H2A.X SMRT-ChIP

It has widely accepted that 5-methylcytosine is the only form of DNA methylation in mammalian genomes, whereas the other forms, such as N6-methyladenine, primarily exist in prokaryotes and only a few eukaryotes. Herein, we demonstrated the surprising presence of N6-methyladenine in mammalian genomes, especially, mouse embryonic stem cells. This modification is enriched at histone variant H2A.X-deposited genomic regions in wild type embryonic stem cells. Our work also showed that a previously unknown DNA demethylase, Alkbh1, is the major demethylase for N6-methyladenine in embryonic stem cells. Increase of N6-methyladenine levels in Alkbh1 deficient cells leads to silencing of genes that regulate embryonic development. Surprisingly, genes located on the X-chromosome, but not the Y-chromosome or autosomes are preferentially silenced by N6-methyladenine. Strikingly, N6-methyladenine in Alkbh1 deficient cells are specifically deposition at young, full-length subfamilies of LINE1 transposons that are strongly enriched on the X chromosome. Furthermore, N6-methyladenine deposition on LINE1s pattern is inversely correlated with their evolutionary age. The deposition of N6-methyladenine results in epigenetic silencing of such L1s, which are otherwise actively transcribed in wild type embryonic stem cells, and the neighboring enhancers and genes. Furthermore, N6-methyladenine induced-silencing resists gene activation signals during embryonic stem cell differentiation. Thus, N6-methyladenine adopts a new function in epigenetic silencing in evolution, distinct from its role in gene activation in other organisms. In summary, our results demonstrate that N6-methyladenine unexpectedly constitutes a crucial component of the epigenetic regulation repertoire in mammalian genomes. First, we used a native-ChIP approach to enrich for DNA molecules residing in H2A.X-deposition regions in mouse ESCs as previously described. Then, co-purified DNA molecules from WT or KO ESCs were subject to SMRT sequencing and data analysis for DNA modifications (Pacific Biosciences). Overall design: H2A.X Native ChIP coupling with SMRT sequencing (separate the short "S" and long "L" part to sequencing)