The BRD3 ET domain recognizes a short peptide motif through a mechanism that is conserved across chromatin remodelers and transcriptional regulators

Abstract

Members of the bromodomain and extra-terminal domain (BET) family of proteins (bromodomain-containing (BRD) 2, 3, 4, and T) are widely expressed and highly conserved regulators of gene expression in eukaryotes. These proteins have been intimately linked to human disease, and more than a dozen clinical trials are currently underway to test BET-protein inhibitors as modulators of cancer. However, although it is clear that these proteins use their bromodomains to bind both histones and transcription factors bearing acetylated lysine residues, the molecular mechanisms by which BET family proteins regulate gene expression are not well defined. In particular, the functions of the other domains such as the ET domain have been less extensively studied. Here, we examine the properties of the ET domain of BRD3 as a protein/protein interaction module. Using a combination of pulldown and biophysical assays, we demonstrate that BRD3 binds to a range of chromatin-remodeling complexes, including the NuRD, BAF, and INO80 complexes, via a short linear "KIKL" motif in one of the complex subunits. NMR-based structural analysis revealed that, surprisingly, this mode of interaction is shared by the AF9 and ENL transcriptional coregulators that contain an acetyl-lysine-binding YEATS domain and regulate transcriptional elongation. This observation establishes a functional commonality between these two families of cancer-related transcriptional regulators. In summary, our data provide insight into the mechanisms by which BET family proteins might link chromatin acetylation to transcriptional outcomes and uncover an unexpected functional similarity between BET and YEATS family proteins.

Keywords: AF9; BET family proteins; acetylation; bromodomain; extraterminal domain; nuclear magnetic resonance (NMR); protein structure; protein/protein interaction; transcriptional coactivator.

Figure 1.

BRD3 can bind to a large number of gene regulatory proteins. A, reducing SDS-PAGE showing proteins pulled down from HEK293 nuclear extract by FLAG-Brd3 immobilized on FLAG-Sepharose beads. The control comprised beads alone. The gel was cut into the four indicated sections (right), and the proteins contained in each section were identified by MS. B, coimmunoprecipitation showing the ability of FLAG-Brd3 to pull CHD4 and MTA2 from HEK293 cells. Western blottings were carried out with the indicated antibodies. C, coimmunoprecipitation showing the ability of FLAG-Brd3 to pull BRG1 and BAF170 from HEK293 cells. Western blots were carried out with the indicated antibodies. D, coimmunoprecipitations mapping the BAF-binding domain of Brd3. Western blots were carried out with the indicated antibodies. E, domain architecture of BET proteins. Residue numbering is based on human proteins (Uniprot IDs: BRD2, P25440; BRD3, Q15059; BRD4, O60885; and BRDT, Q58F21). BD, bromodomain; ET, extra-terminal domain; CTD, C-terminal domain. Regions of BRD2, BRD3, and BRD4 previously identified to interact with full-length CHD4 (46), as well as BRD3 constructs used in this study, are also shown.

Figure 2.

BRD3 binds to a short motif in the N-terminal disordered region of CHD4. A, human CHD4 constructs used in this study. Domains with known structures are indicated. The presence or absence of an interaction with BRD3 is indicated by + or −, respectively. CD = chromodomain. B, BRD3 coimmunoprecipitates with the N-terminal third of CHD4. BRD3-HA (full-length, L or BD) and CHD4-FLAG (full-length, NT, M, or CT) constructs were coexpressed in HEK293 cells and applied to anti-FLAG-agarose beads. Western blottings using anti-HA or anti-FLAG antibodies are shown. C, CHD4(265–310) (fragment Ne) binds BRD3. Bacterially expressed CHD4 fragments immobilized on GSH beads were used to pull down mammalian-expressed HA-BRD3-L. Each Western blotting was visualized using α-HA and α-GST antibodies. PD = pulldown; FT = flow-through; * = unidentified band.

Figure 3.

BRD3-ET binds CHD4(265–310). ¹⁵N HSQC spectra were acquired in 20 mm sodium phosphate, pH 6.5, 50 mm NaCl, 1 mm DTT at 298 K. A, ¹⁵N HSQC spectra of BRD3-L (black) and BRD3-ET (red). Most of BRD3-L outside of the core ET domain appears to be disordered. B, titration of ¹⁵N-BRD3-L with CHD4-Ne. Arrows indicate large chemical shift changes induced by CHD4-Ne. Boxed regions are shown in detail in D. C, ¹⁵N HSQC spectra of ¹⁵N-BRD3-ET alone and in the presence of 2 m eq of CHD4-Ne. Boxed regions are shown in detail in D. D, comparison of CHD4-induced peak movements in BRD3-L and BRD3-ET, showing that BRD3-ET and BRD-L bind CHD4 in a comparable manner.

Figure 4.

BRD3-ET can interact in a conserved manner with motifs from a range of transcriptional coregulators. A, sequence alignment of CHD-Ne with a peptide from MLV-IN that is known to bind the ET domain of Brd4 (50), as well as related sequences found in INO80B, BRG1, NSD3, GLTSCR1, and TAF7. A region of high similarity (representing a potential ET recognition motif) is boxed. Residues that display the largest chemical shift perturbations in the presence of BRD3-ET are indicated by arrows. B, partial ¹⁵N HSQC spectra of BRD3-ET alone (red) and titrated with peptides from (top to bottom) CHD4, BRG1, INO80B, NSD3, and MLV-IN. The number of molar equivalents of each peptide added are indicated in each spectrum. C, chemical shift changes induced in BRD3 ET domain at saturation with CHD4, INO80B, and NSD3. The horizontal red line indicates 1 S.D. above mean chemical shift change. Peaks no longer visible at the titration end point are indicated by vertical dashed lines. Residues that are identical or highly similar between CHD4 and MLV integrase are indicated by asterisks and a colon, respectively.

Figure 5.

¹⁵N HSQC titration of ¹⁵N-labeled CHD4-Ne with BRD3-ET. 200 μm ¹⁵N CHD4-Ne was titrated with BRD3-ET in 20 mm sodium phosphate, pH 6.5, 50 mm NaCl, 1 mm DTT at 298 K. Several peaks that undergo large chemical shift changes are labeled with their assignments.

Figure 6.

SPR-derived binding affinities for interactions between BRD3-ET and KIKL peptides. Sensograms (upper panels) and fits to equilibrium responses from the sensograms (lower panels) are shown for BRD3-ET binding to CHD4 (A), BRG1 (B), INO80B (C), and NSD3 (D) peptides. Data were fitted to a simple Langmuir 1:1 binding isotherm in the Biacore software. Measurements were made in a Biacore T200 at 4 °C in a buffer containing 20 mm HEPES and 150 mm NaCl, pH 7.5.

Figure 7.

Solution NMR structure of the BRD3-ET–CHD4 and BRD3-ET–BRG1 complexes. A, ribbon representation of the ensemble of 20 lowest energy structures for the BRD3-ET–CHD4-Ng complex. N and C termini are labeled. B, surface representation of BRD3-ET with CHD4-Ng shown as sticks. Only ordered CHD4 residues are shown and labeled. BRD3 residues with CHD4-induced chemical shift changes of >1 standard deviation above the mean are indicated in yellow. C, surface representation showing the electrostatic properties of the binding pocket. BRD3-ET residues are labeled. The electrostatic complementarity is clear. D–F, representations of the BRD3-ET·BRG1 structure, as for A–C. BRG1 is shown in magenta.

Figure 8.

KIKL motif in CHD4 is important for the BRD3/CHD4 interaction. Bacterially-expressed GST–BRD3-ET immobilized on GSH beads was used to pull down mammalian-expressed full-length FLAG-CHD4 (WT or K297A/L298A mutant). Western blots using anti-GST and anti-FLAG antibodies are shown.

Figure 9.

Comparison of structures of ET domain complexes. ET-binding peptides, as well as the ET domain of BRD3 (gray) or Brd4 (blue), are shown as ribbons. The BET protein, core peptide sequence, and PDB code are indicated below the structures.

Figure 10.

ET interaction mechanism is conserved in AF9/ENL family proteins. A, comparison of BRD3–CHD4 (left) and AF9–DOT1L (right) complexes showing the ET/AHD as a surface and the partner peptide as cartoon/stick representation. B, top, amino acid sequences of the ET and AHD domains of human BRD3 and AF9, respectively. Identical residues are shown in orange and similar residues in yellow. The sequence that forms a β-sheet with the partner is underlined. Bottom, alignment of the ET-binding regions of CHD4 and BRG1 with the corresponding regions from the AHD-binding proteins CBX8, AF4, and BCoR. Sequences from the human proteins are shown in all cases. C, comparison of the interface residues of ET/AHD domains (left) and of the partner peptides (right); strong conservation is apparent in the former case, whereas the alternating pattern of hydrophobic and basic residues observed in ET-binding sequences is not as strongly conserved in AHD-binding sequences.