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Histone H4 acetylation at Lysine 16 (H4K16ac) is a key epigenetic mark involved in gene regulation, DNA repair and chromatin remodeling, and though it is known to be essential for embryonic development, its role during adult life is still poorly understood. Here we show that this lysine is massively hyperacetylated in peripheral neutrophils. Genome-wide mapping of H4K16ac in terminally differentiated blood cells, along with functional experiments, supported a role for this histone post-translational modification in the regulation of cell differentiation and apoptosis in the hematopoietic system. Furthermore, in neutrophils, H4K16ac was enriched at specific DNA repeats. These DNA regions presented an accessible chromatin conformation and were associated with the cleavage sites that generate the 50 kb DNA fragments during the first stages of programmed cell death. Our results thus suggest that H4K16ac plays a dual role in myeloid cells as it not only regulates differentiation and apoptosis, but it also exhibits a non-canonical structural role in poising chromatin for cleavage at an early stage of neutrophil cell death., Plan Nacional de I+D+I co-funding FEDER [PI15/00892 and PI18/01527 to M.F.F. and A.F.F.; PI16/01318 and PI14/01244 to C.L.]; ISCIII-Subdireccion General de Evaluacion y Fomento de la Investigacion, and Plan Nacional de I+D+I 2008–2011/FEDER [CP11/00131 to A.F.F.]; IUOPA (to G.F.B. and M.I.S.); Fundacion Cientifica de la AECC (to R.G.U.); Ministry of Economy and Competitiveness Juan de la Cierva postdoctoral fellowships [FJCI-2015-26965 to J.R.T., IJCI-2015- 23316 to V.L.]; Fundacion Ramon Areces (to M.F.F); FICYT (to E.G.T., M.G.G., A.C.); Asturias Regional Government [GRUPIN14-052 to M.F.F.]; Gobierno del Principado de Asturias, PCTI-Plan de Ciencia, Tecnologia e Innovacion co-funding Fondos FEDER (grant number IDI/2018/146 to M.F.F. and IDI/2018/144 to C.L.); Deutsche Forschungsgemeinschaft (DFG) [SFB960 to A.V.G., R.D.]; European Research Council [CoG-2014-646903]; Spanish Ministry of Economy and Competitiveness [SAF-SAF2013-43065 to P.M.]; Asociacion Española Contra el Cancer [AECC-CI-2015]; FERO Foundation, and the ISCIII [PI14-01191 to C.B.]; P.M. acknowledges financial support from The Obra Social La Caixa Fundacio Josep Carreras and The Generalitat de Catalunya (SGR330). P.M. an investigator from the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Liberbank-Cajastur, Spain. Funding for open access charge: Plan Nacional de I+D+I co-funding FEDER [PI18/01527].

This work has been financially supported by: the Plan Nacional de IþDþI 2013–2016/FEDER (PI15/00892 to M.F.F. and A.F.F.; RTC2015-3393-1 to A.F.F.); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación, and the Plan Nacional de IþDþI 2008–2011/FEDER (CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); the Fundación Científica de la AECC (to R.G.U.); the Fundación Ramón Areces (to M.F.F); FICYT (to E.G.T., M.G.G. and A.C.); and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). Work in P.M. laboratory is supported by the European Research Council (CoG-2014-646903), the Spanish Ministry of Economy-Competitiveness (SAF-SAF2013-43065), the Obra Social La Caixa-Fundació Josep Carreras, and the Generalitat de Catalunya. P.M. is an investigator in the Spanish Cell Therapy cooperative network (TERCEL). The IUOPA is supported by the Obra Social Cajastur-Liberbank, Spain., Fernandez, A.F., Bayón, G.F., Sierra, M.I., Urdinguio, R.G., Toraño, E.G., García, M.G., Carella, A., López, V., Santamarina, P., Pérez, R.F., Belmonte, T., Tejedor, J.R., Cobo, I., Menendez, P., Mangas, C., Ferrero, C., Rodrigo, L., Astudillo, A., Ortea, I., Díaz, S.C., Rodríguez-Gonzalez, P., Alonso, J.I.G., Mollejo, M., Meléndez, B., Domínguez, G., Bonilla, F., Fraga, M.F.

Aberrant DNA hypermethylation is a hallmark of cancer although the underlying molecular mechanisms are still poorly understood. To study the possible role of 5-hydroxymethylcytosine (5hmC) in this process we analyzed the global and locus-specific genome-wide levels of 5hmC in primary samples from 54 gliomas and 72 colorectal cancer patients. Levels of 5hmC in colorectal cancer were very low and no consistent changes were detected between control tissues and tumors. As expected, levels of 5hmC in non-tumoral brain samples were high and significantly reduced at the 49,601 CpG sites in gliomas. Strikingly, hypo-hydroxymethylation at 4,627 (9.3%) of these CpG sites was associated with aberrant DNA hypermethylation. The DNA regions containing these CpG sites were enriched in H3K4me2, and presented a different genuine chromatin signature to that characteristic of the genes classically aberrantly hypermethylated in cancer. We conclude that this data identifies a novel 5hmC-dependent non-canonical class of aberrant DNA hypermethylation in glioma.

[Background]: Age-associated changes in genomic DNA methylation have been primarily attributed to 5-methylcytosine (5mC). However, the recent discovery of 5-hydroxymethylcytosine (5hmC) suggests that this epigenetic mark might also play a role in the process. [Methods]: Here, we analyzed the genome-wide profile of 5hmc in mesenchymal stem cells (MSCs) obtained from bone-marrow donors, aged 2-89 years. [Results]: We identified 10,685 frequently hydroxymethylated CpG sites in MSCs that were, as in other cell types, significantly associated with low density CpG regions, introns, the histone posttranslational modification H3k4me1 and enhancers. Study of the age-associated changes to 5hmC identified 785 hyper- and 846 hypo-hydroxymethylated CpG sites in the MSCs obtained from older individuals. [Conclusions]: DNA hyper-hydroxymethylation in the advanced-age group was associated with loss of 5mC, which suggests that, at specific CpG sites, this epigenetic modification might play a role in DNA methylation changes during lifetime. Since bone-marrow MSCs have many clinical applications, and the fact that the epigenomic alterations in this cell type associated with aging identified in this study could have associated functional effects, the age of donors should be taken into account in clinical settings., This work has been financially supported by the Plan Nacional de I + D + I 2008–2011/2013–2016/FEDER (PI11/01728 to A.F.F.; PI12/01080 and PI15/00892 to M.F.F.; PI 12/0615 to J.A.R.; PI05/2217 and PI08/0029 to J.G-C); the ISCIII-Subdirección General de Evaluación y Fomento de la Investigación (Miguel Servet contract: CP11/00131 to A.F.F.); IUOPA (to G.F.B. and M.S); Fundacion Cientifica de la AECC (to R.G.U.); Fundación Ramón Areces (to M.F.F); and FICYT (to E.G.T., M.G.G and A.C.);the Madrid Regional Government (S-BIO-02042006 and S2010/BMD-2420 to J.G-C) and the Asturias Regional Government (GRUPIN14-052 to M.F.F.). The IUOPA is supported by the Obra Social Cajastur, Spain.