantification of the mean fluorescence intensity of Hβ1 staining in mitotic cells in relation to DAPI (n =4; 30 mitoses per animal; p* = 0.00004)
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Figure 6: Figure 6. Acetylation of H3K9 is increased in mitotic, Trps1-/- cells. (A–F and M) Immunofluorescent labeling of primary chondrocyte from wild-type (A–C) and Trps1-/- (D–F) mice using α-H3K9ac and α-y-Tubulin antibodies and DAPI counterstaining revealed increased H3K9 acetylation in metaphases of Trps1-/- mutants (D–F). (M) Quantification of the mean fluorescence intensity of H3K9 acetylation in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (G–L) Immunofluorescent staining of wild-type (G–I) and Trps1-/- chondrocytes (J–L) with an α-H3K9acS10p antibody revealed an increased level of acetylated pH3 in mitotic, Trps1-/- cells. Scale bar: 10 µm. (M–R) Immunofluorescent labeling with an α-HP1β antibody as marker for pericentric heterochromatin showed reduced staining in Trps1-/- cells (P–R) compared with wild-type chondrocytes (M–O). (A) Quantification of the mean fluorescence intensity of H3K9 acetylation in mitotic cells in relation to DAPI (n =5; 100 mitoses per animal; p* = 0.000003). (B) Quantification of the mean fluorescence intensity of Hβ1 staining in mitotic cells in relation to DAPI (n =4; 30 mitoses per animal; p* = 0.00004)
Mentions: We next asked if the increased H3 acetylation levels were maintained during mitosis and investigated H3K9 acetylation at distinct mitotic stages in primary chondrocyte cultures after immunofluorescent labeling with an α-H3K9ac antibody. As described for other cell types,6 H3K9 acetylation decreases during prophase and prometaphase, leading to reduced acetylation levels in metaphases of wild-type chondrocytes (Fig. 6A–C). In contrast, in Trps1-/- metaphases, H3K9 acetylation appeared to be increased compared with that in wild-type cells (Fig. 6D–F). To quantify the differences in H3K9 acetylation in mitotic cells, we measured the mean fluorescent intensity of acetylated H3K9 after immunofluorescent labeling in relation to DAPI-stained DNA in 100 mitotic cells per culture of 5 embryos per genotype. In mitotic wild-type chondrocytes, the ratio of acetylated H3K9 to DAPI was 3.55, while it was significantly increased to 4.53 in Trps1-/- cells (Fig. 6S).

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