Figure 4. Naa10p Loss Impairs Dnmt1 Activity and Binding to ICRs/DMRs.
(A and B) Naa10p KO reduces Dnmt1 activity in nuclear extracts of mouse ESCs. Nuclear extracts (A) or Dnmt1 immunoprecipitated (B) from two WT, two Naa10-KO, or Dnmt1-KO ESCs were incubated with hemi-methylated double-stranded DNA oligonucleotides and [3H]-S-adenosyl-L-methionine, followed by determination of radioactivity of the DNA oligonucleotides and western blotting. After subtracting the background signal of Dnmt1-KO cells, the relative Dnmt1 activity from each sample was normalized to the mean activity of WT replicates (A, bottom, and B, right). Data represent the mean ± SD from three independent experiments. Asterisks indicate statistical difference calculated by two-tailed t test: **p < 0.01. IP, immunoprecipitation.
(C) Naa10p KO impairs Dnmt1 binding to multiple ICRs/DMRs during S phase in mouse ESCs. ChIP-qPCR analyses of Dnmt1 occupancy at eight representative ICRs/DMRs in fluorescence-activated cell-sorted WT#1 and KO#1 ESCs are shown. Error bars represent SD of triplicate PCR reactions. Biological repeats using independent ESC clones WT#2 and KO#2 are shown in Figure S4C.
(D) Naa10p KO impairs Dnmt1 binding to multiple ICRs/DMRs in a mouse embryo. A similar experiment as in (C) was performed in mouse embryos. (C and D) Error bars represent SD determined from triplicate PCR reactions. Asterisks indicate a statistical difference calculated by two-tailed t test: *p < 0.05, **p < 0.01.
See also Figure S4.