Figure 2.

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-Dnmt1^wt 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-Dnmt1^ΔTS 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-Dnmt1^Q162E 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-Dnmt1^wt and GFP-Dnmt1^Q162E 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).