Frequent lack of repressive capacity of promoter DNA methylation identified through genome-wide epigenomic manipulation
Open accessAbstract
It is widely assumed that the addition of DNA methylation at CpG rich gene promoters silences gene transcription. However, this conclusion is largely drawn from the observation that promoter DNA methylation inversely correlates with gene expression in natural conditions. The effect of induced DNA methylation on endogenous promoters has yet to be comprehensively assessed. Here, we induced the simultaneous methylation of thousands of promoters in the genome of human cells using an engineered zinc finger-DNMT3A fusion protein, enabling assessment of the effect of forced DNA methylation upon transcription, histone modifications, and DNA methylation persistence after the removal of the fusion protein. We find that DNA methylation is frequently insufficient to transcriptionally repress promoters. Furthermore, DNA methylation deposited at promoter regions associated with H3K4me3 is rapidly erased after removal of the zinc finger-DNMT3A fusion protein. Finally, we demonstrate that induced DNA methylation can exist simultaneously on promoter nucleosomes that possess the active histone modification H3K4me3, or DNA bound by the initiated form of RNA polymerase II. These findings suggest that promoter DNA methylation is not generally sufficient for transcriptional inactivation, with implications for the emerging field of epigenome engineering. One Sentence Summary: Genome-wide epigenomic manipulation of thousands of human promoters reveals that induced promoter DNA methylation is unstable and frequently does not function as a primary instructive biochemical signal for gene silencing and chromatin reconfiguration.
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A recent methylation editing study demonstrated that deposition of 5mC at thousands of gene promoters led to only moderate transcriptional alterations.
“For example, a recent methylation editing study demonstrating that deposition of 5mC at thousands of gene promoters led to only moderate transcriptional alterations (65, 66).”
From Dynamic DNA methylation: In the right place at the right time (2018) · cites this paper as [65]
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“Moreover, Fidanza et al. created the UniSam system, which is an all-in-one vector version of dCas9-SAM technology to maximize post-transfection cell viability by D ow nloaded from https://academ ic.oup.com /nar/advance-article/doi/10.1093/nar/gkaa1000/5983619 by guest on 25 N ovem ber 2020 Zinc-Finger proteins (ZFPs)A…”
BackgroundInfluentialSelf-citationSequence determinants, function, and evolution of CpG islands
Angeloni, A., & Bogdanović, O. (2021) · Biochemical Society Transactions · doi:10.1042/bst20200695 · Cited by 90 · see citing works
Reconstructing Denisovan Anatomy Using DNA Methylation Maps
Gokhman, D., Mishol, N., de Manuel, M., Juan, D., Shuqrun, J., Meshorer, E., Marquès‐Bonet, T., Rak, Y., & Carmel, L. (2019) · Cell · doi:10.1016/j.cell.2019.08.035 · Cited by 90 · see citing works
Understanding the Relevance of DNA Methylation Changes in Immune Differentiation and Disease
de la Calle‐Fabregat, C., Morante-Palacios, O., & Ballestar, E. (2020) · Genes · doi:10.3390/genes11010110 · Cited by 85 · see citing works
“A bioinformatic reanalysis of data from a recent study showed that forced genome-wide hypermethylation can, on its own, repress gene expression and rewire chromatin to an inactive state [19,20].”
BackgroundLi, T., García-Gómez, A., Morante-Palacios, O., Ciudad, L., Özkaramehmet, S., Dijck, E. V., Rodríguez‐Ubreva, J., Vaquero, A., & Ballestar, E. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz1127 · Cited by 69 · see citing works
“Similarly, another study shows that the presence of DNA methylation is insufficient to transcriptionally repress promoters (56).”
BackgroundDNA methylation and de-methylation using hybrid site-targeting proteins
Lei, Y., Huang, Y., & Goodell, M. A. (2018) · Genome biology · doi:10.1186/s13059-018-1566-2 · Cited by 64 · see citing works
Fennell, L., Dumenil, T. D., Wockner, L. F., Härtel, G., Nones, K., Bond, C., Borowsky, J., Liu, C., McKeone, D., Bowdler, L. M., et al. (2019) · Cellular and Molecular Gastroenterology and Hepatology · doi:10.1016/j.jcmgh.2019.04.002 · Cited by 54 · see citing works
“DNA methylation alone can be insufficient to induce transcriptional repression.(13) Gene repression also is associated with repressive histonemarks such as the H3K27me3mark,(14) which is catalyzed by the polycomb-repressor-complex 2.”
BackgroundEnhancer-associated H3K4 methylation safeguards in vitro germline competence
Bleckwehl, T., Crispatzu, G., Schaaf, K., Respuela, P., Bartusel, M., Benson, L. J., Clark, S. J., Dorighi, K. M., Barral, A., Laugsch, M., et al. (2021) · Nature Communications · doi:10.1038/s41467-021-26065-6 · Cited by 53 · see citing works
Epigenetic modification of cytosines fine tunes the stability of i-motif DNA
Wright, E. P., Abdelhamid, M. A. S., Ehiabor, M. O., Grigg, M. C., Irving, K. L., Smith, N. M., & Waller, Z. A. E. (2019) · Nucleic Acids Research · doi:10.1093/nar/gkz1082 · Cited by 48 · see citing works
“%) were differentially methylated in MCF7 vs MCF10A, indicative of the different methylation variants in cancerous compared to non-cancerous cells.”
BackgroundInfluentialMorgan, M. D., & Marioni, J. C. (2018) · Genome biology · doi:10.1186/s13059-018-1461-x · Cited by 41 · see citing works
“Non-CpG island promoters maintain transcription repression by cytosine methylation at CpG dinucleotides [21], though recent evidence demonstrates that CpG methylation alone cannot induce repression [22].”
BackgroundTaylor, K., Smith, G. D., Relton, C. L., Gaunt, T. R., & Richardson, T. G. (2019) · Genome Medicine · doi:10.1186/s13073-019-0613-2 · Cited by 40 · see citing works
Early-life DNA methylation profiles are indicative of age-related transcriptome changes
Hadad, N., Masser, D. R., Blanco-Berdugo, L., Stanford, D. R., & Freeman, W. M. (2019) · Epigenetics & Chromatin · doi:10.1186/s13072-019-0306-5 · Cited by 29 · see citing works
“Additionally, DNA methylation changes do not correspond with transcriptional changes in the CNS during neuronal maturation [43] or following induction of methylation in culture [63].”
BackgroundEvolution of DNA Methylation in Papio Baboons
Vilgalys, T. P., Rogers, J., Jolly, C. J., Analysis, B. G., Mukherjee, S., & Tung, J. (2018) · Molecular Biology and Evolution · doi:10.1093/molbev/msy227 · Cited by 29 · see citing works
Deciphering Plant Chromatin Regulation via CRISPR/dCas9-Based Epigenome Engineering
Dubois, A., & Roudier, F. (2021) · Epigenomes · doi:10.3390/epigenomes5030017 · Cited by 24 · see citing works
“The immediate drawback of this approach, also reported for similar designs in animal cells [37], is that the initial NtDRM effector domain fusion with scFv (which recognizes the GCN4 repeat in the Sun Tag) generated high levels of ectopic CHH methylation all along the plant genome.”
BackgroundPřibylová, A., Čermák, V., Tyč, D., & Fischer, L. (2019) · Epigenetics & Chromatin · doi:10.1186/s13072-019-0299-0 · Cited by 22 · see citing works
“H3K4me3 activation marks coexisted with methylated cytosines also in many human promoters, whose activity was frequently unaffected by the introduction of methylation marks [62].”
Connecting the epigenome, metabolome and proteome for a deeper understanding of disease
Suhre, K., & Zaghlool, S. B. (2021) · Journal of Internal Medicine · doi:10.1111/joim.13306 · Cited by 22 · see citing works
Hatcher, C., Relton, C. L., Gaunt, T. R., & Richardson, T. G. (2019) · Translational Psychiatry · doi:10.1038/s41398-019-0437-2 · Cited by 19 · see citing works
Konwar, C., Asiimwe, R., Inkster, A. M., Merrill, S. M., Negri, G. L., Aristizabal, M. J., Rider, C. F., MacIsaac, J. L., Carlsten, C., & Kobor, M. S. (2021) · Epigenetics & Chromatin · doi:10.1186/s13072-021-00428-1 · Cited by 20 · see citing works
“It is important to note that the correlation between DNA methylation and gene expression is not straightforward [96, 97] and is dependent on genomic context and temporal stage.”
BackgroundPiecing together cis‐regulatory networks: insights from epigenomics studies in plants
Huang, S. C., & Ecker, J. R. (2017) · WIREs Systems Biology and Medicine · doi:10.1002/wsbm.1411 · Cited by 19 · see citing works
“In vivo investigations of these factors are inevitably confounded by the chromatin environment such as histone modifications and binding of other TFs.(182,184) DAP-seq”
BackgroundInfluentialCha, Y., Kim, S. Y., Yeo, H. Y., Baek, J. Y., Choi, M. K., Jung, K. H., Dong, S. M., & Chang, H. J. (2018) · Neoplasia · doi:10.1016/j.neo.2018.11.010 · Cited by 16 · see citing works
“Although DNA hypermethylation in a specific gene has long been regarded to reflect decreased mRNA and protein expression in the corresponding gene, it may not be correlated with repressive gene expression in some cases because of the complex regulation of gene expression besides DNA methylation [42].”
Korthauer, K., & Irizarry, R. A. (2018) · doi:10.1101/381145 · Cited by 14 · see citing works
“Although not evaluated in (Ford et al., 2017), we find a similar but slightly weaker trend for RNA PolII occupancy (Figure 3C and D).”
BackgroundMethodInfluentialHarnessing targeted DNA methylation and demethylation using dCas9
Pflueger, C., Swain, T., & Lister, R. (2019) · Essays in Biochemistry · doi:10.1042/ebc20190029 · Cited by 14 · see citing works
Self-citationFunctional genomic insights into the environmental determinants of mammalian fitness
Snyder‐Mackler, N., & Lea, A. J. (2018) · Current Opinion in Genetics & Development · doi:10.1016/j.gde.2018.08.001 · Cited by 11 · see citing works
“Studies that utilize time course experiments [52], Mendelian Randomization or other causal inference tools [33,53,54], and manipulation of epigenetic marks [55,56] promise to help further our Snyder-Mackler and Lea Page 4”
MethodEpigenetics and postbiotics in Aquaculture: Unlocking sustainable solutions through innovation
Sumon, M. A. A., Khan, S. J., Islam, M. J., Abualreesh, M. H., Mondol, M., Tayyamath, K., Hoseinifar, S. H., Ringø, E., & Doan, H. V. (2025) · Fish & Shellfish Immunology · doi:10.1016/j.fsi.2025.110598 · Cited by 8 · see citing works
Enhancer-associated H3K4 methylation safeguards in vitro germline competence
Bleckwehl, T., Crispatzu, G., Schaaf, K., Respuela, P., Bartusel, M., Benson, L. J., Clark, S. J., Dorighi, K. M., Barral, A., Laugsch, M., et al. (2020) · Kölner Universitäts PublikationsServer (Universität zu Köln) · doi:10.17863/cam.76317 · Cited by 8 · see citing works
Abdalla, M., Abdalla, M., McCarthy, M. I., & Holmes, C. C. (2022) · PLoS Computational Biology · doi:10.1371/journal.pcbi.1010028 · Cited by 7 · see citing works
“This is consistent with experimental evidence suggesting that most genetic variants in DNase footprinted (and other annotated) regulatory regions are silent [43], and the fact that histone modifications (e.g. methylation) alone are frequently insufficient to transcriptionally perturb promoters [44].”
BackgroundAntwi, E., Olins, A. L., Teif, V. B., Bieg, M., Bauer, T., Gu, Z., Brors, B., Eils, R., Olins, D. E., & Ishaque, N. (2020) · Biology Open · doi:10.1242/bio.044222 · Cited by 4 · see citing works
A modular dCas9-based recruitment platform for combinatorial epigenome editingPreprint
Swain, T., Pflueger, C., Freytag, S., Poppe, D., Pflueger, J., Nguyen, T. V., Li, J. K., & Lister, R. (2022) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/2022.07.01.498378 · Cited by 4 · see citing works
“Thus, the SSSavi recruitment platform may enable the precise alteration of particular combinatorial chromatin states using artificial effectors where the combinatorial and interconnected nature of the epigenome may have impeded alteration in the past [54,55].”
BackgroundSelf-citationBioinformatic and Biostatistic Methods for DNA Methylome Analysis of Obesity
Voisin, S. A. C. (2019) · Elsevier eBooks · doi:10.1016/b978-0-12-814513-5.00011-8 · Cited by 1 · see citing works
Early life DNA methylation profiles are indicative of age-related transcriptome changesPreprint
Hadad, N., Masser, D. R., Blanco-Berdugo, L., Stanford, D. R., & Freeman, W. M. (2019) · bioRxiv (Cold Spring Harbor Laboratory) · doi:10.1101/557892
DNA Methylation: Methods and Analyses
Gorrie-Stone, T. J. (2019) · Open Access at Essex (University of Essex)
Evidence of positive and negative selection associated with DNA methylationPreprint
Hatcher, C., Hemani, G., Rodriguez, S., Gaunt, T. R., Lawson, D., & Min, J. L. (2021) · bioRxiv · doi:10.1101/2021.11.25.469994
Investigating Neurodegenerative Disorders
Kishore, B., & Dunuwila, T. S. (2025) · IGI Global eBooks · doi:10.4018/979-8-3693-5464-3.ch008
Du, L., Dong, Y., Yang, J., Zhang, Z., & Liu, Z. (2026) · International Journal of Molecular Sciences · doi:10.3390/ijms27167077
“The reason is that the endogenous transcriptional machinery continues to resist epigenetic inhibition [222,223].”
- Authors
- Ford, E., Grimmer, M. R., Stolzenburg, S., Bogdanovic, O., Mendoza, A. D., Farnham, P. J., Blancafort, P., & Lister, R.
- Affiliations
- The University of Western Australia (AU)
- University of Southern California (US)
- Corresponding author
- Lister, Ryan
- ORCID
- Venue
- bioRxiv (Cold Spring Harbor Laboratory)
- Publisher
- openRxiv
- Published
- 2017-08-16
- OpenAlex
- W2749986987
- Cited by
- 45
- References
- 78
- Citations by year
- 2026: 1 · 2025: 2 · 2022: 2 · 2021: 8 · 2020: 5 · 2019: 15 · 2018: 11 · 2017: 1
- Type
- posted content
- Language
- en
- Open access
- green
- doi.org
- Licence
- cc-by-nc-nd
- Locations
- bioRxiv (Cold Spring Harbor Laboratory) (acceptedVersion, cc-by-nc-nd)
- Queen Mary Research Online (Queen Mary University of London) (submittedVersion)
- Funders
- Raine Medical Research Foundation
- National Institutes of Health
- Medical Research Council (GNT1069830)
- Australian Research Council (CE140100008, FT120100862)
- National Health and Medical Research Council (1069830)
- National Human Genome Research Institute (R21HG006761)
- National Cancer Institute (R01CA170370, P30CA014089)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Epigenetics and DNA Methylation
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Genomics and Chromatin Dynamics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Genetics › Genetic Syndromes and Imprinting
- Keywords
- promoter methylation, transcriptional repression, H3K4me3, histone modifications, RNA polymerase II, nucleosome, gene silencing, human cells
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