The knowledge that haspin phosphorylates H3T3ph in mitosis raises the obvious question of the function of H3T3ph. An understanding of this is likely to require consideration of the context in which H3T3ph is found. Phosphorylation is only one of multiple types of modification that occur on histones. For example, mono, di-, and tri-methylation of lysine residues including K4, K9, and K27, dimethylation of arginine residues including R2 and R8, and acetylation of numerous lysines including K4, K9, and K14 have been reported in histone H3 alone (Kouzarides 2007
; Eot-Houllier et al. 2008
). This has led to a number of more or less stringent definitions of a “histone code” hypothesis stating that different combinations of histone modifications form a code that is translated by effector or “reader” proteins into distinct biological functions (Turner 1993
; Strahl and Allis 2000
; Fischle et al. 2003
; Sims and Reinberg 2008
). Therefore, the function of H3T3ph may be influenced by adjacent modifications on histone tails.
A recent report suggested that H3T3ph is always found within a combinatorial modification pattern with H3K4me3 and H3R8me2 (named “PMM”) in mitotic cells (Markaki et al. 2009
). This assertion was based on a combination of mass spectrometry (MS) and immunochemical data that could not assign modifications with certainty. In contrast, H3T3ph-containing H3 peptides from mitotic cells observed in more direct MS/MS experiments lack H3K4me3 and H3R8me2 though in some cases they contain H3K4me1, H3K9me2, and/or H3S10ph (Garcia et al. 2005
; Bonenfant et al. 2007
). Further, although subject to the caveats of epitope occlusion noted earlier, immunofluorescence experiments indicate that H3K4me2, H3K4me3, and H3T3ph have distinct localizations at centromeres in mitosis (Sullivan and Karpen 2004
; Dai et al. 2006
). In addition, at least in vitro, haspin activity towards H3 peptides is progressively reduced as H3K4 is increasingly methylated (Eswaran et al. 2009
). Casas-Mollano et al. (2008)
also reported an inverse relationship between H3T3ph and H3K4me1 versus H3K4me2 and H3K4me3 at promoters in Chlamydomonas
cells. Therefore, although the PMM may exist in cells (Markaki et al. 2009
), it seems unlikely that H3T3ph is found only in this context. We favor the hypothesis that different (though likely partly overlapping) localizations of modifications such as H3T3ph, H3S10ph, and H3K4 methylation contribute to the creation of distinct zones of chromatin modification within centromeres and elsewhere on chromatin that have different functional attributes in mitosis. Crosstalk between modifications such as an inhibitory effect of H3K4me3 on haspin activity may play a role in establishing such regionality.
At least three (not entirely separable) molecular events that could be regulated by histone phosphorylation can be imagined: (1) phosphorylation induces a physiochemical change that influences interaction between nucleosomes or between histones and DNA. (2) Phosphorylation creates or abrogates a binding site for a histone-binding protein. (3) Phosphorylation influences the generation or functional consequences of other histone modifications. Variations of all three have been proposed for the best studied mark, H3S10ph. This modification was originally implicated in chromosome condensation in Tetrahymena
(Wei et al. 1999
) and it was reported to influence the interaction of H3 tails with DNA, perhaps altering nucleosome packing (Sauve et al. 1999
), an example of a physiochemical mechanism. However, an H3S10 mutation does not prevent mitotic progression in budding yeast (Hsu et al. 2000
) and the effects of Aurora B on condensation in multicellular organisms may be independent of H3S10ph (Adams et al. 2001
; de la Barre et al. 2001
; MacCallum et al. 2002
; Lipp et al. 2007
). An instance in which histone phosphorylation generates a protein-binding site is provided by the H3S10ph-dependent binding of 14-3-3 proteins to H3, an interaction that plays a role in regulation of gene transcription (Macdonald et al. 2005
). More recently, in an example of the third mechanism mentioned above, it has been shown that H3S10ph serves as a component of a “methyl/phospho” switch to regulate the association of histone-binding proteins with chromatin. The binding of heterochromatin protein-1 (HP1) to H3 peptides is strongly enhanced by H3K9me3. The phosphorylation of H3S10 adjacent to H3K9me3, however, prevents HP1 binding and may eject it from pericentromeric heterochromatin during mitosis (Fischle et al. 2005
; Hirota et al. 2005
Similar mechanisms can be envisaged for H3T3ph. Theoretical arguments suggest that H3T3ph could contribute to changes in nucleosomal packing, though experimental approaches will be required to substantiate these ideas (Georgatos et al. 2009
). There are a number of published examples where H3T3ph abrogates the interaction of proteins with histone H3. The direct binding of the inhibitor of acetyltransferases complex (INHAT) to H3 tail peptides is prevented by prior T3 phosphorylation in vitro (Schneider et al. 2004
). However, H3T3ph is not unique in this, as H3S10ph and H3T11ph as well as multisite acetylation are also able to dislodge INHAT. In two other similar in vitro examples, binding of the autoimmune regulator protein AIRE and the methyltransferase-associated WDR5 protein to the tail of H3 are abrogated by either H3R2 methylation or H3T3ph (Couture et al. 2006
; Koh et al. 2008
; Chignola et al. 2009
). In addition, the methylation of H3K4 by mixed-lineage leukemia protein is strongly reduced by H3T3ph (Southall et al. 2009
). Whether H3T3 phosphorylation displaces these proteins from chromatin in cells and if this has functional significance remains to be explored.
The proximity of H3T3 to the H3R2 and H3K4 methylation sites has led to the suggestion that H3T3ph might operate in a methyl/phospho switch mechanism like that at H3K9/H3S10 (Fischle et al. 2003
). Indeed, binding of chromodomain helicase DNA binding protein-1 (CHD1) to H3 methylated at K4 is abrogated by H3T3ph in vitro (Flanagan et al. 2005
). Although no cell-based experiments have yet directly confirmed this idea, it is quite plausible that H3T3ph serves to displace a number of H3-binding transcription and chromatin-regulating factors from chromosomes during mitosis. This process might make way for new chromatin-binding proteins or chromatin rearrangements that are needed to accomplish chromosome segregation, without erasing epigenetic histone modifications that control gene expression. It is also possible that “clearing the deck” in this way provides a means of re-initializing cellular gene expression programs during mitosis, either to prevent the accumulation of errors over multiple cell cycles or to help establish new transcriptional profiles in differentiating cells (Egli et al. 2008
). In this last scenario, H3T3ph functions during mitosis but is not directly required for mitosis itself.
In another example in which phosphorylation alters protein interaction with histones, it was recently suggested that H3T3ph plays a role in activating Aurora B at centromeres (Rosasco-Nitcher et al. 2008
). In this case, prior addition of H3 peptides to inactive recombinant Aurora B/INCENP prevented activation of the kinase by TD-60 and microtubules in vitro. Such inhibition did not occur, however, if the H3 peptides carried H3T3ph, and direct binding experiments revealed that H3T3ph prevented interaction of the H3 peptides with Aurora B/INCENP. These results led to a model in which binding of H3 substrate in the active site of inactive Aurora B prevents activation of the kinase, perhaps by competing with autophosphorylation of the activation segment. Priming phosphorylation of H3T3 by haspin prevents H3 association with Aurora B and allows kinase activation to occur, particularly at centromeres where H3T3ph is concentrated. This hypothesis is currently based almost exclusively on studies of recombinant proteins in vitro, and further experiments are needed to confirm that haspin and/or H3T3ph are required for centromeric Aurora B activity in cells. It will also be of interest to determine if H3T3ph is unique among H3 modifications in its ability to facilitate Aurora B activation, and to determine if H3T3ph has a direct effect on nearby H3S10 phosphorylation by Aurora B.
Finally, though it has yet to be demonstrated, it is appealing to hypothesize the existence of proteins that bind to H3 only when phosphorylated at T3. As for H3S10ph (Hans and Dimitrov 2001
), it has been speculated that H3T3ph might serve to mark chromosomes that have successfully reached metaphase (Markaki et al. 2009
), though the feature(s) of mitotic progression to which H3T3 or H3S10 would be sensitive and what the reader(s) of these marks would be are unknown. Of course, a number of other roles for H3T3ph-mediated protein binding can be imagined. In particular, the centromeric accumulation of H3T3ph suggests that it might recruit proteins that function at inner centromeres. Such proteins might include cohesion factors, chromosomal passenger proteins (Ruchaud et al. 2007
), or proteins that contribute to the structural or mechanical properties of centromeres.