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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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DNA replication progression and H1P are directly related and dependent on Cdk2. CHO cells were synchronized at G1–S using isoleucine and mimosine. (A) Cells were released into S-phase, and 1 h later 250 μM olomoucine, 40 μM roscovitine, or DMSO (top, BrdU) was added. Cells were pulsed with BrdU (15 min) at each time point, followed by analysis of BrdU (FITC) and H1P (Texas red) levels. Equal (long) exposures were obtained for each field of cells. Arrowheads indicate nuclei with coelevated BrdU and H1P levels versus other nuclei in the field. Arrows indicate nuclei that show low H1P levels. a, strong regions; b; weaker regions. (B) Parallel samples were immunoblotted. Normalization was to total protein.
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fig6: DNA replication progression and H1P are directly related and dependent on Cdk2. CHO cells were synchronized at G1–S using isoleucine and mimosine. (A) Cells were released into S-phase, and 1 h later 250 μM olomoucine, 40 μM roscovitine, or DMSO (top, BrdU) was added. Cells were pulsed with BrdU (15 min) at each time point, followed by analysis of BrdU (FITC) and H1P (Texas red) levels. Equal (long) exposures were obtained for each field of cells. Arrowheads indicate nuclei with coelevated BrdU and H1P levels versus other nuclei in the field. Arrows indicate nuclei that show low H1P levels. a, strong regions; b; weaker regions. (B) Parallel samples were immunoblotted. Normalization was to total protein.

Mentions: Cells were synchronized in G0 by isoleucine deprivation and were released into G1 in the presence of mimosine to arrest cells at the G1–S transition (Fig. 6, mim time point). As expected, virtually no BrdU was incorporated during the pulse period in the presence of mimosine (Fig. 6 A, mim sample in top panels). However, control cells released into S-phase after mimosine removal efficiently incorporated BrdU as S-phase progressed (Fig. 6 A, top). In contrast, cells treated with olomoucine or roscovitine at 1 h into S-phase did not efficiently incorporate BrdU at any time thereafter (Fig. 6 A, middle and bottom, samples 2, 4, 6, and 8 h), consistent with Cdk2 being required for S-phase progression (Schutte et al., 1997).


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

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

DNA replication progression and H1P are directly related and dependent on Cdk2. CHO cells were synchronized at G1–S using isoleucine and mimosine. (A) Cells were released into S-phase, and 1 h later 250 μM olomoucine, 40 μM roscovitine, or DMSO (top, BrdU) was added. Cells were pulsed with BrdU (15 min) at each time point, followed by analysis of BrdU (FITC) and H1P (Texas red) levels. Equal (long) exposures were obtained for each field of cells. Arrowheads indicate nuclei with coelevated BrdU and H1P levels versus other nuclei in the field. Arrows indicate nuclei that show low H1P levels. a, strong regions; b; weaker regions. (B) Parallel samples were immunoblotted. Normalization was to total protein.
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Related In: Results  -  Collection

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fig6: DNA replication progression and H1P are directly related and dependent on Cdk2. CHO cells were synchronized at G1–S using isoleucine and mimosine. (A) Cells were released into S-phase, and 1 h later 250 μM olomoucine, 40 μM roscovitine, or DMSO (top, BrdU) was added. Cells were pulsed with BrdU (15 min) at each time point, followed by analysis of BrdU (FITC) and H1P (Texas red) levels. Equal (long) exposures were obtained for each field of cells. Arrowheads indicate nuclei with coelevated BrdU and H1P levels versus other nuclei in the field. Arrows indicate nuclei that show low H1P levels. a, strong regions; b; weaker regions. (B) Parallel samples were immunoblotted. Normalization was to total protein.
Mentions: Cells were synchronized in G0 by isoleucine deprivation and were released into G1 in the presence of mimosine to arrest cells at the G1–S transition (Fig. 6, mim time point). As expected, virtually no BrdU was incorporated during the pulse period in the presence of mimosine (Fig. 6 A, mim sample in top panels). However, control cells released into S-phase after mimosine removal efficiently incorporated BrdU as S-phase progressed (Fig. 6 A, top). In contrast, cells treated with olomoucine or roscovitine at 1 h into S-phase did not efficiently incorporate BrdU at any time thereafter (Fig. 6 A, middle and bottom, samples 2, 4, 6, and 8 h), consistent with Cdk2 being required for S-phase progression (Schutte et al., 1997).

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