Human HIRA, ASF1a, ASF1b and CAF-1 are evolutionally conserved histone chaperones that form multiple functionally distinct chromatin assembly complexes, with roles linked to diverse nuclear process, such as DNA replication and formation of heterochromatin in senescent cells. We report the crystal structure of an ASF1a/HIRA heterodimer and a biochemical dissection of ASF1a's mutually exclusive interactions with HIRA and the p60 subunit of CAF-1. The HIRA B-domain forms an antiparallel β-hairpin that binds perpendicular to the strands of the β-sandwich of ASF1a, via β-sheet, salt-bridge and van der Waals contacts. The N-and C-terminal regions of ASF1a and ASF1b determine the different affinities of these two proteins for HIRA, by contacting regions outside the HIRA B-domain. CAF-1 p60 also employs B-domain-like motifs for binding to ASF1a, thereby competing with HIRA. Together, these studies begin to define the molecular determinants of assembly of functionally diverse macromolecular histone chaperone complexes. KeywordsHistone Deposition; Chromatin Regulation; Histone Chaperones; ASF1; HIRA; CAF-1The basic repeating unit of eukaryotic chromatin is the core nucleosome. Each core nucleosome consists of a histone (H3/H4) 2 heterotetramer, two histone (H2A/H2B) heterodimers and about 147 base pairs of DNA. During chromatin assembly, histone chaperones donate histones to DNA 1 . In human cells, the deposition of the histone H3/H4 complex involves four histone chaperones, Chromatin Assembly Factor 1 (CAF-1), HIstone Regulatory Homolog A (HIRA), and two Anti-Silencing Factor paralogs (ASF1a and ASF1b).CAF-1, an evolutionarily conserved heterotrimeric chaperone comprised of p150, p60 and p48 subunits, mediates DNA synthesis-coupled chromatin assembly in S-phase 1,2 . CAF-1 is also 4 Correspondence should be addressed to R.M. or P.D. A., Ronen Marmorstein, Tel: (215) FAX: (215) marmor@wistar.org, Peter D. Adams, Tel: (215) FAX: (215) 728 3616, Peter.Adams@fccc.edu. 5 Y.T. and M.V.P contributed equally to this work.The coordinates of the ASF1a/HIRA complex structure has been deposited with the Protein Data Bank (PDB) with access code 2I32. COMPETING INTERESTS STATEMENTThe authors declare that they have no competing financial interests. AUTHOR CONTRIBUTIONSY.T. and M.V.P. designed and carried out experiments reported in the manuscript, and prepared manuscript figures and text; K.Z., M.G., A.C. and R.D. carried out preliminary studies that led to experiments reported in the manuscript; P.D.A and R.M. designed and supervised experiments and prepared manuscript text. All authors read and approved the submitted manuscript. NIH Public Access Author ManuscriptNat Struct Mol Biol. Author manuscript; available in PMC 2010 September 6. NIH-PA Author ManuscriptNIH-PA Author Manuscript NIH-PA Author Manuscript required for chromatin assembly coupled to DNA repair 3-8 , formation of specialized chromatin structures, such as transcriptionally silent chromatin at telomeres, rDNA yeast mating loci, and plant gene loci 3,...

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Bromodomains are epigenetic readers that are recruited to acetyllysine residues in histone tails. Recent studies have identified non-acetyl acyllysine modifications, raising the possibility that these might be read by bromodomains. Profiling the nearly complete human bromodomain family revealed that while most human bromodomains bind only the shorter acetyl and propionyl marks, the bromodomains of BRD9, CECR2, and the second bromodomain of TAF1 also recognize the longer butyryl mark. In addition, the TAF1 second bromodomain is capable of binding crotonyl marks. None of the human bromodomains tested binds succinyl marks. We characterized structurally and biochemically the binding to different acyl groups, identifying bromodomain residues and structural attributes that contribute to specificity. These studies demonstrate a surprising degree of plasticity in some human bromodomains but no single factor controlling specificity across the family. The identification of candidate butyryl- and crotonyllysine readers supports the idea that these marks could have specific physiological functions.

Cellular senescence is an irreversible proliferation arrest, tumor suppression process and likely contributor to tissue aging. Senescence is often characterized by domains of facultative heterochromatin, called senescenceassociated heterochromatin foci (SAHF), which repress expression of proliferation-promoting genes. Given its likely contribution to tumor suppression and tissue aging, it is essential to identify all components of the SAHF assembly pathway. Formation of SAHF in human cells is driven by a complex of histone chaperones, namely, HIRA and ASF1a. In yeast, the complex orthologous to HIRA/ASF1a contains two additional proteins, Hpc2p and Hir3p. Using a sophisticated approach to search for remote orthologs conserved in multiple species through evolution, we identified the HIRA-associated proteins, UBN1 and UBN2, as candidate human orthologs of Hpc2p. We show that the Hpc2-related domain of UBN1, UBN2, and Hpc2p is an evolutionarily conserved HIRA/Hir-binding domain, which directly interacts with the N-terminal WD repeats of HIRA/Hir. UBN1 binds to proliferation-promoting genes that are repressed by SAHF and associates with histone methyltransferase activity that methylates lysine 9 of histone H3, a site that is methylated in SAHF. UBN1 is indispensable for formation of SAHF. We conclude that UBN1 is an ortholog of yeast Hpc2p and a novel regulator of senescence.

The biological role played by non-BET bromodomains remains poorly understood, and it is therefore imperative to identify potent and highly selective inhibitors to effectively explore the biology of individual bromodomain proteins. A ligand-efficient nonselective bromodomain inhibitor was identified from a 6-methyl pyrrolopyridone fragment. Small hydrophobic substituents replacing the N-methyl group were designed directing toward the conserved bromodomain water pocket, and two distinct binding conformations were then observed. The substituents either directly displaced and rearranged the conserved solvent network, as in BRD4(1) and TAF1(2), or induced a narrow hydrophobic channel adjacent to the lipophilic shelf, as in BRD9 and CECR2. The preference of distinct substituents for individual bromodomains provided selectivity handles useful for future lead optimization efforts for selective BRD9, CECR2, and TAF1(2) inhibitors.