Limits...
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.

Show MeSH

Related in: MedlinePlus

Cdc45, but not Cdc6, targeting promotes large-scale chromatin decondensation. (A) Control A03_1 cells were transfected with vectors expressing LacI alone, LacI-VP16, LacI alone, or LacI-BRCA1(6c-w). Immunohistochemistry was performed using rabbit anti-LacI (with Texas red) to detect open or closed chromatin. DAPI was used for nuclei. (B) A03_1 cells were transfected with pRcLac-Cdc45 and chromatin decondensation (or lack thereof) was detected as in A. O, open chromatin; C, closed; I, indeterminate. (C) A03_1 cells were transfected with pRcLac-Cdc6 and chromatin decondensation structures were detected. Representative pictures from seven similar experiments are shown.
© Copyright Policy
Related In: Results  -  Collection


getmorefigures.php?uid=PMC2171796&req=5

fig1: Cdc45, but not Cdc6, targeting promotes large-scale chromatin decondensation. (A) Control A03_1 cells were transfected with vectors expressing LacI alone, LacI-VP16, LacI alone, or LacI-BRCA1(6c-w). Immunohistochemistry was performed using rabbit anti-LacI (with Texas red) to detect open or closed chromatin. DAPI was used for nuclei. (B) A03_1 cells were transfected with pRcLac-Cdc45 and chromatin decondensation (or lack thereof) was detected as in A. O, open chromatin; C, closed; I, indeterminate. (C) A03_1 cells were transfected with pRcLac-Cdc6 and chromatin decondensation structures were detected. Representative pictures from seven similar experiments are shown.

Mentions: The HSR can be directly visualized in either fixed or living A03_1 cells by providing the LacI protein or LacI-GFP derivative in trans. In either case, when visualized microscopically via an anti-LacI antibody or the fluorescent tag itself, the HSR assumes a condensed dot-like structure and is heterochromatic (Fig. 1 A, pNYE4-LacI and pRcLac; Li et al., 1998; Tumbar et al., 1999). However, the expression of certain proteins fused to LacI (e.g., VP16 or BRCA1) induces a dramatic decondensation of the HSR via recruitment of chromatin remodeling complexes by the LacI fusion partner (Fig. 1 A, pNYE4-LacI-VP16 and pRcLac-BRCA1(6c-w); Tumbar et al., 1999; Ye et al., 2001).


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

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

Cdc45, but not Cdc6, targeting promotes large-scale chromatin decondensation. (A) Control A03_1 cells were transfected with vectors expressing LacI alone, LacI-VP16, LacI alone, or LacI-BRCA1(6c-w). Immunohistochemistry was performed using rabbit anti-LacI (with Texas red) to detect open or closed chromatin. DAPI was used for nuclei. (B) A03_1 cells were transfected with pRcLac-Cdc45 and chromatin decondensation (or lack thereof) was detected as in A. O, open chromatin; C, closed; I, indeterminate. (C) A03_1 cells were transfected with pRcLac-Cdc6 and chromatin decondensation structures were detected. Representative pictures from seven similar experiments are shown.
© Copyright Policy
Related In: Results  -  Collection

Show All Figures
getmorefigures.php?uid=PMC2171796&req=5

fig1: Cdc45, but not Cdc6, targeting promotes large-scale chromatin decondensation. (A) Control A03_1 cells were transfected with vectors expressing LacI alone, LacI-VP16, LacI alone, or LacI-BRCA1(6c-w). Immunohistochemistry was performed using rabbit anti-LacI (with Texas red) to detect open or closed chromatin. DAPI was used for nuclei. (B) A03_1 cells were transfected with pRcLac-Cdc45 and chromatin decondensation (or lack thereof) was detected as in A. O, open chromatin; C, closed; I, indeterminate. (C) A03_1 cells were transfected with pRcLac-Cdc6 and chromatin decondensation structures were detected. Representative pictures from seven similar experiments are shown.
Mentions: The HSR can be directly visualized in either fixed or living A03_1 cells by providing the LacI protein or LacI-GFP derivative in trans. In either case, when visualized microscopically via an anti-LacI antibody or the fluorescent tag itself, the HSR assumes a condensed dot-like structure and is heterochromatic (Fig. 1 A, pNYE4-LacI and pRcLac; Li et al., 1998; Tumbar et al., 1999). However, the expression of certain proteins fused to LacI (e.g., VP16 or BRCA1) induces a dramatic decondensation of the HSR via recruitment of chromatin remodeling complexes by the LacI fusion partner (Fig. 1 A, pNYE4-LacI-VP16 and pRcLac-BRCA1(6c-w); Tumbar et al., 1999; Ye et al., 2001).

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