Fig. 5: The BAHCC1TTD:H4K20me1 interaction facilitates MCM loading.
From: BAHCC1 binds H4K20me1 to facilitate the MCM complex loading and DNA replication
a, b Immunoblotting to assess the chromatin association and loading of MCM2 and MCM3 in HeLa (a) or MEF cells (b) bearing the BAHCC1TTD-dead mutation (Y1993A for HeLa or Y1998A for MEF) or BAHCC1 KO, compared to WT. MCM loading is inferred from resistance to high salt extraction. GAPDH serves as a chromatin fractionation control and histone H3 as a loading control. c Flow cytometry-based assessment of MCM2 chromatin binding (y-axis) across different cell cycle stages, as determined by EdU incorporation and DNA content (DAPI intensity, x-axis) in HeLa cells with BAHCC1 KO (right) or its TTD-dead mutation (Y1993A; middle), relative to WT (left). Blue, MCM2 positive/EdU-negative (G1 phase); Orange, MCM2 positive/EdU positive (S phase); Gray, MCM2 negative nuclei (mainly G2/M). d Quantification of the intensities of MCM2 chromatin binding in the origin licensing phase (G1) of HeLa cells with BAHCC1 KO (right) or its TTD-dead mutation (Y1993A; middle), relative to WT (left). Error bars indicate mean ± SD; Two-tailed unpaired t-test: ****P < 0.0001; from left to right: rep1, n = 2,936 (WT), 2,766 (Y1993A) and 2,737 (KO); rep2, n = 2,974 (WT), 2,955 (Y1993A) and 2,750 (KO); rep3: n = 4,261 (WT), 2,495 (Y1993A) and 3,235 (KO). e Chromatin binding of MCM2 (y-axis) shown as a function of DAPI intensity (x-axis) and cell cycle stages, discriminated by EdU labeling in MEFs bearing either WT or TTD-dead (Y1998A) Bahcc1. f Chromatin-associated ORC4 levels (y-axis) shown as a function of DAPI intensity (x-axis) and cell cycle stages, discriminated by EdU labeling in HeLa cells bearing either WT (left) or TTD-dead (Y1993A; middle) BAHCC1 or its KO (right). Green and purple colors refer to cells with loaded ORC4 in G1 and S phases, respectively, while gray color designates cells without loaded ORC4. g Quantification of the intensities of chromatin-bound ORC4 in G1 phase cells shown in (f). Error bars indicate mean ± s.d.; Two-tailed unpaired t-test: ****P < 0.0001; from left to right: rep1, n = 3,920 (WT), 3,382 (Y1993A) and 4,096 (KO); rep2, n = 3,617 (WT), 2,803 (Y1993A) and 2,590 (KO). Source data are provided as a Source Data file.
