Mammalian ALKBH1 serves as an N6-mA demethylase of unpairing DNA

Abstract

N 6-methyladenine (N 6-mA) of DNA is an emerging epigenetic mark in mammalian genome. Levels of N 6-mA undergo drastic fluctuation during early embryogenesis, indicative of active regulation. Here we show that the 2-oxoglutarate-dependent oxygenase ALKBH1 functions as a nuclear eraser of N 6-mA in unpairing regions (e.g., SIDD, Stress-Induced DNA Double Helix Destabilization regions) of mammalian genomes. Enzymatic profiling studies revealed that ALKBH1 prefers bubbled or bulged DNAs as substrate, instead of single-stranded (ss-) or double-stranded (ds-) DNAs. Structural studies of ALKBH1 revealed an unexpected "stretch-out" conformation of its "Flip1" motif, a conserved element that usually bends over catalytic center to facilitate substrate base flipping in other DNA demethylases. Thus, lack of a bending "Flip1" explains the observed preference of ALKBH1 for unpairing substrates, in which the flipped N 6-mA is primed for catalysis. Co-crystal structural studies of ALKBH1 bound to a 21-mer bulged DNA explained the need of both flanking duplexes and a flipped base for recognition and catalysis. Key elements (e.g., an ALKBH1-specific α1 helix) as well as residues contributing to structural integrity and catalytic activity were validated by structurebased mutagenesis studies. Furthermore, ssDNA-seq and DIP-seq analyses revealed significant co-occurrence of base unpairing regions with N 6-mA in mouse genome. Collectively, our biochemical, structural and genomic studies suggest that ALKBH1 is an important DNA demethylase that regulates genome N 6-mA turnover of unpairing regions associated with dynamic chromosome regulation.

ALKBH1 deficiency leads to loss of homeostasis in human diploid somatic cells

“…Despite that ALKBH1 has recently been reported as a 6mA demethylase (Xiao et al, 2018;Xie et al, 2018), which is by far the only one identified in human, we did not observe discernible difference in the 6mA levels in hESCs, hMSCs or hVSMCs caused by ALKBH1 deficiency, consistent with the recent report that ALKBH1 fails to eliminate 6mA in mESCs or HEK293T cells (Liu et al, 2020). Given that two studies have shown that ALKBH1 preferably demethylates 6mA on unpaired DNAs (Tian et al, 2020;Zhang et al, 2020a), our results imply that there might be few unpaired DNAs in the normally cultured hESCs, hMSCs and hVSMCs. For example, R-loop, a form of unpaired DNAs, is a known ALKBH1 substrate that only accounts for about 5% of the human genome (Sanz et al, 2016;Zhang et al, 2020a).…”

“…Given that two studies have shown that ALKBH1 preferably demethylates 6mA on unpaired DNAs (Tian et al, 2020;Zhang et al, 2020a), our results imply that there might be few unpaired DNAs in the normally cultured hESCs, hMSCs and hVSMCs. For example, R-loop, a form of unpaired DNAs, is a known ALKBH1 substrate that only accounts for about 5% of the human genome (Sanz et al, 2016;Zhang et al, 2020a). Accordingly, the potential alteration in 6mA levels on R-loop may be undetectable in our study.…”

“…6mA has been reported to associate with multiple physiological processes including embryonic development and tumorigenesis (Greer et al, 2015 ; Zhang et al, 2015 ; Xie et al, 2018 ), yet some controversies exist. In contrast to the findings showing that ALKBH1 (alkB homolog 1) is a primary 6mA demethylase in mouse and human cells (Wu et al, 2016 ; Xiao et al, 2018 ; Xie et al, 2018 ), other studies indicate that ALKBH1 is prone to demethylate 6mA on bubbled or bulged DNAs that are often featured by a locally unpairing region with flanking duplex, such as D-loop, R-loop as well as DNA or RNA stem-loop, and single-stranded DNAs at a lower efficiency, but not double-stranded DNAs (Tian et al, 2020 ; Zhang et al, 2020a ). Stem cell exhaustion is a major causal and risk factor underlying the progressive disruption of physiological integrity during the development of aging-associated disorders, in which epigenetic alterations are closely implicated (Zhang et al, 2020b ).…”

Biochemical and structural basis for YTH domain of human YTHDC1 binding to methylated adenine in DNA

“…Here we characterized, for the first time, the in vitro binding activity of the YTH domain of human YTHDC1 as preferentially targeting DNA, relative to RNA. YTH binds N6mA in the context of ssDNA, an activity comparable to that found in the human enzymatic activities of the MettL3–14 MTase complex and the demethylase Alkbh1, as they each act on damaged or unpaired DNA ( 26 , 37 ). Additional study will be required to address whether YTHDC1 is involved in the recognition of DNA adenine methylation in vivo and, if so, its impact on chromatin organization.…”

“…Human and mouse homologs (Alkbh1 and Alkbh4) of the E. coli repair enzyme AlkB can function as sequence-independent DNA N6mA demethylases ( 9 , 10 , 13 ), and at least the mouse Alkbh1 prefers locally-unpaired DNA substrates ( 37 ). It is intriguing that both the writer (MTase) and eraser (demethylase) of the N6mA methyl mark act on single-stranded and transiently unpaired DNA.…”