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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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Super-resolution imaging of heterochromatin association of GFP-Dnmt1 constructs in late S phase. (A–C) 3D-SIM optical mid sections and z-projections from of C2C12 cells expressing GFP-Dnmt1 wild type and mutant constructs immunostained with antibodies against endogenous PCNA. Profile plots were scaled between minimum and maximum intensity values for each nucleus. (A) GFP-Dnmt1wt co-localizes largely but not strictly with PCNA inside ∼200 nm wide lacunas within otherwise densely packed DAPI-intense chromocenters of clustered pHC (inset a1, arrowheads in profile plot 1 and inset a3). Anti-PCNA-labeled RF outside of chromocenters show only minor or no association of Dnmt1 (inset a2, arrows in profile plot 2 and inset a3). (B) GFP-Dnmt1ΔTS strictly co-localizes with PCNA at RF inside and outside chromocenters (insets b1 + b2 and profile plots 3 + 4). An increased diffuse fraction is visible as small grainy evenly distributed nucleoplasmic background. (C) GFP-Dnmt1Q162E does not strictly co-localize with PCNA, but also associates with adjacent regions of pHC (arrowheads, inset c3 and profile plot 5). No association is detected in RF outside chromocenters (arrows, inset c3 and profile plot 6). (D) Additional replication labeling with a 60-min EdU pulse prior fixation. Association of GFP-Dnmt1wt and GFP-Dnmt1Q162E to chromocenter regions outside of PCNA foci is restricted to the bulk of EdU-labeled postreplicative chromatin (insets, arrows), while unlabeled, presumably not yet replicated chromocenter regions are still void of GFP-Dnmt1 (insets, arrowheads). Scale bars: 5 µm and 1 µm (insets).
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gkt191-F2: Super-resolution imaging of heterochromatin association of GFP-Dnmt1 constructs in late S phase. (A–C) 3D-SIM optical mid sections and z-projections from of C2C12 cells expressing GFP-Dnmt1 wild type and mutant constructs immunostained with antibodies against endogenous PCNA. Profile plots were scaled between minimum and maximum intensity values for each nucleus. (A) GFP-Dnmt1wt co-localizes largely but not strictly with PCNA inside ∼200 nm wide lacunas within otherwise densely packed DAPI-intense chromocenters of clustered pHC (inset a1, arrowheads in profile plot 1 and inset a3). Anti-PCNA-labeled RF outside of chromocenters show only minor or no association of Dnmt1 (inset a2, arrows in profile plot 2 and inset a3). (B) GFP-Dnmt1ΔTS strictly co-localizes with PCNA at RF inside and outside chromocenters (insets b1 + b2 and profile plots 3 + 4). An increased diffuse fraction is visible as small grainy evenly distributed nucleoplasmic background. (C) GFP-Dnmt1Q162E does not strictly co-localize with PCNA, but also associates with adjacent regions of pHC (arrowheads, inset c3 and profile plot 5). No association is detected in RF outside chromocenters (arrows, inset c3 and profile plot 6). (D) Additional replication labeling with a 60-min EdU pulse prior fixation. Association of GFP-Dnmt1wt and GFP-Dnmt1Q162E to chromocenter regions outside of PCNA foci is restricted to the bulk of EdU-labeled postreplicative chromatin (insets, arrows), while unlabeled, presumably not yet replicated chromocenter regions are still void of GFP-Dnmt1 (insets, arrowheads). Scale bars: 5 µm and 1 µm (insets).

Mentions: For a more detailed view on the spatial relationships of wild-type and mutant Dnmt1 and PCNA at RF, we used super-resolution 3D-SIM (31,32). Owing to the ∼8-fold improved volumetric resolution of 3D-SIM (39), we could clearly notice subtle variations in the Dnmt1 localization that escaped detection with conventional imaging (Figure 2 and Supplementary Figure S3). In late S phase, GFP-Dnmt1wt coincides to a large extent with immunofluorescently labeled endogenous PCNA foci in locally decondensed parts of otherwise homogenously compacted chromocenters with some Dnmt1 signal extending slightly (by a few 100 nm) beyond the PCNA signal. Interestingly, RF outside of chromocenters showed almost no enrichment of Dnmt1 (Figure 2A). In contrast, early S phase cells showed a more balanced co-localization at RF (Supplementary Figure S3A). Co-immunostaining of non-transfected cells with the Dnmt1-specific monoclonal antibody 5A10 confirmed the same localization characteristics for the endogenous Dnmt1, hence excluding potential artifacts by the GFP-tagging or overexpression (Supplementary Figure S3B). As opposed to this, GFP-Dnmt1ΔTS precisely co-localized with all PCNA marked RF inside and outside of chromocenters (Figure 2B). GFP-Dnmt1Q162E, similar to the wild type, displayed an enrichment at chromocenter-associated RF but also in the nearby regions of the chromocenters that were more compacted. We further noted these regions to become larger toward the end of late S phase, indicating that TS-mediated binding primarily occurs at postreplicative pHC (Supplementary Figure S3C). Pulse replication labeling with 5-ethenyl-2’-deoxyuridine and co-staining with PCNA confirmed the association of both, wild type and the PBD mutant Dnmt1 to postreplicative pHC (Figure 2D). Hence, we conclude a strict co-localization of the ΔTS mutant with PCNA at replication sites in late S phase, whereas both TS domain-containing constructs (GFP-Dnmt1Q162E stronger than GFP-Dnmt1wt) show a non-strict co-localization and a tendency to bind adjacent postreplicative pHC.Figure 2.


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)

Super-resolution imaging of heterochromatin association of GFP-Dnmt1 constructs in late S phase. (A–C) 3D-SIM optical mid sections and z-projections from of C2C12 cells expressing GFP-Dnmt1 wild type and mutant constructs immunostained with antibodies against endogenous PCNA. Profile plots were scaled between minimum and maximum intensity values for each nucleus. (A) GFP-Dnmt1wt co-localizes largely but not strictly with PCNA inside ∼200 nm wide lacunas within otherwise densely packed DAPI-intense chromocenters of clustered pHC (inset a1, arrowheads in profile plot 1 and inset a3). Anti-PCNA-labeled RF outside of chromocenters show only minor or no association of Dnmt1 (inset a2, arrows in profile plot 2 and inset a3). (B) GFP-Dnmt1ΔTS strictly co-localizes with PCNA at RF inside and outside chromocenters (insets b1 + b2 and profile plots 3 + 4). An increased diffuse fraction is visible as small grainy evenly distributed nucleoplasmic background. (C) GFP-Dnmt1Q162E does not strictly co-localize with PCNA, but also associates with adjacent regions of pHC (arrowheads, inset c3 and profile plot 5). No association is detected in RF outside chromocenters (arrows, inset c3 and profile plot 6). (D) Additional replication labeling with a 60-min EdU pulse prior fixation. Association of GFP-Dnmt1wt and GFP-Dnmt1Q162E to chromocenter regions outside of PCNA foci is restricted to the bulk of EdU-labeled postreplicative chromatin (insets, arrows), while unlabeled, presumably not yet replicated chromocenter regions are still void of GFP-Dnmt1 (insets, arrowheads). Scale bars: 5 µm and 1 µm (insets).
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gkt191-F2: Super-resolution imaging of heterochromatin association of GFP-Dnmt1 constructs in late S phase. (A–C) 3D-SIM optical mid sections and z-projections from of C2C12 cells expressing GFP-Dnmt1 wild type and mutant constructs immunostained with antibodies against endogenous PCNA. Profile plots were scaled between minimum and maximum intensity values for each nucleus. (A) GFP-Dnmt1wt co-localizes largely but not strictly with PCNA inside ∼200 nm wide lacunas within otherwise densely packed DAPI-intense chromocenters of clustered pHC (inset a1, arrowheads in profile plot 1 and inset a3). Anti-PCNA-labeled RF outside of chromocenters show only minor or no association of Dnmt1 (inset a2, arrows in profile plot 2 and inset a3). (B) GFP-Dnmt1ΔTS strictly co-localizes with PCNA at RF inside and outside chromocenters (insets b1 + b2 and profile plots 3 + 4). An increased diffuse fraction is visible as small grainy evenly distributed nucleoplasmic background. (C) GFP-Dnmt1Q162E does not strictly co-localize with PCNA, but also associates with adjacent regions of pHC (arrowheads, inset c3 and profile plot 5). No association is detected in RF outside chromocenters (arrows, inset c3 and profile plot 6). (D) Additional replication labeling with a 60-min EdU pulse prior fixation. Association of GFP-Dnmt1wt and GFP-Dnmt1Q162E to chromocenter regions outside of PCNA foci is restricted to the bulk of EdU-labeled postreplicative chromatin (insets, arrows), while unlabeled, presumably not yet replicated chromocenter regions are still void of GFP-Dnmt1 (insets, arrowheads). Scale bars: 5 µm and 1 µm (insets).
Mentions: For a more detailed view on the spatial relationships of wild-type and mutant Dnmt1 and PCNA at RF, we used super-resolution 3D-SIM (31,32). Owing to the ∼8-fold improved volumetric resolution of 3D-SIM (39), we could clearly notice subtle variations in the Dnmt1 localization that escaped detection with conventional imaging (Figure 2 and Supplementary Figure S3). In late S phase, GFP-Dnmt1wt coincides to a large extent with immunofluorescently labeled endogenous PCNA foci in locally decondensed parts of otherwise homogenously compacted chromocenters with some Dnmt1 signal extending slightly (by a few 100 nm) beyond the PCNA signal. Interestingly, RF outside of chromocenters showed almost no enrichment of Dnmt1 (Figure 2A). In contrast, early S phase cells showed a more balanced co-localization at RF (Supplementary Figure S3A). Co-immunostaining of non-transfected cells with the Dnmt1-specific monoclonal antibody 5A10 confirmed the same localization characteristics for the endogenous Dnmt1, hence excluding potential artifacts by the GFP-tagging or overexpression (Supplementary Figure S3B). As opposed to this, GFP-Dnmt1ΔTS precisely co-localized with all PCNA marked RF inside and outside of chromocenters (Figure 2B). GFP-Dnmt1Q162E, similar to the wild type, displayed an enrichment at chromocenter-associated RF but also in the nearby regions of the chromocenters that were more compacted. We further noted these regions to become larger toward the end of late S phase, indicating that TS-mediated binding primarily occurs at postreplicative pHC (Supplementary Figure S3C). Pulse replication labeling with 5-ethenyl-2’-deoxyuridine and co-staining with PCNA confirmed the association of both, wild type and the PBD mutant Dnmt1 to postreplicative pHC (Figure 2D). Hence, we conclude a strict co-localization of the ΔTS mutant with PCNA at replication sites in late S phase, whereas both TS domain-containing constructs (GFP-Dnmt1Q162E stronger than GFP-Dnmt1wt) show a non-strict co-localization and a tendency to bind adjacent postreplicative pHC.Figure 2.

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
Related in: MedlinePlus