12038 2016 9600 article p 295. - ias

Bromodomain and extra-terminal (BET) family proteins:New therapeutic targets in major diseases

Department of Biophysics, National Institute of Mental Health and Neurosciences (NIMHANS),Bangalore 560 029, India

*Corresponding author (Email, [email protected]; [email protected])

The bromodomains and extra-terminal domain (BET) family proteins recognize acetylated chromatin through theirbromodomains (BDs) and help in regulating gene expression. BDs are chromatin ‘readers’: by interacting withacetylated lysines on the histone tails, they recruit chromatin-regulating proteins on the promoter region to regulategene expression and repression. Extensive efforts have been employed by scientific communities worldwide toidentify and develop potential inhibitors of BET family BDs to regulate protein expression by inhibiting acetylatedhistone (H3/H4) interactions. Several small molecule inhibitors have been reported, which not only have high affinitybut also have high specificity to BET BDs. These developments make BET family proteins an important therapeutictargets for major diseases such as cancer, neurological disorders, obesity and inflammation. Here, we review anddiscuss the structural biology of BET family BDs and their applications in major diseases.

[Padmanabhan B, Mathur S, Manjula R and Tripathi S 2016 Bromodomain and extra-terminal (BET) family proteins: New therapeutic targets inmajor diseases. J. Biosci. 41 295–311] DOI 10.1007/s12038-016-9600-6

1. Introduction

The histone code, defined by combinatorial post-translationmodifications (PTMs) on the histone tails, regulates geneexpression and repression through structural alteration ofthe chromatin and/or association of its trans-acting factors(Strahl and Allis 2000). PTMs generally refer to the additionof a functional group covalently to a protein as in methyla-tion, acetylation, phosphorylation, ubiquitylation andsumoylation. Among PTMs, acetylation of lysine residues

(Kac) on the histone tails and the acetylated-histone recog-nition is a typical hallmark of transcriptionally active genes(Taverna et al. 2007). Although PTMs can affect the chro-matin structure, the overall state of chromatin is eventuallydetermined by combinations of these modifications. Thesecomplex codes are important in maintaining genome integ-rity by means of epigenetic memory, a mechanism thatinherits expression patterns in dividing cells (Jenuwein andAllis 2001). The acetylated N-terminal tails of histones arerecognized by bromodomains (BDs), which act as a reader oflysine acetylation state (Dhalluin et al. 1999; Mujtaba et al.

http://www.ias.ac.in/jbiosci J. Biosci. 41(2), June 2016, 295–311* Indian Academy of Sciences 295

Keywords. BET family bromodomains; drug targets; epigenetics; structural biology; transcription regulation

Abbreviations used: AML, acute myeloid leukaemia; Bcl2, B-cell lymphoma 2; BD, bromodomain; BET, bromodomain and extra-terminal; BRD2, bromodomain containing protein 2; BRD3, bromodomain containing protein 3; BRD4, bromodomain containingprotein 4; BRDT, bromodomain, testis specific; CDK, cyclin-dependent kinase; CREBP, cAMP (adenosine 3′5′ cyclic monophosphate)response element-binding protein; CREBBP, CREB binding protein; EJM1, epilepsy juvenile myoclonic 1; FA, fluorescence anisot-ropy; GHB, γ-hydroxybutyrate; HAT, histone acetyl transferases; HIVAN, HIV-associated neuropathy; HTS, high-throughput screen-ing; mB, motif b; LAC, lung adenocarcinoma; MYCN, V-Myc Avian Myelocytomatosis Viral Oncogene Neuroblastoma DerivedHomolog; NMR, nuclear magnetic resonance; NUT, nuclear protein in testis; PAFc, polymerase associated factor complex; P-TEFb,positive transcription elongation factor; PTM, post-translational modifications; Ptn, Pleotrophin; SEC, super elongation complex; SPR,surface Plasmon resonance; VS, virtual screening

Review

Published online: 29 April 2016

BALASUNDARAM PADMANABHAN*, SHRUTI MATHUR, RAMU MANJULA and SHAILESH TRIPATHI

2007). The bromodomains consist of about 110 residues andare structurally conserved. The BDs are present in manychromatin-associated factors, including nuclear histone ace-tyl transferases (HATs), chromatin remodeling factors andbromodomains and extra terminal (BET) domains familynuclear proteins (Mujtaba et al. 2007; Wu and Chiang2007). The tertiary structure of BD contains an α-helicalbundle formed by four α-helices (αZ, αA, αB, αC)(figure 1a). The substrate binding site (acetylated histonesor non-histones) is defined by an extended long loop ZA,which connects the helices αZ and αA and by another shortloop BC, connecting the helices αB and αC. These two loopsproduce a deep cleft with a cavity at the middle of thepocket. The hydrophobic core cavity of BD is stabilizedmainly by the conserved hydrophobic residues and addition-ally by conserved hydrophilic residues (Mujtaba et al. 2007).

The BET family proteins (BRD2, BRD3, BRD4 andmBRDT) contain two tandem bromodomains (BD1 andBD2) and the C-terminal extra-terminal (ET) domain(figure 1b). In human cells, bromodomain containing protein2 (BRD2) and bromodomain containing protein 3 (BRD3)are known to recognize acetylated chromatin which is sig-nificantly enriched in K5 and K12 acetylated histone H4(LeRoy et al. 2008). It is also shown that BRD2 and bromo-domain containing protein 4 (BRD4) associate with acety-lated chromatin throughout the cell cycle (Dey et al. 2003;Kanno et al. 2004) while other non-BET bromodomainfamily members dissociate from the chromosomes duringmitosis (Martínez-Balbás et al. 1995). The retention ofmitotic chromosomes by BRD2 and BRD4 is postulated tobe important to carry epigenetic memory across cell cycle.This unique feature can be utilized by the human papillomaas well as Kaposi’s sarcoma-associated herpes virus fortethering genomes to the mitotic chromosome of their hostand for propagating them during the cell division (Platt et al.1999; You et al. 2004).

Recent reports have shown the importance of BET familyBDs as therapeutic targets for cancer, neurological disorder,obesity and inflammation (Belkina and Denis 2012, andreferences therein). NUT (nuclear protein in testis) midlinecarcinoma (NMC) is occurred due to the rearrangements ofthe NUT gene. The coding sequence of NUT on chromo-some 15q14 is fused with BRD3 or BRD4, creating chimericgenes that encode BRD-NUT fusion proteins (French 2012).As BRD4 promotes transcription of growth promoting andantiapoptotic genes, it plays a crucial role in promotingseveral types of tumors (Rahl et al. 2010). As BRD2 is alsoexpressed in differentiating neurons (Crowley et al. 2004), itmight be linked to the neural tube closure defects observedin Brd2-knockout mouse embryos as well as to the associa-tion of BRD2 locus with human juvenile myoclonic epilepsy(Gyuris et al. 2009; Shang et al. 2009; Velíšek et al. 2011).Recent studies reported that the growth factor pleiotrophin(Ptn) antagonizes BRD2 during neuronal differentiation;thus, Ptn-mediated BRD2 antagonism acts as a modulation

system accounting for the balance between cell proliferationand differentiation in the vertebrate nervous system (Garcia-Gutierrz et al. 2014). It is also shown that the reducedexpression of BRD2 in mice produced a hypomorphic phe-notype with extreme obesity and hyperinsulinemia, but en-hanced tolerance and low blood glucose (Wang et al. 2010).

In the last few years, both academia and industry haveshown immense interest in the BET family protein targets todiscover and develop inhibitors to regulate gene expressionby inhibiting the acetylated chromatin interaction (Hewingset al. 2012). Here, we review the biological role of BETfamily proteins based on the BD structures to emphasizetheir importance as potential therapeutic targets for diseasessuch as cancer, neurodegenerative disorders, inflammationand obesity.

2. Bromodomain architecture

Recent reviews have discussed different types of BD con-ta in ing pro te ins and the i r in te rac t ing modules(Filippakopoulos and Knapp 2012; Filippakopoulos et al.2010). Based on the structure-based comparison of humanBDs, 61 BD containing proteins have been grouped intoeight families. The BET family proteins are known to rec-ognize mono-, di-, tri- or tetra-acetylated histones H4(Filippakopoulos et al. 2012a; Huang et al. 2007; Liu et al.2008; Umehara et al. 2010a, b). The N-terminal BD (BD1)of BET family possesses more affinity towards acetylatedH4 compared to the C-terminal BD (BD2). Recognition ofacetylated histone H3 by the BD containing proteins havebeen implicated in chromatin remodeling (Dhalluin et al.1999; Thomson et al. 1999). The BD2 of mouse bromodo-main testis-specific protein (mBRDT) has a significant af-finity with the acetylated histone H3 compared to themBRDT-BD1 (Morinière et al. 2009).

The tertiary structure of BD1 and BD2 of BRD2, BRD4and mBRDT have been reported both in the apo-form as wellas in complex with the acetylated histone tails (Morinièreet al. 2009; Umehara et al. 2010a, b; Vollmuth and Geyer2010). The side-chain of Kac sits in the deep hydrophobicpocket, produced by the residues P98, F99, V103, L108,L110, C152 and I162 (BRD2-BD1 numbering). All theseresidues are conserved, except I162 which is replaced byV435 in BD2. The acetyl group of Kac is stabilized by Nζ2of conserved N156 and by Y113 side-chain through a watermolecule. This water molecule is conserved in all BDs(Mujtaba et al. 2007).

The BD1 of BRD2 forms a homodimer both in vitro andin vivo (Nakamura et al. 2007). The substrate binding pock-ets of BD1 are side-by-side (figure 1c). The BRD2-BD1crystal structure (Chung et al. 2011) also possesses identicaldimer arrangement (PDB: 4A9M). Although the dimer pack-ing is slightly different in mouse BRDT-BD1, it also forms a

296 B Padmanabhan et al.

J. Biosci. 41(2), June 2016

similar dimer arrangement via αB and αC interface (PDB:2WP2) (Morinière et al. 2009). The BRD2-BD2 also exhib-its dimerization with relatively weak interactions betweenthe monomers (Umehara et al. 2010b). The weak BD2dimerization is the result of the buried water molecules. InBRD2-BD2 dimer, binding pockets of monomers are nearlyopposite to each other. The existence of BRD2 dimerizationis also present in cell, and it has been recently linked to aprominent role in cell proliferation (Garcia-Gutierrez et al.2012). The conserved motif B of 47 amino acids is alsoshown to be essential for dimerization. The motif B ispositioned between BD2 and the ET domain. The dimeriza-tion of motif B plays an important role during mitosis tomaintain the attachment of BET proteins to chromatin andhelps BDs to recognize hyper-acetylated histones. Removalof this region has been shown to alter the functionality ofBRD2 (Garcia-Gutierrez et al. 2012, and references therein).

In the BRD2-BD1 complex, the BD1 dimer recognizestwo H4K12ac peptides of length corresponding to the region1-15aa in H4. The K8 of H4 is anchored at the dimerinterface. The side chain of K8 binds into the deep cleftproduced by the dimer interface. The K8 anchoring assistsH4K12ac strong peptide binding with BRD2-BD1 (Umeharaet al. 2010a) (figure 1c). In the complex of BRD2-BD2 withthe H4K5ac/H4K12ac diacetylated peptide, K5ac binds toone BD2 molecule, while K12ac of the same peptide bindsto the other BD2 molecule simultaneously (Umehara et al.2010b) (figure 1d). This indicates that a diacetylated H4peptide tethers two BD2 molecules simultaneously.

The BRD4-BD1 and BRDT-BD1 complexes are shownto bind to the diacetylated H4K5acK8ac peptide (PDB:3UVW, 2WP2) (Filippakopoulos et al. 2012a; Morinièreet al. 2009). It showed that ‘a single BD1’ binds to twoacetyl-lysines (K5ac and K8ac) simultaneously, in a cooper-ative manner; one lysine (K5ac) binds to the conventionalKac binding pocket and other lysine (K8ac) binds to nearthis pocket across the WPF (W97, P98 and F99) shelf(figure 1e). The conserved WPF motif lies in the ZA loop(figures 2 and 3b). These observations suggest that BD1 ofBRD2, BRD4 and BRDT may also recognize a diacetylatedH4 tail, when the acetylated lysine residues are separated byfew residues, such as H4K5acK8ac. Moreover, biochemicalstudies suggest that ‘a single BD’ of BRD2, BRD3, BRD4and BRDT also interacts with tri- or tetracetylated H3 or H4tails (Dey et al. 2003; Filippakopoulos et al. 2012a; Kannoet al. 2004; Vollmuth et al. 2009; Vollmuth and Geyer2010). However, structurally how these monomers recognizemultiple acetyl-lysines of H3 and H4, simultaneously arestill not known.

The conserved residues D385, Y428, N429, D432 andH433 in BRD2-BD2 are critical residues to interact with theH4Kac peptide. These residues are absent in non-BET BDs,suggesting that each BD is unique to recognize histones

acetylated at specific lysine(s) (figure 2). Sequence compar-ison between the BET family members has shown thatcorresponding N-terminal and C-terminal BDs (i.e. BD1 vsBD1, and BD2 vs BD2) are highly homologous, as com-pared to the non-equivalent BDs (BD1 vs BD2) (figure 2).The H4-tail interacting residues are highly conserved, sug-gesting that the other members of the BET family (BRD4,BRD3 and BRDT) recognize acetylated histone tails in asimilar manner as in BRD2.

As the BRD2 protein forms a dimer in solution (Garcia-Gutierrez et al. 2012; Nakamura et al. 2007), the BD1 mayassociate with two mono-acetylated H4 tails of the samenucleosome or different nucleosome. Since BD2 also bindsto two independent diacetylated H4 tails, BRD2-BD2 canassociate with two diacetylated H4 tails of the same nucleo-some or different nucleosomes. However, our model analy-sis suggests that BD2 is unlikely to interact with the sameflanking tails of H4, which already associate with BRD2-BD1 (unpublished results). Overall, a full-length BRD2 di-mer may associate with at least three nucleosomessimultaneously.

3. BET family inhibitors

The first crystal structure of BET family BD has beenreported by Yokoyama and co-workers (Nakamura et al.2007). Recently, both pharmaceutical companies and acad-emicians have shown great interest in BET proteins for drugdiscovery and development for treating cancer, inflammationand neurodegenerative diseases. The discovery of first selec-tive inhibitors in a nanomolar range for the tandem BETfamily BDs (Chung et al. 2011), suggests that BDs are goodtherapeutic targets for small-molecule drug discovery, linkedto the above-mentioned diseases. Structures of inhibitors-BET family BDs complexes are shown in tables 1 and 2.

In a pioneering study, NMR screening technique has beenexploited to filter out molecules that could inhibit CREB(cyclic AMP response element binding protein) bindingprotein (CREBBP) (Mujtaba et al. 2004). The Kac mimics,which possess only weak inhibitory activity againstCREBBP BD have been obtained. They are known to mod-ulate p53 stability and its function in response to DNAdamage.

A BRD2-BD1 inhibitor was discovered and checked forits inhibitory function by FRET analysis using the fluores-cent probe, Histac-K12 (Ito et al. 2011). This powerful tooldetects any physical interaction between H4K12ac and BDin vivo. In the crystal structure (PDB Id: 3AQA), the benzenering of the benzimidazole moiety is positioned in the Kacbinding site, while the WPF shelf region is occupied bybenzaimidazole-2-thione (figure 3). The Kd value of above-mentioned BIC1 inhibitor is 28 μM against the H4K12acpeptide.

BET family proteins as therapeutic targets in major diseases 297


JQ1 ((+)-JQ1, triazolobenzodiazepine derivative), PCT/JP2008/073864 (US2010/0286127 A1, Mitsubishi Pharma-ceuticals), (Miyoshi et al. 2009), is a stereo-specific compoundreported to be an anti-tumor agent. This compound has alsobeen developed by structure-based approach against BETfamily BDs (Filippakopoulos et al. 2010). The cue for thecore scaffold has been taken from FDA approved substances,such as alprazolam and triazolam. The compound JQ1 showsan IC50 of 77 nM on binding with BRD4-BD1, as deter-mined by differential scanning fluorimetry and Alpha Screentitrations.

It is recently reported that a bioisostere of peptide lysinenamely, (3, 5-dimethylisoxazole) is capable of displacing theacetylated histone peptides from the BDs (Hewings et al.2011). It has shown IC50 value of <5 μM for BRD2 andBRD4. To generate cell-penetrant chemical probes for BDs,a binding assay has been developed based on protein stabil-ity shift to identify a fragment and small molecule ligandsthat inhibit the BD-histone tail interaction.

Of the two compounds GSK525762A and GSK525768A(discovered by the GSK group), GSK525762A is the potentmolecule against BET BDs (nM range) (Chung et al. 2011).

Figure 1. BET family bromodomains: (a) Overall tertiary structure of BRD2-BD1 in complex with H4K12ac peptide (PDB Id: 2DVQ).The K12ac residue is shown in sticks. The potential residues of BD1 responsible for acetylated histone tail interactions are shown in sticksmodel; (b) schematic representations of the BET proteins. The bromodomains (BD1 and BD2), extra-terminal region (ET) and an additionaldimerization region (mB) are indicated brown, green, violet and cyan boxes, respectively; (c) electrostatic surface charge distribution at theacetyl-lysine binding site of BRD2-BD1 homodimer in complex with H4K12ac peptides (shown in sticks); (d) overall structure of BRD2-BD2 dimer complex that interacts with diacetylated H4 tails (H4K5acK12ac) (PDB Id: 2E3K). The diacetylated peptides (Q and R) areshown as ribbon and side-chains of H4K5ac and H4K12ac are indicated by stick models; and (e) the tertiary structure of the BRD2-BD1complex with H4K5acK8ac diacetylated peptide (PDB Id: 3UVW). The diacetylated peptide is shown as ribbon and the side-chains ofH4K5ac and H4K8ac are indicated by stick models.



It is a pan BET protein inhibitor (BRD2, BRD3, BRD4),capable of directly inhibiting the protein-protein interactionsat the acetyl lysine recognition pocket. Moreover, the mo-lecular mechanism of the above-mentioned compound hasbeen elucidated by correlating the BET protein (BRD2,BRD3, and BRD4) inhibition with upregulation of apolipo-protein A1 based on both in vitro and in vivo assays.

Fragment based drug discovery (FBDD) approach hasalso yielded several low molecular weight compounds

that inhibit BRD2 and BRD4 BDs (table 1). To assessthe strength of interaction of these compounds againstBDs (for example paracetamol), ligand efficiency (LE)parameter was taken into consideration. These compounds(table 1, #16–25) are found to bind to different BDs,such as CREBBP in a consistent mode and could thuspotentially act as generic BD templates. In addition,structure-based optimization of these compounds has ledto the development of a structure-activity relationship of a

Figure 2. Sequence comparison between the BET and non-BET family bromodomains [The name and region of the BET family areindicated in blue (i.e. BD1 and BD2 regions of human BRD2 and BRD4). The amino acid sequence alignment is produced by ClustalW(Thompson et al. 1994) and is manually modified. Red characters indicate the identical residues in the BET family (group 1) of BRD2 andBRD4 bromodomains. Characters on the red background indicate completely conserved amino acids among the BET (group 1) and non-BET (group 2) bromodomains. The figure is generated by ESpript (Gouet et al. 1999).



series of sulfonamide analogues with anti-inflammatoryactivity (μM range) (Bamborough et al. 2012; Chunget al. 2012).

It is suggested that the WPF motif contributes to the selec-tivity of inhibitors specific to the BET family BDs (Chunget al. 2011). As the sequence comparison analysis has revealed

that WPF motif is also present in the hTAFI, hGCN5 andhPCAF BDs (figure 2), additional care is required while de-signing inhibitors specific to BET family BDs.

Based on the known structures of BET BD–inhibitorcomplexes, three key areas of interactions need to be con-sidered – the Kac-binding pocket, WPF shelf region, and ZA

Figure 3. Comparison with representative BET family bromodomain-inhibitor complexes: (a) Superposition of representative inhibitors(shown as sticks) near the acetyl-lysine binding pocket (PDB Id: 4A9M, fire-brick; PDB Id: 3MXF, olive green; PDB Id: 3AQA, red; PDBId: 2YEM, yellow). The side-chain of H4K12ac of the BD1 complex (PDB Id: 2DVQ) is shown in violet sticks; and (b) a close-up view ofBIC1 complex (PDB Id: 3AQA) as shown by sticks near the binding site. The H4K12ac peptide (PDB Id: 2DVQ) is shown in yellowribbon. The WPF motif is coloured in green.



channel (figure 3b). In Kac-binding pocket, a part of inhib-itor compounds mimics Kac-binding mode; the highly con-served residues F99, V103, Y113, N156 and conservedwater molecules contribute interactions with ligand atomsas observed in Kac interaction. The WPF shelf region, which

is predominantly hydrophobic is a potential site for design-ing selective BET family inhibitors. The third region the ZAchannel, where ligand interactions are observed, is anotherpotential site to be considered when designing lead com-pounds. Two crystallographically conserved water

Table 1. BET bromodomains–inhibitor complexes (PDB structures)

S. No. BET bromodomain PDB ID Ligand/Inhibitor References

BRD4-BD1

1 BRD4-BD1 3MXF JQ1 Filippakopoulos et al. 2010

2 BRD4-BD1 2YEM GWX841819X Chung et al. 20113 BRD4-BD1 2YEL GWX841819X

4 BRD4-BD1 3ZYU IBET-151 Dawson et al. 2011

5 BRD4-BD1 3U5J Alprazolam Fedorov et al. 20126 BRD4-BD1 3U5K Midazolam

7 BRD4-BD1 3U5L Benzo-triazepine (BzT-7)

8 BRD4-BD1 3P5O I-BET (GSK525768A) Nicodeme et al. 2010

9 BRD4-BD1 4C66 IBET-762 Mirguet et al. 201310 BRD4-BD1 4BW2 3,5-Dimethylisoxaxole

11 BRD4-BD1 4BW3 3,5-Dimethylisoxaxole

12 BRD4-BD1 4BW4 3,5-Dimethylisoxaxole

BRD2-BD1

13 BRD2-BD1 2YDW GW841819X Chung et al. 201114 BRD2-BD1 2YEK IBET-762 (GSK525762)

15 BRD2-BD1 3AQA B1C1 Ito et al. 2011

16 BRD2-BD1 4ALH 3,5-Dimethyl-4-phenyl-1,2-oxazole

Bamborough et al. 2012

17 BRD2-BD1 4A9O 5-Ethyl-3-methyl-4-phenyl-1,2-oxazole

18 BRD2-BD1 4A9M N-Cyclopentyl-5-(3,5-dimethyl-1,2-oxazol-4-yl)-2-methylbenzene-1-sulfonamide

19 BRD2-BD1 4A9N N-Cyclopropyl-5-(3,5-dimethyl-1,2-oxazol-4-yl)-2-methylbenzene-1-sulfonamide

20 BRD2-BD1 4A9I 3-Methyl-1,2,3,4-tetrahydroquinazolin-2-one

Chung et al. 2012

21 BRD2-BD1 4A9E 3-Methyl-1,2,3,4-tetrahydroquinazolin-2-one

22 BRD2-BD1 4A9F 1-Methylpyrrolidin-2-one

23 BRD2-BD1 4A9H 1-(2-Methyl-1,2,3,4-tetrahydroquinolin-1-yl)ethan-1-one

24 BRD2-BD1 4A9J N-(4-hydroxypheny)-acetamide

25 BRD2-BD1 4A9L 1,3-Dimethyl-6-(morpholine-4-sulfonyl)-1,2,3,4- tetrahydroqunazolin-2-one

26 BRD2-BD1 4AKN IBET-151 Seal et al. 201227 BRD2-BD1 4ALG IBET-151

BRD2-BD2

28 BRD2-BD2 3ONI JQ1 Filippakopoulos et al. 2010

29 BRD2-BD2 4MR5 RVX-208 Picaud et al. 2013b



Table 2. Chemical structures of BET–Inhibitor complexes



Table 2. (continued)



molecules are invariably present in all structures of thecomplexes; hence, these water molecules should also beincluded in docking studies.

4. Biological significance and associated disorders ofBET family proteins

BRD2 and BRD4 play a critical role in cell cycle control ofnormal mammalian cell (Kanno et al. 2004). They affectcellular processes, such as cellular proliferation, apoptosis,and transcription. The presence of BRD4-specific antibodiesleads to cell cycle arrest, suggesting that BRD4 may berequired for the G2-M phase transition during cell cycle(Dey et al. 2000). Since BRD2 and BRD4 remain bound tomitotic chromosomes, it is hypothesized that they carryepigenetic memory across cell division through recognizingacetylated histone tails (Delmore et al. 2011; Dey et al.2009). The P-TEFb recruitment is dependent on BRD4,which is involved in M-G1 post-mitotic phase for transcrip-tion. BRD2 does not recruit P-TEFb; however, it associateswith post-mitotic chromatin. The expression of Aurora Bkinase which is responsible for chromosome separation andcytokinesis during mitosis is dependent on BRD4 (Yan et al.2011; You et al. 2009). Dysregulation of this function leadsto aberrant chromosomal conditions in cancerous cells. Anoutline of the possible clinical implications of BRD4 isdepicted in Table 3.

5. Cancer

BRD2 is generally localized in the nucleus during the stageof cell proliferation and is sequestered in the cytoplasmduring terminal differential stage. Embryonic stem cell stud-ies have shown that BRD2 is essential during the dorsal rootganglia and neural tube closure during the process of em-bryogenesis (Gyuris et al. 2009). In the nucleus, BRD2 isreported to interact with E2F and transactivating promotersof genes encoding cell cycle regulatory proteins, such ascyclin D1, A2 and E, and dihydrofolate reductase (Deniset al. 2006; Guo et al. 2000; Sinha et al. 2005).

Nuclear protein in testis (NUT) encodes a nuclear proteinwhich is expressed only in testis. NUT midline carcinoma(NMC) is a rare, highly lethal and poorly differentiated orundifferentiated epithelial cancer caused by BRD4 translo-cations (Delmore et al. 2011). The BRD4-NUT fused onco-gene might contribute to: (i) reduced expression of longBRD4 isoform, (ii) increased tendency of fused BRD4-NUT which perturbs non-fused BRD4 functions to cellulardifferentiation, finally leading to oncogenic progression inthe highly aggressive NMC (Delmore et al. 2011; Frenchet al. 2003; Yan et al. 2011). The recently discovered BRD4inhibitor JQ1 is found to be highly effective against NMCT

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xenografts in mice and promotes both growth and differen-tiation of NMC cell in vitro (Filippakopoulos et al. 2010).

BRD3 and BRD4 are involved in regulating the proto-oncogene Myc (Zuber et al. 2011). Recently reported BETinhibitor JQ1 has shown potent inhibitory property againstvarious c-Myc related cancer cell as acute myeloid leukemia(AML) (Hewings et al. 2012), Burkitt’s lymphoma (BL)(Hewings et al. 2012) and multiple myeloma (MM)(Delmore et al. 2011). The BET inhibitor I-BET151 hasbeen reported to arrest the cell cycle and apoptosis inmixed-lineage leukemia (MLL) cell line (Dawson et al.2011; Huang et al. 2009).

The testing of JQ1 inhibitor in a large panel of lungadenocarcinoma (LAC) cell line has shown that a subset ofLAC cell lines is acutely susceptible to BET inhibition. Thisis largely mediated by the repression of the oncogenic tran-scription factor FOS-like antigen 1 (FOSL1) and it’s down-stream targets (Lockwood et al. 2012). The BET familyinhibitor GSK1210151A (I-BET151) has shown to inducecell cycle arrest and apoptosis in human and murine MLL-fusion leukaemic cell lines, where it displaces BRD3/4,PAFc (polymerase associated factor complex) and SEC (su-per elongation complex) components from chromatin, sub-sequently inhibiting the transcription of B-cell lymphoma 2(BCL2), c-Myc and cyclin-dependent kinase 6 (CDK6)(Dawson et al. 2011). An isoxazole azepine BRD4 inhibitoris reported to bind (μM range) to Myc locus, thereby reduc-ing mRNA transcription of Myc oncogenes following cellcycle arrest in vivo models of leukemia and lymphoma(Gehling and Hewitt 2013).

6. Inflammation and obesity

A significant association has been found between humansingle nucleotide polymorphisms (SNPs) in the BRD2 locuswith autoimmune and inflammatory process, which subse-quently leads to rheumatoid arthritis. On the other hand,BRD4 is a novel co-activator of NF-κB through direct inter-action with RelA acetylated at K310 (Chen et al. 2002;Mahdi et al. 2009). A recent study suggests that inhibitingthe BET-BD, thereby repressing the NF-κB pro-inflamma-tory genes activity as a novel therapeutic approach for treat-ing NF-κB-mediated inflammation and kidney injury,especially in HIVAN (HIV-associated neuropathy) (Zhanget al. 2012). For example, BET inhibitors such as JQ1 and I-BET-762 possess anti-inflammatory activity by inhibitingthe expression level of several pro-inflammatory cytokines,including IL-1β, IL-16, IL-12a, CXCL9 and CCL12 in awell-established model system of pro-inflammatory cytokineproduction from bone marrow-derived macrophages(Bandukwala et al. 2012; Belkina et al. 2013).

Whole body reduction of BRD2 expression in mice havebeen shown to have induce lifelong obesity (with

hyperinsulinaema, hepatosteatosis, and elevated pro-inflammatory cytokines), but also the symptoms of overallimproved metabolic profile (enhanced glucose tolerance,increased weight of brown adipose tissue, lowered bloodglucose levels, etc) and helped avoid Type 2 diabetes(Wang et al. 2009; Belkina et al. 2010). Insertion of LacZgene near the 5’ transcription initiation site of BRD2 gene,helped in reducing the expression of BRD2 rather thaneliminating. The study may help to provide a new treatmentmodel for obese subjects demonstrating glucose intoleranceand inflammation related complications. The study alsodiversifies the role of bromodomain proteins in diseasesinvolving metabolic disorders.

7. Neurological disorders

7.1 Epilepsy

Idiopathic generalized epilepsy (IGE) constitutes of a varietyof epileptic disorders in humans, including juvenile myo-clonic epilepsy (JME) and juvenile absence epilepsy (JAE).JME is characterized by myoclonic seizures, consisting ofsmall jerks of the arms, shoulders and sometimes legs.Faulty genetic variation in the Brd2 gene in brain, is nowbeing realized to be significant cause of JME (Pal et al.2003). Several linkages and association studies have sup-ported the notion of BRD2 to be the epilepsy juvenilemyoclonic 1 (EJM1) locus. It is observed that the locus forBrd2 gene can be mapped to chromosome 6p21.3 and Brd2gene defects therein correlate with JME. Hence many studieson BRD2 homologues have been conducted, to determinethe exact function of this protein. Studies on human Brd2gene have revealed that certain polymorphisms in non-coding regions, near the BRD2 promoter are associated withJME (Shang et al. 2011).

Increased levels of γ-aminobutyric acid (GABA) havebeen associated with high susceptibility to primarily gener-alized spontaneous seizures (Velíšek et al. 2011). Moreover,treatment of IGE patients with lamotrigine has been shownto reduce glucose metabolism (thereby reduced GABA lev-els) in basal ganglia (including substantia nigra), cerebralcortex and thalamus pathway. The haploinsufficient Brd2gene (chromosome 6p21.3) has been used to study theGABAergic release in mice (Velíšek et al. 2011). TheBrd2 haploinsufficiency condition led to a reduction ofGABAergic neurons along the basal ganglia seizure-controlling pathway. This causes decrease in GABAergicneurons and reduction in their synthesizing enzyme expres-sion (GAD67), thereby increasing susceptibility and sponta-neous seizure development. Overall, they demonstrateincreased susceptibility of JME in the haploinsufficientBrd2 gene condition in mice. Thus, the above-mentionedstudy has provided pioneering evidence for the following



findings: (i) developmentally-related mechanism for seizuresusceptibility of common forms of epilepsy, (ii) specificseizure disorders may involve in specific brain structuresand, (iii) sex-specific increases in seizure susceptibility inhuman JME. However, these findings do not hold true forthe Asian population and are unable to explain a rise incompensatory GABA levels or enhanced activity of gluta-minergic excitatory system in neurons.

Another independent finding highlights the importance ofγ-hydroxybutyrate (GHB) which is a natural brain neuro-modulator and brain regions involved therein (Kemmel et al.2010). A single administration of GHB is found to increasethe release of GABA that significantly affects expression ofseveral genes, including BRD4 BDs. However, these studiesraise questions whether BRD2 interacts directly or indirectlywith GABA. It will be interesting to know, how these find-ings can bear significance to the clinical implications of theBET family proteins.

A far more comprehensive finding to explain the molec-ular mechanism of Brd2 gene defect mediated epilepsy hasbeen put forward by Shang et al (Shang et al. 2011).Theyfound different BRD2 promoters expressing distinct tissue-specific transcripts. These transcripts were found to differ inlengths of 5′-untranslated regions (5′UTR) and ultimatelyaffect the efficiency of resultant protein product. In fact,presence of BRD2 protein was observed only in the cerebralPurkinje, despite occurrence of the functional Brd2 mRNAtranscripts in hippocampus and cerebellum. Evidence is alsosuggestive of a microsatellite (GT sequence) based mecha-nism to account for the premature termination of translationon inclusion of a highly conserved alternative spliced exon(Shang et al. 2011).

7.2 Embryonic neural defects

BRD2 is expressed in a variety of tissues such as mammarygland, uterus, epididymis, ovary and testis (Shang et al.2011). It is also reported in the germ lines of testis andsomatic cells in the ovary. In fact, null mutants of mice havebeen used to illustrate that Brd2 gene deficient embryosaffected neural tube defects and neurogenesis. As the expres-sion of BRD2 is maximum during the neural tube develop-ment, BRD2 is essential for the successful completion ofembryonic stage development and neural tube closure(Shang et al. 2009). Embryos deficient in Brd2 exhibitsmisexpression of multiple genes, which are required fornormal neuronal development, ultimately leading to deathof the embryo within 15 days, unable to cross the mid-gestation period. Similar to the neural tube defect, thehomeosis defect of head and tail region is also observed inDrosophila ortholog fsh1 mutants (Rhee et al. 1998). Thegrowth factor pleiotrophin (Ptn) is abundantly expressed inthe developing nervous systems and its neuroprotective

properties are important for nerve regeneration (Jin et al.2009). Recent studies showed that Ptn directly interacts withBRD2 during neuronal differentiation and thereby Ptnenhances induced neuronal differentiation, by antagonizingBRD2 cell-cycle stimulating activity (Garcia-Gutierrz et al.2014). Thus, these findings hold a key for therapeutic inter-vention in defects of neuronal differentiation and seizuredisorders (epilepsy).

7.3 Memory, neurons and brain

Casein Kinase 2 (CK2) has been previously shown toactivate neuronal stimulation (Schael et al. 2013) andBRD4 activity has been previously shown to be regulat-ed by CK2 (Wu et al, 2013). A direct link betweenneuronal stimulation and transcription activation by his-tone modification was unknown until recently it wasshown that BET family member BRD4 regulates tran-scription activity underlying memory formation (Korbet al. 2015). Blocking BRD4 using JQ1 inhibitor ledto memory impairment in mice. The results are first ofits kinds, suggesting direct role of bromodomain proteinsin memory formation. Neurological disorders like seiz-ures, which are the result of excessive synaptic excit-ability, were further shown to decrease in mice treatedwith JQ1 in the same study. Such an application ofthese small molecule BD inhibitors is very important,considering they are highly specific in binding, as wellas they can cross a highly selective blood-brain barrier(BBB).

Genome wide expression analysis of neuroblastoma can-cer cell lines treated with BET inhibitor, have been shown todownregulate MYCN (V-Myc Avian Myelocytomatosis Vi-ral Oncogene Neuroblastoma Derived Homolog), followedby apoptosis (Puissant et al. 2013). The results also suggestthe BET BDs to be the transcriptional regulators of MYCN.The BET inhibition has also been shown to induce neuronaldifferentiation of neuroblastoma both in vitro and in vivo inmice (Lee et al. 2015). These results all together suggest thatthese BET inhibitors, not only have the potential to providenew promise against various forms of cancer, but they canalso serve as excellent chemical probes for future studies onepigenetic pathways.

8. Conclusion

This review presents an overview of the BET family BDs.The BET family proteins are chromatin readers which rec-ognize specific acetylated histones, thereby regulating chro-matin functions. The established role of BET family proteinsin cell development and cell-cycle control suggests howBET family proteins are being implicated in several inter-



related disease pathways such as neural tube defects, neuro-degenerative disorders as well as cancer, adipogenesis andchronic inflammation-related disorders.

A significant achievement has recently been made indeveloping inhibitors of BET family proteins by integratedapproaches of advanced modern biology. The diverse heter-ocyclics (e.g. benzodiazepines, quinazolines, napthyhy-drines, and phenylisoxazoles) compounds have emerged aspotent inhibitors of the BET family BDs for the treatment ofcancer and inflammation. In fact, continuous efforts arebeing made to synthesize these potent and selective inhib-itors by combining, site directed mutagenesis studies andstructure-based optimization approach. In future, this trendis expected to grow and allow avenues for discovery of yetmore successful chemotypes that can better inhibit the bro-modomain activity and open vistas for drug-development,providing newer drugs for existing/inter-related ailments.

Acknowledgements

BP is grateful to the Department of Biotechnology (DBT),India, for the financial support (102/IFD/SAN/792/2013-14).SM is grateful to DST for DST WOS-A fellowship (SR/WOS-A/LS-100/2013). MR to CSIR for SRF fellowship(091490(0093) 2012 EMR-1).

References

Alsarraj J and Hunter KW 2012 Bromodomain-containing protein4: a dynamic regulator of breast cancer metastasis throughmodulation of the extracellular matrix. Int. J. Breast Can-cer.2012 670632

Bamborough P, Diallo H, Goodacre JD, Gordon L, Lewis A,Seal JT, Wilson DM, Woodrow MD, et al. 2012 Fragment-based discovery of bromodomain inhibitors part 2: optimiza-tion of phenylisoxazole sulfonamides. J. Med. Chem. 55 587–596

Bandukwala HS, Gagnon J, Togher S, Greenbaum JA, LampertiED, Parr NJ, Molesworth AMH, Smithers N, et al. 2012 Selec-tive inhibition of CD4+ T-cell cytokine production and autoim-munity by BET protein and c-Myc inhibitors. Proc. Natl. Acad.Sci. 109 14532–14537

Belkina AC and Denis GV 2012 BET domain co-regulators in obe-sity, inflammation and cancer. Nat. Rev. Cancer. 12 465–477

Belkina AC, Blanton W, Wang F, Liu H and Denis GV 2010 Wholebody Brd2 deficiency protects obese mice from insulin resistanceby creating low inflammatory enironments. Obesity 18 S58

Belkina AC, Nikolajczyk BS and Denis GV 2013 BET proteinfunction is required for inflammation: Brd2 genetic disruptionand BET inhibitor JQ1 impair mouse macrophage inflammatoryresponses. J. Immunol. 190 3670–3678

Chen L, Mu Y and Greene WC 2002 Acetylation of RelA atdiscrete sites regulates distinct nuclear functions of NF-kB.EMBO J. 21 6539–6548

Chung C-W, Coste H, White JH, Mirguet O, Wilde J, Gosmini RL,Delves C, Magny SM, et al. 2011 Discovery and characteriza-tion of small molecule inhibitors of the BET family bromodo-mains. J. Med. Chem. 54 3827–3838

Chung C-W, Dean AW, Woolven JM and Bamborough P 2012Fragment-based discovery of bromodomain inhibitors part 1:inhibitor binding modes and implications for lead discovery.J. Med. Chem. 55 576–586

Crowley T, Brunori W, Rhee K, Wang X and Wolgemuth DJ 2004Change in nuclear-cytoplasmic localization of double-bromodomain protein during proliferation and differentiationof mouse spinal cord and dorsal root ganglia. Brain Res. Dev.Brain Res. 149 93–101

Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, BantscheffM, Chan W-I, Robson SC, Chung C, et al. 2011 Inhibition ofBET recruitment to chromatin as an effective treatment forMLL-fusion leukaemia. Nature 478 529–533

Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM,Kastritis E, Gilpatrick T, et al. 2011 BET bromodomain inhibi-tion as a therapeutic strategy to target c-Myc. Cell 146 904–917

Denis GV, McComb ME, Faller DV, Sinha A, Romesser PB andCostello CE 2006 Identification of transcription complexesthat contain the double bromodomain protein Brd2 and chro-matin remodeling machines. J. Proteome Res. 5 502–511

Dey A, Ellenberg J, Farina A, Coleman AE, Maruyama T,Sciortino S, Lippincott-Schwartz J and Ozato K 2000 Abromodomain protein, MCAP, associates with mitotic chro-mosomes and affects G(2)-to-M transition. Mol. Cell. Biol.20 6537–6549

Dey A, Chitsaz F, Abbasi A, Misteli T and Ozato K 2003 Thedouble bromodomain protein Brd4 binds to acetylated chroma-tin during interphase and mitosis. Proc. Natl. Acad. Sci. USA100 8758–8763

Dey A, Nishiyama A, Karpova T, McNally J and Ozato K 2009Brd4 marks select genes on mitotic chromatin and directs post-mitotic transcription. Mol. Biol. Cell. 20 4899–4909

Dhalluin C, Carlson JE, Zeng L, He C, Aggarwal AK and Zhou M-M 1999 Structure and ligand of a histone acetyltransferasebromodomain. Nature 399 491–496

Fedorov O, Wrobel M, Picaud S, Bracher F, Morgenstern O, KellerM, Filippakopoulos P and Knapp S 2012 Benzodiazepines andbenzotriazepines as protein interaction inhibitors targeting bromo-domains of the BET family. Bioorg. Med. Chem. 20 1878–1886

Filippakopoulos P and Knapp S 2012 The bromodomain interactionmodule. FEBS Lett. 586 2692–2704

Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O,Morse EM, Keates T, et al. 2010 Selective inhibition of BETbromodomains. Nature 468 1067–1073

Filippakopoulos P, Picaud S, Mangos M, Keates T, Lambert J-P,Barsyte-Lovejoy D, Felletar I, Volkmer R, et al. 2012a Histonerecognition and large-scale structural analysis of the humanbromodomain family. Cell 149 214–231



Filippakopoulos P, Picaud S, Fedorov O, Keller M, Wrobel M,Morgenstern O, Bracher F and Knapp S 2012b Benzodiazepinesand benzotriazepines as protein interaction inhibitors targetingbromodomains of the BET family. Bioorg. Med. Chem. 201878–1886

French CA 2012 Pathogenesis of NUT midline carcinoma. Annu.Rev. Pathol. 7 247–265

French CA, Miyoshi I, Kubonishi I, Grier HE, Perez-atayde AR andFletcher JA 2003 BRD4-NUT Fusion Oncogene: A novel mech-anism in aggressive carcinoma. Cancer Res. 304–307

Gagnon D, Joubert S, Sénéchal H, Fradet-Turcotte A, Torre Sand Archambault J 2009 Proteasomal degradation of the pap-illomavirus E2 protein is inhibited by overexpression ofbromodomain-containing protein 4. J. Virol. 83 4127–4139

Garcia-Gutierrez P, Mundi M and Garcia-Dominguez M 2012Association of bromodomain BET proteins with chromatinrequires dimerization through the conserved motif B. J. CellSci. 125 3671–3680

Garcia-Gutierrz P, Juarez-Vicente F, Wolgemuth DJ and Garcia-Dominguez M 2014 Pleiotrophin antagonizes Brd2 during neu-ronal differentiation. J. Cell Sci. 127 2554–2564

Gehling V and Hewitt M 2013 Discovery, design, and optimizationof isoxazole azepine BET inhibitors. ACS Med. Chem. Lett. 4835–840

Gouet P, Courcelle E, Stuart DI and Metoz F 1999 ESPript: anal-ysis of multiple sequence alignments in PostScript. Bioinfor-matics. 15 305–308

Guo N, Faller DV and Denis GV 2000 Activation-induced nucleartranslocation of RING3. J. Cell Sci. 113 3085–3091

Gyuris A, Donovan DJ, Seymour KA, Lovasco LA, SmilowitzNR, Halperin ALP, Klysik JE and Freiman RN 2009 Thechromatin-targeting protein Brd2 is required for neural tubeclosure and embryogenesis. Biochim. Biophys. Acta.1789413–421

Hewings DS, Wang M, Philpott M, Fedorov O, Uttarkar S, Fili-ppakopoulos P, Picaud S, Vuppusetty C, et al. 2011 3,5-Dime-thylisoxazoles act as acetyl-lysine-mimetic. J. Med. Chem.546761–6770

Hewings DS, Rooney TPC, Jennings LE, Hay DA, Schofield CJ,Brennan PE, Knapp S and Conway SJ 2012 Progress in thedevelopment and application of small molecule inhibitors ofbromodomain-acetyl-lysine interactions. J. Med. Chem. 559393–9413

Huang H, Zhang J, Shen W, Wang X, Wu JJ and Shi Y 2007Solution structure of the second bromodomain of Brd2 and itsspecific interaction with acetylated histone tails. BMC Struct.Biol. 7 1–17

Huang B, Yang X-D, Zhou M-M, Ozato K and Chen L-F 2009Brd4 coactivates transcriptional activation of NF-kB via specificbinding to acetylated RelA. Mol. Cell. Biol. 29 1375–1387

Ito T, Umehara T, Sasaki K, Nakamura Y, Nishino N, Terada T,Shirouzu M, Padmanabhan B, et al. 2011 Real-time imaging ofhistone H4K12-specific acetylation determines the modes ofaction of histone deacetylase and bromodomain inhibitors.Chem. Biol. 18 495–507

Jenuwein T and Allis CD 2001 Translating the histone code.Science 293 1074–1080

Jin L, Jianghai C, Juan L and Hao K 2009 Pleiotrophin andperipheral nerve injury. Neurosurg. Rev. 32 387–393

Kanno T, Kanno Y, Siegel RM, Jang MK, Lenardo MJ and OzatoK 2004 Selective recognition of acetylated histones by bromo-domain proteins visualized in living cells. Mol. Cell. 13 33–43

Kemmel V, Klein C, Dembélé D, Jost B, Taleb O, Aunis D,Mensah-nyagan AG, Maitre M, et al. 2010 A single acutepharmacological dose of -hydroxybutyrate modifies multiplegene expression patterns in rat hippocampus and frontal cortex.Physiol. Genomics 41 146–160

Korb E, Herre M, Zucker-Scharff I, Darnell RB and Allis CD 2015BET protein BRD4 activates transcription in neurons and BETinhibitor Jq1 blocks memory in mice. Nat. Neurosci. 18 1464–1473

Lee S, Rellinger EJ, Kim KW, Craig BT, Romain CV, Qiao J andChung DH 2015 Bromodomain and extraterminal inhibitionblocks tumor progression and promotes differentiation in neuro-blastoma. Surgery 158 819–826

LeRoy G, Rickards B and Flint SJ 2008 The double bromodomainproteins Brd2 and Brd3 couple histone acetylation to transcrip-tion. Mol. Cell. 30 51–60

Liu Y, Wang X, Zhang J, Huang H, Ding B, Wu J and Shi Y 2008Structural basis and binding properties of the second bromodomainof Brd4 with acetylated histone tails. Biochemistry.47 6403–6417

Lockwood WW, Zejnullahu K, Bradner JE and Varmus H 2012Sensitivity of human lung adenocarcinoma cell lines to targetedinhibition of BET epigenetic signaling proteins. Proc. Natl.Acad. Sci. USA 109 19408–19413

Mahdi H, Fisher BA, Källberg H, Plant D, Malmström V, Rönnelid J,Charles P, Ding B, et al. 2009 Specific interaction between geno-type, smoking and autoimmunity to citrullinated alpha-enolase inthe etiology of rheumatoid arthritis. Nat. Genet. 41 1319–1324

Martínez-Balbás MA, Dey A, Rabindran SK, Ozato K and Wu C1995 Displacement of sequence-specific transcription factorsfrom mitotic chromatin. Cell 83 29–38

Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S,Mele DA, Bergeron L and Sims RJ 2011 Targeting MYCdependence in cancer by inhibiting BET bromodomains. Proc.Natl. Acad. Sci. USA 108 16669–16674

Mirguet O, Gosmini R, Toum J, Clément CA, Barnathan M, BrusqJ-M, Mordaunt JE, Grimes RM, et al. 2013 Discovery of epige-netic regulator I-BET762: lead optimization to afford a clinicalcandidate ihibitor of the BET Bromodomains. J. Med. Chem. 567501–7515

Miyoshi S, Ooike S, Iwata K, Hikawa H and Sughara K Antitumoragent, PCT/JP2008/073864(WO/2009/084693), 2009

Morinière J, Rousseaux S, Steuerwald U, Soler-López M, Curtet S,Vitte A-L, Govin J, Gaucher J, et al. 2009 Cooperative bindingof two acetylation marks on a histone tail by a single bromodo-main. Nature 461 664–668

Mujtaba S, He Y, Zeng L, Yan S, Plotnikova O, Sachchidanand SR,Zeleznik-Le NJ, et al. 2004 Structural mechanism of the bromo-domain of the coactivator CBP in p53 transcriptional activation.Mol. Cell. 13 251–263



Mujtaba S, Zeng L and Zhou M-M 2007 Structure and acetyl-lysinerecognition of the bromodomain. Oncogene 26 5521–5527

Nakamura Y, Umehara T, Nakano K, Jang MK, Shirouzu M,Morita S, Uda-Tochio H, Hamana H, et al. 2007 Crystal struc-ture of the human BRD2 bromodomain: insights into dimeriza-tion and recognition of acetylated histone H4. J. Biol. Chem. 2824193–4201

Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S,Chung C-W, Chandwani R, Marazzi I, et al. 2010 Suppres-sion of inflammation by a synthetic histone mimic. Nature468 1119–1123

Ottinger M, Pliquet D, Christalla T, Frank R, Stewart JP and SchulzTF 2009 The interaction of the gammaherpesvirus 68 orf73protein with cellular BET proteins affects the activation of cellcycle promoters. J. Virol. 83 4423–4434

Pal DK, Evgrafov OV, Tabares P, Zhang F, Durner M and Green-berg DA 2003 BRD2 (RING3) is a probable major susceptibilitygene for common juvenile myoclonic epilepsy. Am. J. Hum.Genet. 73 261–270

Picaud S, Da Costa D, Thanasopoulou A, Filippakopoulos P, FishPV, Philpott M, Fedorov O, Brennan P, et al. 2013a PFI-1, ahighly selective protein interaction inhibitor, targeting BETBromodomains. Cancer Res. 73 3336–3346

Picaud S, Wells C, Felletar I, Brotherton D, Martin S, Savitsky P,Diez-Dacal B, Philpott M, et al. 2013b RVX-208, an inhibitor ofBET transcriptional regulators with selectivity for the secondbromodomain. Proc. Natl. Acad. Sci. USA.110 19754–19759

Platt GM, Simpson GR, Mittnacht S and Schulz TF 1999 Latentnuclear antigen of Kaposi’s sarcoma-associated herpesvirusinteracts with RING3, a homolog of the Drosophila femalesterile homeotic (fsh) gene. J. Virol. 73 9789–9795

Puissant A, Frumm SM, Alexe G, Bassil CF, Qi J, ChantheryYH, Nekirtz EA, Zeid R, et al. 2013 Targeting MYCN inneuroblastoma by BET bromodomain inhibition. CancerDiscov. 3 308–323

Rahl PB, Lin CY, Seila AC, Flynn RA, McCuine S, Burge CB,Sharp PA and Young RA 2010 c-Myc regulats transcriptionpause release. Cell 141 432–445

Rhee K, Brunori M, Besset V, Trousdale R and Wolgemuth DJ1998 Expression and potential role of Fsrg1, a murinebromodomain-containing homologue of the Drosophila genefemale sterile homeotic. J. Cell Sci. 111 3541–3550

Schael S, Nuchel J, Muller S, Petermann P, Kormann J, Perez-Otano I, Martinez SM, Paulsson M, et al. 2013 Casein kinase 2phosphorylation of protein kinase C and casein kinase 2 sub-strate in neurons (PACSIN) 1 protein regulates neuronal spineformation. J. Biol. Chem. 288 9303–9312

Seal J, Lamotte Y, Donche F, Bouillot A, Mirguet O, Gellibert F,Nicodeme E, Krysa G, et al. 2012 Identification of a novel seriesof BET family bromodomain inhibitors: binding mode andprofile of I-BET151 (GSK1210151A). Bioorg. Med. Chem. Lett.22 2968–2972

Shang E, Wang X, Wen D, Greenberg DA and Wolgemuth DJ2009 Double bromodomain-containing gene Brd2 is essentialfor embryonic development in mouse. Dev. Dyn. Off. Publ. Am.Assoc. Anat. 238 908–917

Shang E, Cui Q, Wang X, Beseler C, Greenberg DA and Wolge-muth DJ 2011 The bromodomain-containing gene BRD2 isregulated at transcription, splicing, and translation levels.J. Cell. Biochem. 112 2784–2793

Sinha A, Faller DV and Denis GV 2005 Bromodomain analysis ofBrd2-dependent transcriptional activation of cyclin A. Biochem.J. 387 257–269

Strahl BD and Allis CD 2000 The language of covalent histonemodifications. Nature 403 41–45

Taverna SD, Li H, Ruthenburg AJ, Allis CD and Patel DJ 2007How chromatin-binding modules interpret histone modifica-tions: lessons from professional pocket pickers. Nat. Struct.Mol. Biol. 14 1025–1040

Thompson JD, Higgins DG and Gibson TJ 1994 CLUSTAL W:improving the sensitivity of progressive multiple sequencealignment through sequence weighting, position-specific gappenalties and weight matrix choice. Nucleic Acids Res. 224673–4680

Thomson S, Clayton AL, Hazzalin CA, Rose S, Barratt MJ andMahadevan LC 1999 The nucleosomal response associated withimmediate-early gene induction is mediated via alternative MAPkinase cascades: MSK1 as a potential histone H3/HMG-14kinase. Eur. Mol. Biol. Organ J. 18 4779–4793

Umehara T, Nakamura Y, Jang MK, Nakano K, Tanaka A, OzatoK, Padmanabhan B and Yokoyama S 2010a Structural basis foracetylated histone H4 recognition by the human BRD2 bromo-domain. J. Biol. Chem. 285 7610–7618

Umehara T, Nakamura Y, Wakamori M, Ozato K, Yokoyama S andPadmanabhan B 2010b Structural implications for K5/K12-di-acetylated histone H4 recognition by the second bromodomainof BRD2. FEBS Lett. 584 3901–3908

Velíšek L, Shang E, Velíšková J, Chachua T, Macchiarulo S,Maglakelidze G, Wolgemuth DJ and Greenberg DA 2011GABAergic neuron deficit as an idiopathic generalized epilepsymechanism: the role of BRD2 haploinsufficiency in JuvenileMyoclonic Epilepsy. PLoS One 6 e23656

Vollmuth F and Geyer M 2010 Interaction of propionylated andbutyrylated histone H3 lysine marks with Brd4 bromodomains.Angew. Chem. Int. Ed. 39 6768–6772

Vollmuth F, Blankenfeldt W and Geyer M 2009 Structures of thedual bromodomains of the P-TEFb-activating protein Brd4 atatomic resolution. J. Biol. Chem. 284 36547–36556

Wang F, Liu H, Blanton WP, Belkina A, Lebrasseur NK and DenisGV 2010 Brd2 disruption in mice causes severe obesity withoutType diabetes. Biochem. J. 425 71–83

Wu S-Y and Chiang C-M 2007 The double bromodomain-containing chromatin adaptor Brd4 and transcriptional regula-tion. J. Biol. Chem. 282 13141–13145

Wu S-Y, Lee A-Y, Lai H-T, Zhang H and Chaing C-M 2013Phospho switch triggers Brd4 chromatin binding and activatorrecruitment for gene-specific targetting. Mol. Cell. 49 843–857

Yan J, Diaz J, Jiao J, Wang R and You J 2011 Perturbation ofBRD4 protein function by BRD4-NUT protein abrogates cellu-lar differentiation in NUT midline carcinoma. J. Biol. Chem. 28627663–27675

Yang Z, He N and Zhou Q 2008 Brd4 recruits P-TEFb to chromo-somes at late mitosis to promote G1 gene expression and cellcycle progression. Mol. Cell. Biol. 28 967–976



You J, Croyle JL, Nishimura A, Ozato K and Howley PM 2004Interaction of the bovine papillomavirus E2 protein with Brd4tethers the viral DNA to host mitotic chromosomes. Cell. 117349–360

You J, Li Q, Wu C, Kim J, Ottinger M and Howley PM 2009Regulation of aurora B expression by the bromodomain proteinBrd4. Mol. Cell. Biol. 29 5094–5103

Zhang G, Liu R, Zhong Y, Plotnikov AN, Zhang W, Zeng L,Rusinova E, Gerona-Nevarro G, et al. 2012 Down-regulation

of NF-κB transcriptional activity in HIV-associated kidney dis-ease by BRD4 inhibition. J. Biol. Chem. 287 28840–28851

Zhou M, Huang K, Jung KJ, Cho WK, Klase Z, Kashanchi F, Pise-Masison CA and Brady JN 2009 Bromodomain protein Brd4regulates human immunodeficiency virus transcription throughphosphorylation of CDK9 at threonine 29. J. Virol. 83 1036–1044

Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA,Magoon D, Qi J, et al. 2011 RNAi screen identifies Brd4 as atherapeutic target in acute myeloid leukaemia. Nature 478 1–7

MS received 11 March 2015; accepted 05 February 2016

Corresponding editor: PRIM B SINGH



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