A Trans-tail Histone Code Defined by Monomethylated H4 Lys-20 and H3 Lys-9 Demarcates Distinct Regions of Silent Chromatin

Abstract

The specific post-translational modifications of the histone proteins are associated with specific DNA-templated processes, such as transcriptional activation or repression. To investigate the biological role(s) of histone H4 lysine 20 (H4 Lys-20) methylation, we created a novel panel of antibodies that specifically detected mono-, di-, or trimethylated H4 Lys-20. We report that the different methylated forms of H4 Lys-20 are compartmentalized within visually distinct, transcriptionally silent regions in the mammalian nucleus. Interestingly, direct comparison of methylated H4 Lys-20 with the different methylated states of histone H3 lysine 9 (H3 Lys-9) revealed significant overlap and exclusion between the specific groups of methyl modifications. Trimethylated H4 Lys-20 and H3 Lys-9 were both selectively enriched within pericentric heterochromatin. Similarly, monomethylated H4 Lys-20 and H3 Lys-9 partitioned together and the dimethylated forms partitioned together within the chromosome arms; however, the mono-and dimethylated modifications were virtually exclusive. These findings strongly suggest that the combinatorial presence or absence of the different methylated states of H4 Lys-20 and H3 Lys-9 define particular types of silent chromatin. Consistent with this, detailed analysis of monomethylated H4 Lys-20 and H3 Lys-9 revealed that both were preferentially and selectively enriched within the same nucleosome particle in vivo. Collectively, these findings define a novel trans-tail histone code involving monomethylated H4 Lys-20 and H3 Lys-9 that act cooperatively to mark distinct regions of silent chromatin within the mammalian epigenome.Within eukaryotic nuclei, DNA associates with nuclear proteins to form chromatin. The most fundamental structural unit of chromatin, the nucleosome, is composed of ϳ146 bp of DNA wrapped around an octamer of the core histone proteins H2A, H2B, H3, and H4 (1). Although much has been elucidated about chromatin in the last decade, it remains unclear how distinct functional domains of chromatin are established and maintained within living cells. It is well known that chromatin function is modulated, at least in part, by enzymes that posttranslationally modify specific amino acids on the histone proteins (2). Such post-translational modifications include acetylation, phosphorylation, ubiquitination, and methylation (3). Increasing evidence indicates that specific covalent modifications, alone or in combination, directly participate in specific downstream nuclear processes including transcription, replication, and repair (4). Thus, it is theorized that this "histone code" may serve to establish and maintain distinct functional domains that are epigenetically transmitted (5-7).One histone modification that has received increased attention is the methylation of lysine 20 on histone H4 (H4 Lys-20).2 Although this methylated histone lysine residue was identified over 40 years ago, the biological significance of this modification has remained enigmatic (8). Investigations into H4 Lys-20 ...

Histone H4 lysine 20 monomethylation promotes transcriptional repression by L3MBTL1

“…This implies that L3MBTL1 mediates repression in the context of an H4K20me1 mark in facultative heterochromatin (that lacks acetylation). The nuclear distribution of L3MBTL1 and H4K20me1, and the identification of HP1g, a protein linked to facultative heterochromatin, in the L3MBTL1 complex further support our inference (Sims et al, 2006;Dialynas et al, 2007;Trojer et al, 2007).…”

“…Initial reports highlighted the importance of H4K20me1 in 'facultative' heterochromatin and Xinactivation (Fang et al, 2002;Nishioka et al, 2002;Kohlmaier et al, 2004;Karachentsev et al, 2005;Martens et al, 2005;Sims et al, 2006). Recently, this exclusivity has been questioned by studies that suggest a role in transcriptional competence and elongation (Talasz et al, 2005;Vakoc et al, 2006;Barski et al, 2007;Pesavento et al, 2008).…”

“…Furthermore, the methylation status (mono-, di-or tri-(me1/me2/ me3)) of specific lysines, extends the options for signaling to chromatin (Peters et al, 2003;Rice et al, 2003) as exemplified for H4K20 methylation. H4K20me1 is associated with facultative heterochromatin (Fang et al, 2002;Martens et al, 2005;Sims et al, 2006), and the inactive X chromosome (Kohlmaier et al, 2004). In contrast, H4K20me3 marks constitutive heterochromatin (Schotta et al, 2004;Mikkelsen et al, 2007).…”

Catalytic Function of the PR-Set7 Histone H4 Lysine 20 Monomethyltransferase Is Essential for Mitotic Entry and Genomic Stability

“…Immunofluorescence-Indirect immunofluorescence of mouse embryonic fibroblasts was performed as previously described (32). Antibody dilutions were used as follows: H4K20 mono-(1:1,000), di-(1:500), and trimethyl (1:1,000) (LP Biologicals); ␥H2AX (Millipore; 1:500); and ␥-tubulin (Sigma; 1:400).…”

“…To further investigate this, we first employed a panel of H4K20 methyl-specific antibodies to determine that only the monomethylated form of H4K20 displayed a distinct cell cycle profile: low at G 1 , increased during late S, and highest at G 2 /M, identical to PR-Set7 (32). Furthermore, we also defined that the cell cycle-dependent changes in monomethylated H4K20 occurred locally at specific genomic regions.…”