Reduced Bisulfite Sequencing: Quantitative Base-Resolution Sequencing of 5-Formylcytosine

Overview

Journal Methods Mol Biol

Specialty Molecular Biology

Date 2021 May 19

PMID 34009605

Citations 1

Authors

Michael J Booth

Shankar Balasubramanian

Affiliations

Soon will be listed here.

Abstract

The generation of tools to study mammalian epigenetics is vital to understanding normal biological function and to identify how it is dysregulated in disease. The well-studied epigenetic DNA modification 5-methylcytosine can be enzymatically oxidized to 5-formylcytosine (5fC) in vivo. 5fC has been demonstrated to be an intermediate in demethylation, but recent evidence suggests that 5fC may have an epigenetic function of its own. We have developed reduced bisulfite sequencing (redBS-seq), which can quantitatively locate 5fC bases at single-base resolution in genomic DNA. In bisulfite sequencing (BS-seq), 5fC is converted to uracil, as happens to unmodified cytosine (C), and thus cannot be discriminated from C. However, in redBS-seq, a specific reduction of 5fC to 5-hydroxymethylcytosine (5hmC) stops this conversion, allowing its discrimination from C. 5fC levels are inferred by comparison of a redBS-Seq run with a BS-seq run.

Citing Articles

Correlation of ChREBP Gene Methylation with Pathological Characteristics of Type 2 Diabetes Mellitus.

Wang X, Huangfu W, Zhao F Appl Biochem Biotechnol. 2023; 196(6):3076-3087.

PMID: 37615853 DOI: 10.1007/s12010-023-04714-4.

References

Hayatsu H, Wataya Y, Kai K, Iida S . Reaction of sodium bisulfite with uracil, cytosine, and their derivatives. Biochemistry. 1970; 9(14):2858-65. DOI: 10.1021/bi00816a016. View

Tahiliani M, Koh K, Shen Y, Pastor W, Bandukwala H, Brudno Y . Conversion of 5-methylcytosine to 5-hydroxymethylcytosine in mammalian DNA by MLL partner TET1. Science. 2009; 324(5929):930-5. PMC: 2715015. DOI: 10.1126/science.1170116. View

Kriaucionis S, Heintz N . The nuclear DNA base 5-hydroxymethylcytosine is present in Purkinje neurons and the brain. Science. 2009; 324(5929):929-30. PMC: 3263819. DOI: 10.1126/science.1169786. View

Ito S, Shen L, Dai Q, Wu S, Collins L, Swenberg J . Tet proteins can convert 5-methylcytosine to 5-formylcytosine and 5-carboxylcytosine. Science. 2011; 333(6047):1300-3. PMC: 3495246. DOI: 10.1126/science.1210597. View

Pfaffeneder T, Hackner B, Truss M, Munzel M, Muller M, Deiml C . The discovery of 5-formylcytosine in embryonic stem cell DNA. Angew Chem Int Ed Engl. 2011; 50(31):7008-12. DOI: 10.1002/anie.201103899. View

Maiti A, Drohat A . Thymine DNA glycosylase can rapidly excise 5-formylcytosine and 5-carboxylcytosine: potential implications for active demethylation of CpG sites. J Biol Chem. 2011; 286(41):35334-35338. PMC: 3195571. DOI: 10.1074/jbc.C111.284620. View

Raiber E, Beraldi D, Ficz G, Burgess H, Branco M, Murat P . Genome-wide distribution of 5-formylcytosine in embryonic stem cells is associated with transcription and depends on thymine DNA glycosylase. Genome Biol. 2012; 13(8):R69. PMC: 3491369. DOI: 10.1186/gb-2012-13-8-r69. View

Song C, Szulwach K, Dai Q, Fu Y, Mao S, Lin L . Genome-wide profiling of 5-formylcytosine reveals its roles in epigenetic priming. Cell. 2013; 153(3):678-91. PMC: 3657391. DOI: 10.1016/j.cell.2013.04.001. View

Wu X, Zhang Y . TET-mediated active DNA demethylation: mechanism, function and beyond. Nat Rev Genet. 2017; 18(9):517-534. DOI: 10.1038/nrg.2017.33. View

10.

Iurlaro M, Ficz G, Oxley D, Raiber E, Bachman M, Booth M . A screen for hydroxymethylcytosine and formylcytosine binding proteins suggests functions in transcription and chromatin regulation. Genome Biol. 2013; 14(10):R119. PMC: 4014808. DOI: 10.1186/gb-2013-14-10-r119. View

11.

Iurlaro M, McInroy G, Burgess H, Dean W, Raiber E, Bachman M . In vivo genome-wide profiling reveals a tissue-specific role for 5-formylcytosine. Genome Biol. 2016; 17(1):141. PMC: 4928330. DOI: 10.1186/s13059-016-1001-5. View

12.

Bachman M, Uribe-Lewis S, Yang X, Burgess H, Iurlaro M, Reik W . 5-Formylcytosine can be a stable DNA modification in mammals. Nat Chem Biol. 2015; 11(8):555-7. PMC: 5486442. DOI: 10.1038/nchembio.1848. View

13.

Su M, Kirchner A, Stazzoni S, Muller M, Wagner M, Schroder A . 5-Formylcytosine Could Be a Semipermanent Base in Specific Genome Sites. Angew Chem Int Ed Engl. 2016; 55(39):11797-800. DOI: 10.1002/anie.201605994. View

14.

Kellinger M, Song C, Chong J, Lu X, He C, Wang D . 5-formylcytosine and 5-carboxylcytosine reduce the rate and substrate specificity of RNA polymerase II transcription. Nat Struct Mol Biol. 2012; 19(8):831-3. PMC: 3414690. DOI: 10.1038/nsmb.2346. View

15.

Zhu C, Gao Y, Guo H, Xia B, Song J, Wu X . Single-Cell 5-Formylcytosine Landscapes of Mammalian Early Embryos and ESCs at Single-Base Resolution. Cell Stem Cell. 2017; 20(5):720-731.e5. DOI: 10.1016/j.stem.2017.02.013. View

16.

Huang Y, Pastor W, Shen Y, Tahiliani M, Liu D, Rao A . The behaviour of 5-hydroxymethylcytosine in bisulfite sequencing. PLoS One. 2010; 5(1):e8888. PMC: 2811190. DOI: 10.1371/journal.pone.0008888. View

17.

Cokus S, Feng S, Zhang X, Chen Z, Merriman B, Haudenschild C . Shotgun bisulphite sequencing of the Arabidopsis genome reveals DNA methylation patterning. Nature. 2008; 452(7184):215-9. PMC: 2377394. DOI: 10.1038/nature06745. View

18.

Meissner A, Gnirke A, Bell G, Ramsahoye B, Lander E, Jaenisch R . Reduced representation bisulfite sequencing for comparative high-resolution DNA methylation analysis. Nucleic Acids Res. 2005; 33(18):5868-77. PMC: 1258174. DOI: 10.1093/nar/gki901. View

19.

Quinlivan E, Gregory 3rd J . DNA digestion to deoxyribonucleoside: a simplified one-step procedure. Anal Biochem. 2007; 373(2):383-5. PMC: 2239294. DOI: 10.1016/j.ab.2007.09.031. View