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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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Cdc45-promoted chromatin decondensation is associated with H1 phosphorylation and Cdk2 recruitment. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed for open structures and colocalizing modified core histones. Open structures were detected with mouse anti-LacI (and FITC), and modified histones with anti–acetyl-H3/H4 and anti–methyl-K4 (and Texas red). (B and C) Open chromatin structures promoted by Cdc45 were analyzed as in A. H1P colocalization was detected with anti-H1P (and Texas red). Arrows indicate open chromatin structures. Arrowheads indicate nuclei displaying a high level of H1P, suggesting G2 or M staging. (D) Open chromatin structures promoted by Cdc45 were visualized with rabbit anti-LacI (and Texas red). Cdk2 colocalization was detected with mouse anti-Cdk2 (and FITC). Colocalization is shown in the figure by yellow in merge.
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fig2: Cdc45-promoted chromatin decondensation is associated with H1 phosphorylation and Cdk2 recruitment. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed for open structures and colocalizing modified core histones. Open structures were detected with mouse anti-LacI (and FITC), and modified histones with anti–acetyl-H3/H4 and anti–methyl-K4 (and Texas red). (B and C) Open chromatin structures promoted by Cdc45 were analyzed as in A. H1P colocalization was detected with anti-H1P (and Texas red). Arrows indicate open chromatin structures. Arrowheads indicate nuclei displaying a high level of H1P, suggesting G2 or M staging. (D) Open chromatin structures promoted by Cdc45 were visualized with rabbit anti-LacI (and Texas red). Cdk2 colocalization was detected with mouse anti-Cdk2 (and FITC). Colocalization is shown in the figure by yellow in merge.

Mentions: Using modification-specific antibodies, we were not able to detect an increase in H3 or H4 acetylation or methylation at sites of Cdc45-decondensed chromatin (Fig. 2 A, top; and Table II). An important caveat is that failure to demonstrate colocalization of modified core histones with Cdc45-decondensed chromatin could result from the possible transience of such alterations or to the particular choice of antisera. However, colocalizing core histone modifications were detected when VP16 was targeted to the HSR (Fig. 2 A, bottom; and Table II), which is consistent with other published observations (Tumbar et al., 1999; Ye et al., 2001). Thus, it appears that Cdc45-induced chromatin decondensation does not involve these particular modifications to core histones. Importantly, the latter data argue against the possibility that tethering Cdc45 to LacI unmasks a cryptic transactivation domain that is actually responsible for chromatin decondensation.


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

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

Cdc45-promoted chromatin decondensation is associated with H1 phosphorylation and Cdk2 recruitment. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed for open structures and colocalizing modified core histones. Open structures were detected with mouse anti-LacI (and FITC), and modified histones with anti–acetyl-H3/H4 and anti–methyl-K4 (and Texas red). (B and C) Open chromatin structures promoted by Cdc45 were analyzed as in A. H1P colocalization was detected with anti-H1P (and Texas red). Arrows indicate open chromatin structures. Arrowheads indicate nuclei displaying a high level of H1P, suggesting G2 or M staging. (D) Open chromatin structures promoted by Cdc45 were visualized with rabbit anti-LacI (and Texas red). Cdk2 colocalization was detected with mouse anti-Cdk2 (and FITC). Colocalization is shown in the figure by yellow in merge.
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Related In: Results  -  Collection

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getmorefigures.php?uid=PMC2171796&req=5

fig2: Cdc45-promoted chromatin decondensation is associated with H1 phosphorylation and Cdk2 recruitment. (A) A03_1 cells expressing LacI-Cdc45 or LacI-VP16 were analyzed for open structures and colocalizing modified core histones. Open structures were detected with mouse anti-LacI (and FITC), and modified histones with anti–acetyl-H3/H4 and anti–methyl-K4 (and Texas red). (B and C) Open chromatin structures promoted by Cdc45 were analyzed as in A. H1P colocalization was detected with anti-H1P (and Texas red). Arrows indicate open chromatin structures. Arrowheads indicate nuclei displaying a high level of H1P, suggesting G2 or M staging. (D) Open chromatin structures promoted by Cdc45 were visualized with rabbit anti-LacI (and Texas red). Cdk2 colocalization was detected with mouse anti-Cdk2 (and FITC). Colocalization is shown in the figure by yellow in merge.
Mentions: Using modification-specific antibodies, we were not able to detect an increase in H3 or H4 acetylation or methylation at sites of Cdc45-decondensed chromatin (Fig. 2 A, top; and Table II). An important caveat is that failure to demonstrate colocalization of modified core histones with Cdc45-decondensed chromatin could result from the possible transience of such alterations or to the particular choice of antisera. However, colocalizing core histone modifications were detected when VP16 was targeted to the HSR (Fig. 2 A, bottom; and Table II), which is consistent with other published observations (Tumbar et al., 1999; Ye et al., 2001). Thus, it appears that Cdc45-induced chromatin decondensation does not involve these particular modifications to core histones. Importantly, the latter data argue against the possibility that tethering Cdc45 to LacI unmasks a cryptic transactivation domain that is actually responsible for chromatin decondensation.

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