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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 4. Trps1 interacts with Hdac1 and Hdac4 and increases their activity. (A) Immunoprecipitation with an α-Hdac4 or α-Trps1 antibody followed by western blot analysis demonstrates an interaction of the endogenous proteins in ATDC5 cells. (B and C) Overexpression of Trps1 and Flag-tagged Hdac1 (B) or Hdac2 (C) in HEK293 EBNA cells, followed by immunoprecipitation with an α-Flag οr α-Trps1 antibody identifies an interaction of Trps1 with Hdac1, but not Hdac2. (In, input; Sn, supernatant; IP, immunopreciptiation). (D and E) Trps1 and Hdac4 were overexpressed in HEK293 EBNA cells and Hdac activity was analyzed using a fluorometric assay. (D) Western blot analysis confirms overexpression of both proteins. (E) Overexpression of Trps1 and Hdac4 increases Hdac activity by 9% and 15%, respectively, compared with an empty vector. Co-transfection of Hdac4 and Trps1 augments Hdac activity by 20%. TSA treatment reduced Hdac activity to 88%, representing loss of all class 1 and class 2 Hdac activity in the protein extracts (blue brackets); (n =3; p* < 0.05). (F) Analysis of Hdac activity in protein extracts of E16.5 limbs revealed reduced Hdac activity in Trps1-/- mutants compared with wild-type mice in vivo (n =4; p* < 0.03). (G) Primary chondrocytes were treated with TSA to block Hdac activity, and cell cycle distribution was measured by flow cytometry. TSA treatment decreases the numbers of cells in S- and increases the proportion of cells in G2/M-phase (n =3; p*S-phase < 0.004, p*G2/M-phase = 0.035).
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Figure 4: Figure 4. Trps1 interacts with Hdac1 and Hdac4 and increases their activity. (A) Immunoprecipitation with an α-Hdac4 or α-Trps1 antibody followed by western blot analysis demonstrates an interaction of the endogenous proteins in ATDC5 cells. (B and C) Overexpression of Trps1 and Flag-tagged Hdac1 (B) or Hdac2 (C) in HEK293 EBNA cells, followed by immunoprecipitation with an α-Flag οr α-Trps1 antibody identifies an interaction of Trps1 with Hdac1, but not Hdac2. (In, input; Sn, supernatant; IP, immunopreciptiation). (D and E) Trps1 and Hdac4 were overexpressed in HEK293 EBNA cells and Hdac activity was analyzed using a fluorometric assay. (D) Western blot analysis confirms overexpression of both proteins. (E) Overexpression of Trps1 and Hdac4 increases Hdac activity by 9% and 15%, respectively, compared with an empty vector. Co-transfection of Hdac4 and Trps1 augments Hdac activity by 20%. TSA treatment reduced Hdac activity to 88%, representing loss of all class 1 and class 2 Hdac activity in the protein extracts (blue brackets); (n =3; p* < 0.05). (F) Analysis of Hdac activity in protein extracts of E16.5 limbs revealed reduced Hdac activity in Trps1-/- mutants compared with wild-type mice in vivo (n =4; p* < 0.03). (G) Primary chondrocytes were treated with TSA to block Hdac activity, and cell cycle distribution was measured by flow cytometry. TSA treatment decreases the numbers of cells in S- and increases the proportion of cells in G2/M-phase (n =3; p*S-phase < 0.004, p*G2/M-phase = 0.035).

Mentions: So far, our experiments showed that loss of Trps1 leads to a decreased proportion of cells in S-phase combined with increased numbers of cells in mitosis. A similar phenotype has been observed after inhibition of histone deacetylases (Hdacs) in established cell lines.25,26 To test if inhibition of Hdac activity induces a comparable mitotic arrest in primary chondrocytes, we treated wild-type and Trps1-/- chondrocytes with the Hdac inhibitor Trichostatin A (TSA). In accordance with earlier studies,27,26 TSA treatment reduced the proportion of primary chondrocytes in S-phase from 23.7 to 11.5%, while the percentage of cells in G2/M-phase was increased from 12.2 to 21.1% (Fig. 4G). Hdac inhibition thus induced a similar cell cycle defect in wild-type chondrocytes as observed in Trps1-/- mutants. We next asked if Trps1 might interact with Hdac proteins. Hdac4 expression overlaps with that of Trps1 in proliferating and prehypertrophic chondrocytes, interacts, and represses Runx2 similar to Trps1 and, therefore, seemed to be a good candidate for such an interaction.29-31 First, we confirmed the endogenous expression of both proteins in the chondrogenic cell line, ATDC5 (Fig. 4A, input). After immunoprecipitation using either an α-Hdac4 or α-Trps1 antibody, western blot analysis demonstrated an interaction of Trps1 with Hdac4 in a protein complex (Fig. 4A). Next, we asked if other Hdacs might similarly interact with Trps1. Hdac1 and Hdac2 are ubiquitously expressed and share redundant functions in most tissues.32 Co-expression of Flag-tagged Hdac1 or Flag-tagged Hdac2 protein with Trps1 in HEK293 EBNA cells followed by immunoprecipitation with either an α-Flag or an α-Trps1 antibody revealed an interaction of Trps1 with Hdac1 (Fig. 4B) but not with Hdac2 (Fig. 4C), demonstrating the specificity of the interaction. As Trps1 interacts with Hdac1, but not with the highly homologous Hdac2 protein, we ask which protein domain mediates the interaction with Trps1. Surprisingly, immunoprecipitation of GFP-tagged deletion constructs of Hdac1 and Hdac2 and Flag-tagged Trps1 revealed that the N-terminal region, containing the conserved Hdac domain but not the C-terminal domain of Hdac1, is required for the interaction with Trps1 (Fig. S2C, E, and F). In contrast the N-terminal region of Hdac2 did not interact with Trps1 (Fig. S2D).


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 4. Trps1 interacts with Hdac1 and Hdac4 and increases their activity. (A) Immunoprecipitation with an α-Hdac4 or α-Trps1 antibody followed by western blot analysis demonstrates an interaction of the endogenous proteins in ATDC5 cells. (B and C) Overexpression of Trps1 and Flag-tagged Hdac1 (B) or Hdac2 (C) in HEK293 EBNA cells, followed by immunoprecipitation with an α-Flag οr α-Trps1 antibody identifies an interaction of Trps1 with Hdac1, but not Hdac2. (In, input; Sn, supernatant; IP, immunopreciptiation). (D and E) Trps1 and Hdac4 were overexpressed in HEK293 EBNA cells and Hdac activity was analyzed using a fluorometric assay. (D) Western blot analysis confirms overexpression of both proteins. (E) Overexpression of Trps1 and Hdac4 increases Hdac activity by 9% and 15%, respectively, compared with an empty vector. Co-transfection of Hdac4 and Trps1 augments Hdac activity by 20%. TSA treatment reduced Hdac activity to 88%, representing loss of all class 1 and class 2 Hdac activity in the protein extracts (blue brackets); (n =3; p* < 0.05). (F) Analysis of Hdac activity in protein extracts of E16.5 limbs revealed reduced Hdac activity in Trps1-/- mutants compared with wild-type mice in vivo (n =4; p* < 0.03). (G) Primary chondrocytes were treated with TSA to block Hdac activity, and cell cycle distribution was measured by flow cytometry. TSA treatment decreases the numbers of cells in S- and increases the proportion of cells in G2/M-phase (n =3; p*S-phase < 0.004, p*G2/M-phase = 0.035).
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Figure 4: Figure 4. Trps1 interacts with Hdac1 and Hdac4 and increases their activity. (A) Immunoprecipitation with an α-Hdac4 or α-Trps1 antibody followed by western blot analysis demonstrates an interaction of the endogenous proteins in ATDC5 cells. (B and C) Overexpression of Trps1 and Flag-tagged Hdac1 (B) or Hdac2 (C) in HEK293 EBNA cells, followed by immunoprecipitation with an α-Flag οr α-Trps1 antibody identifies an interaction of Trps1 with Hdac1, but not Hdac2. (In, input; Sn, supernatant; IP, immunopreciptiation). (D and E) Trps1 and Hdac4 were overexpressed in HEK293 EBNA cells and Hdac activity was analyzed using a fluorometric assay. (D) Western blot analysis confirms overexpression of both proteins. (E) Overexpression of Trps1 and Hdac4 increases Hdac activity by 9% and 15%, respectively, compared with an empty vector. Co-transfection of Hdac4 and Trps1 augments Hdac activity by 20%. TSA treatment reduced Hdac activity to 88%, representing loss of all class 1 and class 2 Hdac activity in the protein extracts (blue brackets); (n =3; p* < 0.05). (F) Analysis of Hdac activity in protein extracts of E16.5 limbs revealed reduced Hdac activity in Trps1-/- mutants compared with wild-type mice in vivo (n =4; p* < 0.03). (G) Primary chondrocytes were treated with TSA to block Hdac activity, and cell cycle distribution was measured by flow cytometry. TSA treatment decreases the numbers of cells in S- and increases the proportion of cells in G2/M-phase (n =3; p*S-phase < 0.004, p*G2/M-phase = 0.035).
Mentions: So far, our experiments showed that loss of Trps1 leads to a decreased proportion of cells in S-phase combined with increased numbers of cells in mitosis. A similar phenotype has been observed after inhibition of histone deacetylases (Hdacs) in established cell lines.25,26 To test if inhibition of Hdac activity induces a comparable mitotic arrest in primary chondrocytes, we treated wild-type and Trps1-/- chondrocytes with the Hdac inhibitor Trichostatin A (TSA). In accordance with earlier studies,27,26 TSA treatment reduced the proportion of primary chondrocytes in S-phase from 23.7 to 11.5%, while the percentage of cells in G2/M-phase was increased from 12.2 to 21.1% (Fig. 4G). Hdac inhibition thus induced a similar cell cycle defect in wild-type chondrocytes as observed in Trps1-/- mutants. We next asked if Trps1 might interact with Hdac proteins. Hdac4 expression overlaps with that of Trps1 in proliferating and prehypertrophic chondrocytes, interacts, and represses Runx2 similar to Trps1 and, therefore, seemed to be a good candidate for such an interaction.29-31 First, we confirmed the endogenous expression of both proteins in the chondrogenic cell line, ATDC5 (Fig. 4A, input). After immunoprecipitation using either an α-Hdac4 or α-Trps1 antibody, western blot analysis demonstrated an interaction of Trps1 with Hdac4 in a protein complex (Fig. 4A). Next, we asked if other Hdacs might similarly interact with Trps1. Hdac1 and Hdac2 are ubiquitously expressed and share redundant functions in most tissues.32 Co-expression of Flag-tagged Hdac1 or Flag-tagged Hdac2 protein with Trps1 in HEK293 EBNA cells followed by immunoprecipitation with either an α-Flag or an α-Trps1 antibody revealed an interaction of Trps1 with Hdac1 (Fig. 4B) but not with Hdac2 (Fig. 4C), demonstrating the specificity of the interaction. As Trps1 interacts with Hdac1, but not with the highly homologous Hdac2 protein, we ask which protein domain mediates the interaction with Trps1. Surprisingly, immunoprecipitation of GFP-tagged deletion constructs of Hdac1 and Hdac2 and Flag-tagged Trps1 revealed that the N-terminal region, containing the conserved Hdac domain but not the C-terminal domain of Hdac1, is required for the interaction with Trps1 (Fig. S2C, E, and F). In contrast the N-terminal region of Hdac2 did not interact with Trps1 (Fig. S2D).

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