The Oncoprotein BRD4-NUT Generates Aberrant Histone Modification Patterns

Defects in chromatin proteins frequently manifest in diseases. A striking case of a chromatin-centric disease is NUT-midline carcinoma (NMC), which is characterized by expression of NUT as a fusion partner most frequently with BRD4. ChIP-sequencing studies from NMC patients revealed that BRD4-NUT (B4N) covers large genomic regions and elevates transcription within these domains. To investigate how B4N modulates chromatin, we performed affinity purification of B4N when ectopically expressed in 293-TREx cells and quantified the associated histone posttranslational modifications (PTM) using proteomics. We observed significant enrichment of acetylation particularly on H3 K18 and of combinatorial patterns such as H3 K27 acetylation paired with K36 methylation. We postulate that B4N complexes override the preexisting histone code with new PTM patterns that reflect aberrant transcription and that epigenetically modulate the nucleosome environment toward the NMC state

Structural and Atropisomeric Factors Governing the Selectivity of Pyrimido-benzodiazipinones as Inhibitors of Kinases and Bromodomains

This is the author accepted manuscript. The final version is available from American Chemical Society via the DOI in this recordBromodomains have been pursued intensively over the past several years as emerging targets for the devel-opment of anti-cancer and anti-inflammatory agents. It has recently been shown that some kinase inhibitors are able to potently inhibit the bromodomains of BRD4. The clinical activities of PLK inhibitor BI-2536 and JAK2-FLT3 inhibitor TG101348 have been attributed to this unexpected poly-pharmacology, indicating that dual-kinase/bromodomain activity may be advantageous in a therapeutic context. However, for target validation and biological investigation, a more selec-tive target profile is desired. Here we report that benzo[e]pyrimido-[5,4-b]diazepine-6(11H)-ones, versatile ATP-site di-rected kinase pharmacophores utilized in the development of inhibitors of multiple kinases including a number of previ-ously reported kinase chemical probes, are also capable of exhibiting potent BRD4-dependent pharmacology. Using a dual kinase-bromodomain inhibitor of the kinase domains of ERK5 and LRRK2, and the bromodomain of BRD4 as a case study, we define the structure-activity relationships required to achieve dual kinase/BRD4 activity as well as how to di-rect selectivity towards inhibition of either ERK5 or BRD4. This effort resulted in identification of one of the first report-ed kinase-selective chemical probes for ERK5 (JWG-071), a BET selective inhibitor with 1 μM BRD4 IC50 (JWG-115), and additional inhibitors with rationally designed polypharmacology (JWG-047, JWG-069). Co-crystallography of seven representative inhibitors with the first bromodomain of BRD4 demonstrate that distinct atropisomeric conformers rec-ognize the kinase ATP-site and the BRD4 acetyl lysine binding site, conformational preferences supported by rigid dock-ing studies.This work was supported by NIH (Grant No. U54HL127365, to N.S.G. and J.W.; No. NIH P50 GM107618, to X.X. and S.C.B.; Nos. NIH U54 HD093540 and P01 CA066996, to J.Q.), the Medical Research Council (No. MC_UU_12016/2, to D.R.A.), the Spanish Ministerio de Economia y Competitividad (MINECO) (Grant No. SAF2015-60268R, to J.M.L.), and Fondo Europeo de Desarrollo Regional (FEDER) funds (to J.M.L.). D.L.B. was supported as a Merck Fellow of Damon Runyon Cancer Research Foundation (No. DRG-2196-14)

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Experimental design to determine complex-specific histone PTMs.

<p>The BioTAP affinity tag, which combines both Protein A and a biotinylation acceptor sequence, was cloned into the N-terminus of the three different cDNAs. Tissue culture cells expressing the tagged baits were crosslinked with formaldehyde and sonicated prior to affinity pulldowns and on-bead derivatization.</p

Quantification of H3 K18 and K23 acetylation enrichment.

<p>(A) Relative levels of unmodified, mono-acetylation, and di-acetylation of the H3 peptide spanning K18 and K23 in genomic and immunoprecipitated histones. Note that the mono-acetylation values combine K18_ac1K23_un and K18_unK23_ac1 together. Error bars of genomic and BRD4_short samples represent standard deviation of 4 replicates. Error bars of B4N sample represent range of 2 replicates. Asterisk denotes significant difference in ac2 levels between input and BRD4_short (p < 0.05). (B) Resolving the mono-acetylation patterns in Fig 2A into the two isoforms for each sample. Relative levels are provided below each bar corresponding to K18ac1 (top bold) and K23ac1 (bottom) and are the same scale as in panel A. For instance, the 30% H3K23ac1 in the genomic sample is relative to unmodified, H3K18ac1, and H3K18K23ac2 as seen in Panel A. Note that most of the mono-acetylated in the samples is K23ac1. 293 refers to genomic histones, L3 refers to L3MBTL3 associated histones, and B4 refers to BRD4_short associated histones.</p

Model for ectopic histone PTM activation by the B4N complex.

<p>(A) Wild type BRD4 recognizes regions that are already acetylated. (B) B4N complex binds to regions already acetylated. These regions may also be already marked with H3K79 mono-methylation. With the EP300 binding partner, the B4N complex targets adjacent chromatin for additional acetylation, for instance at H3K18. This creates additional binding sites for B4N, allowing B4N to spread farther than wild type BRD4 and potentially into regions containing H3K36 methylation. Wild type BRD4 can also co-localize with B4N as megadomains are established.</p

Quantification of variant H3.3 K27 and K36 modification enrichment.

<p>Relative levels of combinatorially modified forms of K27 and K36 methylation and acetylation. Error bar of genomic and BRD4_short sample represents standard deviation of 4 replicates. Note the significant enrichment for K27 mono-methylation paired with K36 di- and tri-methylation by B4N, as well as K27 acetylation paired with K36 mono-methylation.</p

Quantification of H4 K5, K8, K12, and K16 acetylation enrichment.

<p>(A) Relative levels of unmodified, mono-acetylation, di-acetylation, tri-acetylation, and tetra-acetylation of the H4 peptide spanning K5, K8, K12, and K16 in genomic and immunoprecipitated histones. Error bars of genomic and BRD4_short samples represent standard deviation of 4 replicates. Error bar of B4N sample represents range of 2 replicates. Asterisks denote significant difference in ac3 and ac4 levels between input and BRD4_short (p < 0.05). (B) Resolving the tri-acetylation patterns in Fig 4A into the four isoforms for each sample. The two most abundant tri-acetylated isoforms in the B4N samples are K5K8K12ac3 and K5K8K16ac3. Abbreviations are the same as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163820#pone.0163820.g002" target="_blank">Fig 2B</a>.</p

Formation of nuclear foci enriched in H3K18 acetylation by B4N and not BRD4_short.

<p>Confocal microscopy of 293-TREx cells with inducible HA-B4N and HA-BRD4_short stained with anti-HA (green), which labels the bait protein, and anti-H3K18ac (red). Scale bar represents 10 micrometers. Zoomed-in images are provided to illustrate difference between H3K18_ac granules in BRD4_short cells and larger H3K18_ac foci in B4N cells. See <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163820#pone.0163820.s005" target="_blank">S5 Fig</a> for non-confocal imaging and <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0163820#pone.0163820.s006" target="_blank">S6 Fig</a> for separate green and red confocal layers of the same wide-field image of B4N-expressing cells.</p