In one of modern cinema’s greatest movies, Jedi Master Yoda proclaims “No…there is another…”. He was talking about the presumed existence of only one Jedi, much as many mammalian biologists speak about 5-methylcytosine (5-mC) as being the only type of DNA methylation.
In a ground breaking new study, Andrew Z. Xiao (Yale School of Medicine, USA) has taken the role of Yoda and uncovered the “another” of the mammalian DNA methylation world – N6-methyladenine (N6-mA). N6-mA appears in mainly in prokaryotes and a limited number of eukaryotes, but only a single report in the 1980s had found evidence of N6-mA in mammals.
The revelation of the “another” came after the combination of clever thinking and new chromatin immunoprecipitation (ChIP) technology by a team of determined researchers.
- The study first immunoprecipitated DNA associated with histone variant H2AX from mouse ESCs
- H2AX deposition is strongly associated with cell fate transitions in mammals
- Variant histones also form altered nucleosome structures that may accommodate variations in DNA structures, such as chemical modifications
- They then subjected this DNA to a newly developed single molecular real-time sequencing of chromatin immunoprecipitation-enriched DNA (SMRT ChIP)
- Identifies modified bases based on inter-pulse duration ratios in the sequencing data
- This identified N6-mA sites in H2AX deposition regions with high confidence at intergenic, but not gene-rich regions.
Further investigation of N6-mA then uncovered more interesting findings:
- Alkbh1 acts as an N6-mA demethylase
- contains the conserved Fe2+ ion and 2-oxo-glutarate-dependent, dioxygenase domain required for demethylation and shares the strongest similarity to bacteria demethylase Alkb
- Alkbh1 deficiency leads to embryonic lethality signifying a critical role in early development
- Alkbh1 knockout by CRISPR caused a significant increase in N6-mA levels in ESCs
- The subsequent accumulation of N6-mA levels led to the repression of transcription, particularly on the X chromosome
- Specifically, N6-mA targets young (just 1.5 million years old!) full-length long interspersed element 1 (LINE-1 or L1) retrotransposons and protein-coding genes located on X chromosome
- Therefore, N6-mA deposition may silence transposons and neighboring DNA elements to resist gene activation signals that occur during ESC differentiation
The authors posit that their paradigm breaking study supports the view of N6-mA as a key element in epigenetic silencing in developing mammals. Interestingly, a brand new study now suggests that N6-mA is also detectable in adult mouse tissues and so may be more widespread than previously thought.
So will studies on this “another” proliferate until we have more new papers on N6-mA than Star Wars sequels, prequels, origins stories, and spin-offs? Until we get that answer, check out the Jedi-like moves behind this great new study at Nature, March 2016.