Mitotic inheritance of the DNA methylome is a challenging task for the maintenance of cell identity. Whether DNA methylation pattern in different genomic contexts can all be faithfully maintained is an open question. A replication-coupled DNA methylation maintenance model was proposed decades ago, but some observations suggest that a replication-uncoupled maintenance mechanism exists. However, the capacity and the underlying molecular events of replication-uncoupled maintenance are unclear. By measuring maintenance kinetics at the single-molecule level and assessing mutant cells with perturbation of various mechanisms, we found that the kinetics of replication-coupled maintenance are governed by the UHRF1–Ligase 1 and PCNA–DNMT1 interactions, whereas nucleosome occupancy and the interaction between UHRF1 and methylated H3K9 specifically regulate replication-uncoupled maintenance. Surprisingly, replication-uncoupled maintenance is sufficiently robust to largely restore the methylome when replication-coupled maintenance is severely impaired. However, solo-WCGW sites and other CpG sites displaying aging- and cancer-associated hypomethylation exhibit low maintenance efficiency, suggesting that although quite robust, mitotic inheritance of methylation is imperfect and that this imperfection may contribute to selective hypomethylation during aging and tumorigenesis.
Subscribe to Journal
Get full journal access for 1 year
only $9.92 per issue
All prices are NET prices.
VAT will be added later in the checkout.
Tax calculation will be finalised during checkout.
Rent or Buy article
Get time limited or full article access on ReadCube.
All prices are NET prices.
HeLa OK-seq data were obtained from NCBI SRA under the accession number SRP065949.49 Histone H3 ChIP-seq of HeLa S3 was obtained from GSM788634.96 Aging associated DNA methylation change data was taken from pulication.68 All high throughput data generated in this study have been deposited at NCBI GEO under accession number: GSE131098. Custom scripts for data analysis used in this data are available upon reasonable requests.
Jones, P. A. Functions of DNA methylation: islands, start sites, gene bodies and beyond. Nat. Rev. Genet. 13, 484–492 (2012).
Li, E. & Zhang, Y. DNA methylation in mammals. Cold Spring Harb. Perspect. Biol. 6, a019133 (2014).
Bird, A. DNA methylation patterns and epigenetic memory. Genes Dev. 16, 6–21 (2002).
Araujo, F. D., Knox, J. D., Szyf, M., Price, G. B. & Zannis-Hadjopoulos, M. Concurrent replication and methylation at mammalian origins of replication. Mol. Cell Biol. 18, 3475–3482 (1998).
Gruenbaum, Y., Szyf, M., Cedar, H. & Razin, A. Methylation of replicating and post-replicated mouse L-cell DNA. Proc. Natl. Acad. Sci. USA 80, 4919–4921 (1983).
Kappler, J. W. The kinetics of DNA methylation in cultures of a mouse adrenal cell line. J. Cell Physiol. 75, 21–31 (1970).
Xu, C. & Corces, V. G. Nascent DNA methylome mapping reveals inheritance of hemimethylation at CTCF/cohesin sites. Science 359, 1166–1170 (2018).
Adams, R. L. The relationship between synthesis and methylation of DNA in mouse fibroblasts. Biochim Biophys. Acta 254, 205–212 (1971).
Liang, G. et al. Cooperativity between DNA methyltransferases in the maintenance methylation of repetitive elements. Mol. Cell Biol. 22, 480–491 (2002).
Woodcock, D. M. et al. Delayed DNA methylation is an integral feature of DNA replication in mammalian cells. Exp. Cell Res. 166, 103–112 (1986).
Charlton, J. et al. Global delay in nascent strand DNA methylation. Nat. Struct. Mol. Biol. 25, 327–332 (2018).
Bestor, T. H. The DNA methyltransferases of mammals. Hum. Mol. Genet. 9, 2395–2402 (2000).
Jeltsch, A. On the enzymatic properties of Dnmt1: specificity, processivity, mechanism of linear diffusion and allosteric regulation of the enzyme. Epigenetics 1, 63–66 (2006).
Song, J., Rechkoblit, O., Bestor, T. H. & Patel, D. J. Structure of DNMT1-DNA complex reveals a role for autoinhibition in maintenance DNA methylation. Science 331, 1036–1040 (2011).
Song, J., Teplova, M., Ishibe-Murakami, S. & Patel, D. J. Structure-based mechanistic insights into DNMT1-mediated maintenance DNA methylation. Science 335, 709–712 (2012).
Bashtrykov, P. et al. Specificity of Dnmt1 for methylation of hemimethylated CpG sites resides in its catalytic domain. Chem. Biol. 19, 572–578 (2012).
Bashtrykov, P., Ragozin, S. & Jeltsch, A. Mechanistic details of the DNA recognition by the Dnmt1 DNA methyltransferase. FEBS Lett. 586, 1821–1823 (2012).
Fatemi, M., Hermann, A., Pradhan, S. & Jeltsch, A. The activity of the murine DNA methyltransferase Dnmt1 is controlled by interaction of the catalytic domain with the N-terminal part of the enzyme leading to an allosteric activation of the enzyme after binding to methylated DNA. J. Mol. Biol. 309, 1189–1199 (2001).
Li, Y. et al. Stella safeguards the oocyte methylome by preventing de novo methylation mediated by DNMT1. Nature 564, 136–140 (2018).
Gowher, H. & Jeltsch, A. Mammalian DNA methyltransferases: new discoveries and open questions. Biochem Soc. Trans. 46, 1191–1202 (2018).
Chuang, L. S. et al. Human DNA-(cytosine-5) methyltransferase-PCNA complex as a target for p21WAF1. Science 277, 1996–2000 (1997).
Schermelleh, L. et al. Dynamics of Dnmt1 interaction with the replication machinery and its role in postreplicative maintenance of DNA methylation. Nucleic Acids Res. 35, 4301–4312 (2007).
Spada, F. et al. DNMT1 but not its interaction with the replication machinery is required for maintenance of DNA methylation in human cells. J. Cell Biol. 176, 565–571 (2007).
Xie, S. & Qian, C. The growing complexity of UHRF1-mediated maintenance DNA methylation. Genes (Basel) 9, 600 (2018).
Karagianni, P., Amazit, L., Qin, J. & Wong, J. ICBP90, a novel methyl K9 H3 binding protein linking protein ubiquitination with heterochromatin formation. Mol. Cell Biol. 28, 705–717 (2008).
Rothbart, S. B. et al. Association of UHRF1 with methylated H3K9 directs the maintenance of DNA methylation. Nat. Struct. Mol. Biol. 19, 1155–1160 (2012).
Zhao, Q. et al. Dissecting the precise role of H3K9 methylation in crosstalk with DNA maintenance methylation in mammals. Nat. Commun. 7, 12464 (2016).
Feinberg, A. P. & Tycko, B. The history of cancer epigenetics. Nat. Rev. Cancer 4, 143–153 (2004).
Day, K. et al. Differential DNA methylation with age displays both common and dynamic features across human tissues that are influenced by CpG landscape. Genome Biol. 14, R102 (2013).
Heyn, H. et al. Distinct DNA methylomes of newborns and centenarians. Proc. Natl Acad. Sci. USA 109, 10522–10527 (2012).
Yuan, T. et al. An integrative multi-scale analysis of the dynamic DNA methylation landscape in aging. PLoS Genet. 11, e1004996 (2015).
Bollati, V. et al. Decline in genomic DNA methylation through aging in a cohort of elderly subjects. Mech. Ageing Dev. 130, 234–239 (2009).
Figueiredo, J. C. et al. Global DNA hypomethylation (LINE-1) in the normal colon and lifestyle characteristics and dietary and genetic factors. Cancer Epidemiol. Biomark. Prev. 18, 1041–1049 (2009).
Bocklandt, S. et al. Epigenetic predictor of age. PLoS One 6, e14821 (2011).
Horvath, S. DNA methylation age of human tissues and cell types. Genome Biol. 14, R115 (2013).
Hannum, G. et al. Genome-wide methylation profiles reveal quantitative views of human aging rates. Mol. Cell 49, 359–367 (2013).
Ciccarone, F., Tagliatesta, S., Caiafa, P. & Zampieri, M. DNA methylation dynamics in aging: how far are we from understanding the mechanisms? Mech. Ageing Dev. 174, 3–17 (2018).
Field, A. E. et al. DNA methylation clocks in aging: categories, causes, and consequences. Mol. Cell 71, 882–895 (2018).
Klutstein, M., Nejman, D., Greenfield, R. & Cedar, H. DNA methylation in cancer and aging. Cancer Res. 76, 3446–3450 (2016).
Zhou, W. et al. DNA methylation loss in late-replicating domains is linked to mitotic cell division. Nat. Genet. 50, 591–602 (2018).
Laird, C. D. et al. Hairpin-bisulfite PCR: assessing epigenetic methylation patterns on complementary strands of individual DNA molecules. Proc. Natl. Acad. Sci. USA 101, 204–209 (2004).
Zhao, L. et al. The dynamics of DNA methylation fidelity during mouse embryonic stem cell self-renewal and differentiation. Genome Res. 24, 1296–1307 (2014).
Jones, P. A. & Baylin, S. B. The epigenomics of cancer. Cell 128, 683–692 (2007).
Hermann, A., Goyal, R. & Jeltsch, A. The Dnmt1 DNA-(cytosine-C5)-methyltransferase methylates DNA processively with high preference for hemimethylated target sites. J. Biol. Chem. 279, 48350–48359 (2004).
Goyal, R., Reinhardt, R. & Jeltsch, A. Accuracy of DNA methylation pattern preservation by the Dnmt1 methyltransferase. Nucleic Acids Res. 34, 1182–1188 (2006).
Vilkaitis, G., Suetake, I., Klimasauskas, S. & Tajima, S. Processive methylation of hemimethylated CpG sites by mouse Dnmt1 DNA methyltransferase. J. Biol. Chem. 280, 64–72 (2005).
Petryk, N. et al. MCM2 promotes symmetric inheritance of modified histones during DNA replication. Science 361, 1389–1392 (2018).
Yu, C. et al. A mechanism for preventing asymmetric histone segregation onto replicating DNA strands. Science 361, 1386–1389 (2018).
Petryk, N. et al. Replication landscape of the human genome. Nat. Commun. 7, 10208 (2016).
Bostick, M. et al. UHRF1 plays a role in maintaining DNA methylation in mammalian cells. Science 317, 1760–1764 (2007).
Sharif, J. et al. The SRA protein Np95 mediates epigenetic inheritance by recruiting Dnmt1 to methylated DNA. Nature 450, 908–912 (2007).
Xie, S., Jakoncic, J. & Qian, C. UHRF1 double tudor domain and the adjacent PHD finger act together to recognize K9me3-containing histone H3 tail. J. Mol. Biol. 415, 318–328 (2012).
Rothbart, S. B. et al. Multivalent histone engagement by the linked tandem Tudor and PHD domains of UHRF1 is required for the epigenetic inheritance of DNA methylation. Genes Dev. 27, 1288–1298 (2013).
Qin, W. et al. DNA methylation requires a DNMT1 ubiquitin interacting motif (UIM) and histone ubiquitination. Cell Res. 25, 911–929 (2015).
Rajakumara, E. et al. PHD finger recognition of unmodified histone H3R2 links UHRF1 to regulation of euchromatic gene expression. Mol. Cell 43, 275–284 (2011).
Nishiyama, A. et al. Uhrf1-dependent H3K23 ubiquitylation couples maintenance DNA methylation and replication. Nature 502, 249–253 (2013).
Ishiyama, S. et al. Structure of the Dnmt1 reader module complexed with a unique two-mono-ubiquitin mark on histone h3 reveals the basis for dna methylation maintenance. Mol. Cell 68, 350–360 e357 (2017).
Li, T. et al. Structural and mechanistic insights into UHRF1-mediated DNMT1 activation in the maintenance DNA methylation. Nucleic Acids Res. 46, 3218–3231 (2018).
Esteve, P. O. et al. Direct interaction between DNMT1 and G9a coordinates DNA and histone methylation during replication. Genes Dev. 20, 3089–3103 (2006).
Liu, X. et al. UHRF1 targets DNMT1 for DNA methylation through cooperative binding of hemi-methylated DNA and methylated H3K9. Nat. Commun. 4, 1563 (2013).
Ferry, L. et al. Methylation of DNA ligase 1 by G9a/GLP recruits UHRF1 to replicating DNA and regulates DNA methylation. Mol. Cell 67, 550–565 e555 (2017).
Lambrus, B. G., Moyer, T. C. & Holland, A. J. Applying the auxin-inducible degradation system for rapid protein depletion in mammalian cells. Methods Cell Biol. 144, 107–135 (2018).
Nishimura, K., Fukagawa, T., Takisawa, H., Kakimoto, T. & Kanemaki, M. An auxin-based degron system for the rapid depletion of proteins in nonplant cells. Nat. Methods 6, 917–922 (2009).
Mu, Z. M., Le, X. F., Vallian, S., Glassman, A. B. & Chang, K. S. Stable overexpression of PML alters regulation of cell cycle progression in HeLa cells. Carcinogenesis 18, 2063–2069 (1997).
Kaufman, P. D., Kobayashi, R., Kessler, N. & Stillman, B. The p150 and p60 subunits of chromatin assembly factor I: a molecular link between newly synthesized histones and DNA replication. Cell 81, 1105–1114 (1995).
Huang, H. et al. A unique binding mode enables MCM2 to chaperone histones H3-H4 at replication forks. Nat. Struct. Mol. Biol. 22, 618–626 (2015).
Gan, H. et al. The Mcm2-Ctf4-polalpha axis facilitates parental histone H3-H4 transfer to lagging strands. Mol. Cell 72, 140–151 e143 (2018).
Johansson, A., Enroth, S. & Gyllensten, U. Continuous aging of the human DNA Methylome throughout the human lifespan. PLoS One 8, e67378 (2013).
Dmitrijeva, M., Ossowski, S., Serrano, L. & Schaefer, M. H. Tissue-specific DNA methylation loss during ageing and carcinogenesis is linked to chromosome structure, replication timing and cell division rates. Nucleic Acids Res. 46, 7022–7039 (2018).
Hon, G. C. et al. Global DNA hypomethylation coupled to repressive chromatin domain formation and gene silencing in breast cancer. Genome Res. 22, 246–258 (2012).
Berman, B. P. et al. Regions of focal DNA hypermethylation and long-range hypomethylation in colorectal cancer coincide with nuclear lamina-associated domains. Nat. Genet. 44, 40–46 (2011).
Costantini, M. & Bernardi, G. Replication timing, chromosomal bands, and isochores. Proc. Natl. Acad. Sci. USA 105, 3433–3437 (2008).
Dennis, K., Fan, T., Geiman, T., Yan, Q. & Muegge, K. Lsh, a member of the SNF2 family, is required for genome-wide methylation. Genes Dev. 15, 2940–2944 (2001).
Eckhardt, F. et al. DNA methylation profiling of human chromosomes 6, 20 and 22. Nat. Genet. 38, 1378–1385 (2006).
Jackson, D. A. & Pombo, A. Replicon clusters are stable units of chromosome structure: evidence that nuclear organization contributes to the efficient activation and propagation of S phase in human cells. J. Cell Biol. 140, 1285–1295 (1998).
Sogo, J. M., Stahl, H., Koller, T. & Knippers, R. Structure of replicating simian virus 40 minichromosomes. The replication fork, core histone segregation and terminal structures. J. Mol. Biol. 189, 189–204 (1986).
Casillas, M. A. Jr, Lopatina, N., Andrews, L. G. & Tollefsbol, T. O. Transcriptional control of the DNA methyltransferases is altered in aging and neoplastically-transformed human fibroblasts. Mol. Cell Biochem 252, 33–43 (2003).
Ronn, T. et al. Impact of age, BMI and HbA1c levels on the genome-wide DNA methylation and mRNA expression patterns in human adipose tissue and identification of epigenetic biomarkers in blood. Hum. Mol. Genet. 24, 3792–3813 (2015).
Valentini, E. et al. Analysis of the machinery and intermediates of the 5hmC-mediated DNA demethylation pathway in aging on samples from the MARK-AGE Study. Aging (Albany NY) 8, 1896–1922 (2016).
Berger S. L., Sassone-Corsi P. Metabolic signaling to chromatin. Cold Spring Harb. Perspect. Biol. 8, a019463 (2016).
Alisch, R. S. et al. Age-associated DNA methylation in pediatric populations. Genome Res. 22, 623–632 (2012).
Shipony, Z. et al. Dynamic and static maintenance of epigenetic memory in pluripotent and somatic cells. Nature 513, 115–119 (2014).
Levine, M. E. et al. An epigenetic biomarker of aging for lifespan and healthspan. Aging (Albany NY) 10, 573–591 (2018).
De Cecco, M. et al. L1 drives IFN in senescent cells and promotes age-associated inflammation. Nature 566, 73–78 (2019).
Presolski, S. I., Hong, V. P. & Finn, M. G. Copper-catalyzed azide-alkyne click chemistry for bioconjugation. Curr. Protoc. Chem. Biol. 3, 153–162 (2011).
Ramachandran, S. & Henikoff, S. Transcriptional regulators compete with nucleosomes post-replication. Cell 165, 580–592 (2016).
Sanjana, N. E., Shalem, O. & Zhang, F. Improved vectors and genome-wide libraries for CRISPR screening. Nat. Methods 11, 783–784 (2014).
Haeussler, M. et al. Evaluation of off-target and on-target scoring algorithms and integration into the guide RNA selection tool CRISPOR. Genome Biol. 17, 148 (2016).
Xu, H. et al. Sequence determinants of improved CRISPR sgRNA design. Genome Res. 25, 1147–1157 (2015).
Natsume, T., Kiyomitsu, T., Saga, Y. & Kanemaki, M. T. Rapid protein depletion in human cells by auxin-inducible degron tagging with short homology donors. Cell Rep. 15, 210–218 (2016).
Adey, A. et al. The haplotype-resolved genome and epigenome of the aneuploid HeLa cancer cell line. Nature 500, 207–211 (2013).
Urich, M. A., Nery, J. R., Lister, R., Schmitz, R. J. & Ecker, J. R. MethylC-seq library preparation for base-resolution whole-genome bisulfite sequencing. Nat. Protoc. 10, 475–483 (2015).
Sun, M. A., Velmurugan, K. R., Keimig, D. & Xie, H. HBS-tools for hairpin bisulfite sequencing data processing and analysis. Adv. Bioinforma. 2015, 760423 (2015).
Heintzman, N. D. et al. Histone modifications at human enhancers reflect global cell-type-specific gene expression. Nature 459, 108–112 (2009).
Paternoster, R., Brame, R., Mazerolle, P. & Piquero, A. Using the correct statistical test for the equality of regression coefficients. Criminology 36, 859–866 (1998).
Ray-Gallet, D. et al. Dynamics of histone H3 deposition in vivo reveal a nucleosome gap-filling mechanism for H3.3 to maintain chromatin integrity. Mol. Cell 44, 928–941 (2011).
We thank J.Y. Jia and S. Sun for technical assistance in FACS analysis. This work was primarily supported by the National Natural Science Foundation of China (31530047). This work was also supported by the Ministry of Science and Technology of China (2016YFA0100400), the Chinese Academy of Sciences (XDB39000000 and QYZDY-SSW-SMC031). Z.Z. is sponsored by the Youth Innovation Promotion Association (2017133) of the Chinese Academy of Sciences.
The authors declare no competing interests.
About this article
Cite this article
Ming, X., Zhang, Z., Zou, Z. et al. Kinetics and mechanisms of mitotic inheritance of DNA methylation and their roles in aging-associated methylome deterioration. Cell Res 30, 980–996 (2020). https://doi.org/10.1038/s41422-020-0359-9
Clinical Epigenetics (2021)
Simultaneously measuring the methylation of parent and daughter strands of replicated DNA at the single-molecule level by Hammer-seq
Nature Protocols (2021)
Nature Reviews Genetics (2021)
Nature Structural & Molecular Biology (2021)
The histone and non-histone methyllysine reader activities of the UHRF1 tandem Tudor domain are dispensable for the propagation of aberrant DNA methylation patterning in cancer cells
Epigenetics & Chromatin (2020)