Extended Data Figure 3. Alkbh1 is a specific N6-mA demethylase in vivo and in vitro.

a, Top: schematic of the CRISPR–Cas9 approach. Alkbh1 KO alleles don’t contain the XmaI site at exon 3. Bottom left: PCR-DNA digestion approach indicating the homozygosity of the knockout alleles, which are resistant to Xma1 digestion. Bottom right: western blotting did not detect any ALKBH1 proteins in the KO cells. b, Three additional Alkbh1 knockout ES cell clones show similar levels of N6-mA upregulation. Shown are dot blot results. c, Validating the specificity of anti-N6-mA antibodies with synthetic oligonucleotides. d, Validating the specificity of anti-N6-mA antibodies with DNA samples of different N6-mA/dA ratio. 125 ng of genomic DNA (MEFs) which does not contain any endogenous N6-mA was spiked with N6-mA containing oligonucleotides at the indicated concentration. e, Tandem mass spectrometric analysis shows the lack of H2AK118/119 methylation in wild-type or Alkbh1 knockout ES cells. Spectral counts for H2A peptides containing K118/119 revealed that H2AK118/119 is predominately non-methylated at similar levels between wild-type and Alkbh1 knockout ES cells. Spectral counts are reported as an average with standard deviation from biological triplicate analyses. K118/119: no methylation; K118/119me1: K118/119 monomethylation. f, MS analysis showed that the co-purified factors with recombinant ALKBH1 proteins are mainly heat shock proteins. g, ALKBH1 proteins don’t have noticeable activities towards to dual- or hemi-methylated double-stranded oligonucleotide substrates. h, ALKBH1 activities are dependent on Fe²⁺ and α-KG. Error bars: standard deviation of triplicates. i, Ectopic expression of wild-type, but not mutant, Alkbh1 (D233A) at the catalytic motif, can rescue the aberrant increase of N6-mA level in Alkbh1 knockout ES cells. The wild-type and mutant Alkbh1 were expressed at similar levels. j, Quantification of three independent rescue experiments in i. P value as labelled, determined by t-test; error bars, s.d. for three biological replicates. k, The demethylation activity of N6-mA by recombinant D233A mutant protein is much reduced in comparison with the wild-type counterpart. l, No significant activities were detected with increasing concentrations of recombinant D233A mutant proteins in demethylation reaction. Error bars, s.d. of triplicates.