Histones are nuclear proteins that package and order the DNA into nucleosomes . Five major families of histones exist: H1 (H5), H2A, H2B, H3, and H4. Two copies of the core histones H2A, H2B, H3 and H4 form the octameric nucleosome core particles . Unlike the other histones, only one copy of the linker histone H1 is present and stabilizes the DNA, which is wrapped around the core nucleosome . Linker histones bind to both the nucleosome and the linker DNA region (approximately 20 to 80 nucleotides in length) between nucleosomes. The interaction of H1 with the nucleosome and additional DNA stretches at the entry/exit of the nucleosome forms the chromatosome and leads to higher order chromatin structure . Many experiments addressing H1 function have been performed with purified, processed chromatin under low-salt conditions, but the in vivo role of H1 is less clear. Cellular studies have shown that overexpression of H1 can cause aberrant nuclear morphology and chromatin structure and, depending on the gene, H1 can serve as either a positive or a negative regulator of transcription . Similar to the core histones, H1 is composed of three domains . The N-terminus is a short, flexible segment rich in basic amino acids, the central domain exhibits a globular structure composed of a winged helix motif  and the C-terminus is predominantly composed of lysine, alanine and proline residues and is the main determinant for H1 binding to chromatin . Among the five histone families of the chromatosome, the linker histone H1 is the least conserved. In the human genome, 11 genes encoding H1 variants have been identified and are transcribed either ubiquitously or in a cell type-specific manner [4, 8]. The study described here focuses on histone H1.4, a histone variant that is expressed in somatic cells during S phase. Together with H1.2 it is the predominant histone variant in most cell types. Similar to the core histones, linker histones are subject to extensive post-translational modifications (PTMs), including phosphorylation, methylation and acetylation .
SET7/9 (also SET7, SET9, SETD7 or KMT7) is a mono-methyltransferase for the lysine residue at position 4 of histone H3 (H3K4) [10, 11] that was linked to transcriptional activation. It methylates the consensus motif [K>R][S>KYARTPN][Kme] and prefers lysine residues within positively charged regions . However, SET7/9 exhibits only weak lysine methyltransferase activity towards H3 in nucleosomes in vitro, suggesting that additional factors may affect SET7/9-dependent H3K4 methylation in vivo, or that histone proteins are not the main substrates of SET7/9. Lysine methylation can be reversed by demethylases of the lysine-specific demethylase (LSD) family or the Jumonji-C domain family of proteins [13, 14].
In contrast to the canonical PTMs of the histone code, adenosine diphosphate (ADP)-ribosylation is much less studied. ADP-ribosylation comprises the transfer of the ADP-ribose moiety from the co-substrate nicotinamide adenine dinucleotide (NAD+) onto specific amino acid side chains of acceptor proteins or to pre-existing protein-linked ADP-ribose units by ADP-ribosyltransferases (ARTs). Mammalian ARTs can be divided into two groups according to their similarity to the bacterial diphtheria and cholera toxins - the ARTDs (also known as poly(ADP-ribose) polymerases (PARPs)) and ARTCs, respectively . ARTD1 (PARP1) is the best-studied member of the ARTD family and represents a highly abundant (on average 1 × 106 molecules per cell), chromatin-associated enzyme that is responsible for most (about 90%) of the cellular PAR generation [16, 17]. It is implicated in many cellular processes such as the genotoxic stress response, cell cycle regulation, gene expression, differentiation and aging [18, 19]. The major modification target of ARTD1 is ARTD1 itself, but it also modifies other nuclear proteins including all five histone proteins in vitro and in vivo. In native chromatin, histone H1 is the main ADP-ribose acceptor, but depending on the chromatin composition and the accessibility of different histones, the ADP-ribosylation pattern of histones varies [21, 22]. Mass spectrometry and electron-transfer dissociation (ETD) identified for the first time K13 of histone H2A, K30 of H2B, K27 and K37 of H3 as well as K16 of H4 as ADP-ribose acceptor sites (catalyzed by ARTD1) .
Crosstalk between different PTMs occurs directly by competition for acceptor sites or indirectly by changes in the accessibility of chromatin for modifying enzymes. The observation that specific lysine residues serve as ADP-ribose acceptors is important because the same amino acid residues are potential acetylation and methylation sites . It is therefore likely that competition for acceptor sites between different histone PTMs such as ADP-ribosylation, acetylation, methylation and phosphorylation causes crosstalk . This has been demonstrated by the finding that acetylation of lysine residue K16 of histone H4 inhibits ADP-ribosylation in vitro, which suggests that different crosstalk likely exists in vivo as well. Similarly, H1.4 K26 dimethylation and AuroraB-mediated phosphorylation of S27 have been reported to interfere with each other . Whether or not other modifications of the histone code such as methylation or phosphorylation also crosstalk with ADP-ribosylation has not been studied before.
Here, we define the linker histone H1.4 as a novel target of SET7/9-dependent methylation, identify lysines K121, K129, K159, K171, K177 and K192 as methyl acceptor sites and describe crosstalk between H1.4 methylation and ADP-ribosylation as well as competition with histone H3 methylation.