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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 2.Trps1-/- chondrocytes display aberrant mitosis. (A–C) Representative flow cytometry of primary chondrocyte cultures from wild-type (A) and Trps1-/- mice (B) stained with α-pH3 antibody and Propidium Iodide (PI) revealed increased numbers of pH3-positive cells in Trps1-/- mutants. (C) Statistic evaluation of pH3 measurement (n =5; p* = 0.018). (D and E) Hematoxylin-Eosin (HE) staining of paraffin sections of E16.5 wild-type and Trps1-/- ulnae showing the region of proliferating chondrocytes. Parallel sections (F and G) were used for immunofluorescent detection of pH3-positive, mitotic cells (green) in wild-type (F and H) and Trps1-/- mice (G–I). (J) The statistic evaluation of the pH3-positive cells in vivo revealed increased numbers of mitotic cells (green, arrows) in Trps1-/- mutants. (n =5; p* = 0.025). (H and I) DAPI counterstaining of this sections showed asymmetric chromatin segregation in mitotic chondrocytes of Trps1-/- mutants (I, arrow). (K and L) Giemsa stained chromosome spreads of wild-type (K) and Trps1-/- mice (L) revealed increased numbers of aneuploid cells in Trps1-/- mutants. (M) Statistic evaluation of chromosome numbers in mitotic cells (n =4, 50 mitoses each; p* = 0.0028). Scale bar: 100 µm, (F and G), 10 µm, (I and J).
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Figure 2: Figure 2.Trps1-/- chondrocytes display aberrant mitosis. (A–C) Representative flow cytometry of primary chondrocyte cultures from wild-type (A) and Trps1-/- mice (B) stained with α-pH3 antibody and Propidium Iodide (PI) revealed increased numbers of pH3-positive cells in Trps1-/- mutants. (C) Statistic evaluation of pH3 measurement (n =5; p* = 0.018). (D and E) Hematoxylin-Eosin (HE) staining of paraffin sections of E16.5 wild-type and Trps1-/- ulnae showing the region of proliferating chondrocytes. Parallel sections (F and G) were used for immunofluorescent detection of pH3-positive, mitotic cells (green) in wild-type (F and H) and Trps1-/- mice (G–I). (J) The statistic evaluation of the pH3-positive cells in vivo revealed increased numbers of mitotic cells (green, arrows) in Trps1-/- mutants. (n =5; p* = 0.025). (H and I) DAPI counterstaining of this sections showed asymmetric chromatin segregation in mitotic chondrocytes of Trps1-/- mutants (I, arrow). (K and L) Giemsa stained chromosome spreads of wild-type (K) and Trps1-/- mice (L) revealed increased numbers of aneuploid cells in Trps1-/- mutants. (M) Statistic evaluation of chromosome numbers in mitotic cells (n =4, 50 mitoses each; p* = 0.0028). Scale bar: 100 µm, (F and G), 10 µm, (I and J).

Mentions: As loss of Trps1 leads to an accumulation of cells in G2/M-phase, we next investigated the phosphorylation of histone H3 at serine 10 (pH3), which serves as specific marker for mitotic cells.24 Flow cytometry of cells labeled with an α-pH3 antibody revealed that 3.5% wild-type chondrocytes in culture were pH3-positive (Fig. 2A and C), while in cultures of Trps1-/- mutants, the proportion of mitotic cells was increased to 7.3% (Fig. 2B and C). To examine if loss of Trps1 leads to a similar increase in mitotic cells in vivo, we quantified pH3-positive cells in the region of proliferating chondrocytes of E16.5 mouse ulnae (Fig. 2F–I). Comparable to our in vitro data, the number of pH3-positive cells was increased to 2.9% in Trps1-/- mutants, while 1.3% pH3-positive cells can be found in wild-type littermates (Fig. 2J). Trps1 thus activates mitotic progression in vitro and in vivo. Interestingly, at high magnification, irregular chromatin distribution could be observed in a subset of pH3-positive, Trps1-/- chondrocytes, likely reflecting disturbed chromosome segregation (Fig. 2I, arrow). We next investigated chromosome spreads of E12.5 primary chondrocyte cultures from Trps1-/- (Fig. 2L) and wild-type mice (Fig. 2K), and found the proportion of aneuploid cells with high chromosome numbers to be increased from 10.5% in wild-type mice to 22% in Trps1-/- mutants (Fig. 2M).


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 2.Trps1-/- chondrocytes display aberrant mitosis. (A–C) Representative flow cytometry of primary chondrocyte cultures from wild-type (A) and Trps1-/- mice (B) stained with α-pH3 antibody and Propidium Iodide (PI) revealed increased numbers of pH3-positive cells in Trps1-/- mutants. (C) Statistic evaluation of pH3 measurement (n =5; p* = 0.018). (D and E) Hematoxylin-Eosin (HE) staining of paraffin sections of E16.5 wild-type and Trps1-/- ulnae showing the region of proliferating chondrocytes. Parallel sections (F and G) were used for immunofluorescent detection of pH3-positive, mitotic cells (green) in wild-type (F and H) and Trps1-/- mice (G–I). (J) The statistic evaluation of the pH3-positive cells in vivo revealed increased numbers of mitotic cells (green, arrows) in Trps1-/- mutants. (n =5; p* = 0.025). (H and I) DAPI counterstaining of this sections showed asymmetric chromatin segregation in mitotic chondrocytes of Trps1-/- mutants (I, arrow). (K and L) Giemsa stained chromosome spreads of wild-type (K) and Trps1-/- mice (L) revealed increased numbers of aneuploid cells in Trps1-/- mutants. (M) Statistic evaluation of chromosome numbers in mitotic cells (n =4, 50 mitoses each; p* = 0.0028). Scale bar: 100 µm, (F and G), 10 µm, (I and J).
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Figure 2: Figure 2.Trps1-/- chondrocytes display aberrant mitosis. (A–C) Representative flow cytometry of primary chondrocyte cultures from wild-type (A) and Trps1-/- mice (B) stained with α-pH3 antibody and Propidium Iodide (PI) revealed increased numbers of pH3-positive cells in Trps1-/- mutants. (C) Statistic evaluation of pH3 measurement (n =5; p* = 0.018). (D and E) Hematoxylin-Eosin (HE) staining of paraffin sections of E16.5 wild-type and Trps1-/- ulnae showing the region of proliferating chondrocytes. Parallel sections (F and G) were used for immunofluorescent detection of pH3-positive, mitotic cells (green) in wild-type (F and H) and Trps1-/- mice (G–I). (J) The statistic evaluation of the pH3-positive cells in vivo revealed increased numbers of mitotic cells (green, arrows) in Trps1-/- mutants. (n =5; p* = 0.025). (H and I) DAPI counterstaining of this sections showed asymmetric chromatin segregation in mitotic chondrocytes of Trps1-/- mutants (I, arrow). (K and L) Giemsa stained chromosome spreads of wild-type (K) and Trps1-/- mice (L) revealed increased numbers of aneuploid cells in Trps1-/- mutants. (M) Statistic evaluation of chromosome numbers in mitotic cells (n =4, 50 mitoses each; p* = 0.0028). Scale bar: 100 µm, (F and G), 10 µm, (I and J).
Mentions: As loss of Trps1 leads to an accumulation of cells in G2/M-phase, we next investigated the phosphorylation of histone H3 at serine 10 (pH3), which serves as specific marker for mitotic cells.24 Flow cytometry of cells labeled with an α-pH3 antibody revealed that 3.5% wild-type chondrocytes in culture were pH3-positive (Fig. 2A and C), while in cultures of Trps1-/- mutants, the proportion of mitotic cells was increased to 7.3% (Fig. 2B and C). To examine if loss of Trps1 leads to a similar increase in mitotic cells in vivo, we quantified pH3-positive cells in the region of proliferating chondrocytes of E16.5 mouse ulnae (Fig. 2F–I). Comparable to our in vitro data, the number of pH3-positive cells was increased to 2.9% in Trps1-/- mutants, while 1.3% pH3-positive cells can be found in wild-type littermates (Fig. 2J). Trps1 thus activates mitotic progression in vitro and in vivo. Interestingly, at high magnification, irregular chromatin distribution could be observed in a subset of pH3-positive, Trps1-/- chondrocytes, likely reflecting disturbed chromosome segregation (Fig. 2I, arrow). We next investigated chromosome spreads of E12.5 primary chondrocyte cultures from Trps1-/- (Fig. 2L) and wild-type mice (Fig. 2K), and found the proportion of aneuploid cells with high chromosome numbers to be increased from 10.5% in wild-type mice to 22% in Trps1-/- mutants (Fig. 2M).

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