Quantitative LC–MS Provides No Evidence for m⁶dA or m⁴dC in the Genome of Mouse Embryonic Stem Cells and Tissues

Abstract

Abstract:Until recently,i tw as believed that the genomes of higher organisms contain, in addition to the four canonical DNAb ases,o nly 5-methyl-dC (m 5 dC) as am odified base to control epigenetic processes.I nr ecent years,t his view has changed dramatically with the discovery of 5-hydroxymethyldC (hmdC), 5-formyl-dC (fdC), and 5-carboxy-dC (cadC) in DNAfrom stem cells and brain tissue.N 6 -methyldeoxyadenosine (m 6 dA) is the most recent base reported to be present in the genome of various eukaryotic organisms.T his base,t ogether with N 4 -methyldeoxycytidine (m 4 dC), was first reported to be ac omponent of bacterial genomes.I nt his work, we investigated the levels and distribution of these potentially epigenetically relevant DNAb ases by using an ovel ultrasensitive UHPLC-MS method. We further report quantitative data for m 5 dC,h mdC,f dC,a nd cadC,b ut we were unable to detect either m 4 dC or m 6 dA in DNAisolated from mouse embryonic stem cells or brain and liver tissue,w hichc alls into question their epigenetic relevance.The genetic material of living organisms is constructed from the four canonical nucleobases dA, dC,d G, and dT,w hich establish the sequence information that, in multicellular organisms,i ss tored in the nucleus of every cell ( Figure 1). In addition to the canonical bases,the methylated dC base 5-methyldeoxycytidine (m 5 dC) is frequently found.[1] The presence or absence of this base in specific promoter segments determines whether the gene is actively transcribed or silenced.[1] Thec ell-type-specific distribution of m 5 dC thus determines the identity of ag iven cell. Recently,5 -hydroxymethyldeoxycytidine (hmdC) was found as as ixth base of the genetic system [2,3] and in 2011, 5-formyldeoxycytidine (fdC) [4,5] and 5-carboxydeoxycytidine (cadC) [5,6] were also discovered, particularly in DNAisolated from stem cells,but also in brain DNA. It is currently believed that fdC and cadC are intermediates in an active demethylation process that allows cells to change the methylation pattern and hence the activity state of specific genes. [7,8] ForfdC,separate epigenetic functions are also envisaged. [9] While the genomes of bacteria are known to also contain N 4 -methyldeoxycytidine (m 4 dC) [10] and N 6 -methyldeoxyadenosine (m 6 dA), [11] attempts to detect these bases in the DNA of higher organisms have failed until recently. [12][13][14][15] m 6 dA has now been found in algae (0.4 mol %m 6 dA/A), [12] fruit flies (0.001 %-0.07 %m 6 dA/A), [14] and C. elegans (0.01 %-0.4 % m 6 dA/A), [13] and its presence has even been reported in the DNAo fv ertebrates (0.00009 %i nX. laevis [16] and 0.00019-0.003 %o fd Ai nm urine cells and tissue [17] ). These discoveries,e specially concerning the DNAo fv ertebrates,h ave spurred aw orldwide research interest in unraveling the function of these new bases in human genomic DNA. [18][19][20] In this study,w ed eveloped an ultrasensitive triple quadrupole mass spectrometry (QQQ-MS) method, which in combination with ultra-high-pressure chromatograph...

A reassessment of DNA immunoprecipitation-based genomic profiling

“…Using this approach, we found that up to 15% of reads in published samples of 6mA-DIP-seq in mammals mapped to the Mycoplasma genome 31 . Taken together with the results of a recent study that was unable to detect 6mA in mammalian cells using mass spectrometry and our results showing clear enrichment for STRs using the 6mA antibody, a re-evaluation of the extent and origin of 6mA in mammalian studies is advisable 34 . As many bacteria and viruses contain abundant amounts of modified bases including 5mC, 5hmC and 6mA, controlling for contaminating DNA in all DIP-seq assays requires more rigorous application, particularly when the genomic content of target modifications in mammals becomes increasingly rare.…”

“…2f). Contamination of these samples may explain the earlier detection of 6mA in mammalian samples by mass spectrometry 31 and the subsequent failure of more recent attempts to detect 6mA in mammalian DNA using ultrasensitive UHPLC-MS 28,34 .…”

Non‐canonical Bases in the Genome: The Regulatory Information Layer in DNA

“…[145][146][147][148][149] While the levels of m 6 dA are high in unicellular eukaryotes (ciliated protozoa and green algae), they are extremely low in all higher eukaryotes studied so far. They are so low that ar ecent ultrasensitive LC-MS 2 based approach, using an isotopically labelled m 6 dA standard, was unable to confirm the presence of m 6 dA in vertebrates.T his result questions whether m 6 dA is af requently present and epigenetically relevant base, [150] but it cannot be excluded that m 6 dA is formed as aD NA lesion or during certain developmental phases.T he idea that m 6 dA is formed as al esion could explain the requirement for an m 6 dA demethylase. [144,147] One study showed recently as harp peak of m 6 dA content in early embryos of zebrafish and pig, [148] hinting indeed at an internal source of m 6 dA.…”

No evidence for DNA N 6 -methyladenine in mammals

“…Although most studies have reported vanishingly low 6mdA levels in mammals (0.0001 to 0.01% 6mdA/adenine), the range of reported 6mdA abundance between mammalian species and tissues is considerable (0.0001 to 1% 6mdA/dA; range, ~10,000-fold) (4,(6)(7)(8)(9)(10). In contrast, several other studies have been unable to detect 6mdA in mammals using highly sensitive methods (11)(12)(13). While the function of 6mdA in mammalian DNA is currently unknown, reports have suggested a potential role for 6mdA in lineage specification (8,9), similar to the role of its RNA counterpart, N 6 -methyladenosine (m 6 A) (14).…”