Although the acetylation of histones has a well-documented regulatory role in transcription, its role in other chromosomal functions remains largely unexplored. Here we show that distinct patterns of histone H4 acetylation are essential in two separate pathways of double-strand break repair. A budding yeast strain with mutations in wild-type H4 acetylation sites shows defects in nonhomologous end joining repair and in a newly described pathway of replication-coupled repair. Both pathways require the ESA1 histone acetyl transferase (HAT), which is responsible for acetylating all H4 tail lysines, including ectopic lysines that restore repair capacity to a mutant H4 tail. Arp4, a protein that binds histone H4 tails and is part of the Esa1-containing NuA4 HAT complex, is recruited specifically to DNA double-strand breaks that are generated in vivo. The purified Esa1-Arp4 HAT complex acetylates linear nucleosomal arrays with far greater efficiency than circular arrays in vitro, indicating that it preferentially acetylates nucleosomes near a break site. Together, our data show that histone tail acetylation is required directly for DNA repair and suggest that a related human HAT complex may function similarly.

Nucleosomes impose a block to transcription that can be overcome in vivo by remodeling complexes such as SNF/SWI and histone modification complexes such as SAGA. Mutations in the major core histones relieve transcriptional repression and bypass the requirement for SNF/SWI and SAGA. We have found that the variant histone H2A.Z regulates gene transcription, and deletion of the gene encoding H2A.Z strongly increases the requirement for SNF/SWI and SAGA. This synthetic genetic interaction is seen at the level of single genes and acts downstream of promoter nucleosome reorganization. H2A.Z is preferentially crosslinked in vivo to intergenic DNA at the PH05 and GAL1 loci, and this association changes with transcriptional activation. These results describe a novel pathway for regulating transcription using variant histones to modulate chromatin structure.

No abstract

Apoptosis is a highly coordinated cell suicide mechanism in vertebrates. Phosphorylation of serine 14 of histone H2B, catalyzed by Mst1 kinase, has been linked to chromatin compaction during apoptosis. We extend these results to unicellular eukaryotes by demonstrating that H2B is specifically phosphorylated at serine 10 (S10) in a hydrogen peroxide-induced cell death pathway in S. cerevisiae. H2B S10A mutants are resistant to cell death elicited by H(2)O(2) while H2B S10E phospho-site mimics promote cell death and induce the "constitutive" formation of condensed chromatin. Ste20 kinase, a yeast homolog of mammalian Mst1 kinase, translocates into the nucleus in a caspase-independent fashion and directly phosphorylates H2B at S10. Conservation of targeted H2B phosphorylation and the enzyme system responsible for the process point to an ancient mechanism of chromatin remodeling that likely plays an important role in governing cellular homeostasis in a wide range of organisms.

The initiation of DNA replication is tightly regulated in eukaryotic cells to ensure that the genome is precisely duplicated once and only once per cell cycle. This is accomplished by controlling the assembly of a prereplicative complex (pre-RC) which involves the sequential binding to replication origins of the origin recognition complex (ORC), Cdc6/Cdc18, Cdt1, and the minichromosome maintenance complex (Mcm2-Mcm7, or Mcm2-7). Several mechanisms of pre-RC regulation are known, including ATP utilization, cyclindependent kinase levels, protein turnover, and Cdt1 binding by geminin. Histone acetylation may also affect the initiation of DNA replication, but at present neither the enzymes nor the steps involved are known. Here, we show that Hbo1, a member of the MYST histone acetyltransferase family, is a previously unrecognized positive regulatory factor for pre-RC assembly. When Hbo1 expression was inhibited in human cells, Mcm2-7 failed to associate with chromatin even though ORC and Cdc6 loading was normal. When Xenopus egg extracts were immunodepleted of Xenopus Hbo1 (XHbo1), chromatin binding of Mcm2-7 was lost, and DNA replication was abolished. The binding of Mcm2-7 to chromatin in XHbo1-depleted extracts could be restored by the addition of recombinant Cdt1.