Limits...
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.

Show MeSH

Related in: MedlinePlus

Figure 1. Trps1 regulates two independent steps of the cell cycle. (A–C) Representative flow cytometry after labeling primary chondrocytes from wild-type (A) and Trps1-/- (B) mice with BrdU and 7-AAD identifies significantly decreased numbers of cells in S-phase, while the proportion of cells in G2/M-phase is increased in Trps1-/- mutants. (C) Statistic evaluation, n =6; p*S-phase = 0.001, p*G2/M-phase = 0.0021). Western blot (D and E) and flow cytometry (F) of HEK293 EBNA cells transfected with Trps1 specific or unspecific (mock) siRNA. The level of Trps1 protein was quantified in relation to histone H3 protein (n =6; p* = 0.000066 in (E). (F) Similar to Trps1-/- mutants, knockdown of Trps1 in HEK293 EBNA cells decreases the number of cells in S-phase and increases the number of cells in G2/M-phase (n =3; p* = 0.007 in (F). (G and H) Statistical evaluation derived from flow cytometric analyses of Aphidicolin- and Nocodazole-treated Trps1-/- (red) and wild-type (blue) cells compared with control cells. (G) During Aphidicolin treatment, Trps1-/- and wild-type chondrocytes are similarly arrested prior to S-phase. One h after release from Aphidicolin 5.8% wild-type chondrocytes have entered S-phase, while few Trps1-/- cells progress into S-phase (n =3; p* < 0.05). (H) Trps1-/-−/− cells are less sensitive to Nocodazole induced mitotic block (n =6; p* < 0.005).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC3755072&req=5

Figure 1: Figure 1. Trps1 regulates two independent steps of the cell cycle. (A–C) Representative flow cytometry after labeling primary chondrocytes from wild-type (A) and Trps1-/- (B) mice with BrdU and 7-AAD identifies significantly decreased numbers of cells in S-phase, while the proportion of cells in G2/M-phase is increased in Trps1-/- mutants. (C) Statistic evaluation, n =6; p*S-phase = 0.001, p*G2/M-phase = 0.0021). Western blot (D and E) and flow cytometry (F) of HEK293 EBNA cells transfected with Trps1 specific or unspecific (mock) siRNA. The level of Trps1 protein was quantified in relation to histone H3 protein (n =6; p* = 0.000066 in (E). (F) Similar to Trps1-/- mutants, knockdown of Trps1 in HEK293 EBNA cells decreases the number of cells in S-phase and increases the number of cells in G2/M-phase (n =3; p* = 0.007 in (F). (G and H) Statistical evaluation derived from flow cytometric analyses of Aphidicolin- and Nocodazole-treated Trps1-/- (red) and wild-type (blue) cells compared with control cells. (G) During Aphidicolin treatment, Trps1-/- and wild-type chondrocytes are similarly arrested prior to S-phase. One h after release from Aphidicolin 5.8% wild-type chondrocytes have entered S-phase, while few Trps1-/- cells progress into S-phase (n =3; p* < 0.05). (H) Trps1-/-−/− cells are less sensitive to Nocodazole induced mitotic block (n =6; p* < 0.005).

Mentions: Trps1-deficient (Trps1-/-) mice display a reduced size of the skeletal elements, which is, at least in part, due to a decreased chondrocyte proliferation rate.14,19,20 To characterize cell cycle progression, we analyzed chondrogenic precursor cells isolated from E12.5 limb mesenchyme (primary chondrocytes) by flow cytometry after labeling cells in S-phase with bromodesoxyuridine (BrdU) and DNA with 7-aminoactinomycin (7-AAD) (Fig. 1A and B). In agreement with previous reports,14,19,20 we found that the proportion of cells in S-phase was decreased from 20.5% in wild-type cultures to 14.4% in cultures of Trps1-/- mutants (Fig. 1C). Surprisingly, although a lower percentage of Trps1-/- cells replicated their DNA, the proportion of cells in G2/M-phase was significantly increased to 20.4%, while in wild-type cultures, 16.7% of chondrocytes were in G2/M-phase. To support a role of Trps1 in regulating cell cycle progression we knocked-down Trps1 in HEK293 EBNA cells using siRNA, which resulted in a reduction of Trps1 protein to 36% (Fig. 1D and E). In agreement with our data obtained in primary chondrocytes, siRNA-mediated knockdown of Trps1 in HEK293 EBNA reduced the numbers of cells in S-phase and increased the number of cells in G2/M-phase in HEK293 EBNA cells (Fig. 1F).


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 1. Trps1 regulates two independent steps of the cell cycle. (A–C) Representative flow cytometry after labeling primary chondrocytes from wild-type (A) and Trps1-/- (B) mice with BrdU and 7-AAD identifies significantly decreased numbers of cells in S-phase, while the proportion of cells in G2/M-phase is increased in Trps1-/- mutants. (C) Statistic evaluation, n =6; p*S-phase = 0.001, p*G2/M-phase = 0.0021). Western blot (D and E) and flow cytometry (F) of HEK293 EBNA cells transfected with Trps1 specific or unspecific (mock) siRNA. The level of Trps1 protein was quantified in relation to histone H3 protein (n =6; p* = 0.000066 in (E). (F) Similar to Trps1-/- mutants, knockdown of Trps1 in HEK293 EBNA cells decreases the number of cells in S-phase and increases the number of cells in G2/M-phase (n =3; p* = 0.007 in (F). (G and H) Statistical evaluation derived from flow cytometric analyses of Aphidicolin- and Nocodazole-treated Trps1-/- (red) and wild-type (blue) cells compared with control cells. (G) During Aphidicolin treatment, Trps1-/- and wild-type chondrocytes are similarly arrested prior to S-phase. One h after release from Aphidicolin 5.8% wild-type chondrocytes have entered S-phase, while few Trps1-/- cells progress into S-phase (n =3; p* < 0.05). (H) Trps1-/-−/− cells are less sensitive to Nocodazole induced mitotic block (n =6; p* < 0.005).
© Copyright Policy - open-access
Related In: Results  -  Collection

License
Show All Figures
getmorefigures.php?uid=PMC3755072&req=5

Figure 1: Figure 1. Trps1 regulates two independent steps of the cell cycle. (A–C) Representative flow cytometry after labeling primary chondrocytes from wild-type (A) and Trps1-/- (B) mice with BrdU and 7-AAD identifies significantly decreased numbers of cells in S-phase, while the proportion of cells in G2/M-phase is increased in Trps1-/- mutants. (C) Statistic evaluation, n =6; p*S-phase = 0.001, p*G2/M-phase = 0.0021). Western blot (D and E) and flow cytometry (F) of HEK293 EBNA cells transfected with Trps1 specific or unspecific (mock) siRNA. The level of Trps1 protein was quantified in relation to histone H3 protein (n =6; p* = 0.000066 in (E). (F) Similar to Trps1-/- mutants, knockdown of Trps1 in HEK293 EBNA cells decreases the number of cells in S-phase and increases the number of cells in G2/M-phase (n =3; p* = 0.007 in (F). (G and H) Statistical evaluation derived from flow cytometric analyses of Aphidicolin- and Nocodazole-treated Trps1-/- (red) and wild-type (blue) cells compared with control cells. (G) During Aphidicolin treatment, Trps1-/- and wild-type chondrocytes are similarly arrested prior to S-phase. One h after release from Aphidicolin 5.8% wild-type chondrocytes have entered S-phase, while few Trps1-/- cells progress into S-phase (n =3; p* < 0.05). (H) Trps1-/-−/− cells are less sensitive to Nocodazole induced mitotic block (n =6; p* < 0.005).
Mentions: Trps1-deficient (Trps1-/-) mice display a reduced size of the skeletal elements, which is, at least in part, due to a decreased chondrocyte proliferation rate.14,19,20 To characterize cell cycle progression, we analyzed chondrogenic precursor cells isolated from E12.5 limb mesenchyme (primary chondrocytes) by flow cytometry after labeling cells in S-phase with bromodesoxyuridine (BrdU) and DNA with 7-aminoactinomycin (7-AAD) (Fig. 1A and B). In agreement with previous reports,14,19,20 we found that the proportion of cells in S-phase was decreased from 20.5% in wild-type cultures to 14.4% in cultures of Trps1-/- mutants (Fig. 1C). Surprisingly, although a lower percentage of Trps1-/- cells replicated their DNA, the proportion of cells in G2/M-phase was significantly increased to 20.4%, while in wild-type cultures, 16.7% of chondrocytes were in G2/M-phase. To support a role of Trps1 in regulating cell cycle progression we knocked-down Trps1 in HEK293 EBNA cells using siRNA, which resulted in a reduction of Trps1 protein to 36% (Fig. 1D and E). In agreement with our data obtained in primary chondrocytes, siRNA-mediated knockdown of Trps1 in HEK293 EBNA reduced the numbers of cells in S-phase and increased the number of cells in G2/M-phase in HEK293 EBNA cells (Fig. 1F).

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