The incorporation of histone variants into chromatin is one of the mechanisms used by the eukaryotic cell to increase the potential diversity of the conformation of and recruitment to the chromatin template. Though the SWR1 complex has been identified as the primary effector by which histone variant H2A.Z is deposited into chromatin, the exact nature of the binding interface between H2A.Z and the SWR1 complex is poorly understood. In this work, we have shown that specific residues contained within the acidic patch of H2A.Z confer the ability to be deposited into chromatin to provide normal function. Through an unbiased genetic screen, we have shown that two evolutionarily conserved amino acid residues of the acidic patch of H2A.Z are important for its correct function. Mutation of two of these residues confers strong phenotypes that stem from defective function, because these mutant histones are not deposited in appropriate patterns in chromatin at the PHO5
locus. These findings can be thought of as a fine-structure mapping of the deposition function of the H2A.Z molecule. D99 and E101 represent candidate residues for direct interaction with the SWR1 complex, either contacting the scaffold Swr1 directly or possibly the Swc2 subunit (Wu et al. 2005
). Further studies aimed at confirming and characterizing this interaction will shed considerable light on the nature of the histone exchange reaction carried out by the SWR1 complex.
A common technique for identifying important amino acid residues of a protein is to systematically mutate each individual residue to alanine. Alanine is a neutral amino acid and is fairly small, so it would not be expected to cause serious ionic or structural distortions. It should be noted that if alanine scanning had been applied to the acidic patch to uncover important residues, it would have identified D99 as being important for Htz1 function. However, it would have missed E101 as an important residue due to its lack of serious effect in the alanine mutant. Our results are in concordance with the general genetic concept that site-directed and random methods are most successful when utilized together. We first performed a random screen for HTZ1 mutations, and then applied thorough systematic techniques to the region highlighted by that screen. The obvious charge difference of the E101K mutation originally identified in htz1-21 (as well as the presence of a more conservative mutation in htz1-27) suggested to us to construct our mutant panel taking into account mutating each acidic residue to a neutral as well as a basic amino acid. This allowed us to characterize the importance of both D99 and E101.
Previous domain swapping experiments have established the importance of the M6 domain of H2A.Z for function (Clarkson et al. 1999
; Wu et al. 2005
). Through the use of an unbiased genetic screen, we have also defined a region of functional importance in H2A.Z even more specific than the M6 domain. In H2A.Z, three consecutive conserved acidic residues of H2A (DDE) are extended to four out of five residues (DDELD). Given the importance of H2A.Z function as being distinct from H2A (Jackson and Gorovsky 2000
), it might be expected that a crucial residue in the acidic patch that specifies a function for H2A.Z would be D103, since there is no corresponding residue in H2A (). Surprisingly, mutation of D103 was without effect in our studies (, ); we have shown that the important residues of the acidic patch are actually among those shared with the major H2A (D99 and E101). Importantly, not every residue in the patch conferred the strong phenotypes. Singly mutating two of three conserved residues (D99 or E101) was sufficient to generate phenotypes, but mutating the conserved residue immediately between them (D100) was without serious effect. In addition, changing acidic E101 to neutral alanine (a more conservative change) was without serious effect, but changing it to basic lysine (a less conservative change) was sufficient to generate the phenotypes in question. These results essentially describe a fine-structure mapping of the acidic patch region of H2A.Z within a sequence of amino acid residues that are crucial for binding.
Studies in the Tremethick laboratory have been performed on the acid patch region of H2A.Z in higher eukaryotes. In one study, mutations in the acidic patch region of X. laevis
H2A.Z including the equivalent of yeast D103 were sufficient to confer an embryonic developmental defect (Ridgway et al. 2004
). In the other study, those mutations conferred to H2A.Z nucleosomal arrays a folding property not different than that of H2A-containing arrays, pointing to a role for the acid patch residues of H2A.Z in the compaction of chromatin (Fan et al. 2004
). The patch itself is highly conserved, but these studies are not incompatible with our findings, particularly the absence of any serious effect in our D103 yeast mutant strains. These other experiments were performed using H2A.Z from a multicellular organism in which H2A.Z is essential for viability. Since yeast does not require H2A.Z to survive, D103 likely acts in a pathway in X. laevis
that is distinct from that of yeast.
There is also considerable structural support for the array of effects we have observed. The NMR structure of H2A.Z-specific histone chaperone Chz1 in a heterotrimer complexed with histones H2A.Z and H2B (the CZB complex) was recently published (Zhou et al. 2008
). As H2A.Z–H2B dimers are expected to interact with the SWR1 complex in a chaperone-bound form (Luk et al. 2007
), the structural predictions made are directly applicable to our observations. The structure indicates that D99 and E101, the two acidic residues that demonstrated strong phenotypes when mutated, are exposed in a groove on one side of the CZB complex (Zhou et al. 2008
). In addition, D100 and D103, the two acidic residues that did not demonstrate strong phenotypes when mutated, are not in this groove. D100 is located to the side of this groove, and D103 is behind the groove. We do not know the exact locations of the binding partners on the SWR1 complex, but it is tempting to speculate that this groove represents the binding surface for the SWR1 complex on the H2A.Z molecule. The differential locations of the four acidic residues could explain why D99 and E101 specifically demonstrate strong effects, and D100 and D103 do not. It is also possible that if D99 interacted with its binding partner through short-range hydrophobic interactions, and E101 interacted with its binding partner through long-range electrostatic interactions, then this could potentially explain the different effects of the alanine substitutions in these two residues. Mutation to alanine would then be better tolerated at E101, but still clash with its binding partner at D99. It should be noted that the disruption of SWR1 complex binding in the D99A/K and E101K mutations might also be due to an indirect effect, as D99 and E101 do not appear to be completely solvent-accessible. These mutations, rather than directly disrupting the interface between H2A.Z and the SWR1 complex, could instead simply alter the internal structure of the Chz1–H2A.Z–H2B heterotrimer so that the SWR1 complex becomes unable to bind to it.
Finally, while this manuscript was in revision, another study on H2A.Z incorporation by the SWR1 complex was published (Luk et al. 2010
). This elegant series of experiments dissected the nature of the histone exchange reaction carried out by the SWR1 complex in great detail. Using immobilized nucleosome arrays, the Wu lab determined that the SWR1 complex deposits H2A.Z–H2B dimers into chromatin in a stepwise, unidirectional fashion (Luk et al. 2010
). They observed that one canonical H2A–H2B dimer is removed from the nucleosome, generating a heterotypic intermediary nucleosome containing one H2A.Z–H2B dimer and the remaining H2A–H2B dimer. The second H2A–H2B dimer is then finally exchanged, generating a homotypic nucleosome containing two H2A.Z–H2B dimers as an end product. H2A-containing nucleosomes, H2A.Z–H2B dimers, and ATP are all required for this exchange reaction (Luk et al. 2010
). However, these experiments do not address the molecular determinants of the H2A.Z–H2B dimer that are specifically recognized by the SWR1 complex. Specific acidic residues within H2A.Z may represent this molecular determinant.