Dissecting the Molecular Roles of Histone Chaperones in Histone Acetylation by Type B Histone Acetyltransferases (HAT-B)

Allison Haigney,M.Daniel Ricketts,Ronen Marmorstein

doi:10.1074/jbc.m115.688523

Allison Haigney, M.Daniel Ricketts + Show 1 more

Open Access

https://doi.org/10.1074/jbc.m115.688523

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Abstract

The HAT-B enzyme complex is responsible for acetylating newly synthesized histone H4 on lysines K5 and K12. HAT-B is a multisubunit complex composed of the histone acetyltransferase 1 (Hat1) catalytic subunit and the Hat2 (rbap46) histone chaperone. Hat1 is predominantly localized in the nucleus as a member of a trimeric NuB4 complex containing Hat1, Hat2, and a histone H3-H4 specific histone chaperone called Hif1 (NASP). In addition to Hif1 and Hat2, Hat1 interacts with Asf1 (anti-silencing function 1), a histone chaperone that has been reported to be involved in both replication-dependent and -independent chromatin assembly. To elucidate the molecular roles of the Hif1 and Asf1 histone chaperones in HAT-B histone binding and acetyltransferase activity, we have characterized the stoichiometry and binding mode of Hif1 and Asf1 to HAT-B and the effect of this binding on the enzymatic activity of HAT-B. We find that Hif1 and Asf1 bind through different modes and independently to HAT-B, whereby Hif1 binds directly to Hat2, and Asf1 is only capable of interactions with HAT-B through contacts with histones H3-H4. We also demonstrate that HAT-B is significantly more active against an intact H3-H4 heterodimer over a histone H4 peptide, independent of either Hif1 or Asf1 binding. Mutational studies further demonstrate that HAT-B binding to the histone tail regions is not sufficient for this enhanced activity. Based on these data, we propose a model for HAT-B/histone chaperone assembly and acetylation of H3-H4 complexes.

Highlights

Taken together with our earlier observations that full-length H3-H4 histone heterodimer is a better substrate for HAT-B than an H4 peptide, these data suggest that HAT-B binding to the histone tail regions is not sufficient for the preferential acetylation activity of HAT-B for the H3-H4 histone heterodimer over the histone H4 tail
The HAT-B histone acetyltransferase is the enzyme complex responsible for acetylation of newly synthesized histone H4 molecules on K5 and K12 (9 –13), an activity that is evolutionarily conserved from yeast to human
D, enzyme kinetics of HAT-B with either H3-H4 compared with Hat2 mutants on tail-less H3tl-H4 substrates as described for C

Summary

Experimental Procedures

Protein Preparation—Asf and the full-length histone H3-H4 heterodimers were generated as described previously [25, 28]. To prepare NuB4/H3-H4, a molar excess of H3-H4 was incubated on ice with NuB4 for 10 min This complex was subjected to Superdex 200 gel filtration chromatography (in 20 mM Tris, pH 7.9, 500 mM NaCl). To prepare HAT-B/Asf1/H3H4, a molar excess of Asf1/H3-H4 was incubated on ice with HAT-B for 10 min To prepare NuB4/Asf1/H3-H4, a molar excess of Asf1/H3-H4 was incubated on ice with NuB4 for 10 min All reactions were performed in duplicate, and radioactive counts were converted to molar units using a standard curve generated by spotting known concentrations of 14C-ACoA onto P81 paper and allowing the papers to dry before being placed into scintillation fluid for counting

Results

Proposed stoichiometry

Molecular mass of proposed

Discussion