Chromatin is an essential platform for almost all DNA-templated processes, including replication, repair, recombination and transcription1-3
. Nucleosomes, the fundamental unit of chromatin, consist of 147 base pairs (bp) of DNA wrapped around an octamer of histones consisting of two H2A-H2B dimers and a single H3-H4 tetramer, forming two nearly symmetrical halves in their tertiary structure4
. The core histones are highly conserved proteins from yeast to humans, indicating the importance of each amino acid residue within the histones. Nucleosomes present a degree of structural constraint, and to circumvent these restrictions chromatin structure may be locally or globally altered either through the post-translational modification of histones1-3
, interactions with other proteins such as ATP-dependent chromatin-remodeling complexes, or by replacement of core histones with histone variants5-7
. Covalent modifications of histones include acetylation, methylation, ubiquitination, sumoylation and phosphorylation8,9
. Most of the identified histone modifications are located in the histone tail, where modifying enzymes or modification-targeting proteins can gain relatively easy access. However, post-translational modifications within the core histones have also been reported2,10-14
A prevalent modification of histones that is associated with active transcription is H3K4 methylation15-24
. More importantly, the lysine residues on histones can be mono-, di- or trimethylated by histone methyltransferases (HMTases), with each pattern of modification having a specific biological outcome2,17,23
. Chromatin immunoprecipitation (ChIP)-based studies revealed that COMPASS and H3K4 trimethylation is found at the 5′ end of the coding region15,16
. Several studies also demonstrated that the acetylation of H3K9 and H3K14 are also found at the 5′ ends of actively transcribed genes25,26
. However, the relationship between H3 acetylation and H3 methylation is yet to be determined.
Methylation of H3K4 is catalyzed by the enzymatic activity of the macromolecular complex COMPASS, which contains the methyltransferase Set1 (ref. 2
). Following the identification of Set1-COMPASS as the first H3K4 HMTase15,18,19,21
, it was demonstrated that the human homologs of Set1, the mixed lineage leukemia (MLL) proteins, MLL2-4, and Set1A and Set1B, were also found in COMPASS-like complexes capable of methylating H3K4 (ref. 27
). Previously, we, and others, demonstrated that monoubiquitination of histone H2B on lysine 123 (H2BK123) is required for the proper methylation of H3K4 by COMPASS28,29
. We now know that H2B monoubiquitination regulates H3K4 methylation via regulation of COMPASS catalytic activity. The molecular mechanism identified in yeast that regulates the implementation and removal of H3K4 methylation is conserved in humans30-32
. Therefore, the lessons learned from yeast chromatin are highly valuable in defining the chromatin and transcriptional machinery in humans.
Not only is H2B monoubiquitination required for H3K4 methylation by COMPASS, but methylation of histone H3 arginine 2 (H3R2) also has an important role in regulating COMPASS’s activity33,34
. To determine how many other residues within the histones are required for proper H3 methylation by COMPASS, we systematically generated a library of alanine mutants of all residues of the four core histones in yeast S. cerevisiae
. We call this library the scanning histone mutagenesis with alanine (SHIMA) library. This is an unbiased approach, which will facilitate determination of the importance and functional significance of all of the residues within histones. Given the conservation of residues between histones from yeast to humans, we initially predicted that many mutated residues within the histones would be required for viability, and therefore, be lethal. To our surprise, only 18 residues were found to be essential for viability on complete growth medium, three of which represented an extreme slow-growth phenotype. Recently, Matsubara and colleagues developed a strategy called global analysis of surfaces by point mutation (GLASP), where they mutated the surface residues of the four histones35
. Their study resulted in the identification of eight essential residues within the four histones. The identification of additional essential residues here suggests the utility of SHIMA as a comprehensive and systematic mutant collection that will allow characterization of the functional importance of almost all residues within the core histones.
Using this entire comprehensive histone-mutant collection and our global proteomic screen (GPS) in S. cerevisiae
, we then explored the network of histone cross-talk between histone H3K4 methylation and other residues within the histones36
. With GPS, we have examined the extracts from the histone-mutant collection by western analysis using antibodies directed toward modified histones. We have identified several previously uncharacterized residues within histones acting either in cis
to regulate proper H3K4 methylation. Here we provide evidence for the existence of possible cis
-cross-talk between histone H3K4 trimethylation and histone H3K14 acetylation. We have also identified residues that act in trans
to regulate the pattern of histone H3K4 methylation, and they map to a patch on nucleosomes. This patch contains His112 and Arg119 of histone H2B, and Glu65, Leu66, Asn69 and Asp73 of histone H2A, all of which reside near H2BK123 when visualized on the three-dimensional structure of the nucleosome. Two of the residues are required for H2BK123 monoubiquitination, whereas the other residues may regulate COMPASS’s activity independently of H2B monoubiquitination. This comprehensive library of histone mutants has been instrumental in defining the global regulation of histone cross-talk for H3K4 methylation. This collection will be a useful resource to the chromatin community for defining the role of histone residues in numerous processes involving chromatin.