Crystal Structure of the Human BRD2 Bromodomain

Nakamura, Yoshihiro; Umehara, Takashi; Nakano, Kazumi; Jang, Moon Kyoo; Shirouzu, Mikako; Morita, Satoshi; Uda-Tochio, Hiroko; Hamana, H.; Terada, Takaho; Adachi, Naruhiko; Matsumoto, Takehisa; Tanaka, Akiko; Horikoshi, Masami; Ozato, Keiko; Padmanabhan, Balasundaram; Yokoyama, Shigeyuki

doi:10.1074/jbc.m605971200

Cited by 103 publications

(99 citation statements)

References 48 publications

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“…This suggests that the glutamic acid might be too constrained to form a stable salt bridge with the arginine over a larger separation distance. The selective nature of the observed electro-static effect in peptides having charged groups near the acetyl-lysine supports the observations from structural data that the BrD-histone interface encompasses multiple amino acids in addition to the acetyl-lysine [37,[39][40][41][42][43][44][45][46]. where ΔA np and ΔA p are the changes in nonpolar and polar surface area, respectively.…”

supporting

confidence: 63%

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

Thompson

Chandrasekaran

2008

Analytical Biochemistry

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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. KeywordsAcetyl-lysine; Histone acetylation; Polybromo; Histone code; Protein-protein interactions; Thermodynamics Histone acetylation is a key regulator of transcription; however, it is still a matter of debate as to whether the regulatory features are influenced by modification at specific lysine sites or it is the cumulative electrostatic consequences of random lysine acetylation. Mounting evidence indicates that large multiprotein chromatin remodeling complexes regulate transcription by localizing to certain chromatin sites through the interaction of bromodomain (BrD) 1 proteins with acetylated nucleosomes [1][2][3][4][5][6][7]. The BrD is a protein motif, approximately 100 amino acids in length, primarily associated with targeting subunits of chromatin remodeling complexes [8]. The biological role of the BrD centers on its ability to discriminate the acetylation state of lysine residues in histone proteins. In fact, there is growing evidence that the position of the acetyl-lysine is not random, but instead, the BrD requires acetylation at specific sites for highaffinity binding [9]. For example, fluorescence resonance energy transfer assays were used to show that the bromodomain protein Brd2 requires the interaction between its bromodomain and an acetyl-histone to amplify transcription in vivo [10]. To activate transcription after SAGA (Spt-Ada-Gcn5 acetyl-transferase) complex directed acetylation at specific nucleosomal locations, BrD proteins of the Swi2/Snf2 complex are required to displace acetyl-histones [11]. Likewise, the yeast RSC (remodels the structure of chromatin) complex requires multiple BrD...

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supporting

confidence: 63%

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

Thompson

Chandrasekaran

2008

Analytical Biochemistry

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“…Indeed, the N-terminal bromodomain of Brd2 was recently shown to form an intact homodimer; thus, presenting two acetyl-lysine-binding pockets in close proximity (38). Together with a negatively charged secondary binding pocket, produced at the dimer interface between the two bromodomains, this unique feature was suggested to selectively recognize acetylated H4 histone tail motifs.…”

Section: Discussionmentioning

confidence: 99%

“…Size Exclusion Chromatography of Brd4 BromodomainsThe Brd2 BD1 bromodomain was recently shown to form an intact homodimer presenting two acetyl-lysine-binding pockets in close proximity (38). The crystallographic assembly of the BD1 bromodomains of Brd2 and Brd4, however, appeared very different with opposing binding sites in the crystal mates, despite a sequence identity of 79% over 112 residues (Fig.…”

Section: Volume 284 • Number 52 • December 25 2009mentioning

confidence: 99%

Structures of the Dual Bromodomains of the P-TEFb-activating Protein Brd4 at Atomic Resolution

Vollmuth¹,

Blankenfeldt

Geyer

2009

Journal of Biological Chemistry

116

118

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Brd4 is a member of the bromodomains and extra terminal domain (BET) family of proteins that recognize acetylated chromatin structures through their bromodomains and act as transcriptional activators. Brd4 functions as an associated factor and positive regulator of P-TEFb, a Cdk9-cyclin T heterodimer that stimulates transcriptional elongation by RNA polymerase II. Here, the crystal structures of the two bromodomains of Brd4 (BD1 and BD2) were determined at 1.5 and 1.2 Å resolution, respectively. Complex formation of BD1 with a histone H3 tail polypeptide encompassing residues 12-19 showed binding of the N-acetylated lysine 14 to the conserved asparagine 140 of Brd4. In contrast, in BD2 the N-terminal linker sequence was found to interact with the binding site for acetylated lysines of the adjacent molecule to form continuous strings in the crystal lattice. This assembly shows for the first time a different binding ligand than acetylated lysine indicating that also other sequence compositions may be able to form similar interaction networks. Isothermal titration calorimetry revealed best binding of BD1 to H3 and of BD2 to H4 acetylated lysine sequences, suggesting alternating histone recognition specificities. Intriguingly, an acetylated lysine motif from cyclin T1 bound similarly well to BD2. Whereas the structure of Brd2 BD1 suggested its dimer formation, both Brd4 bromodomains appeared monomeric in solution as shown by size exclusion chromatography and mutational analyses.The transition from transcription initiation to productive transcription elongation depends on the phosphorylation of the C-terminal domain of the RNA polymerase II by the positive transcription elongation factor P-TEFb (1, 2). Active P-TEFb is composed of the kinase Cdk9 and its cyclin-partner cyclin T or K. In its inhibited form, P-TEFb is bound to a heterotrimeric complex formed by the small nuclear RNA 7SK and the proteins Hexim and Larp7 (3-5). This negatively regulated complex can be converted into the active form by the bromodomain (BD) 3 -containing protein 4 (Brd4) by a yet unknown mechanism (6 -8). It is thought that Brd4 couples the P-TEFb complex to chromatin structures via binding of its bromodomains to acetylated lysines in the histone H3 and H4 tail sequences.In eukaryotic cells, the DNA templates are condensed into chromatin structures that consist of repetitive units of nucleosomes. In each nucleosome, the DNA is wrapped around an octamer of core histones that consist of two H2A/H2B heterodimers and an H3/H4 tetramer (9). The N-terminal tail regions of histones H3 and H4 are flexible in the nucleosome and rich in lysine, arginine, and serine residues. They are thus accessible to enzymatic modifications such as acetylation, methylation, and phosphorylation (10). The variable patterns generated by these covalent modifications define the histone code, which represents a fundamental regulatory mechanism of gene expression and repression (11). Histone acetylation of lysines in H3 and H4 is mediated by acetyltransferases including ...

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“…[122][123][124][125] This is also exemplified by the Brg-1 component of remodeling complex BAF, which contains two bromodomains (BD 1 and BD 2 ) with capacity to trigger transcription through association with transcription factor E2F, transcription mediators such as CDK8 and TRAP220, RNAP II, 119 and with ability to dock to acetylated histone tails. 126 Other human BRD proteins, such as Brd4, are also recruited to acetylated H3/H4 tails or phosphorylated H3S10 and, in turn, recruit enzymes that phosphorylate RNAP II and recruit P-TEFb to transcriptional start sites. 127 The nucleosomal reposition machinery, therefore, not only enforces ejection of histones, but also recruits transcriptional machinery and provides the torsional stress on chromatin required to open a DNA loop out of the helix, which is further mediated in part by RAD54 in Swi2/Snf2 (yeast) in co-operation with Rad51, which destabilizes nucleosomes along stretches of DNA associated with eviction.…”

Section: Heterochromatin Proteinsmentioning

confidence: 99%