Metastatic melanoma remains a mostly incurable disease. Although newly approved targeted therapies are efficacious in a subset of patients, resistance and relapse rapidly ensue. Alternative therapeutic strategies to manipulate epigenetic regulators and disrupt the transcriptional program that maintains tumor cell identity are emerging. Bromodomain and extraterminal domain (BET) proteins are epigenome readers known to exert key roles at the interface between chromatin remodeling and transcriptional regulation. Here, we report that BRD4, a BET family member, is significantly upregulated in primary and metastatic melanoma tissues compared with melanocytes and nevi. Treatment with BET inhibitors impaired melanoma cell proliferation in vitro and tumor growth and metastatic behavior in vivo, effects that were mostly recapitulated by individual silencing of BRD4. RNA sequencing of BET inhibitor–treated cells followed by Gene Ontology analysis showed a striking impact on transcriptional programs controlling cell growth, proliferation, cell-cycle regulation, and differentiation. In particular, we found that, rapidly after BET displacement, key cell-cycle genes (SKP2, ERK1, and c-MYC) were downregulated concomitantly with the accumulation of cyclin-dependent kinase (CDK) inhibitors (p21 and p27), followed by cell-cycle arrest. Importantly, BET inhibitor efficacy was not influenced by BRAF or NRAS mutational status, opening the possibility of using these small-molecule compounds to treat patients for whom no effective targeted therapy exists. Collectively, our study reveals a critical role for BRD4 in melanoma tumor maintenance and renders it a legitimate and novel target for epigenetic therapy directed against the core transcriptional program of melanoma.
Viruses recruit host proteins to secure viral genome maintenance and replication. However, whether they modify host histones directly to interfere with chromatin-based transcription is unknown. Here we report that Paramecium bursaria chlorella virus 1 (PBCV-1) encodes a functional SET domain histone Lys methyltransferase (HKMTase) termed vSET, which is linked to rapid inhibition of host transcription after viral infection. We show that vSET is packaged in the PBCV-1 virion, and that it contains a nuclear localization signal and probably represses host transcription by methylating histone H3 at Lys 27 (H3K27), a modification known to trigger gene silencing in eukaryotes. We also show that vSET induces cell accumulation at the G2/M phase by recruiting the Polycomb repressive complex CBX8 to the methylated H3K27 site in a heterologous system. vSET-like proteins that have H3K27 methylation activity are conserved in chlorella viruses. Our findings suggest a viral mechanism to repress gene transcription by direct modification of chromatin by PBCV-1 vSET.
Twist is a key transcription activator of epithelial-mesenchymal transition (EMT). It remains unclear how Twist induces gene expression. Here we reported a mechanism by which Twist recruits BRD4 to direct WNT5A expression in basal-like breast cancer (BLBC). Twist contains a “histone H4 mimic” GK-X-GK motif that is di-acetylated by Tip60. The di-acetylated Twist binds the second bromodomain of BRD4, whose first bromodomain interacts with acetylated H4, thereby constructs an activated Twist/BRD4/P-TEFb/RNA-PolII complex at the WNT5A promoter and enhancer. Pharmacologic inhibition of the Twist-BRD4 association reduced WNT5A expression and suppressed invasion, cancer stem cell (CSC)-like properties, and tumorigenicity of BLBC cells. Our study indicates that the interaction with BRD4 is critical for the oncogenic function of Twist in BLBC.
Human immunodeficiency virus (HIV) when integrated into a host chromosome exists in a transcriptionally inactive but replication-competent state. Such latent infection represents a major challenge to HIV eradication efforts because a permanent virus reservoir resided in the infected cell is able to spike the viral load on immune suppression or during interruption of highly active anti-retroviral therapy. Understanding the molecular mechanisms that control HIV proviral latency and its reactivation could provide new perspectives on host factors as therapeutic targets for abolishing cellular reservoirs of dormant HIV. Although the control of HIV latency is multifactorial, chromatin structure and the chromatin-associated transcriptional machinery are known to be important factors. For instance, transcription initiation of the HIV provirus involves a complex molecular interplay between chromatin-associated proteins and the virus-encoded trans-activator, Tat. The first part of this review discusses our current understanding of the elements involved in HIV transcriptional activation and viral mRNA elongation, mainly post-translational modifications of HIV Tat and its interactions with host chromatin-modifying enzymes and chromatin-remodeling complexes. The second part highlights new experimental therapeutic approaches aimed at administrating activators of HIV gene expression to reduce or eliminate the pool of latently HIV-infected cells.
HIV; Tat; Transcription; Histone acetylation
BRD4, characterized by two acetyl-lysine binding bromodomains and an extra-terminal (ET) domain, is a key chromatin organizer that directs gene activation in chromatin through transcription factor recruitment, enhancer assembly, and pause release of the RNA polymerase II complex for transcription elongation. BRD4 has been recently validated as a new epigenetic drug target for cancer and inflammation. Our current knowledge of the functional differences of the two bromodomains of BRD4, however, is limited, hindered by the lack of selective inhibitors. Here, we report our structure-guided development of diazobenzene-based small molecule inhibitors for the BRD4 bromodomains that have over 90% sequence identity at the acetyl-lysine binding site. Our lead compound MS436, through a set of water-mediated interactions, exhibits low nanomolar affinity (estimated Ki of 30–50 nM) with preference for the first bromodomain over the second. We demonstrated that MS436 effectively inhibits BRD4 activity in NF-κB-directed production of nitric oxide and pro-inflammatory cytokine interleukin-6 in murine macrophages. MS436 represents a new class of bromodomain inhibitors and will facilitate further investigation of the biological functions of the two bromodomains of BRD4 in gene expression.
Rapid advances in biomedical sciences in recent years have drastically accelerated the discovery of the molecular basis of human diseases. The great challenge is how to translate the newly acquired knowledge into new medicine for disease prevention and treatment. Drug discovery is a long and expensive process and the pharmaceutical industry has not been very successful at it despite its enormous resources and spending on the process. It is increasingly realized that academic biomedical research institutions ought to be engaged in early stage drug discovery, especially when it can be coupled to their basic research. To leverage the productivity of new drug development a substantial acceleration in validation of new therapeutic targets is required, which would require small molecules that can precisely control target functions in complex biological systems in a temporal and dose-dependent manner. In this review, we describe a process of integration of small molecule discovery and chemistry in academic biomedical research, which will ideally bring together the elements of innovative approaches to new molecular targets; existing basic and clinical research; screening infrastructure; and synthetic and medicinal chemistry to follow-up on small molecule hits. Such integration of multi-disciplinary resources and expertise will enable academic investigators to discover novel small molecules that are expected to facilitate their efforts in both mechanistic research and new drug target validation. More broadly academic drug discovery should contribute new entities to therapy for intractable human diseases especially for orphan diseases, and hopefully stimulate and synergize with the commercial sector.
Small molecule; drug discovery; chemical screening; medicinal chemistry
The Polybromo (PB) protein functions as a key component of the human PBAF chromatin remodeling complex in regulation of gene transcription. PB is made up of modular domains including six bromodomains that are known as acetyl-lysine binding domains. However, histone-binding specificity of the bromodomains of PB has remained elusive. In this study, we report biochemical characterization of all six PB bromodomains’ binding to a suite of lysine-acetylated peptides derived from known acetylation sites on human core histones. We demonstrate that bromodomain 2 of PB preferentially recognizes acetylated lysine 14 of histone H3 (H3K14ac), a post-translational mark known for gene transcriptional activation. We further describe the molecular basis of the selective H3K14ac recognition of bromodomain 2 by solving the protein structures in both the free and bound forms using X-ray crystallography and NMR, respectively.
NMR; crystallography; bromodomain; chromatin; transcription regulator
This paper describes a computer program named Dockres that is designed to analyze and summarize results of virtual screening of small molecules. The program is supplemented with utilities that support the screening process. Foremost among these utilities are scripts that run the virtual screening of a chemical library on a large number of processors in parallel.
Dockres and some of its supporting utilities are written Fortran-77; other utilities are written as C-shell scripts. They support the parallel execution of the screening. The current implementation of the program handles virtual screening with Autodock-3 and Autodock-4, but can be extended to work with the output of other programs.
Analysis of virtual screening by Dockres led to both active and selective lead compounds.
Analysis of virtual screening was facilitated and enhanced by Dockres in both the authors' laboratories as well as laboratories elsewhere.
We describe a computer program named Pspace designed to a) obtain a reliable basis for the description of three-dimensional structures of a given protein family using homology modeling through selection of an optimal subset of the protein family whose structure would be determined experimentally; and b) aid in the search of orthologs by matching two sets of sequences in three different ways.
The prioritization is established dynamically as new sequences and new structures are becoming available through ranking proteins by their value in providing structural information about the rest of the family set. The matching can give a list of potential orthologs or it can deduce an overall optimal matching of two sets of sequences.
The various covering strategies and ortholog searches are tested on the bromodomain family.
The possibility of extending this approach to the space of all proteins is discussed.
vSET (a viral SET domain protein) is an attractive Polycomb Repressive Complex 2 (PRC2) surrogate to study the effect of histone H3 lysine 27 (H3K27) methylation on gene transcription as both catalyze histone H3K27 tri-methylation. In order to control the enzymatic activity of vSET in vivo with an engineered S-adenosyl-L-methionine (SAM) analogue as methyl donor cofactor, we have carried out structure-guided design, synthesis and characterization of orthogonal vSET methyltransferase mutant/SAM analogue pairs using a “bump-and-hole” strategy.
PHD (plant homeodomain) zinc fingers are structurally conserved modules found in proteins that modify chromatin as well as mediate molecular interactions in gene transcription. The original discovery of their role in gene transcription is attributed to the recognition of lysine-methylated histone H3. Recent studies show that PHD fingers have a sophisticated histone sequence reading capacity that is modulated by the interplay between different histone modifications. These studies underscore the functional versatility of PHD fingers as epigenome readers that control gene expression through molecular recruitment of multi-protein complexes of chromatin regulators and transcription factors. Moreover, they reinforce the concept that evolutionary changes in amino acids surrounding ligand binding sites on a conserved structural fold impart great functional diversity upon this family of proteins.
Histone lysine acetylation is central to epigenetic control of gene transcription. Bromodomains of chromosomal proteins function as acetyl-lysine (Kac) binding domains. However, how bromodomains recognize site-specific histones remains unanswered. Here, we report three three-dimensional solution structures of the bromodomains of the human transcriptional coactivators CREB-binding protein (CBP) and p300/CBP-associated factor (PCAF) bound to peptides derived from histone acetylation sites at lysines 36 and 9 in H3, and lysine 20 in H4. From structural and biochemical binding analyses, we determine consensus histone recognition by the bromodomains of PCAF and CBP, which represent two different subgroups of the bromodomain family. Through bromodomain residues in the ZA and BC loops, PCAF prefers acetylation sites with a hydrophobic residue at (Kac+2) position and a positively charged or aromatic residue at (Kac+3), whereas CBP favors bulky hydrophobic residues at (Kac+1) and (Kac+2), a positively charged residue at (Kac−1), and an aromatic residue at (Kac−2).
As a master transcription factor in cellular responses to external stress, tumor suppressor p53 is tightly regulated. Excessive p53 activity during myocardial ischemia causes irreversible cellular injury and cardiomyocyte death. p53 activation is dependent on lysine acetylation by the lysine acetyltransferase and transcriptional co-activator CBP (CREB-binding protein) and on acetylation-directed CBP recruitment for p53 target gene expression. Here, we report a small molecule ischemin, developed with a structure-guided approach to inhibit the acetyl-lysine binding activity of the bromodomain of CBP. We show that ischemin alters post-translational modifications on p53 and histones, inhibits p53 interaction with CBP and transcriptional activity in cells, and prevents apoptosis in ischemic cardiomyocytes. Our study suggests small molecule modulation of acetylation-mediated interactions in gene transcription as a new approach to therapeutic interventions of human disorders such as myocardial ischemia.
The tandem PHD finger–bromodomain, found in many chromatin-associated proteins, has an important role in gene silencing by the human co-repressor KRAB-associated protein 1 (KAP1). Here we report the three-dimensional solution structure of the tandem PHD finger–bromodomain of KAP1. The structure reveals a distinct scaffold unifying the two protein modules, in which the first helix, αZ, of an atypical bromodomain forms the central hydrophobic core that anchors the other three helices of the bromodomain on one side and the zinc binding PHD finger on the other. A comprehensive mutation-based structure-function analysis correlating transcriptional repression, ubiquitin-conjugating enzyme 9 (UBC9) binding and SUMOylation shows that the PHD finger and the bromodomain of KAP1 cooperate as one functional unit to facilitate lysine SUMOylation, which is required for KAP1 co-repressor activity in gene silencing. These results demonstrate a previously unknown unified function for the tandem PHD finger–bromodomain as an intramolecular small ubiquitin-like modifier (SUMO) E3 ligase for transcriptional silencing.
Histone methylation recognition is accomplished by a number of evolutionarily conserved protein domains, including those belonging to the methylated lysine-binding Royal family of structural folds. One well-known member of the Royal family, the chromodomain, is found in the HP1/Chromobox and CHD subfamilies of proteins, in addition to a small number of other proteins that are involved in chromatin remodeling and gene transcriptional silencing. Here we discuss the structure and function of the chromodomain within these proteins as histone methylated lysine binders, and how the functions of these chromodomains can be modulated by additional post-translational modifications or binding to nucleic acids.
Expression of the INK4b/ARF/INK4a tumor suppressor locus in normal and cancerous cell growth is controlled by methylation of histone H3 at lysine 27 (H3K27me) as directed by the Polycomb group proteins. The antisense non-coding RNA ANRIL of the INK4b/ARF/INK4a locus is also important for expression of the protein-coding genes in cis, but its mechanism has remained elusive. Here we report that chromobox 7 (CBX7) within the Polycomb Repressive Complex 1 binds to ANRIL, and both CBX7 and ANRIL are found at elevated levels in prostate cancer tissues. In concert with H3K27me recognition, binding to RNA contributes to CBX7 function and disruption of either interaction impacts the ability of CBX7 to repress the INK4b/ARF/INK4a locus and control senescence. Structure-guided analysis reveals the molecular interplay between non-coding RNA and H3K27me as mediated by the conserved chromodomain. Our study suggests a new mechanism by which non-coding RNA participates directly in epigenetic transcriptional repression.
Ever since their existence, there has been an everlasting arms race between viruses and their host cells. Host cells have developed numerous strategies to silence viral gene expression whereas viruses always find their ways to overcome these obstacles. Recent studies show that viruses have also evolved to take full advantage of existing cellular chromatin components to activate or repress its own genes when needed. While in most cases viruses encode certain proteins to recruit or inhibit cellular factors through physical interactions, growing examples show that viral encoded enzymes affect host chromatin structure through post-translationally modifying histones or other cellular proteins important for chromatin function. The most well studied example is vSET encoded by paramecium bursaria chlorella virus 1. vSET specifically methylates histone H3 at lysine 27, causing genome-wide silencing of Polycomb target genes upon infection, thus mimicking the function of Polycomb repressive complex 2 (PRC2) in eukaryotes. Other examples include BGLF4 from Epstein-Barr virus that affects both condensin and topoisomerase II activity and Us3 from Herpes Simplex virus 1 that inhibits HDAC1 function through phosphorylation.
Histone lysine acetylation and methylation are important during gene transcription in a chromatin context1,2. Our knowledge about the types of protein modules that can interact with acetyl-lysine has so far been limited to bromodomains1. Recently, a tandem PHD (plant homeodomain) finger3 (PHD12) of human DPF3b, which functions in association with the BAF chromatin remodelling complex to initiate transcription in the heart and muscle development, was reported to bind histones H3 and H4 in an acetylation sensitive manner4, making it a first alternative to bromodomains for acetyl-lysine binding5. Here, we report the structural mechanism of acetylated histone binding by the double PHD fingers of DPF3b. Our three-dimensional solution structures and biochemical analysis of DPF3b illuminate the molecular basis of the integrated tandem PHD finger, which acts as one functional unit in the sequence-specific recognition of lysine 14-acetylated histone H3 (H3K14ac). Whereas the interaction with H3 is promoted by acetylation at lysine 14, it is inhibited by methylation at lysine 4, and these opposing influences are important during transcriptional activation of DPF3b target genes Pitx2 and Jmjd1c. Binding of this tandem protein module to chromatin can thus be regulated by different histone modifications during the initiation of gene transcription.
Human autoimmune regulator (AIRE) functions to control thymic expression of tissue-specific antigens via sequence-specific histone H3 recognition by its plant homeodomain (PHD) finger. Mutations in the AIRE PHD finger have been linked to autoimmune polyendocrinopathy-candidiasis-ectodermal dystrophy (APECED). Here we report the three-dimensional solution structure of the first PHD finger of human AIRE bound to a histone H3 peptide. The structure reveals a detailed network of interactions between the protein and the amino-terminal residues of histone H3, and particularly key electrostatic interactions of a conserved aspartic acid 297 in AIRE with the unmodified lysine 4 of histone H3 (H3K4). NMR binding study with H3 peptides carrying known post-translational modifications flanking H3K4 confirms that transcriptional regulation by AIRE through its interactions with histone H3 is confined to the first N-terminal eight residues in H3. Our study offers a molecular explanation for the APECED mutations and helps define a subclass of the PHD finger family proteins that recognize histone H3 in a sequence-specific manner.
Transcription factors and chromatin-remodeling complexes are key determinants of embryonic stem cell (ESC) identity. Here, we demonstrate that BRD4, a member of the bromodomain and extraterminal domain (BET) family of epigenetic readers, regulates the self-renewal ability and pluripotency of ESCs. BRD4 inhibition resulted in induction of epithelial-tomesenchymal transition (EMT) markers and commitment to the neuroectodermal lineage while reducing the ESC multidifferentiation capacity in teratoma as-says. BRD4 maintains transcription of core stem cell genes such as OCT4 and PRDM14 by occupying their super-enhancers (SEs), large clusters of regulatory elements, and recruiting to them Mediator and CDK9, the catalytic subunit of the positive transcription elongation factor b (P-TEFb), to allow Pol-II-dependent productive elongation. Our study describes a mechanism of regulation of ESC identity that could be applied to improve the efficiency of ESC differentiation.
The acetylation of histone lysine is central to providing the dynamic regulation of chromatin-based gene transcription. The bromodomain (BRD), which is the conserved structural module in chromatin-associated proteins and histone acetyltranferases, is the sole protein domain known to recognize acetyl-lysine residues on proteins. Structural analyses of the recognition of lysine-acetylated peptides derived from histones and cellular proteins by BRDs have provided new insights into the differences between and unifying features of the selectivity that BRDs exhibit in binding biological ligands. Recent research has highlighted the importance of BRD/acetyl-lysine binding in orchestrating molecular interactions in chromatin biology and regulating gene transcription. These studies suggest that modulating BRD/acetyl-lysine interactions with small molecules may provide new opportunities for the control of gene expression in human health and disease.
Acetyl-lysine recognition; bromodomain; gene transcription; lysine acetylation
It has been shown that molecular interactions between site-specific chemical modifications such as acetylation and methylation on DNA-packing histones and conserved structural modules present in transcriptional proteins are closely associated with chromatin structural changes and gene activation. Unlike methyl-lysine that can interact with different protein modules including chromodomains, Tudor and MBT domains, as well as PHD fingers, acetyl-lysine (Kac) is known thus far to be recognized only by bromodomains. While histone lysine acetylation plays a crucial role in regulation of chromatin-mediated gene transcription, a high degree of sequence variation of the acetyl-lysine binding site in the bromodomains has limited our understanding of histone binding selectivity of the bromodomain family. Here, we report a systematic family-wide analysis of 14 yeast bromodomains binding to 32 lysine-acetylated peptides derived from known major acetylation sites in four core histones that are conserved in eukaryotes.
The histone binding selectivity of purified recombinant yeast bromodomains was assessed by using the native core histones in an overlay assay, as well as N-terminally biotinylated lysine-acetylated histone peptides spotted on streptavidin-coated nitrocellulose membrane in a dot blot assay. NMR binding analysis further validated the interactions between histones and selected bromodomain. Structural models of all yeast bromodomains were built using comparative modeling to provide insights into the molecular basis of their histone binding selectivity.
Our study reveals that while not all members of the bromodomain family are privileged to interact with acetylated-lysine, identifiable sequence features from those that bind histone emerge. These include an asparagine residue at the C-terminus of the third helix in the 4-helix bundle, negatively charged residues around the ZA loop, and preponderance of aromatic amino acid residues in the binding pocket. Further, while bromodomains exhibit selectivity for different sites in histones, individual interactions are of modest affinity. Finally, electrostatic interactions appear to be a primary determining factor that guides productive association between a bromodomain and a lysine-acetylated histone.
In eukaryotes, different chromatin states facilitate or repress gene expression and restrict the activity of transposable elements. Post-translational modifications (PTMs) of amino acid residues on the N-terminal tails of histones are suggested to define such states. The histone lysine methyltransferase (HKMTase) SU(VAR)3-9 RELATED4 (SUVR4) of Arabidopsis thaliana functions as a repressor of transposon activity. Binding of ubiquitin by the WIYLD domain facilitates the addition of two methyl groups to monomethylated lysine 9 of histone H3. By using nuclear magnetic resonance (NMR) spectroscopy, we identified SUVR4 WIYLD (S4WIYLD) as a domain with a four-helix bundle structure, in contrast to three-helix bundles of other ubiquitin binding domains. NMR titration analyses showed that residues of helix α1 (Q38, L39, and D40) and helix α4 (N68, T70, A71, V73, D74, I76, S78, and E82) of S4WIYLD and residues between the first and second β-strands (T9 and G10) and on β-strands 3 (R42, G47, K48, and Q49) and 4 (H68, R72, and L73) undergo significant chemical shift changes when the two proteins interact. A model of the complex, generated using HADDOCK, suggests that the N-terminal and C-terminal parts of S4WIYLD constitute a surface that interacts with charged residues close to the hydrophobic patch of ubiquitin. The WIYLD domains of the closely related SUVR1 and SUVR2 Arabidopsis proteins also bind ubiquitin, indicating that this is a general feature of this domain. The question of whether SUVR proteins act as both readers of monoubiquitinated H2B and writers of histone PTMs is discussed.
The therapeutic potential of pharmacologic inhibition of bromodomain and extraterminal (BET) proteins has recently emerged in hematological malignancies and chronic inflammation. We find that BET inhibitor compounds (JQ1, I-Bet, I-Bet151 and MS417) reactivate HIV from latency. This is evident in polyclonal Jurkat cell populations containing latent infectious HIV, as well as in a primary T-cell model of HIV latency. Importantly, we show that this activation is dependent on the positive transcription elongation factor p-TEFb but independent from the viral Tat protein, arguing against the possibility that removal of the BET protein BRD4, which functions as a cellular competitor for Tat, serves as a primary mechanism for BET inhibitor action. Instead, we find that the related BET protein, BRD2, enforces HIV latency in the absence of Tat, pointing to a new target for BET inhibitor treatment in HIV infection. In shRNA-mediated knockdown experiments, knockdown of BRD2 activates HIV transcription to the same extent as JQ1 treatment, while a lesser effect is observed with BRD4. In single-cell time-lapse fluorescence microscopy, quantitative analyses across ~2,000 viral integration sites confirm the Tat-independent effect of JQ1 and point to positive effects of JQ1 on transcription elongation, while delaying re-initiation of the polymerase complex at the viral promoter. Collectively, our results identify BRD2 as a new Tat-independent suppressor of HIV transcription in latently infected cells and underscore the therapeutic potential of BET inhibitors in the reversal of HIV latency.
HIV; latency; Tat; JQ1; MS417; I-BET; I-BET151; P-TEFb; BRD4; BRD2