CpG island hypermethylation and global genomic hypomethylation are common epigenetic features of cancer cells. Less attention has been focused on histone modifications in cancer cells. We characterized post-translational modifications to histone H4 in a comprehensive panel of normal tissues, cancer cell lines and primary tumors. Using immunodetection, high-performance capillary electrophoresis and mass spectrometry, we found that cancer cells had a loss of monoacetylated and trimethylated forms of histone H4. These changes appeared early and accumulated during the tumorigenic process, as we showed in a mouse model of multistage skin carcinogenesis. The losses occurred predominantly at the acetylated Lys16 and trimethylated Lys20 residues of histone H4 and were associated with the hypomethylation of DNA repetitive sequences, a well-known characteristic of cancer cells. Our data suggest that the global loss of monoacetylation and trimethylation of histone H4 is a common hallmark of human tumor cells.

Histone lysine methylation is a central modification to mark functionally distinct chromatin regions. In particular, H3-K9 trimethylation has emerged as a hallmark of pericentric heterochromatin in mammals. Here we show that H4-K20 trimethylation is also focally enriched at pericentric heterochromatin. Intriguingly, H3-K9 trimethylation by the Suv39h HMTases is required for the induction of H4-K20 trimethylation, although the H4 Lys 20 position is not an intrinsic substrate for these enzymes. By using a candidate approach, we identified Suv4-20h1 and Suv4-20h2 as two novel SET domain HMTases that localize to pericentric heterochromatin and specifically act as nucleosomal H4-K20 trimethylating enzymes. Interaction of the Suv4-20h enzymes with HP1 isoforms suggests a sequential mechanism to establish H3-K9 and H4-K20 trimethylation at pericentric heterochromatin. Heterochromatic H4-K20 trimethylation is evolutionarily conserved, and in Drosophila, the Suv4-20 homolog is a novel PEV modifier to regulate position-effect variegation. Together, our data indicate a function for H4-K20 trimethylation in gene silencing and further suggest H3-K9 and H4-K20 trimethylation as important components of a repressive pathway that can index pericentric heterochromatin.[Keywords: Histone code; histone H4 Lys 20; mono-, di-, trimethylation; Suv4-20h HMTases; heterochromatin; combinatorial histone methyl marks] Supplemental material is available at http://www.genesdev.org.

Su(var)3±9 is a dominant modi®er of heterochromatin-induced gene silencing. Like its mammalian and Schizosaccharomyces pombe homologues, Su(var) 3±9 encodes a histone methyltransferase (HMTase), which selectively methylates histone H3 at lysine 9 (H3-K9). In Su(var)3±9 null mutants, H3-K9 methylation at chromocentre heterochromatin is strongly reduced, indicating that SU(VAR)3±9 is the major heterochromatin-speci®c HMTase in Drosophila. SU (VAR)3±9 interacts with the heterochromatin-associated HP1 protein and with another silencing factor, SU(VAR)3±7. Notably, SU(VAR)3±9±HP1 interaction is interdependent and governs distinct localization patterns of both proteins. In Su(var)3±9 null mutants, concentration of HP1 at the chromocentre is nearly lost without affecting HP1 accumulation at the fourth chromosome. By contrast, in HP1 null mutants SU(VAR)3±9 is no longer restricted at heterochromatin but broadly dispersed across the chromosomes. Despite this interdependence, Su(var)3±9 dominates the PEV modi®er effects of HP1 and Su(var)3±7 and is also epistatic to the Y chromosome effect on PEV. Finally, the human SUV39H1 gene is able to partially rescue Su(var)3±9 silencing defects. Together, these data indicate a central role for the SU(VAR)3±9 HMTase in heterochromatin-induced gene silencing in Drosophila.

H4K20 methylation is a broad chromatin modification that has been linked with diverse epigenetic functions. Several enzymes target H4K20 methylation, consistent with distinct mono-, di-, and trimethylation states controlling different biological outputs. To analyze the roles of H4K20 methylation states, we generated conditional null alleles for the two Suv4-20h histone methyltransferase (HMTase) genes in the mouse. Suv4-20h-double-null (dn) mice are perinatally lethal and have lost nearly all H4K20me3 and H4K20me2 states. The genome-wide transition to an H4K20me1 state results in increased sensitivity to damaging stress, since Suv4-20h-dn chromatin is less efficient for DNA double-strand break (DSB) repair and prone to chromosomal aberrations. Notably, Suv4-20h-dn B cells are defective in immunoglobulin class-switch recombination, and Suv4-20h-dn deficiency impairs the stem cell pool of lymphoid progenitors. Thus, conversion to an H4K20me1 state results in compromised chromatin that is insufficient to protect genome integrity and to process a DNA-rearranging differentiation program in the mouse.[Keywords: H4K20 methylation; Suv4-20h enzymes; DNA repair; genome integrity; B-cell differentiation; class-switch recombination] Supplemental material is available at http://www.genesdev.org. Received February 18, 2008; revised version accepted May 30, 2008. Histone lysine methylation is a central epigenetic modification in eukaryotic chromatin. Five major positions for lysine methylation exist in the histone N termini, each with distinct regulatory functions. The repressive methyl marks H3K9, H3K27, and H4K20 are involved in constitutive heterochromatin formation and gene repression, X inactivation, and Polycomb silencing, and in DNA damage repair, mitotic chromosome condensation, and gene regulation (Allis et al. 2007). Additional complexity arises through the fact that histone methylation can be present in three distinct states (mono, di, or tri), which may have different biological readouts depending on the association with specific binding partners. Although there has been significant insight in histone lysine methylation pathways, we still know very little about how the diverse methylation states affect chromatin biology.H4K20 methylation is evolutionarily conserved from Schizosaccharomyces pombe to man . In mammalian cells, H4K20me1 is exclusively induced by the PrSet7/KMT5A histone methyltransferase (HMTase) (Fang et al. 2002;Nishioka et al. 2002), where it has been linked with transcriptional repression (Karachentsev et al. 2005) and X inactivation (Kohlmaier et al. 2004). More recently, genome-wide profiling of H4K20me1 also revealed enrichment of this mark across actively transcribed genes (Papp and Muller 2006;Vakoc et al. 2006). H4K20me1 is very dynamic throughout the cell cycle and becomes highly enriched during S phase (Jorgensen et al. 2007;Tardat et al. 2007;Huen et al. 2008