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Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.

Schneider K, Fuchs C, Dobay A, Rottach A, Qin W, Wolf P, Álvarez-Castro JM, Nalaskowski MM, Kremmer E, Schmid V, Leonhardt H, Schermelleh L - Nucleic Acids Res. (2013)

Bottom Line: We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s).In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions.We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich (LMU), 82152 Planegg-Martinsried, Germany.

ABSTRACT
DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

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Domain structure and subnuclear localization of GFP-Dnmt1 constructs. (A) Dnmt1 consists of a large N-terminal regulatory domain containing PBD, TS domain and a CXXC zinc finger (ZnF) domain, and a conserved C-terminal catalytic domain. The point mutation of a highly conserved glutamine to glutamic acid introduced within the PBD eliminates interaction with the replication machinery (GFP-Dnmt1Q162E). A deletion in the central part of the TS domain (GFP-Dnmt1ΔTS) was introduced to abolish interaction with constitutive heterochromatin. Furthermore a construct containing both mutations was generated (GFP-Dnmt1Q162E/ΔTS). GFP alone is used as a control for a non-binding protein. (B) Spinning disk confocal mid sections of GFP-Dnmt1 wild type (wt) and mutant constructs in live mouse C2C12 cells co-expressing RFP-PCNA to mark RF. In early S phase, GFP-Dnmt1wt accumulates at RF, whereas PCNA-binding deficient GFP-Dnmt1Q162E is diffusely distributed throughout the nucleus. GFP-Dnmt1ΔTS is still associated with RF, but not as prominently as GFP-Dnmt1wt. In late S phase GFP-Dnmt1wt, GFP-Dnmt1Q162E and GFP-Dnmt1ΔTS accumulate at larger RF of late replicating pHC, although with slightly less strong enrichment observed for both mutants. GFP-Dnmt1Q162E/ΔTS is distributed diffusely in the nucleus throughout interphase. Scale bar: 5 µm.
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gkt191-F1: Domain structure and subnuclear localization of GFP-Dnmt1 constructs. (A) Dnmt1 consists of a large N-terminal regulatory domain containing PBD, TS domain and a CXXC zinc finger (ZnF) domain, and a conserved C-terminal catalytic domain. The point mutation of a highly conserved glutamine to glutamic acid introduced within the PBD eliminates interaction with the replication machinery (GFP-Dnmt1Q162E). A deletion in the central part of the TS domain (GFP-Dnmt1ΔTS) was introduced to abolish interaction with constitutive heterochromatin. Furthermore a construct containing both mutations was generated (GFP-Dnmt1Q162E/ΔTS). GFP alone is used as a control for a non-binding protein. (B) Spinning disk confocal mid sections of GFP-Dnmt1 wild type (wt) and mutant constructs in live mouse C2C12 cells co-expressing RFP-PCNA to mark RF. In early S phase, GFP-Dnmt1wt accumulates at RF, whereas PCNA-binding deficient GFP-Dnmt1Q162E is diffusely distributed throughout the nucleus. GFP-Dnmt1ΔTS is still associated with RF, but not as prominently as GFP-Dnmt1wt. In late S phase GFP-Dnmt1wt, GFP-Dnmt1Q162E and GFP-Dnmt1ΔTS accumulate at larger RF of late replicating pHC, although with slightly less strong enrichment observed for both mutants. GFP-Dnmt1Q162E/ΔTS is distributed diffusely in the nucleus throughout interphase. Scale bar: 5 µm.

Mentions: Our aim was to analyze the S phase–dependent regulation of the Dnmt1-binding behavior. To that end, we investigated four GFP fusions: wild type Dnmt1 (GFP-Dnmt1wt), the full-length Dnmt1 carrying a point mutation (GFP-Dnmt1Q162E) within the PBD (6), a Dnmt1 mutant carrying a deletion of a highly conserved part of the TS domain comprising the amino acids 459–501 (GFP-Dnmt1ΔTS) and a Dnmt1 double mutant containing both mutations (GFP-Dnmt1Q162E/ΔTS) (Figure 1A). To identify different cell cycle stages, we first co-expressed GFP-Dnmt1 constructs with PCNA fused to monomeric red fluorescent protein (RFP-PCNA) in mouse C2C12 myoblast cells and acquired confocal mid sections of the living cells (Figure 1B). As previously described (6), GFP-Dnmt1wt co-localized with RFP-PCNA at RF in early S phase. Co-localization with RF was also apparent in late S phase, when DNA of pericentromeric heterochromatin (pHC) is replicated. In contrast, the double mutant GFP-Dnmt1Q162E/ΔTS was diffusely distributed within nuclei throughout interphase, suggesting a deficiency to target RF during S phase. GFP-Dnmt1ΔTS still accumulated at RF in early and late S phase, but showed a slightly weaker association compared with GFP-Dnmt1wt, indicating the activity of PBD-mediated targeting in all S phase stages, independent of the presence of the TS domain (Figure 1B and Supplementary Figure S2). As previously reported, GFP-Dnmt1Q162E showed a diffuse nuclear distribution in early S phase but notable association to pHC replicating in late S phase (6). Together, this suggests that the PBD-mediated interaction with PCNA is necessary for the Dnmt1 localization in early S phase, but evidently not for the association at pHC in late S phase.Figure 1.


Dissection of cell cycle-dependent dynamics of Dnmt1 by FRAP and diffusion-coupled modeling.

Schneider K, Fuchs C, Dobay A, Rottach A, Qin W, Wolf P, Álvarez-Castro JM, Nalaskowski MM, Kremmer E, Schmid V, Leonhardt H, Schermelleh L - Nucleic Acids Res. (2013)

Domain structure and subnuclear localization of GFP-Dnmt1 constructs. (A) Dnmt1 consists of a large N-terminal regulatory domain containing PBD, TS domain and a CXXC zinc finger (ZnF) domain, and a conserved C-terminal catalytic domain. The point mutation of a highly conserved glutamine to glutamic acid introduced within the PBD eliminates interaction with the replication machinery (GFP-Dnmt1Q162E). A deletion in the central part of the TS domain (GFP-Dnmt1ΔTS) was introduced to abolish interaction with constitutive heterochromatin. Furthermore a construct containing both mutations was generated (GFP-Dnmt1Q162E/ΔTS). GFP alone is used as a control for a non-binding protein. (B) Spinning disk confocal mid sections of GFP-Dnmt1 wild type (wt) and mutant constructs in live mouse C2C12 cells co-expressing RFP-PCNA to mark RF. In early S phase, GFP-Dnmt1wt accumulates at RF, whereas PCNA-binding deficient GFP-Dnmt1Q162E is diffusely distributed throughout the nucleus. GFP-Dnmt1ΔTS is still associated with RF, but not as prominently as GFP-Dnmt1wt. In late S phase GFP-Dnmt1wt, GFP-Dnmt1Q162E and GFP-Dnmt1ΔTS accumulate at larger RF of late replicating pHC, although with slightly less strong enrichment observed for both mutants. GFP-Dnmt1Q162E/ΔTS is distributed diffusely in the nucleus throughout interphase. Scale bar: 5 µm.
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Related In: Results  -  Collection

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gkt191-F1: Domain structure and subnuclear localization of GFP-Dnmt1 constructs. (A) Dnmt1 consists of a large N-terminal regulatory domain containing PBD, TS domain and a CXXC zinc finger (ZnF) domain, and a conserved C-terminal catalytic domain. The point mutation of a highly conserved glutamine to glutamic acid introduced within the PBD eliminates interaction with the replication machinery (GFP-Dnmt1Q162E). A deletion in the central part of the TS domain (GFP-Dnmt1ΔTS) was introduced to abolish interaction with constitutive heterochromatin. Furthermore a construct containing both mutations was generated (GFP-Dnmt1Q162E/ΔTS). GFP alone is used as a control for a non-binding protein. (B) Spinning disk confocal mid sections of GFP-Dnmt1 wild type (wt) and mutant constructs in live mouse C2C12 cells co-expressing RFP-PCNA to mark RF. In early S phase, GFP-Dnmt1wt accumulates at RF, whereas PCNA-binding deficient GFP-Dnmt1Q162E is diffusely distributed throughout the nucleus. GFP-Dnmt1ΔTS is still associated with RF, but not as prominently as GFP-Dnmt1wt. In late S phase GFP-Dnmt1wt, GFP-Dnmt1Q162E and GFP-Dnmt1ΔTS accumulate at larger RF of late replicating pHC, although with slightly less strong enrichment observed for both mutants. GFP-Dnmt1Q162E/ΔTS is distributed diffusely in the nucleus throughout interphase. Scale bar: 5 µm.
Mentions: Our aim was to analyze the S phase–dependent regulation of the Dnmt1-binding behavior. To that end, we investigated four GFP fusions: wild type Dnmt1 (GFP-Dnmt1wt), the full-length Dnmt1 carrying a point mutation (GFP-Dnmt1Q162E) within the PBD (6), a Dnmt1 mutant carrying a deletion of a highly conserved part of the TS domain comprising the amino acids 459–501 (GFP-Dnmt1ΔTS) and a Dnmt1 double mutant containing both mutations (GFP-Dnmt1Q162E/ΔTS) (Figure 1A). To identify different cell cycle stages, we first co-expressed GFP-Dnmt1 constructs with PCNA fused to monomeric red fluorescent protein (RFP-PCNA) in mouse C2C12 myoblast cells and acquired confocal mid sections of the living cells (Figure 1B). As previously described (6), GFP-Dnmt1wt co-localized with RFP-PCNA at RF in early S phase. Co-localization with RF was also apparent in late S phase, when DNA of pericentromeric heterochromatin (pHC) is replicated. In contrast, the double mutant GFP-Dnmt1Q162E/ΔTS was diffusely distributed within nuclei throughout interphase, suggesting a deficiency to target RF during S phase. GFP-Dnmt1ΔTS still accumulated at RF in early and late S phase, but showed a slightly weaker association compared with GFP-Dnmt1wt, indicating the activity of PBD-mediated targeting in all S phase stages, independent of the presence of the TS domain (Figure 1B and Supplementary Figure S2). As previously reported, GFP-Dnmt1Q162E showed a diffuse nuclear distribution in early S phase but notable association to pHC replicating in late S phase (6). Together, this suggests that the PBD-mediated interaction with PCNA is necessary for the Dnmt1 localization in early S phase, but evidently not for the association at pHC in late S phase.Figure 1.

Bottom Line: We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s).In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions.We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

View Article: PubMed Central - PubMed

Affiliation: Department of Biology and Center for Integrated Protein Science, Ludwig Maximilians University Munich (LMU), 82152 Planegg-Martinsried, Germany.

ABSTRACT
DNA methyltransferase 1 (Dnmt1) reestablishes methylation of hemimethylated CpG sites generated during DNA replication in mammalian cells. Two subdomains, the proliferating cell nuclear antigen (PCNA)-binding domain (PBD) and the targeting sequence (TS) domain, target Dnmt1 to the replication sites in S phase. We aimed to dissect the details of the cell cycle-dependent coordinated activity of both domains. To that end, we combined super-resolution 3D-structured illumination microscopy and fluorescence recovery after photobleaching (FRAP) experiments of GFP-Dnmt1 wild type and mutant constructs in somatic mouse cells. To interpret the differences in FRAP kinetics, we refined existing data analysis and modeling approaches to (i) account for the heterogeneous and variable distribution of Dnmt1-binding sites in different cell cycle stages; (ii) allow diffusion-coupled dynamics; (iii) accommodate multiple binding classes. We find that transient PBD-dependent interaction directly at replication sites is the predominant specific interaction in early S phase (residence time Tres ≤ 10 s). In late S phase, this binding class is taken over by a substantially stronger (Tres ∼22 s) TS domain-dependent interaction at PCNA-enriched replication sites and at nearby pericentromeric heterochromatin subregions. We propose a two-loading-platform-model of additional PCNA-independent loading at postreplicative, heterochromatic Dnmt1 target sites to ensure faithful maintenance of densely methylated genomic regions.

Show MeSH