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.

p53, the tumour suppressor and transcriptional activator, is regulated by numerous post-translational modifications, including lysine methylation. Histone lysine methylation has recently been shown to be reversible; however, it is not known whether non-histone proteins are substrates for demethylation. Here we show that, in human cells, the histone lysine-specific demethylase LSD1 (refs 3, 4) interacts with p53 to repress p53-mediated transcriptional activation and to inhibit the role of p53 in promoting apoptosis. We find that, in vitro, LSD1 removes both monomethylation (K370me1) and dimethylation (K370me2) at K370, a previously identified Smyd2-dependent monomethylation site. However, in vivo, LSD1 shows a strong preference to reverse K370me2, which is performed by a distinct, but unknown, methyltransferase. Our results indicate that K370me2 has a different role in regulating p53 from that of K370me1: K370me1 represses p53 function, whereas K370me2 promotes association with the coactivator 53BP1 (p53-binding protein 1) through tandem Tudor domains in 53BP1. Further, LSD1 represses p53 function through the inhibition of interaction of p53 with 53BP1. These observations show that p53 is dynamically regulated by lysine methylation and demethylation and that the methylation status at a single lysine residue confers distinct regulatory output. Lysine methylation therefore provides similar regulatory complexity for non-histone proteins and for histones.

Mouse primordial germ cells (PGC) undergo sequential epigenetic changes and genome-wide DNA demethylation to reset the epigenome for totipotency. Here, we demonstrate that erasure of CpG methylation (5mC) in PGCs occurs via conversion to 5-hydroxymethylcytosine (5hmC), driven by high levels of TET1 and TET2. Global conversion to 5hmC initiates asynchronously among PGCs at embryonic day (E) 9.5-E10.5 and accounts for the unique process of imprint erasure. Mechanistically, 5hmC enrichment is followed by its protracted decline thereafter at a rate consistent with replication-coupled dilution. The conversion to 5hmC is a significant component of parallel redundant systems that drive comprehensive reprogramming in PGCs. Nonetheless, we identify rare regulatory elements that escape systematic DNA demethylation in PGCs, providing a potential mechanistic basis for transgenerational epigenetic inheritance.Specification of primordial germ cells (PGCs) from epiblast cells at ~E6.25 is linked with extensive epigenetic reprogramming, including global DNA demethylation, chromatin reorganisation and imprint erasure, that is vital for generating totipotency (1, 2). The erasure of CpG methylation (5mC) is a key component of this program, but the dynamics and underlying mechanisms of the process remain unclear (3). Here we report a comprehensive analysis of PGCs by combining immunofluorescence, genome-wide (h)meDIP-seq, single cell RNA-seq, bisulfite-seq and functional analyses to address the mechanistic basis of epigenetic reprogramming in PGCs.We investigated Tet expression using single cell RNA-seq, which revealed that Tet1 and Tet2 are expressed in PGCs and peak between E10.5-E11.5, but that Tet3 is undetectable (Fig. 1A). Immunofluorescence (IF) showed that TET1 and TET2 are nuclear and expressed at significantly higher levels in PGCs than neighbouring somatic cells between E9.5-E11.5 (Fig. 1B & S1-S2). This suggests that erasure of 5mC in PGCs could occur through conversion to 5-hydroxymethylcytosine (5hmC) by TET1/TET2 (4, 5).We pursued this possibility by IF and found a progressive reduction of 5mC in PGCs between E9.5-E10.5, until it became undetectable by E11.5 (Fig. 1C). The loss of 5mC occurs concurrently with a global enrichment of 5hmC in PGCs between E9.5-E10.5, † To whom correspondence should be addressed. a.surani@gurdon.cam.ac.uk.