TIGS-1164; No. of Pages 1

Letter

On the meaning of the word ‘epimutation’: a comment Da-Hai Yu1, Robert A. Waterland1,2, and Lanlan Shen1 1 2

Department of Pediatrics, Baylor College of Medicine, USDA/ARS Children’s Nutrition Research Center, Houston, TX 77030, USA Department of Molecular & Human Genetics, Baylor College of Medicine, Houston, TX 77030, USA

We appreciate that Oey and Whitelaw highlighted our recent study [1] in their Forum article [2]. In our opinion, however, their comments include several misleading assertions, which we wish to address. First, we agree with them regarding the definition of the word ‘epimutation’, which was originally proposed by Robin Holliday [3]. But Oey and Whitelaw failed to mention that, in his prescient article [3], Holliday emphasized that epimutations are epigenetic defects that accumulate with aging. Accordingly, unlike the rare examples elaborated by Oey and Whitelaw (germline epimutations and metastable epialleles), Holliday proposed that epimutations may be relatively common, occurring at much higher frequency than genetic mutations. Indeed, there is evidence that such age-associated epigenetic defects occur spontaneously at the p16 promoter, both in humans [4] and in mice [5]. The goal of our study, in which we inserted an Alu-related cis-element [6] upstream of p16 ( p16cis), was to accelerate this natural process and assess consequences for tumorigenesis. Second, Oey and Whitelaw criticize the fact that the p16 methylation change is initiated by ‘a DNA mutation (the insertion of a fragment of DNA)’. They also suggest that epigenetic heritability cannot be demonstrated at the p16cis locus because it is ‘inextricably linked’ to a heritable transgene. We would argue that these exact same statements also hold true for the Avy allele, which Oey and Whitelaw characterize as a good example of a primary epimutation. The insertional mutation of an intracisternal A particle is the ‘initiating event’ of and inextricably linked to epigenetic metastability at the Avy locus [7]. Just as at Avy, however, variation in methylation at p16cis is not correlated with genetic variation. The same cis-element leads to a range of p16 methylation depending on the individual, tissue, and age. Our model, inspired by Holliday’s inheritance of epigenetic defects [3], is that, once methylation is established at the locus, it is mitotically inherited and, therefore, accumulates with age. Consistent with this model, we found that age-associated methylation increases at the p16cis promoter were greatest in highly

proliferative tissues [1]. (It is worth clarifying that when Holliday referred to inheritance of epimutations [3] he was principally referring to mitotic heritability in somatic tissues and/or cells.) Lastly, Oey and Whitelaw propose that the transcriptional silencing in p16cis mice is due simply to insertion effects. Our paper [1] presented several lines of evidence against this: (i) controlled knock-in experiments showed that the insertion itself does not affect transcription; (ii) time-course studies showed that in no case did transcriptional silencing precede hypermethylation; rather, we observed that methylation increases either coincided with (i.e., in spleen, liver, and colon) or preceded (i.e., lung) decreases in gene expression; and (iii) pharmacologic intervention experiments in cells with p16cis methylation documented transcriptional reactivation of p16 by the DNA hypomethylating agent 5-aza-20 -deoxycytidine. In summary, our results clearly show that p16 gene activity is controlled by DNA methylation, and aberrant methylation precedes the development of tumors. Hence, we stand by our original assertion that targeted p16 epimutation causes tumorigenesis in mice. References 1 Yu, D.H. et al. (2014) Targeted p16(Ink4a) epimutation causes tumorigenesis and reduces survival in mice. J. Clin. Invest. 124, 3708–3712 2 Oey, H. and Whitelaw, E. (2014) On the meaning of the word ‘epimutation’. Trends Genet. Published online October 6, 2014, http:// dx.doi.org/10.1016/j.tig.2014.08.005. 3 Holliday, R. (1987) The inheritance of epigenetic defects. Science 238, 163–170 4 Nishida, N. et al. (2008) Aberrant methylation of multiple tumor suppressor genes in aging liver, chronic hepatitis, and hepatocellular carcinoma. Hepatology 47, 908–918 5 Maegawa, S. et al. (2010) Widespread and tissue specific age-related DNA methylation changes in mice. Genome Res. 20, 332–340 6 Shen, L. et al. (2007) Genome-wide profiling of DNA methylation reveals a class of normally methylated CpG island promoters. PLoS Genet. 3, 2023–2036 7 Waterland, R.A. and Jirtle, R.L. (2003) Transposable elements: targets for early nutritional effects on epigenetic gene regulation. Mol. Cell. Biol. 23, 5293–5300

Corresponding author: Shen, L. ([email protected]). Keywords: epigenetic; DNA methylation; aging; tumorigenesis; p16. 0168-9525/ ß 2014 Elsevier Ltd. All rights reserved. http://dx.doi.org/10.1016/j.tig.2014.11.001

Trends in Genetics xx (2014) 1–1

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On the meaning of the word 'epimutation': a comment.

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