Sequencing 5‐Hydroxymethyluracil at Single‐Base Resolution

Abstract

5-hydroxymethyluracil (5hmU) is formed through oxidation of thymine both enzymatically and non-enzymatically in various biological systems.A lthough 5hmU has been reported to affect biological processes such as protein-DNA interactions,the consequences of 5hmU formation in genomes have not been yet fully explored. Herein, we report amethod to sequence 5hmU at single-base resolution. We employ chemical oxidation to transform 5hmU to 5-formyluracil (5fU), followed by the polymerase extension to induce T-to-C base changes owingt ot he inherent ability of 5fUt of orm 5fU:G base pairing. In combination with the Illumina next generation sequencing technology,w ed eveloped polymerase chain reaction (PCR) conditions to amplify the T-to-C base changes and demonstrate the method in three different synthetic oligonucleotide models as well as part of the genome of a5 hmU-rich eukaryotic pathogen. Our method has the potential capability to map 5hmU in genomic DNAa nd thus will contribute to promote the understanding of this modified base. DNA-base modifications can profoundly influence biologyand an umber of modified bases have been identified in the genomes of av ariety of organisms.[1] 5-hydroxymethyluracil (5hmU) is produced through oxidation of thymine both enzymatically and non-enzymatically [2] and can influence the binding of proteins to DNA. [2b,3] It has also been suggested that 5hmU might lead to genomic instability as it can be removed by DNAr epair enzymes to create potentially mutagenic lesions [4] and affect the stability of DNA duplexes.[5] When incorporated at some promoter sites, 5hmU has been shown to affect transcription by bacterial RNAP,therefore it may have asignificant effect on microbial biology. [6] In mammals,reported levels of 5hmU vary by celland tissue types. [2b, 7] Increased levels of 5hmU autoantibodies have been reported in cancer cases, [8] and blood 5hmU mononucleoside levels have been studied as am arker of cancer risks and invasiveness.[9] We previously reported am ethod to map 5hmU at moderate resolution by chemical enrichment of 5hmU-containing DNAfragments followed by sequencing.[10] Amethod for single-base sequencing of 5hmU would enable the identification of individual modification sites in genomes.Asingle-molecule real-time (SMRT) sequencing approach could in principle be applied to map 5hmU at single-base resolution, however the intrinsic signature signal for 5hmU is rather weak unless the base is further modified.[11] Mapping 5hmU at single-base resolution is aw orthy challenge that could transform genome-wide analysis of 5hmU.H erein, we describe ac hemical approach for single base-resolution sequencing of 5hmU and demonstrate its utility in various sequence contexts.Thec onceptual basis for sequencing 5hmU involves chemical oxidation of 5hmU to 5fU, which ionizes under mild alkaline pH owing to the electron-withdrawing exocyclic aldehyde (pK a at N3 = 8.1 for 5fUv s. 9.3 for 5hmU).[12] The ionized form of 5fUcan base-pair with G (Figure 1), causing aT -to-C base chan...

Highly Selective 5-Formyluracil Labeling and Genome-wide Mapping Using (2-Benzimidazolyl)Acetonitrile Probe

“…Aldehydes present in 5fU are more active than 5fC ( Habgood et al., 2011 , Hardisty et al., 2015 ), which might also conjugate histone to yield DNA-protein cross-linking and influence transcriptional regulation and chromatin remodeling. Although the method we proposed could not realize single-base-resolution analysis of 5fU, we believe that this goal can be realized in the future, according to the recent report of single-base-resolution detection method of 5hmU proposed by Balasubramanian's group ( Kawasaki et al., 2018 ). Further studying and applying this method will advance our understanding of the role played by 5fU modification in genomics.…”

“…In addition, it is unavoidable for nonspecific enrichment of DNA during pull-down process. We are currently in the process of realizing the single-base-resolution analysis of 5fU, as per the recent report by Balasubramanian's group ( Kawasaki et al., 2018 ). Combining with the single-base-resolution analysis, the nonspecific enrichment of DNA can be ruled out.…”

“…Recently, 5-formylcytosine (5fC), the modified cytosine counterpart of 5fU, has been identified as a vital epigenetic modification involved in gene regulation ( Kitsera et al., 2017 , Song et al., 2013 , Wang et al., 2018b ), cell differentiation, and development ( Wagner et al., 2015 , Zhu et al., 2017 ). 5fU may be an oxidation product of 5-hydroxymethyluracil (5hmU) in vivo , and 5hmU has been identified as not only an oxidized nucleobase but also an essential epigenetic mediator that influences transcription factors, changes the physical properties of local DNA duplex in the genome, and helps in binding of chromatin remodeling proteins ( Kawasaki et al., 2017a , Kawasaki et al., 2018 , Modrzejewska et al., 2016 , Pfaffeneder et al., 2014 ). Whether 5fU also acts as an epigenetic mediator like 5fC and 5hmU remains an open question.…”

Bisulfite-free, single base-resolution analysis of 5-hydroxymethylcytosine in genomic DNA by chemical-mediated mismatch

“…First, we extracted the genomic DNA from mESCs. Then the genomic DNA was incubated with hydroxylamine and subjected to oxidation as described by the protocol reported previously,28,40 followed by labeling with azi-BP and PCR. The PCR products were verified by agarose gel electrophoresis analysis (Fig.…”

Cellular macromolecules-tethered DNA walking indexing to explore nanoenvironments of chromatin modifications

“…In the past decade, the chromatin immunoprecipitation (ChIP) deep sequencing and its derivatives have been well established to analyze genomic positioning of histone PTMs 10 , 12 , 15 . Different sequencing strategies have also been developed for genome-wide profiling of DNA modifications including 5mC, 5hmC, 5fC, or 5hmU 4 – 7 , 9 , 16 . These methods reported chemical or chemoenzymatic labeling of modified bases, and revealed their important biological functions.…”