Fig 2: Summary of Tet1 and Mbd1 localization and 5hmC formation. Summary of Tet1 (red) and Mbd1 (green) localization and 5-hydroxymethylcytosine (5hmC, blue) formation. At an early time point, Mbd1 localized at heterochromatin recruits Tet1 to heterochromatic DNA and this further enhances 5hmC formation. However, once 5-methylcytosines (5mC) are modified by Tet1, both, Mbd1 and Tet1 progressively lose binding ability to heterochromatic DNA. Solid lines represent levels in cells with both Mbd1 and Tet1. Dashed lines represent levels in cells with either Mbd1 or Tet1 alone.

Fig 3: Tet1 displaces Mbd1 binding to heterochromatic DNA. (A) Mobility of Mbd1 and Mbd1?CXXC3. MEF cells were transfected with Mbd1-GFP or GFP-Mbd1?CXXC3. Eight hours after transfection, the mobility of Mbd proteins was measured by FRAP. The mean values are plotted as a recovery curve and the cell number is indicated on the plot. T-half values were extracted from mean exponential fitting. Solid and dashed lines indicate recovery curve of Mbd1?CXXC3 and Mbd1, respectively. (B) Mobility of Mbd1 (right) and Mbd1?CXXC3 (left) in the presence of Tet1CD. MEF cells co-expressing mCherry-Tet1CD and GFP-Mbd1 or GFP-Mbd1?CXX3 were used for FRAP analysis. One heterochromatic region was photobleached with a 488 nm laser, and after photobleaching the intensities of GFP and mCherry were measured. The mean values are plotted as a recovery curve and cell numbers are indicated on the plot. T-half values were extracted from the mean exponential fitting. T-half values for single cells are shown as box plots and P-values (Wilcoxon test) are indicated. At least two independent experiments were performed. (C) Scheme summarizing the Mbd1 and Tet1 binding modes to heterochromatic DNA. Black and white circles indicate methylated and unmethylated CpG, respectively.

Fig 4: The interaction of Mbd1 and Tet1 enhances Tet1 localization to heterochromatic DNA. (A) Dnmt1-/- MEF (p53-/- and Dnmt1-/-) cells were transfected with mCherry-Tet1CD and Mbd1-GFP or Mbd2-GFP. Eight hours after transfection, the cells were imaged using confocal microscopy. The left panel shows the schematic of the experiment and the right panel shows exemplary cells with Tet1 and Mbd1/Mbd2 expression. (B) Accumulation of Mbd1/Mbd2 and Tet1 proteins to heterochromatic DNA measured as in Figure 2. (C) Dnmt1-/- MEF cells were triple-transfected with mCherry-Tet1CD, GBP-MaSat (GFP binding protein, polydactyl zinc finger major satellite binder) and Mbd1-GFP or Mbd2-GFP plasmids. Eight hours after transfection, cells were imaged using confocal microscopy. The left panel shows the schematic of the experiment and how GBP-MaSat targets GFP-tagged proteins to heterochromatic DNA. The right panel shows exemplary cells with Tet1 and Mbd1/Mbd2 expression in the presence of GBP-MaSat. (D) Accumulation of Tet1 and Mbd1/2 proteins to heterochromatic DNA. Cell number and P-value of t-test are indicated. (E) Interaction between Tet1CD and subdomains of Mbd1. HEK (human embryonic kidney) cells co-expressing mCherry-Tet1CD and GFP tagged Mbds were used for co-immunoprecipitation assays using immobilized RFP binding protein. The input and bound GFP signals on the membrane were detected using a GFP-specific antibody. Left panels are input for mCherry and GFP fusion proteins and rightmost panel shows interaction bands. The middle panel depicts the structures of Mbd1 fusions. The right panel summarizes the results of two independent biological replicates.

Fig 5: Mbd1 enhances Tet1-mediated 5hmC formation. (A) Experimental rationale. Mouse tail fibroblasts (MTF) were transfected with mCherry-Tet1CD (catalytic domain of Tet1) or mCherry-Tet1FL (full length Tet1) together with Mbd1-GFP (methyl-CpG binding domain protein 1), Mbd2-GFP or GFP. Twenty-four hours after transfection the cells were immunostained with a 5-hydroxymethylcytosine (5hmC) specific antibody. The signals of Tet1, Mbd/GFP and 5hmC were detected by high content screening microscopy and low Tet1 expressing cells were used for plotting. (B) Effect of Mbd1, Mbd2 and GFP on Tet1CD-mediated 5hmC formation. The cells were first grouped according to Tet1 protein levels, then, in each group, cells were further binned by Mbd/GFP protein levels and single cell intensities were plotted. The 5hmC intensities in the cells with high levels of Mbds/GFP and high levels of Tet1CD were used to perform student's t-test and P-values are indicated. Experiments were repeated at least three times. Cell numbers for Tet1CD + Mbd1, Tet1CD + GFP and Tet1CD + Mbd2 are 10817, 7271 and 4375, respectively. (C) Effect of Mbd1 and GFP on Tet1FL-mediated 5hmC formation in mouse embryonic fibroblast cells (MEF). Intensities of single cells are plotted and the P-value of student's t-test is indicated. Experiments were repeated at least two times. Cell numbers for Tet1FL + Mbd1 and Tet1FL + GFP are 7515 and 3429, respectively. (D) Effect of Mbd1 and GFP on Tet1-mediated 5-carboxylcytosine (5caC) formation in MTF cells. MTF cells were transfected with mCherry-Tet1CD together with Mbd1-GFP or GFP. Twenty-four hours after transfection, the cells were immunostained with an 5caC antibody. The average intensity of single cells was plotted according to Tet1CD protein levels. Experiments were repeated twice and mean values were plotted. Cell numbers for Tet1CD + Mbd1 and Tet1CD + GFP are 1770 and 582, respectively. The 5caC intensities in the cells with high levels of Tet1CD (dashed line box) were used to perform student's t-test and the P-value is indicated. 95% confidence intervals were also indicated in the plot.