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.