Abstract
The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4. By performing histone acetyltransferase (HAT) assays on various synthetic H4 N-terminal peptides, we have examined the interactions between Hat1 and the H4 tail domain. It was found that acetylation requires the presence of positively charged amino acids at positions 8 and 16 of H4, positions that are normally occupied by lysine; however, lysine per se is not essential and can be replaced by arginine. In contrast, replacing Lys-8 and -16 of H4 with glutamines reduces acetylation to background levels. Similarly, phosphorylation of Ser-1 of the H4 tail depresses acetylation by both yeast Hat1p and the human HAT-B complex. These results strongly support the model proposed by Ramakrishnan and colleagues for the interaction between Hat1 and the H4 tail (Dutnall, R. N., Tafrov, S. T., Sternglanz, R., and Ramakrishnan, V. (1998) Cell 94, 427-438) and may have implications for the genetic analysis of histone acetylation. It was also found that Lys-12 of H4 is preferentially acetylated by human HAT-B, in further agreement with the proposed model of H4 tail binding. Finally, we have demonstrated that deletion of the hat1 gene from the fission yeast Schizosaccharomyces pombe causes increased sensitivity to the DNA-damaging agent methyl methanesulfonate in the absence of any additional mutations. This is in contrast to results obtained with a Saccharomyces cerevisiae hat1Delta strain, which must also carry mutations of the acetylatable lysines of H3 for heightened methyl methanesulfonate sensitivity to be observed. Thus, although the role of Hat1 in DNA damage repair is evolutionarily conserved, the ability of H3 acetylation to compensate for Hat1 deletion appears to be more variable.
Highlights
The Hat1 histone acetyltransferase catalyzes the acetylation of H4 at lysines 5 and 12, the same sites that are acetylated in newly synthesized histone H4
Acetylation by Hat1 Requires Positively Charged Amino Acids at Positions 8 and 16 of the H4 Tail—In a previous study, we determined that prior acetylation of H4 tail peptides at Lys-8 and/or Lys-16 significantly reduced the ability of both the human histone acetyltransferase (HAT)-B complex and recombinant yeast Hat1p (yHat1p) to acetylate lysines 5 and 12 of H4 [24]
In the original description of the crystal structure of yeast Hat1p, it was postulated that Lys-8 and Lys-16 of the H4 tail could engage in electrostatic interactions with two negatively charged regions adjacent to the catalytic site (Fig. 1) [22, 23]
Summary
Between Lys-8/Lys-16 and the negatively charged patches serve to hold the H4 tail in place during the acetylation of Lys-12 [22]. In support of the model, our results demonstrated that prior acetylation of the H4 tail at Lys-8 and -16 dramatically reduced the ability of recombinant yeast Hat1p and native human HAT-B to acetylate Lys-12 in vitro [24]. To further explore the interaction of the H4 N-terminal domain with Hat, we have extended our analysis to include amino acid substitutions of the H4 tail at lysines 8 and 16. We found that the presence of lysine at these sites is not absolutely required for enzyme activity, but that a positive charge at Lys8/Lys-16 is critical. Our results implicate the N-terminal serine of H4 in the enzyme-substrate interaction. It is further demonstrated that Lys-12 of H4 is the preferred substrate for the human cytosolic HAT-B complex and that Lys-12 is typically acetylated prior to Lys-5 in generating diacetylated H4. In contrast to results obtained with S. cerevisiae, we have shown that hat1⌬ Schizosaccharomyces pombe cells exhibit heightened sensitivity to DNA damage in the absence of concurrent mutations of H3 N-terminal domain
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