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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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Replication foci and H1P foci colocalize in vivo. (A–F) Log CHO cells were labeled with BrdU for 30 min and processed for immunohistochemistry. Confocal imaging of BrdU (with FITC) and H1P foci (Texas red) was performed on various stages of S-phase nuclei. Arrowheads in A–F note particular regions of biologically interesting overlap (see text). The arrowhead in E also points to the region enlarged and circled in F. (G) CHO cells were synchronized with mimosine at the G1–S transition, followed by release into S-phase and pulse-labeling with BrdU for 30 min in late S-phase (8 h after release). Control cells were fixed immediately to verify that the population was in late S-phase by the presence of stage 5 patterns (not depicted). Experimental samples were chased for 8 h without BrdU, putting them in late G1/early S-phase (verified by BrdU pulse-labeling a control sample after the chase; ∼85% of cells in early S-phase and 15% unlabeled/G1 [not depicted]). Immunohistochemistry was performed as in A–F.
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fig5: Replication foci and H1P foci colocalize in vivo. (A–F) Log CHO cells were labeled with BrdU for 30 min and processed for immunohistochemistry. Confocal imaging of BrdU (with FITC) and H1P foci (Texas red) was performed on various stages of S-phase nuclei. Arrowheads in A–F note particular regions of biologically interesting overlap (see text). The arrowhead in E also points to the region enlarged and circled in F. (G) CHO cells were synchronized with mimosine at the G1–S transition, followed by release into S-phase and pulse-labeling with BrdU for 30 min in late S-phase (8 h after release). Control cells were fixed immediately to verify that the population was in late S-phase by the presence of stage 5 patterns (not depicted). Experimental samples were chased for 8 h without BrdU, putting them in late G1/early S-phase (verified by BrdU pulse-labeling a control sample after the chase; ∼85% of cells in early S-phase and 15% unlabeled/G1 [not depicted]). Immunohistochemistry was performed as in A–F.

Mentions: An important concern is whether or not the chromatin remodeling system recapitulates what normally occurs in vivo. To address this issue, we asked if H1P foci colocalize with replication foci during S-phase. As shown by confocal imaging in Fig. 5 (and in additional examples shown in Fig. S1 E), there is significant colocalization between replicating DNA and H1P foci at all five stages of S-phase (indicated by the yellow signal in the merge column; Alexandrow and Hamlin, 2004). This is most obvious in late S-phase (stages 4 and 5) when the replication foci are distinctive, and the number of foci is small (Fig. 5, A–C, note arrowheads). In middle S-phase (stages 2 and 3) when the number of replication foci is larger and displays a perinuclear pattern, the H1P signals are visible in the regions of the nucleus undergoing BrdU labeling, particularly at the periphery (Fig. 5 D, arrowheads). Also evident in early S-phase nuclei (stage 1) are similar spatial patterns of some groups of foci, and distinct shapes of some of the individual foci, with the H1P and BrdU stains (Fig. 5 F, circled region [an enlargement of the region shown by arrowhead in E]).


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

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

Replication foci and H1P foci colocalize in vivo. (A–F) Log CHO cells were labeled with BrdU for 30 min and processed for immunohistochemistry. Confocal imaging of BrdU (with FITC) and H1P foci (Texas red) was performed on various stages of S-phase nuclei. Arrowheads in A–F note particular regions of biologically interesting overlap (see text). The arrowhead in E also points to the region enlarged and circled in F. (G) CHO cells were synchronized with mimosine at the G1–S transition, followed by release into S-phase and pulse-labeling with BrdU for 30 min in late S-phase (8 h after release). Control cells were fixed immediately to verify that the population was in late S-phase by the presence of stage 5 patterns (not depicted). Experimental samples were chased for 8 h without BrdU, putting them in late G1/early S-phase (verified by BrdU pulse-labeling a control sample after the chase; ∼85% of cells in early S-phase and 15% unlabeled/G1 [not depicted]). Immunohistochemistry was performed as in A–F.
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getmorefigures.php?uid=PMC2171796&req=5

fig5: Replication foci and H1P foci colocalize in vivo. (A–F) Log CHO cells were labeled with BrdU for 30 min and processed for immunohistochemistry. Confocal imaging of BrdU (with FITC) and H1P foci (Texas red) was performed on various stages of S-phase nuclei. Arrowheads in A–F note particular regions of biologically interesting overlap (see text). The arrowhead in E also points to the region enlarged and circled in F. (G) CHO cells were synchronized with mimosine at the G1–S transition, followed by release into S-phase and pulse-labeling with BrdU for 30 min in late S-phase (8 h after release). Control cells were fixed immediately to verify that the population was in late S-phase by the presence of stage 5 patterns (not depicted). Experimental samples were chased for 8 h without BrdU, putting them in late G1/early S-phase (verified by BrdU pulse-labeling a control sample after the chase; ∼85% of cells in early S-phase and 15% unlabeled/G1 [not depicted]). Immunohistochemistry was performed as in A–F.
Mentions: An important concern is whether or not the chromatin remodeling system recapitulates what normally occurs in vivo. To address this issue, we asked if H1P foci colocalize with replication foci during S-phase. As shown by confocal imaging in Fig. 5 (and in additional examples shown in Fig. S1 E), there is significant colocalization between replicating DNA and H1P foci at all five stages of S-phase (indicated by the yellow signal in the merge column; Alexandrow and Hamlin, 2004). This is most obvious in late S-phase (stages 4 and 5) when the replication foci are distinctive, and the number of foci is small (Fig. 5, A–C, note arrowheads). In middle S-phase (stages 2 and 3) when the number of replication foci is larger and displays a perinuclear pattern, the H1P signals are visible in the regions of the nucleus undergoing BrdU labeling, particularly at the periphery (Fig. 5 D, arrowheads). Also evident in early S-phase nuclei (stage 1) are similar spatial patterns of some groups of foci, and distinct shapes of some of the individual foci, with the H1P and BrdU stains (Fig. 5 F, circled region [an enlargement of the region shown by arrowhead in E]).

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