Polybromo-1-bromodomains bind histone H3 at specific acetyl-lysine positions

The human polybromo-1 protein is thought to localize the Polybromo, BRG1-associated factors chromatin-remodeling complex to kinetochores during mitosis via direct interaction of its six tandem bromodomains with acetylated nucleosomes. Bromodomains are acetyl-lysine binding modules roughly 100 amino acids in length originally found in chromatin associated proteins. Previous studies verified acetyl-histone binding by each bromodomain, but site-specificity, a central tenet of the histone code hypothesis, was not examined. Here, the acetylation site-dependence of bromodomain-histone interactions was examined using steady-state fluorescence anisotropy. Results indicate that single bromodomains bind specific acetyl-lysine sites within the histone tail with sub-micromolar affinity. Identification of duplicate target sites suggests that native Pb1 interacts with both copies of histone H3 upon nucleosome assembly. Quantitative analysis of single bromodomain-histone interactions can be used to develop hypotheses regarding the histone acetylation pattern that acts as the binding target of the native polybromo-1 protein.     

Tandem bromodomains in the chromatin remodeler RSC recognize acetylated histone H3 Lys14

The coordination of chromatin remodeling with chromatin modification is a central topic in gene regulation. The yeast chromatin remodeling complex RSC bears multiple bromodomains, motifs for acetyl-lysine and histone tail interaction. Here, we identify and characterize Rsc4 and show that it bears tandem essential bromodomains. Conditional rsc4 bromodomain mutations were isolated, and were lethal in combination with gcn5Delta, whereas combinations with esa1 grew well. Replacements involving Lys14 of histone H3 (the main target of Gcn5), but not other H3 or H4 lysine residues, also conferred severe growth defects to rsc4 mutant strains. Importantly, wild-type Rsc4 bound an H3 tail peptide acetylated at Lys14, whereas a bromodomain mutant derivative did not. Loss of particular histone deacetylases suppressed rsc4 bromodomain mutations, suggesting that Rsc4 promotes gene activation. Furthermore, rsc4 mutants displayed defects in the activation of genes involved in nicotinic acid biosynthesis, cell wall integrity, and other pathways. Taken together, Rsc4 bears essential tandem bromodomains that rely on H3 Lys14 acetylation to assist RSC complex for gene activation.     

Thermodynamic analysis of acetylation-dependent Pb1 bromodomain-histone H3 interactions

An acetyl-histone peptide library was used to determine the thermodynamic parameters that define acetylation-dependent bromodomain-histone interactions. Bromodomains interact with histones by binding acetylated lysines. The bromodomain used in this study, BrD3, is derived from the polybromo-1 protein, which is a subunit of the PBAF chromatin remodeling complex. Steady-state fluorescence anisotropy was used to examine the variations in specificity and affinity that drive molecular recognition. Temperature and salt concentration dependence studies demonstrate that the hydrophobic effect is the primary driving force, consistent with lysine acetylation being required for binding. An electrostatic effect was observed in only two complexes where the acetyl-lysine was adjacent to an arginine. The large change in heat capacity determined for the specific complex suggests that the dehydrated BrD3-histone interface forms a tightly bound, high-affinity complex with the target site. These explorations into the thermodynamic driving forces that confer acetylation site-dependent BrD3-histone interactions improve our understanding of how individual bromodomains work in isolation. Furthermore, this work will permit the development of hypotheses regarding how the native Pb1, and the broader class of bromodomain proteins, directs multisubunit chromatin remodeling complexes to specific acetyl-nucleosome sites in vivo.     

Acetylation-dependent chromatin reorganization by BRDT,a testis-specific bromodomain-containing protein

The association between histone acetylation and replacement observed during spermatogenesis prompted us to consider the testis as a source for potential factors capable of remodelling acetylated chromatin. A systematic search of data banks for open reading frames encoding testis-specific bromodomain-containing proteins focused our attention on BRDT, a testis-specific protein of unknown function containing two bromodomains. BRDT specifically binds hyperacetylated histone H4 tail depending on the integrity of both bromodomains. Moreover, in somatic cells, the ectopic expression of BRDT triggered a dramatic reorganization of the chromatin only after induction of histone hyperacetylation by trichostatin A (TSA). We then defined critical domains of BRDT involved in its activity. Both bromodomains of BRDT, as well as flanking regions, were found indispensable for its histone acetylation-dependent remodelling activity. Interestingly, we also observed that recombinant BRDT was capable of inducing reorganization of the chromatin of isolated nuclei in vitro only when the nuclei were from TSA-treated cells. This assay also allowed us to show that the action of BRDT was ATP independent, suggesting a structural role for the protein in the remodelling of acetylated chromatin. This is the first demonstration of a large-scale reorganization of acetylated chromatin induced by a specific factor.     

Synthesis of Nε-acetyl-L-homolysine by the Lossen rearrangement and its application for probing deacetylases and binding modules of acetyl-lysine

Lysine acetylation is a posttranslational protein modification mediating protein–protein interactions by recruitment of bromodomains. Investigations of bromodomains have focused so far on the sequence context of the modification site and acyl-modifications installed at lysine side chains. In contrast, there is only little information about the impact of the lysine residue that carries the modification on bromodomain binding. Here, we report a synthesis strategy for L-acetyl-homolysine from L-2-aminosuberic acid by the Lossen rearrangement. Peptide probes containing acetylated homolysine, lysine, and ornithine were generated and used for probing the binding preferences of four bromodomains from three different families. Tested bromodomains showed distinct binding patterns, and one of them bound acetylated homolysine with similar efficiency as the native substrate containing acetyl-lysine. Deacetylation assays with a bacterial sirtuin showed a strong preference for acetylated lysine, despite a broad specificity for N-acyl modifications.     

Site-specific antibodies to human erythropoietin: immunoaffinity purification of urinary and recombinant hormone

The 19-amino acid domain Ala111----Pro129 of human erythropoietin was identified as an accessible surface antigen based on the binding of radio-iodinated and of unmodified hormone to antibodies prepared against a synthetic peptide of homologous sequence. The specificity and affinity of this binding was sufficient to provide for the use of anti-peptide antibodies in the preparation of an immunosorbent for the purification of urinary, and of recombinant human erythropoietin. Immobilization of anti-peptide antibodies using agarose activated either with CNBr or with N-hydroxysuccinimido groups largely inactivated binding sites for erythropoietin. In contrast, antibodies crosslinked to N-acetyl-DL-homocysteine agarose through the hetero-bifunctional reagent succinimidyl 4-(N-maleimidomethyl)cyclohexane-1-carboxylate retained their antigen-binding capacity virtually completely and provided a superior immunosorbent for hormone. Urinary erythropoietin with a specific bioactivity of 100 U/A280 was prepared initially by chromatography on phenyl-Sepharose. Subsequent immunoaffinity chromatography resulted in a 350-fold purification with 46.2% recovery yielding erythropoietin with a specific bioactivity of 35,200 U/A280 (44,300 U/mg). Radioiodination of this purified protein and subsequent SDS-polyacrylamide gel electrophoresis indicated that this preparation contained a single major component (Mr 30,000) which co-migrated in gels with unmodified biologically active hormone. Recombinant erythropoietin, which was prepared by the cloning of the human erythropoietin gene and its expression in COS cells using the SV40-derived vector pSV2, was purified by the same scheme. Chromatography on phenyl-Sepharose of medium derived from transfected cells (400 U/ml, 170 U/A280) provided for a 3.6-fold purification of recombinant hormone with an apparent recovery of 122%. This erythropoietin bound to the anti-peptide antibody gel and was purified to a specific bioactivity of 10,370 U/A280 with 55% recovery. The procedure described here for attaching antibodies to a solid support maximizes their antigen-binding capacity and is generally applicable. The development of an anti-peptide immunosorbant for human erythropoietin provides a valuable means for isolating hormone for use in studies of its receptor and its presently unresolved mechanism of action.