NORMAL GENOMIC

• Huang S, Qiu Y, Shi Y, Xu Z, Brandt SJ
• P/CAF-mediated acetylation regulates the function of the basic helix-loop-helix transcription factor TAL1/SCL.
• EMBO J. 2000; 19: 6792-803
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• The basic helix-loop-helix transcription factor TAL1 (or SCL) is a critical regulator of hematopoietic and vascular development and is misexpressed in the majority of patients with T-cell acute lymphoblastic leukemia. We found previously that TAL1 could interact with transcriptional co-activator and co-repressor complexes possessing histone acetyltransferase and deacetylase activities, respectively. Here, we report that TAL1 is subject to acetylation in vivo and can be acetylated by p300 and the p300/CBP-associated factor P/CAF in vitro. P/CAF-mediated acetylation, which mapped to a lysine-rich motif in the loop region, increased TAL1 binding to DNA while selectively inhibiting its interaction with the transcriptional co-repressor mSin3A. Furthermore, P/CAF protein, TAL1-P/CAF interaction and TAL1 acetylation increased significantly in murine erythroleukemia cells induced to differentiate in culture, while enforced expression of an acetylation-defective P/CAF mutant inhibited endogenous TAL1 acetylation, TAL1 DNA-binding activity, TAL1-directed transcription and terminal differentiation of these cells. These results reveal a novel mechanism by which TAL1 activity is regulated and implicate acetylation of this transcription factor in promotion of erythroid differentiation.

• Jacobson RH, Ladurner AG, King DS, Tjian R
• Structure and function of a human TAFII250 double bromodomain module.
• Science. 2000; 288: 1422-5
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• TFIID is a large multiprotein complex that initiates assembly of the transcription machinery. It is unclear how TFIID recognizes promoters in vivo when templates are nucleosome-bound. Here, it is shown that TAFII250, the largest subunit of TFIID, contains two tandem bromodomain modules that bind selectively to multiply acetylated histone H4 peptides. The 2.1 angstrom crystal structure of the double bromodomain reveals two side-by-side, four-helix bundles with a highly polarized surface charge distribution. Each bundle contains an Nepsilon-acetyllysine binding pocket at its center, which results in a structure ideally suited for recognition of diacetylated histone H4 tails. Thus, TFIID may be targeted to specific chromatin-bound promoters and may play a role in chromatin recognition.

• Owen DJ et al.
• The structural basis for the recognition of acetylated histone H4 by the bromodomain of histone acetyltransferase gcn5p.
• EMBO J. 2000; 19: 6141-9
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• The bromodomain is an approximately 110 amino acid module found in histone acetyltransferases and the ATPase component of certain nucleosome remodelling complexes. We report the crystal structure at 1.9 A resolution of the Saccharomyces cerevisiae Gcn5p bromodomain complexed with a peptide corresponding to residues 15-29 of histone H4 acetylated at the zeta-N of lysine 16. We show that this bromodomain preferentially binds to peptides containing an N:-acetyl lysine residue. Only residues 16-19 of the acetylated peptide interact with the bromodomain. The primary interaction is the N:-acetyl lysine binding in a cleft with the specificity provided by the interaction of the amide nitrogen of a conserved asparagine with the oxygen of the acetyl carbonyl group. A network of water-mediated H-bonds with protein main chain carbonyl groups at the base of the cleft contributes to the binding. Additional side chain binding occurs on a shallow depression that is hydrophobic at one end and can accommodate charge interactions at the other. These findings suggest that the Gcn5p bromodomain may discriminate between different acetylated lysine residues depending on the context in which they are displayed.

• Trievel RC, Li FY, Marmorstein R
• Application of a fluorescent histone acetyltransferase assay to probe the substrate specificity of the human p300/CBP-associated factor.
• Anal Biochem. 2000; 287: 319-28
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• Histone N-acetyltransferases (HATs) are a group of enzymes which acetylate specific lysine residues in the N-terminal tails of nucleosomal histones to promote transcriptional activation. Recent structural and enzymatic work on the GCN5/PCAF HAT family has elucidated the structure of their catalytic domain and mechanism of histone acetylation. However, the substrate specificity of these enzymes has not been quantitatively investigated. Utilizing a novel microplate fluorescent HAT assay which detects the enzymatic production of coenzyme A (CoA), we have compared the activities of the HAT domains of human PCAF and its GCN5 homologue from yeast and Tetrahymena and found that they have similar kinetic parameters. PCAF was further assayed with a series of different length histone H3 peptide substrates, which revealed that the determinants for substrate recognition lie within a 19-residue sequence. Finally, we evaluated the acetylation of three putative PCAF substrates, histones H3 and H4 and the transcription factor p53, and have determined that histone H3 is significantly preferred over the histone H4 and p53 substrates. Taken together, the fluorescent acetyltransferase assay presented here should be widely applicable to other HAT enzymes, and the results obtained with PCAF demonstrate a strong substrate preference for the N-terminal residues of histone H3.

• Herrera JE, West KL, Schiltz RL, Nakatani Y, Bustin M
• Histone H1 is a specific repressor of core histone acetylation in chromatin.
• Mol Cell Biol. 2000; 20: 523-9
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• Although a link between histone acetylation and transcription has been established, it is not clear how acetylases function in the nucleus of the cell and how they access their targets in a chromatin fiber containing H1 and folded into a highly condensed structure. Here we show that the histone acetyltransferase (HAT) p300/CBP-associated factor (PCAF), either alone or in a nuclear complex, can readily acetylate oligonucleosomal substrates. The linker histones, H1 and H5, specifically inhibit the acetylation of mono- and oligonucleosomes and not that of free histones or histone-DNA mixtures. We demonstrate that the inhibition is due mainly to steric hindrance of H3 by the tails of linker histones and not to condensation of the chromatin fiber. Cellular PCAF, which is complexed with accessory proteins in a multiprotein complex, can overcome the linker histone repression. We suggest that linker histones hinder access of PCAF, and perhaps other HATs, to their target acetylation sites and that perturbation of the linker histone organization in chromatin is a prerequisite for efficient acetylation of the histone tails in nucleosomes.

• Li S, Aufiero B, Schiltz RL, Walsh MJ
• Regulation of the homeodomain CCAAT displacement/cut protein function by histone acetyltransferases p300/CREB-binding protein (CBP)-associated factor and CBP.
• Proc Natl Acad Sci U S A. 2000; 97: 7166-71
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• The CCAAT displacement protein/cut homologue (CDP/cut) is a divergent homeodomain protein that is highly conserved through evolution and has properties of a potent transcriptional repressor. CDP/cut contains three conserved cut-repeat domains and a conserved homeobox, each involved in directing binding specificity to unique nucleotide sequence elements. Furthermore, CDP/cut may play a role as a structural component of chromatin through its direct interaction with nucleosomal DNA and association with nuclear matrix attachment regions. CDP/cut is cell-cycle regulated through interactions with Rb, p107, specific kinases and phosphatases directing the transcriptional activity of CDP/cut on such genes encoding p21(WAF1,CIP1), c-myc, thymidine kinase, and histones. Our previous studies indicate that CDP/cut is associated with histone deacetylase activity and is associated with a corepressor complex through interactions with histone deacetylases. Here, we report the interaction of CDP/cut with CBP and p300/CREB-binding protein-associated factor (PCAF) along with the modification of CDP/cut by the histone acetyltransferase PCAF. Acetylation of CDP/cut by PCAF is directed at conserved lysine residues near the homeodomain region and regulates CDP/cut function. These observations are consistent with the ability of CDP/cut to regulate genes as a transcriptional repressor, suggesting acetylation as a mechanism that regulates CDP/cut function.