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Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation.

Alexandrow MG, Hamlin JL - J. Cell Biol. (2005)

Bottom Line: We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation.Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation.Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.

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Cdk2 mediates Cdc45-promoted chromatin decondensation. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed as in Fig. 1. 25 μM U0126, 250 μM olomoucine, or DMSO were added at the time of transfection, or 6 h afterward, and all cells were fixed 24 h after transfection. p21 inhibitor was cotransfected at a 3:1 ratio with LacI-Cdc45 or -VP16 (far right). Chromatin structures were quantified from 110–180 cells per condition. (B and C) A03_1 cells expressing LacI-Cdk2-wt (left) or LacI-Cdk2-DN (right) were analyzed for open chromatin structures with rabbit anti-LacI (and Texas red). Chromatin structure percentages were determined after examining more than 140 cells for each Cdk2 allele, and the data plotted in C. O, open chromatin; C, closed; I, indeterminate. (D) Confocal imaging of H1P and Cdk2-wt or -DN was performed using mouse anti-LacI (FITC) and anti-H1P (Texas red). Equal exposures were obtained, and lack of bleed-through of emission signals was verified as described in Materials and methods. (A–C) O and red columns, open; C and black columns, closed; I and blue columns, indeterminate.
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fig3: Cdk2 mediates Cdc45-promoted chromatin decondensation. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed as in Fig. 1. 25 μM U0126, 250 μM olomoucine, or DMSO were added at the time of transfection, or 6 h afterward, and all cells were fixed 24 h after transfection. p21 inhibitor was cotransfected at a 3:1 ratio with LacI-Cdc45 or -VP16 (far right). Chromatin structures were quantified from 110–180 cells per condition. (B and C) A03_1 cells expressing LacI-Cdk2-wt (left) or LacI-Cdk2-DN (right) were analyzed for open chromatin structures with rabbit anti-LacI (and Texas red). Chromatin structure percentages were determined after examining more than 140 cells for each Cdk2 allele, and the data plotted in C. O, open chromatin; C, closed; I, indeterminate. (D) Confocal imaging of H1P and Cdk2-wt or -DN was performed using mouse anti-LacI (FITC) and anti-H1P (Texas red). Equal exposures were obtained, and lack of bleed-through of emission signals was verified as described in Materials and methods. (A–C) O and red columns, open; C and black columns, closed; I and blue columns, indeterminate.

Mentions: We found that Cdk2 colocalizes with Cdc45-promoted decondensed HSRs in ∼25% of cells that showed a decondensed structure (Fig. 2 D and Table II), but was not detectable in the small percentage of cells with condensed HSRs (Table II). Because this value is lower than the number of open HSRs displaying H1P costaining, it is conceivable that the kinase associates only transiently with Cdc45 during the decondensation process. We also found that coexpression of LacI-Cdc45 with the Cdk2 inhibitor p21 reduced Cdc45-dependent chromatin unfolding to less than half its normal value and concomitantly increased the number of condensed HSRs detected (Fig. 3 A). Additionally, the Cdk2-specific inhibitor olomoucine (Schutte et al., 1997) reduced the ability of Cdc45 to promote decondensation (Fig. 3 A, time 0). These observations are consistent with a direct role for Cdk2 in mediating chromatin unfolding promoted by Cdc45 targeting.


Chromatin decondensation in S-phase involves recruitment of Cdk2 by Cdc45 and histone H1 phosphorylation.

Alexandrow MG, Hamlin JL - J. Cell Biol. (2005)

Cdk2 mediates Cdc45-promoted chromatin decondensation. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed as in Fig. 1. 25 μM U0126, 250 μM olomoucine, or DMSO were added at the time of transfection, or 6 h afterward, and all cells were fixed 24 h after transfection. p21 inhibitor was cotransfected at a 3:1 ratio with LacI-Cdc45 or -VP16 (far right). Chromatin structures were quantified from 110–180 cells per condition. (B and C) A03_1 cells expressing LacI-Cdk2-wt (left) or LacI-Cdk2-DN (right) were analyzed for open chromatin structures with rabbit anti-LacI (and Texas red). Chromatin structure percentages were determined after examining more than 140 cells for each Cdk2 allele, and the data plotted in C. O, open chromatin; C, closed; I, indeterminate. (D) Confocal imaging of H1P and Cdk2-wt or -DN was performed using mouse anti-LacI (FITC) and anti-H1P (Texas red). Equal exposures were obtained, and lack of bleed-through of emission signals was verified as described in Materials and methods. (A–C) O and red columns, open; C and black columns, closed; I and blue columns, indeterminate.
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Related In: Results  -  Collection

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fig3: Cdk2 mediates Cdc45-promoted chromatin decondensation. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed as in Fig. 1. 25 μM U0126, 250 μM olomoucine, or DMSO were added at the time of transfection, or 6 h afterward, and all cells were fixed 24 h after transfection. p21 inhibitor was cotransfected at a 3:1 ratio with LacI-Cdc45 or -VP16 (far right). Chromatin structures were quantified from 110–180 cells per condition. (B and C) A03_1 cells expressing LacI-Cdk2-wt (left) or LacI-Cdk2-DN (right) were analyzed for open chromatin structures with rabbit anti-LacI (and Texas red). Chromatin structure percentages were determined after examining more than 140 cells for each Cdk2 allele, and the data plotted in C. O, open chromatin; C, closed; I, indeterminate. (D) Confocal imaging of H1P and Cdk2-wt or -DN was performed using mouse anti-LacI (FITC) and anti-H1P (Texas red). Equal exposures were obtained, and lack of bleed-through of emission signals was verified as described in Materials and methods. (A–C) O and red columns, open; C and black columns, closed; I and blue columns, indeterminate.
Mentions: We found that Cdk2 colocalizes with Cdc45-promoted decondensed HSRs in ∼25% of cells that showed a decondensed structure (Fig. 2 D and Table II), but was not detectable in the small percentage of cells with condensed HSRs (Table II). Because this value is lower than the number of open HSRs displaying H1P costaining, it is conceivable that the kinase associates only transiently with Cdc45 during the decondensation process. We also found that coexpression of LacI-Cdc45 with the Cdk2 inhibitor p21 reduced Cdc45-dependent chromatin unfolding to less than half its normal value and concomitantly increased the number of condensed HSRs detected (Fig. 3 A). Additionally, the Cdk2-specific inhibitor olomoucine (Schutte et al., 1997) reduced the ability of Cdc45 to promote decondensation (Fig. 3 A, time 0). These observations are consistent with a direct role for Cdk2 in mediating chromatin unfolding promoted by Cdc45 targeting.

Bottom Line: We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation.Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation.Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1.

View Article: PubMed Central - PubMed

Affiliation: Department of Biochemistry and Molecular Genetics, University of Virginia School of Medicine, Charlottesville, Virginia 22908, USA.

ABSTRACT
Cdc45 is required for initiation of DNA replication and fork progression, but its function in these processes remains unknown. We show that targeting Cdc45 to specific chromosomal sites in mammalian cells results in large-scale chromatin decondensation that strongly correlates with histone H1 phosphorylation. Cdk2 is recruited to sites of Cdc45 decondensation, and Cdk2 inhibitors reduce the level of decondensation. Targeting wild-type Cdk2, but not kinase-defective Cdk2, to chromatin is also effective at inducing decondensation involving phospho-H1. Cdc45, Cdk2, Cyclin A, and phospho-H1 associate with chromatin during S-phase, and Cdc45, Cdk2, and an active H1 kinase physically interact. Replicating DNA and phospho-H1 foci colocalize in vivo, and S-phase progression and H1 phosphorylation are directly related and Cdk2 dependent. Because Cdk2 colocalizes with replication foci and H1 regulates higher-order chromatin, we suggest a model in which Cdc45 recruits Cdk2 to replication foci, resulting in H1 phosphorylation, chromatin decondensation, and facilitation of fork progression.

Show MeSH
Related in: MedlinePlus