Figure 6.

Acute treatment with 5-azaC decreases the mobility of a subset of DNMT1. (A) CpG methylation levels in cells treated with 5-azaC. Cells were treated with 4 μM 5-azaC for 4 or 24 h, gDNA was extracted and subjected to nanopore sequencing. The % methylation difference between the DMSO- and 5-azaC-treated cells is shown. (B) Cells were treated with DMSO for 4 h or 4 μM 5-azaC for 4, 24 or 48 h, harvested using Laemmli buffer and proteins separated on a 3–8% Tris-acetate gel. DNMT1-Halo protein was visualized by incubating cells with 500 nM JF646 Halo-ligand for 30 min before harvesting. Coomassie staining of ∼ 205 kDa region is shown as a loading control. (C–F) Cells were treated with either DMSO or 4 μM 5-azaC for 4 h. Images were acquired using HiLo microscopy at ∼ 97 fps over a 10 s period with no delay. (D-F) Spot-on analysis. Following 5-azaC treatment, the mobility of ∼ 50% of the slower diffusing DNMT1 molecules was significantly decreased, corresponding to an increase in the bound fraction of DNMT1. Violin plots showing the distribution of DNMT1-Halo 32–40 cells across two replicates. Student t-test, two-tailed, ** P < 0.01, ns = not significant. (G-I) 5-azaC treatment relocalizes DNMT1 from the nucleoplasm to chromatin. (G) E8 (+/+) cells treated with either DMSO or 5-azaC were fractionated into nucleoplasm and chromatin fractions. DNMT1-Halo protein is shown by JF646 fluorescent gel or immunoblot against FLAG. Note that the level of DNMT1 in the nucleoplasm decreases following 5-azaC treatment, consistent with total levels shown in (B). PARP1 and histones (from Ponceau gel) are used as protein markers for each compartment. (H) Relative DNMT1 intensity, normalized to total protein loading, from JF646 fluorescent gel, error bars represent standard error of the mean between three replicates, see Supplementary Fig. S10A and B. (I) DNMT1 intensity in the chromatin fraction divided by the nucleoplasmic fraction, error bars represent standard error of the mean.