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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, Cdk2, Cyclin A, and H1P associate with chromatin during G0-, G1-, and S-phases, and Cdc45 interacts with Cdk2 in cells. (A) CHO cells were synchronized in G0 by isoleucine deprivation and released into G1 and S. Total cell extract (TCE), S1, and P3-chromatin fractions were isolated. Log CHO cells were used as a reference. BrdU labeling (30 min) at each time point verified synchrony and indicated that S-phase occurred from 9 to 18 h (not depicted). Immunoblotting was performed with indicated antibodies. (B) HeLa cells were subjected to immunoprecipitation with rabbit anti-Cdc45 (lane 1), goat anti-Cdc45 (lane 2), or nonspecific serum + beads (Ser). Immunoprecipitation (lanes 1, 2, and Ser) and HeLa TCE (lane 3) samples were immunoblotted with rabbit anti-Cdc45 (top) or mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 2, far bottom). Heavy (H) and light (L) chains of immunoprecipitating antibodies are indicated. (C) HeLa cells were subjected to immunoprecipitation with mouse anti-Cdk2 (lane 4). Immunoprecipitation (lane 4) and HeLa TCE (lane 5) samples were immunoblotted with mouse anti-Cdk2 (top) or rabbit anti-Cdc45 (bottom). (D) CHO cells expressing HA-Cdc45 were immunoprecipitated with mouse anti-HA. HA-Cdc45 (lane 6) and endogenous Cdc45 from CHO TCE (lane 7) were detected with rabbit anti-Cdc45 (top). Coimmunoprecipitating (lane 6) and endogenous (lane 7) Cdk2 were detected with mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 6, far bottom).
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fig4: Cdc45, Cdk2, Cyclin A, and H1P associate with chromatin during G0-, G1-, and S-phases, and Cdc45 interacts with Cdk2 in cells. (A) CHO cells were synchronized in G0 by isoleucine deprivation and released into G1 and S. Total cell extract (TCE), S1, and P3-chromatin fractions were isolated. Log CHO cells were used as a reference. BrdU labeling (30 min) at each time point verified synchrony and indicated that S-phase occurred from 9 to 18 h (not depicted). Immunoblotting was performed with indicated antibodies. (B) HeLa cells were subjected to immunoprecipitation with rabbit anti-Cdc45 (lane 1), goat anti-Cdc45 (lane 2), or nonspecific serum + beads (Ser). Immunoprecipitation (lanes 1, 2, and Ser) and HeLa TCE (lane 3) samples were immunoblotted with rabbit anti-Cdc45 (top) or mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 2, far bottom). Heavy (H) and light (L) chains of immunoprecipitating antibodies are indicated. (C) HeLa cells were subjected to immunoprecipitation with mouse anti-Cdk2 (lane 4). Immunoprecipitation (lane 4) and HeLa TCE (lane 5) samples were immunoblotted with mouse anti-Cdk2 (top) or rabbit anti-Cdc45 (bottom). (D) CHO cells expressing HA-Cdc45 were immunoprecipitated with mouse anti-HA. HA-Cdc45 (lane 6) and endogenous Cdc45 from CHO TCE (lane 7) were detected with rabbit anti-Cdc45 (top). Coimmunoprecipitating (lane 6) and endogenous (lane 7) Cdk2 were detected with mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 6, far bottom).

Mentions: Mediation of Cdc45-promoted chromatin decondensation by Cdk2 and H1P predicts that all of these proteins should be associated with chromatin during S-phase. To test this prediction, CHO cells were synchronized by arrest in G0 and then released into G1 and S-phases. Total cell extracts, detergent-sensitive soluble cytoplasmic and nucleoplasmic proteins (S1), and detergent-resistant (P3) proteins (operationally defined as chromatin-bound) were collected at the times indicated in Fig. 4. Antitubulin and antilamin immunoblotting showed that the fractionation was effective (Fig. 4 A, S1 and P3 samples), whereas anti–Cyclin A verified the effectiveness of the synchronization regimen (Fig. 4 A, TCE samples for Cyclin A) as did BrdU labeling of S-phase cells (not depicted). After verifying that two different anti-Cdc45 antisera recognize endogenous Cdc45 (as a doublet) as well as Cdc45 tagged with the LacI domain (Fig. S1, A and B; and not depicted), we determined the kinetics of Cdc45 chromatin binding.


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

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

Cdc45, Cdk2, Cyclin A, and H1P associate with chromatin during G0-, G1-, and S-phases, and Cdc45 interacts with Cdk2 in cells. (A) CHO cells were synchronized in G0 by isoleucine deprivation and released into G1 and S. Total cell extract (TCE), S1, and P3-chromatin fractions were isolated. Log CHO cells were used as a reference. BrdU labeling (30 min) at each time point verified synchrony and indicated that S-phase occurred from 9 to 18 h (not depicted). Immunoblotting was performed with indicated antibodies. (B) HeLa cells were subjected to immunoprecipitation with rabbit anti-Cdc45 (lane 1), goat anti-Cdc45 (lane 2), or nonspecific serum + beads (Ser). Immunoprecipitation (lanes 1, 2, and Ser) and HeLa TCE (lane 3) samples were immunoblotted with rabbit anti-Cdc45 (top) or mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 2, far bottom). Heavy (H) and light (L) chains of immunoprecipitating antibodies are indicated. (C) HeLa cells were subjected to immunoprecipitation with mouse anti-Cdk2 (lane 4). Immunoprecipitation (lane 4) and HeLa TCE (lane 5) samples were immunoblotted with mouse anti-Cdk2 (top) or rabbit anti-Cdc45 (bottom). (D) CHO cells expressing HA-Cdc45 were immunoprecipitated with mouse anti-HA. HA-Cdc45 (lane 6) and endogenous Cdc45 from CHO TCE (lane 7) were detected with rabbit anti-Cdc45 (top). Coimmunoprecipitating (lane 6) and endogenous (lane 7) Cdk2 were detected with mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 6, far bottom).
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fig4: Cdc45, Cdk2, Cyclin A, and H1P associate with chromatin during G0-, G1-, and S-phases, and Cdc45 interacts with Cdk2 in cells. (A) CHO cells were synchronized in G0 by isoleucine deprivation and released into G1 and S. Total cell extract (TCE), S1, and P3-chromatin fractions were isolated. Log CHO cells were used as a reference. BrdU labeling (30 min) at each time point verified synchrony and indicated that S-phase occurred from 9 to 18 h (not depicted). Immunoblotting was performed with indicated antibodies. (B) HeLa cells were subjected to immunoprecipitation with rabbit anti-Cdc45 (lane 1), goat anti-Cdc45 (lane 2), or nonspecific serum + beads (Ser). Immunoprecipitation (lanes 1, 2, and Ser) and HeLa TCE (lane 3) samples were immunoblotted with rabbit anti-Cdc45 (top) or mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 2, far bottom). Heavy (H) and light (L) chains of immunoprecipitating antibodies are indicated. (C) HeLa cells were subjected to immunoprecipitation with mouse anti-Cdk2 (lane 4). Immunoprecipitation (lane 4) and HeLa TCE (lane 5) samples were immunoblotted with mouse anti-Cdk2 (top) or rabbit anti-Cdc45 (bottom). (D) CHO cells expressing HA-Cdc45 were immunoprecipitated with mouse anti-HA. HA-Cdc45 (lane 6) and endogenous Cdc45 from CHO TCE (lane 7) were detected with rabbit anti-Cdc45 (top). Coimmunoprecipitating (lane 6) and endogenous (lane 7) Cdk2 were detected with mouse anti-Cdk2 (bottom). An H1 kinase assay was performed in parallel (lane 6, far bottom).
Mentions: Mediation of Cdc45-promoted chromatin decondensation by Cdk2 and H1P predicts that all of these proteins should be associated with chromatin during S-phase. To test this prediction, CHO cells were synchronized by arrest in G0 and then released into G1 and S-phases. Total cell extracts, detergent-sensitive soluble cytoplasmic and nucleoplasmic proteins (S1), and detergent-resistant (P3) proteins (operationally defined as chromatin-bound) were collected at the times indicated in Fig. 4. Antitubulin and antilamin immunoblotting showed that the fractionation was effective (Fig. 4 A, S1 and P3 samples), whereas anti–Cyclin A verified the effectiveness of the synchronization regimen (Fig. 4 A, TCE samples for Cyclin A) as did BrdU labeling of S-phase cells (not depicted). After verifying that two different anti-Cdc45 antisera recognize endogenous Cdc45 (as a doublet) as well as Cdc45 tagged with the LacI domain (Fig. S1, A and B; and not depicted), we determined the kinetics of Cdc45 chromatin binding.

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