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news and views dependent on the presence of vitamin C in the medium: in the absence of the vitamin, reprogramming was suppressed in Tet1-null cells, but, in its presence, Tet1-null cells were reprogrammed more efficiently compared to wild-type cells. The authors showed that these effects of vitamin C were a consequence of its modulation of the action of TET1 at a defined set of loci involved in MET, a key event in the early stages of fibroblast reprogramming to pluripotency (Fig. 1). These results further our understanding of the reprogramming process and the role of TET enzymes in epigenetic regulation. However, many questions remain concerning the role of TET enzymes in these processes. There are important differences between the function of the three forms of TET found in mammalian cells (reviewed in ref. 9), leaving open the possibility that the enzymes have very different and complementary roles. Moreover, it is clear that TET proteins can affect gene expression through several mechanisms, as the biological effects of knockdown or overexpression do not always relate in a simple way to the catalytic activity of the enzymes
or to global DNA demethylation8. Thus, TET proteins may regulate gene expression through multiple pathways, and the results of Chen et al.2 show that loss of a particular form of TET can apparently result in quite specific patterns of epigenetic change. Finally, as mice lacking both Tet1 and Tet2 undergo normal early embryonic development10, the roles of these enzymes in development in vivo remain to be elucidated. Epigenetics and microenvironment The study by Chen et al.2 and other recent studies show the importance of the context of the cellular microenvironment for epigenetic regulation and cell fate. Many studies have highlighted the instability of the epigenome of human pluripotent stem cells in vitro (reviewed in ref. 11). This epigenetic instability has profound implications for the potential use of pluripotent cells in the study of human development, functional genomics and disease modeling and may affect potential applications in cell therapy.It will therefore be important to gain a better understanding of the roles
that iron-dependent oxidoreductases and the factors that modulate their activity have in epigenetic regulation in cultured cells. These enzymes represent a point of intersection of internal epigenetic regulatory mechanisms with the environment. Elucidation of the role of these enzymes in the epigenetic basis of disease and in early human development will be important areas for future investigation. COMPETING FINANCIAL INTERESTS The author declares no competing financial interests. 1. Monfort, A. & Wutz, A. EMBO Rep. 14, 337–346 (2013). 2. Chen, J. et al. Nat. Genet. 45, 1504–1509 (2013). 3. Chung, T.L. et al. Stem Cells 28, 1848–1855 (2010). 4. Esteban, M.A. et al. Cell Stem Cell 6, 71–79 (2010). 5. Wang, T. et al. Cell Stem Cell 9, 575–587 (2011). 6. Wu, H. et al. Nature 473, 389–393 (2011). 7. Gao, Y. et al. Cell Stem Cell 12, 453–469 (2013). 8. Costa, Y. et al. Nature 495, 370–374 (2013). 9. Kinney, S.R. & Pradhan, S. Adv. Exp. Med. Biol. 754, 57–79 (2013). 10. Dawlaty, M.M. et al. Dev. Cell 24, 310–323 (2013). 11. Lund, R.J., Narva, E. & Lahesmaa, R. Nat. Rev. Genet. 13, 732–744 (2012).
Recurrent H3.3 alterations in childhood tumors Anders M Lindroth & Christoph Plass Comprehensive sequencing of benign and malignant tumors has recently uncovered new driver mutations in childhood tumors. A new report now describes frequent histone H3.3 alterations in chondroblastoma and giant cell tumor of bone, emphasizing the importance of this histone variant in pediatric cancers. The genetic mechanisms underlying tumorigenesis constitute a complex process. Focal mutations alter genes responsible for cell proliferation, DNA repair, chromosome integrity, immune response and/or epigenetic regulation, overturning the normal function of the cell. Some of these mutations may initiate complex genetic rearrangements (mainly translocations) that lead to a failure to uphold the epigenetic states essential for maintaining and directing transcriptional activity. Accumulation of genetic aberrations is a slow and progressive phenomenon, which is why tumor incidence is largely related to age. Striking exceptions Anders M. Lindroth and Christoph Plass are in the Division of Epigenomics and Cancer Risk Factors at the German Cancer Research Center (DKFZ), Heidelberg, Germany. e-mail:
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to these genetic processes are evident in childhood tumors that do not show complex cytogenetic alterations and progress extremely rapidly. A prominent example of driver alterations for childhood tumors is provided by the recent identification of mutations in H3F3A, encoding histone variant H3.3, in pediatric glioblastoma1,2, with highly recurrent mutations affecting the N-terminal tail of H3.3 and causing amino acid substitution of lysine 27 to methionine (p.Lys27Met) and of glycine 34 to arginine or valine (p.Gly34Arg or p.Gly34Val). On page 1479 of this issue, Adrienne Flanagan and colleagues3 report on two other earlyonset cancers—chondroblastoma and giant cell tumor of bone—that show recurrent mutations in H3.3 genes (Fig. 1). These tumors occur more frequently in children, adolescents and young adults, suggesting that the identified driver mutations are developmentally associated.
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H3.3 and tumorigenesis In contrast to the genes for canonical histones, the H3.3 genes are expressed and processed much like normal genes4. The two main H3.3 genes in metazoans, H3F3A and H3F3B, encode an identical protein and appear to be differentially expressed, although a comprehensive analysis has not been performed in mice4,5. The chromosomal deposition of the encoded protein, which occurs primarily in a replicationindependent manner, is surprisingly specific and is dependent on the histone chaperones HIRA, ATRX and DAXX5. Although chaperones control the deposition of H3.3 for both active and repressive transcription, the identification of mutations in both H3F3A and H3F3B by Behjati et al.3 suggests that differential expression rather than deposition is the determining factor for tumorigenesis. The new study further highlights the importance of H3.3 glycine 34 in childhood tumors. Although Behjati et al.3 did not explore functional 1413
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Figure 1 Distinct H3.3 alterations in childhood tumors. Mutations in H3F3A have previously been found in pediatric glioblastoma1,2. The new study by Flanagan and colleagues3 reports frequent H3F3A and H3F3B mutations in giant cell tumor of bone and chondroblastoma. The mutations show a high degree of specificity across the different tumor types.
mechanisms, a recent study of glycine 34 substitutions in glioblastoma showed that they affect the distribution of trimethylation on lysine 36 (H3K36me3) without affecting absolute global levels6. This finding may explain why glycine 34 substitutions involve a range of amino acids, from basic to hydrophobic residues (arginine, valine, tryptophan or leucine), in different tumors (Fig. 1), potentially changing the specificity of H3K36me3 placement by SETD2. Furthermore, the study identified MYCN, a known driver of glioblastoma in a mouse model7, as the most highly upregulated gene in tumors with glycine 34 substitutions6. Whether MYCN is a tumorigenic driver in chondroblastoma remains to be shown. Mechanistic consequences An obvious question is why H3.3 genes are so frequently mutated in childhood tumors. Although most aspects of the underlying mechanisms remain unknown, three studies have recently addressed the mechanistic consequences of mutations in these genes8–10. All three studies stem from the observation that, in probing tissue microarrays of p.Lys27Metpositive glioma biopsies, there was a near complete absence of trimethylated lysine 27 (H3K27me3) for all isoforms of histone H3, in contrast to the expected focal loss of H3K27me3 from H3.3. As the mutation leading to the p.Lys27Met substitution only occurs in H3F3A and in a heterozygous state, the p.Lys27Met alteration apparently has dominant negative characteristics. In binding and activity assays, a p.Lys27Met peptide was shown to interfere with the function of EZH2, which is known to catalyze the addition of H3K27me3 marks. The catalytic domain of EZH2 consists of a SET domain that is carried by all enzymes
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that methylate lysine residues in histones and other proteins. By using histone transgenes encoding lysine-to-methionine substitutions at lysine residues known to be methylated by other SET domain–containing proteins, David Allis and colleagues nicely demonstrated that these substitutions also produce global loss of lysine methylation at their respective peptidyllysine residue8. Similar to p.Lys27Met inhibition of EZH2, p.Lys9Met and p.Lys36Met blocked the activity of their cognate enzymes and reduced the levels of histone dimethylation and trimethylation at lysine 9 and lysine 36, respectively.Because the main function of H3K27me3 is to repress transcriptional activity, global loss of Lys27 methylation led to the upregulation of hundreds of genes, mainly those associated with neurogenic development9,10. Interestingly, both studies observed a gain of Lys27 methylation in some genomic regions in parallel with the global loss. This observation suggests that the homologous EZH1 protein compensates for the loss of EZH2 activity but generates an altered methylation pattern with local increases in H3K27me3 density at some sites. As the global reshaping of the H3K27me3 landscape is so profound, it also likely affects the placement of other epigenetic modifications, particularly DNA methylation. Bender et al.9 further describe the effect of p.Lys27Met on DNA methylation and suggest that an intimate relationship exists between these repressive epigenetic modifications. Indeed, a relationship between these marks has previously been described in mouse models and human breast cancer11–13. Another question relates to the striking tumor type specificity of the patterns of H3.3 alteration. Whereas pediatric brain tumors predominantly show H3F3A mutations
resulting in p.Lys27Met and p.Gly34Arg or p3Gly34Val substitutions, mutations in chondroblastoma only produce p.Lys36Met substitutions and only occur in H3F3B, and giant cell tumors of bone harbor p.Gly34Trp and p.Gly34Leu substitutions caused by mutations in H3F3A. Although the reasons for this distribution of mutations are unknown, differential expression of the two H3.3 genes is a plausible basis for the observed tumor type specificity. Aggressive tumors in children with H3.3 alterations are likely explained by two factors. First, the gain-of-function alterations observed in H3.3 interfere with key epigenetic factors instrumental for fine-tuning gene expression during developmental processes. Second, these alterations will subsequently change the landscape of other epigenetic modifications in a way that affects chromosomal integrity and proliferative activity. In a similar case, p.Arg132His substitutions of the isocitrate dehydrogenases IDH1 and IDH2 result in the production by these enzymes of the oncometabolite 2-hydroxyglutarate, which interferes with the function of the deaminases implicated in the removal or conversion of histone and DNA methylation14. Notably, childhood tumors develop in a relatively normal genetic background with minimal or absent cytogenetic aberrations, which is in stark contrast to the genetic landscape in more common adult tumors. This genetic context in childhood tumors provides a unique opportunity to identify key drivers and to understand the minimal prerequisites for neoplastic growth, motivating early screening for oncogenic mutations and more effective development of strategies for drug intervention. COMPETING FINANCIAL INTERESTS The authors declare no competing financial interests. 1. Schwartzentruber, J. et al. Nature 482, 226–231 (2012). 2. Wu, G. et al. Nat. Genet. 44, 251–253 (2012). 3. Behjati, S. et al. Nat. Genet. 45, 1479–1482 (2013). 4. Ederveen, T.H., Mandemaker, I.K. & Logie, C. Biochim. Biophys. Acta 1809, 577–586 (2011). 5. Elsaesser, S.J. & Allis, C.D. Cold Spring Harb. Symp. Quant. Biol. 75, 27–34 (2010). 6. Bjerke, L. et al. Cancer Discov. 3, 512 (2013). 7. Swartling, F.J. et al. Cancer Cell 21, 601–613 (2012). 8. Lewis, P.W. et al. Science 340, 857–861 (2013). 9. Bender, S. et al. Cancer Cell doi:10.1016/ j.ccr.2013.10.006 (31 October 2013). 10. Chan, K.M. et al. Genes Dev. 27, 985–990 (2013). 11. Lindroth, A.M. et al. PLoS Genet. 4, e1000145 (2008). 12. Hon, G.C. et al. Genome Res. 22, 246–258 (2012). 13. Reddington, J.P. et al. Genome Biol. 14, R25 (2013). 14. Ward, P.S. & Thompson, C.B. Cancer Cell 21, 297–308 (2012).
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