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. Author manuscript; available in PMC: 2015 Jul 17.
Published in final edited form as: Chem Biol. 2014 Jun 19;21(7):841–854. doi: 10.1016/j.chembiol.2014.05.009

Selective Chemical Modulation of Gene Transcription Favors Oligodendrocyte Lineage Progression

Mar Gacias 1,#, Guillermo Gerona-Navarro 2,3,#, Alexander N Plotnikov 2, Guangtao Zhang 2, Lei Zeng 2, Jasbir Kaur 1, Gregory Moy 1, Elena Rusinova 2, Yoel Rodriguez 2,4, Bridget Matikainen 1, Adam Vincek 2, Jennifer Joshua 2, Patrizia Casaccia 1,*, Ming-Ming Zhou 2,*
PMCID: PMC4104156  NIHMSID: NIHMS602357  PMID: 24954007

SUMMARY

Lysine acetylation regulates gene expression through modulating protein-protein interactions in chromatin. Chemical inhibition of acetyl-lysine binding bromodomains of the major chromatin regulators BET (bromodomain and extra-terminal domain) proteins, has been shown to effectively block cell proliferation in cancer and inflammation. However, whether selective inhibition of individual BET bromodomains has distinctive functional consequences, remains only partially understood. In this study, we show that selective chemical inhibition of the first bromodomain of BET proteins using our newly designed small molecule inhibitor, Olinone, accelerated the progression of mouse primary oligodendrocyte progenitors towards differentiation, while inhibition of both bromodomains of BET proteins hindered differentiation. This effect was target-specific, as it was not detected in cells treated with inactive analogues and independent of any effect on proliferation. Therefore, selective chemical modulation of individual bromodomains, rather than use of broad-based inhibitors may enhance regenerative strategies in disorders characterized by myelin loss such as aging and neurodegeneration.

INTRODUCTION

Lysine acetylation plays an essential role in gene transcriptional regulation. The evolutionarily conserved bromodomain (BrD) functions as the acetyl-lysine binding domain (Dhalluin et al., 1999) for acetylated histones and transcription factors, which is required for ordered gene transcription in chromatin (Sanchez and Zhou, 2009). BRD4 is a representative member of the BET family of proteins, characterized by two tandem bromodomains (BrD1 and BrD2) followed by an extra-terminal (ET) domain. Through its bromodomain/acetyl-lysine binding, BRD4 functions to facilitate recruitment of transcription factors to target genes, assembly of the mediator complex at enhancer sites, as well as activation of paused RNA polymerase II complexes for productive transcriptional elongation (Chiang, 2009). Numerous studies reported that broad chemical inhibition of both BET bromodomains effectively blocked genome-wide transcription. This was particularly true for genes regulating proliferation of cancer cells, including NUT midline carcinoma (Filippakopoulos et al., 2010), acute myeloid leukemia (Zuber et al., 2011), MLL-fusion leukemia (Dawson et al., 2011), and neuroblastoma (Puissant et al., 2013). It was also suggested that by modulating gene transcription in immune cells, BrD inhibition has a therapeutic role in inflammatory diseases (Nicodeme et al., 2010; Zhang et al., 2012a). However, the use of selective inhibitors of single bromodomain could have distinctive functional features.

We addressed this question in oligodendrocyte lineage cells, the myelin-forming-cells of the central nervous system whose differentiation has been previously shown to require cell cycle exit (Casaccia 2003 and Magri et al. 2014a and Magri et al, 2014b) and histone deacetylase activity (Marin-Husstege et al., 2002; Shen et al. 2008). In this lineage, the early progenitor stage is characterized by global protein lysine acetylation and decreased global histone acetylation, was previously identified as critical for the proper onset of oligodendrocyte differentiation (Shen et al., 2008; Wu et al., 2012; Ye et al., 2009). Therefore, we reasoned that oligodendrocyte lineage cells would be a suitable biological system to test the functional consequences of BET protein bromodomains inhibition using chemical inhibitors selective for only one or both bromodomains of BET proteins.

Notably, previous studies reported distinctive functions of the two bromodomains of BET proteins, possibly consequent to the interaction with lysine-acetylated histones or with transcriptional proteins (Gamsjaeger et al., 2011; Huang et al., 2009; Jang et al., 2005; Lamonica et al., 2011; Schroder et al., 2012; Shi et al., 2014; Yang et al., 2005; Zhang et al., 2012a). In the case of human BRD4, the first bromodomain appears dedicated to anchoring this molecule and its associated proteins to target gene promoter and enhancer sites in chromatin, through binding to di-acetylated H4K5ac/K8ac (a mark for gene transcriptional activation); while the second bromodomain was associated with the recruitment of non-histone proteins (i.e. transcription factors and the pTEFb complex) to target genes. In the case of BRD3, however, it is the first bromodomain that binds to the hematopoietic transcription factor GATA1, (Gamsjaeger et al., 2011; Lamonica et al., 2011), thereby suggesting context dependent different functions of the two bromodomains of the BET proteins in regulation of ordered gene transcription in chromatin.

This distinctive and unique ligand binding selectivity of the two bromodomains has been attributed to few amino acid residues that distinguish the first and second bromodomains within each BET protein, while they all share nearly identical residues at the corresponding acetyl-lysine binding pocket. In an effort to understand specific molecular functions of the individual bromodomains of BET proteins, we developed small molecule chemical inhibitors that are capable of selectively modulating acetyl-lysine binding activity of the first and/or second bromodomains of BET proteins, and evaluated their effects on the progression of oligodendrocyte progenitor cells towards differentiation.

RESULTS AND DISCUSSION

Structure-Guided Development of Selective BET BrD Inhibitor, Olinone

We employed a structure-guided design strategy to develop selective small molecule inhibitors for the BET bromodomains (Figure 1A). Our rational design of new BET-specific BrD inhibitors started with a chemical scaffold of tetrahydro-pyrido indole that was present in an NMR-based screen hit (MS7972) and showed modest activity as an inhibitor of the CBP bromodomain (Figure 1B) (Sachchidanand et al., 2006). This chemical scaffold is amendable to varying chemical modifications that can be synthetically added on to optimize its interactions with a target protein. Guided by the structural insights of MS7972 bound to the CBP bromodomain, we designed a series of 1-substituted-2,3,4,5-tetrahydro-pyrido-[4,3-b]indol-1-ones as new inhibitors for the BET BrDs. These molecules extend from the key features present on MS7972 for the binding to the BET BrDs to contain a longer acetamidoalkyl substituent at the N-indole as well as an endocyclic peptide bond to enhance interactions with the BrDs (Figure 1B).

Figure 1. Structure-guided design of Olinone, a selective inhibitor for the BET BrD1.

Figure 1

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(A) Schematic illustration of domain organization of the members of the BET protein family (BrD: bromodomain; ET: extra-terminal domain).

(B) Structure and binding affinity of Olinone and its analogs for the BRD4-BrD1, determined by using isothermal titration calorimetry (ITC) method. Results are representative of three independent experiments and the error is SD.

(C) ITC determination of binding affinity of Olinone to the two individual BrDs of BRD4, BRD3, and BRD2.

(D) Selectivity binding of Olinone to BRD4-BrD1 vs. BRD4-BrD2, as illustrated by 2D 1H-15N HSQC spectra of a given bromodomain in the free form (black) and in the presence of Olinone (red); as well as by isothermal titration calorimetry.

(E) Phylogenetic tree of the human bromodomain family. Dots indicate the BrDs for which Olinone binding affinity was evaluated in the assay described in B, C. Dots are color-coded for high affinity (red), modest affinity (blue), or no binding (gray).

(F) Structural basis of selective binding of Olinone to BRD4-BrD1. Upper left, crystal structure of Olinone (yellow) bound to BRD4-BrD1, depicted in a ribbon diagram. The Fo-Fc electron density map was computed after simulated annealing with the compound omitted from the atomic model and shown in blue mesh (contoured to 1.0 σ). Upper right, structural comparison of BRD4-BrD1 and BRD4-BrD2 (pdb: 2YEM), showing steric clash between Olinone with His435 in BRD4-BrD2. Lower left and right, superimposition of crystal structures revealing how Olinone (yellow) and an H4K5ac/K8ac peptide (green) are bound to the BRD4-BrD1 in the protein/ligand complex, depicted in ribbon diagram or electrostatic surface representation (right). Side chains of key amino acid residues at the ligand-binding site in the protein are color-coded by atom type, and bound water molecules are shown as red spheres.

See also Figure S1, Figure S2, and Table S1.

The synthesis of the newly designed compounds was achieved in four steps (see Figure S1 in Supplemental Information). Phenylhydrazone 1 was prepared by reaction of phenyl-hydrazine with 2,4-piperidinedione in water (with 10% acetic acid) under nitrogen atmosphere. Next, the pyrido-indole scaffold was constructed by the Fisher indole synthesis from treatment of phenyl-hydrazone 1 with sulfuric acid (70%) at 0°C (Rodriguez and Temprano, 1989). The resultant 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]indol-1-one 2 was N-alkylated with a corresponding phthalimido protected alkyl bromide in the presence of lithium bis(trimethylsilyl) amide as a base (Coldham et al., 2007). Finally, treatment of N-phthalimido protected substituted pyrido-indoles with hydrazine hydrate and subsequent acetylation afforded the final compounds 4a-d with good yields. Similar procedures were used to generate compounds 5 and 6 (Figure 1B).

We assessed binding affinity of 4a-d, 5 and 6 to BrDs of BET proteins using isothermal titration calorimetry (ITC) technique (Figure 1C and Figure S2A). The structure-activity relationship data revealed that the length of the N-alkylated chain plays an important role in binding to BRD4-BrD1. An N-alkylated chain of four methylene units as in 4b seems to be optimal in binding to BRD4-BrD1, with binding dissociation constant Kd of 3.4 μM. This represents a greater than 15-fold improvement in binding affinity as compared to those analogs that comprise three, five or six methylene groups, i.e. 4a, 4c and 4d, respectively (Figure 1B). Further, elimination of the acetyl group (5) or the entire N-acetylated group (6) resulted in a dramatic loss in binding affinity. We named the best compound of the series 4b, Olinone, for its cellular activity as described below. Consistently, we observed that Olinone exhibits preferred binding for the BrD1 over the BrD2 from all three BET proteins BRD4, BRD3 and BRD2, as assessed by ITC measurements (Figure 1C).

We next profiled Olinone binding selectivity to different bromodomains using 2D 1H-15N HSQC NMR spectroscopy (Figures 1D and Figure S2B). The NMR method offers a reliable assessment for ligands that bind to target proteins with tens-to-hundreds micromolar affinity, which is complementary to ITC that allows quantitative measurements of ligand binding in low micromolar affinity. Notably, Olinone showed modest binding to the bromodomains from CBP and PHIP with Kd of 33 μM and 103.4 μM, respectively, very weak binding to BRD4-BrD2 with Kd >300 μM, and almost no detectable binding to the bromodomains from many other proteins including BRD7, PCAF, ASH1L, TAF1L, SMARCA4, BAZ1B, BAZ2B, ATAD2, BPTF and TRIM24 that represent different subgroups of the human bromodomain family (Figure 1E and Figure S2C). The selectivity for the BrD1 over the BrD2 of the BET proteins was further confirmed using a fluorescence anisotropy competition binding assay (Figure S2D). Moreover, through characterization of Olinone binding to a set of wild type or acetyl-lysine binding deficient mutants of the tandem BrDs of BRD4 proteins, we observed that the two BET BrDs function almost independently of one another, consistent with a long linker sequence that connects them (Figure S2D). Collectively, these results demonstrate that Olinone is a highly selective inhibitor against the first bromodomain of the BET proteins.

Molecular Basis of Selective Recognition of BRD4 BrD1 by Olinone

The molecular basis of Olinone recognition of the BrD1 of BET proteins was revealed by our new X-ray crystal structure of the BRD4 BrD1/Olinone complex, determined at 0.94Å resolution (Supplemental Table 1). Olinone forms a chair-like conformation in the bound state (Figure 1F, upper left); its tri-heterocyclic moiety as the seat packs against the side chains of Trp81 and Pro82 in the one-turn helix Z’, and Ile146 in helix C, and interacts with Asp144 at the opening of the acetyl-lysine binding pocket formed between the ZA and BC loops. Asp144 is one of a few residues that are different between BRD4-BrD1 and BrD2 in the acetyl-lysine binding site; its corresponding residue in BrD2 is His435, which would clash with the indole moiety of Olinone (Figure 1F, upper right).

This major contribution to the overall ligand binding by Asp144 was confirmed in our detailed structural analysis of the BRD4-BrD1/Olinone complex. The acetyl chain of Olinone, as its back, intercalates into a hydrophobic pocket lined with Val87, Leu92, Leu94, and Tyr139 of the ZA loop. The carbonyl oxygen of the acetyl group of Olinone forms a hydrogen bond (2.9Å) to the amide nitrogen of the highly conserved Asn140 in BRD4-BrD1. Notably, the acetyl chain of Olinone adopted an almost identical conformation and position as that of the acetylated-K5 side chain of histone H4K5ac/K8ac peptide when bound in BRD4-BrD1, whereas the piperidone ring of Olinone filled the site for K8ac binding in the H4 peptide (Figure 1F, lower left and right). This explains the optimal length of the acetyl side chain of Olinone in its chemical series, and Olinone’s effective inhibition of BRD4-BrD1 binding to the di-acetylated H4K5ac/K8ac peptide. The binding affinity of the latter interaction, which is important for BRD4 functions in gene transcriptional regulation (Chiang, 2009; Filippakopoulos et al., 2012), is estimated to be Ki of 26 μM. Given that a few ligand binding residues in BRD4-BrD1 such as Ile146 and Asp144 are not conserved outside the BET proteins in the human BrD family proteins (Sanchez and Zhou, 2009), this new structure explains how Olinone is selective for the first BrD of the BET proteins over other bromodomains from different transcriptional proteins.

It is important to note that Olinone is the first reported bromodomain inhibitor (BrDi) with a superior selectivity between the two bromodomains of BET proteins, even as compared to the recently developed: MS436, a diazobenzene compound (Zhang et al., 2013), or BET-specific BrDi, MS417, which is a thienotriazolo-diazepine compound (Zhang et al., 2012b) and structurally related to (+)-JQ1 (Filippakopoulos et al., 2010) and I-BET (Nicodeme et al., 2010). These BET-specific BrDis, particularly MS417 and (+)-JQ1 have high affinity for bromodomain (Kd of 20-100 nM) but do not discriminate between the first and second BrDi of the BET proteins, due to greater than 95% sequence identity at the acetyl-lysine binding pocket.

Selective BET-BrD1 Inhibition favors, while broad BET BrD Inhibition prevents differentiation of oligodendrocyte progenitors

To begin evaluating the differential effect of selective inhibitors of the first bromodomain (such as Olinone) and compare it to that of broad BET inhibitors (i.e. MS417 or JQ1), we measured the levels of expression of the BET protein-encoding genes in the developing white matter tracts of the mouse brain. Of the analyzed molecules, Brd2 was the most abundant BET transcript in the neonatal brain (Figure 2A). However, it is worth noting that the levels of all the BETs (i.e. Brd3, Brd4 long, and Brd4 short isoform) progressively declined in developing white matter tracts during a temporal window that was coincident with the differentiation of progenitors into myelin forming oligodendrocytes, as detected by the expression of the myelin gene Mbp (Figure 2A).

Figure 2. Developmental expression of BET family members in corpus callosum and characterization of BrD inhibition effect in oligodendroglial cells.

Figure 2

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(A) qRT-PCR showing the transcript levels of Brd2, Brd3 and Brd4-long and -short isoforms in developing corpus callosum relative to 18S expression (n=3 animals per age group. One-Way ANOVA with Bonferroni’s post test, *** P<0.01 vs P0).

(B) MTT assay to measure cell viability in primary cultured mouse OPCs, primary cultured mouse astrocytes and Olineu cells after 72 hours of treatment with BrDi at indicated doses (μM). Red lines indicate the dose range matching with the Kd of the inhibitors, showing not toxicity at indicated concentrations. The right panel shows Olinone and MS417 Kd for the first and second bromodomain of BRD4 (n=3; One-Way ANOVA with Dunnett’s post test, *P<0.05, ***P<0.001).

(C) Selective BET-BrD inhibition decreases histone acetylation. Top panel, experimental timeline. Bottom panel, automated high throughput confocal screening for histone acetylation. The graph shows the quantification of the average nuclear residue-specific histone acetylation staining intensity referred to DMSO (n=20 sections; n=3) (two tailed t-test **P<0.01, ***P<0.001). Error bars, ± s.e.m.

(D) Effect of selective BET-BrD inhibition during the early differentiation phase of mOPCs. qRT-PCR showing the expression levels of differentiation markers after 24 hours of BrDi treatment (Olinone at 1 μM and MS417 at 0.05 μM). Results were normalized to 18S with relative mRNA levels in DMSO treated control cells set to 1 (n=4; One-Way ANOVA with Dunnett’s post test, ***P<0.001).

See also Figure S3, and Table S2.

To assess the ability of Olinone to modulate oligodendrocyte progenitor differentiation in mouse cultures, we first determined the effect of Olinone and MS417 on cell viability after 72-hour treatment with increasing concentrations of BrDi, using a MTT assay (Figure 2B). No significant cytotoxicity was observed for Olinone or its inactive analog Compound 5, up to a concentration of 10 μM. In contrast, treatment with MS417 was toxic at doses of 500 nM and above (Figure 2B). Therefore we opted to use Olinone at 1 μM and MS417 at 0.05 μM for most of the cellular studies.

We had previously reported that global histone acetylation characterizes proliferating progenitors and that deacetylation is necessary to initiate a differentiation program (Marin-Husstege et al., 2002; Shen et al., 2005). We therefore asked whether treatment with the BrD1 specific inhibitor Olinone or with the BrD1/BrD2 inhibitor MS417 would similarly affect histone acetylation and gene transcription in immortalized oligodendrocyte progenitors (Olineu). The first experiment was conducted in the presence of mitogens, which represent a strong stimulus towards the persistence of global acetylation in these cells (Wu et al., 2012). Interestingly, in these cells, Olinone treatment resulted in an average reduction of histone H3 global acetylation, detected primarily at residues H3K14ac and H3K36ac, with little changes at H4K5ac and H4K12ac (Figure 2C). Treatment with MS417, in contrast, was less effective at reducing the histone acetylation levels at H3K14, although it decreased the levels of H3K36ac (Figure 2C). Despite the similar effect on histone deacetylation and on the expression of oligodendrocyte specific genes (Figure 2D), treatment of cells with Olinone consistently increased the transcript levels of myelin genes associated with the differentiated phenotype while treatment with MS417 decreased the levels of these genes while increasing transcripts characteristic of the progenitor stage (Figure 2D). These results suggested that BrD1 specific inhibition promoted differentiation, while inhibition of both BrD1 and BrD2 prevented it and retained the cells at a progenitor stage.

Since an obligatory relationship exists between cell cycle exit and differentiation in oligodendrocyte lineage cells (Swiss and Casaccia, 2010), and BrDis have been reported to inhibit proliferation of cancer cells, we asked whether the differential effect on differentiation observed after treatment with Olinone or MS417 could be due to a distinct modulation of the cell cycle. To address this question we adopted three different approaches including: immunohistochemistry with antibodies for the cell cycle marker Ki67 (Figure 3A), expression levels of cell cycle genes (Figure 3B) and FACS analysis (Figure 3C). A detailed quantification of Olinone or MS417 treated cells using immunoreactivity for Ki67 in order to identify proliferating cells, did not reveal any difference among treatment groups (Figure 3A). Gene expression data analysis revealed an overall interesting effect of MS417 - but not Olinone - on the transcript levels of cell cycle genes (Figure 3B). Consistent with the inhibitory effect of MS 417, treatment with this broad BET inhibitor decreased the expression of the gene encoding for p21Waf1, which had been previously shown to positively impact oligodendrocyte differentiation (Zezula et al., 2001) while increasing the levels of cell cycle genes preventing differentiation, such as E2F1 (Magri et al., 2014b) and c-Myc (Magri et al., 2014a). Finally, a measure of the DNA content by FACS analysis did not reveal significant differences in the relative proportion of cells in the different stages of the cell cycle (Figure 3C). Taken together, these data suggest that the pro-differentiation effect of the BrD1 inhibitor Olinone could not be interpreted as the consequence of precocious cell-cycle exit.

Figure 3. Treatment with BrDi does not affect proliferation of primary cultured mouse OPCs and cell cycle exit upon differentiation stimuli.

Figure 3

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(A) Representative confocal images of proliferating mOPCs stained with Ki67 antibody (green) and DAPI (blue). Scale bar: 50 mm. The graph represents the % of mean pixel intensity. Results shown are relative DMSO treated control cells (set to 100%). (n=3; One-Way ANOVA with Dunnett’s post test).

(B) qRT-PCR showing the expression levels of cell cycle genes after 24 hours of BrDi (Olinone at 1 μM and MS417 at 0.05 μM). Results were normalized to Gapdh, with relative mRNA levels in DMSO treated control cells set to 1 (n=3; One-Way ANOVA with Dunnett’s post test, ***P<0.001). Error bars, ± s.e.m.

(C) Cell cycle analysis of BrDi treated mOPCs for 24 hours as indicated. Color bars represent % of cells in each cell cycle phase (n=2; two tailed t-test). Error bars, ± s.e.m.

Temporal control of BrDi effectiveness on oligodendrocyte progenitor differentiation

To gain a better understanding of the effect of the BrD1 specific or broad BET bromodomains inhibitors on differentiation, we conducted a dose/response experiment and measured the transcript levels of late differentiation genes. In both cases we detected a dose-dependent response, corresponding to either MS417-mediated decrease or Olinone-induced increase of myelin gene transcripts at a dose comparable to the in vitro binding affinity to the individual BrDs of BET proteins. This effect was clearly detectable as early as the first 24-hours of differentiation (Figure S3).

To further determine the window of effectiveness of BrDi treatment, we treated cells with either Olinone or MS417 for different amount of time in the presence of mitogens or in their absence. A 24-hour treatment in the presence of mitogens was not sufficient to detect the effects of MS417 or Olinone (Figure 4A). In contrast, treatment for 72 hours starting after the removal of mitogens was sufficient to alter the expression of the late differentiation genes (Figure 4B). Thus inhibition of bromodomain binding was not sufficient to remove the inhibitory block of differentiation caused by mitogen stimulation and was only detected after the cells exited the cell cycle. The first 24 hours following cell cycle exit represent an important critical window for the onset of a complex transcriptional program of differentiation. We therefore asked whether bromodomain inhibition during this critical period was sufficient to induce long-lasting changes. For this reason, after the first 24 hours of treatment, we washed out the drugs and allowed the cells to recover in differentiation medium prior to measuring the transcript levels of differentiation genes (Figure 4B). In these conditions, the levels of gene transcripts measured after washout did not reveal any change, which indicated that the effect of blocking bromodomains was not stable, but reversible over time.

Figure 4. Temporal control of BrD inhibition on OPC differentiation.

Figure 4

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(A) BrD inhibitors do not have broad effects on the expression of oligodendrocytic lineage markers in proliferating conditions. Top panel, experimental time line. Bottom panel, qRT-PCR showing the expression levels of differentiation markers after 24 hours of BrDi treatment (Olinone at 1 μM, and MS417 at 0.05 μM) in the presence of mitogens. Results were normalized to 18S with relative mRNA levels in DMSO treated control cells set to 1 (n=4; One-Way ANOVA with Dunnett’s post test, ***P<0.001).

(B) Top panel, different experimental paradigms of BrDi treatment during mOPC differentiation. Bottom panel, qRT-PCR results showing the transcript levels of late differentiation markers (Mag, Mog, Mbp, Plp) in mOPC differentiated for 72 hours and treated with BrDi as indicated: I) during 72 hours or II) during the first 24 hours of differentiation and then kept in DM with no BrDi. Doses used: Olinone 1 μM and MS417 0.05 μM. (n=3; One-Way ANOVA with Dunnett’s post test, *P<0.05; **P<0.01; ***P<0.001).

(C) Top panel, different experimental paradigms of BrDi treatment. Bottom panel, qRT-PCR results showing the transcript levels of late differentiation markers (Mag, Mog, Mbp, Plp) in mOPC differentiated for 72 hours and treated with BrDi as indicated: I) during 72 hours or III) during the last 36 hours of differentiation. Doses used: Olinone 1 μM and MS417 0.05 μM. (n=3; One-Way ANOVA with Dunnett’s post test, *P<0.05; **P<0.01; ***P<0.001). Error bars, ± s.e.m.

The next question was to define whether inhibition of one or both BrDi would also be detected if cells were treated after the program of oligodendrocyte differentiation had started (i.e. after at least 48 hours in mitogen-free differentiation medium (Figure 4C). In this case the broad BET inhibition by MS417 still significantly decreased the expression of late differentiation genes, even though to a lesser extent than continuous treatment, while the pro-differentiation effect of blocking only the first BrD1 by Olinone was dramatically reduced, with the exception for Mog levels (Figure 4C). Collectively, these data suggest that BET activity in oligodendrocyte lineage cells is differentially affected by inhibitors of the first BrD1 or of both bromodomains of BET proteins.

Specificity and off-target effects

We then examined the specificity of the effect of BrDi on oligodendrocyte differentiation by treating progenitors with structurally related but inactive compounds and conducted a detailed immunophenotypic (Figure 5A) and transcriptional (Figure 5B) characterization of mouse primary oligodendrocyte progenitor differentiation. Immunoreactivity for the proteoglycan NG2 was used to identify early progenitors, O4 to label cells expressing lipid sulfatides at an intermediate stage of maturation, and myelin basic protein (MBP) to identify differentiated oligodendrocytes. In untreated cultures, the progression of progenitors to mature oligodendrocytes was characterized by the progressive decrease of NG2 and the gradual acquisition of O4 immunoreactivity, followed by the acquisition of a myelinating phenotype, characterized by the extension of myelin membranes recognized by both O4 and MBP antibodies.

Figure 5. Olinone specifically promotes the expression of the oligodendrocyte intermediate differentiation marker O4.

Figure 5

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(A) Top panel, experimental timeline. Bottom panel, confocal image of mOPCs treated for 24h with DMSO, Olinone (1 μM), Compound 5 (1 μM), MS417 (0.05 μM) and MS566 (0.05 μM) in differentiation medium (DM) and stained with NG2 (green), O4 (Red) antibodies and DAPI (Blue). Scale bar: 50 mm.

(B) The graph shows the quantification of the percentage of NG2+/O4+ staining in treated cells relative to DMSO (n=5-7 sections per condition, n=3 independent experiments. One-Way ANOVA with Dunnett’s post test, **P<0.01). c) qRT-PCR showing the expression levels of differentiation markers after 24 hours of BrDi (Olinone at 1 μM, Compound 5 at 1 μM, MS417 at 0.05 μM, MS566 at 0.05 μM). Results were normalized to Gapdh, with relative mRNA levels in DMSO treated control cells set to 1 (n=4; One-Way ANOVA with Dunnett’s post test, ***P<0.001).

Treatment of primary oligodendrocyte progenitors with the BrD1 specific inhibitor Olinone, promoted a shift towards the differentiated phenotype, as documented by the increased percentage of O4+ cells (Figure 5B), compared to DMSO treated cells. This effect was not detected in cells that were treated with the inactive Olinone analog, Compound 5. Treatment of primary oligodendrocyte progenitors in the exact same conditions with the broader BET inhibitor MS417, in contrast, generated consistently opposite effects as those induced by Olinone. MS417 antagonized the progression towards a mature phenotype and reduced the number of NG2+/O4+ cells (Figure 5B). The effect was highly specific as the inactive enantiomer of MS417 (Zhang et al., 2012a) called MS566, was incapable of eliciting the same biological effect (Figure 5B). At a transcriptional level, the pro-differentiation effect of Olinone, but not Compound 5 was also detected by the increased transcript levels for the late-differentiation markers Mag, Mog, Plp and Mbp (Figure 5C), whereas MS417 treatment, decreased the expression of these late-differentiation markers in a dose-dependent fashion, while increasing the expression of the transcriptional inhibitor of myelin genes, Hes5 (Figure 5C).

Selective inhibition of the BrD1 enhances, while inhibition of both BET BrDs prevents differentiation of oligodendrocyte progenitors

To begin understanding whether the inhibitory effect of MS 417 was consequent to the inhibition of both BrDs of BET proteins, we used two distinct approaches. First, we asked whether Brd2 in cultured progenitors was also the most prominent BET protein to be detected in the oligodendrocyte lineage cells (Figure 6A), which was consistent with our in vivo studies in developing myelinating tracts (Figure 2A). We then expressed either a wild type eGFP-tagged Brd2 (eGFP-Brd2wt) molecule or a mutant form of Brd2 (eGFP-Brd2mut) lacking the acetyl-lysine binding domains in the first and second BrD of the protein molecule (Hnilicová J et al, 2013) in Olineu cells and asked whether the two molecules were differentially distributed within the nucleus of transfected cells. While the eGFP-Brd2wt transfected cells were characterized by a nuclear diffuse distribution, those transfected with the mutant eGFP-Brd2mut - lacking the ability to bind acetyl-lysine - showed a very distinctive punctate distribution (Figure 6B). Treatment with the BrD1 specific inhibitor Olinone did not alter the distribution of eGFP-Brd2wt, whereas treatment of MS417 induced a punctate distribution of the fusion protein, which resembled that observed in eGFP-Brd2mut transfected cells (Figure 6B). Taken together, these data suggest that the second bromodomain of Brd2 is important for its subcellular localization since Olinone, which inhibits only the first bromodomain, did not have any effect on eGFP-Brd2wt, while MS417 (inhibiting both the first and second bromodomains of Brd2) as well as a mutant Brd2 in both bromodomains showed altered nuclear distribution.

Figure 6. Selective BrDi inhibition differentially affects Brd2 nuclear localization and the progression of primary OPCs towards a fully differentiated phenotype.

Figure 6

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(A) qRT-PCR showing the relative expression levels of Brd2, Brd3 and the long and short isoforms of Brd4 in mouse primary cultured OPCs. Brd2 is the highly expressed isoform in mOPCs. Olig2 (as a marker of oligodendrocyte lineage), Mbp (as a marker of differentiated oligodendrocytes) and Gfap levels (as a marker of astrocytes) were also determined as controls. Results were normalized to 18S (n=3. Error bars, ± s.e.m).

(B) Changes in Brd2 nuclear localization after BrDi treatment ± TSA. Olineu cells transfected with Brd2-eGFP or mutBrd2-eGFP (with two BrDs mutated) in the presence or absence of BrDi (Olinone 1 μM, MS417 0.05 μM), DMSO is used as control. TSA (30 μM) was added after 24 hours of BrDi treatment in order to reduce rapid histone acetylation. Cells were fixed at the indicated time points (1 hour of TSA treatment and 6 hours of TSA treatment). Scale bar= 50 mm. (n=3). Error bars, ± s.e.m.

(C) Top panel, experimental timeline. Transcript levels of late differentiation markers (Mag, Mog, Mbp, Plp, Sirt2) and the myelin gene transcriptional inhibitor (Hes5) in response to 72 hours of BrDi treatment (Olinone at 1 μM, Compound 5 at 1 μM, MS417 at 0.05 μM, MS566 at 0.05 μM, MS611 and MS765/RVS208 at 0.5 μM). Results were normalized to Gapdh with relative mRNA level in DMSO treated control cells set to 1 (n=8; One-Way ANOVA with Dunnett’s post test, *P<0.05; **P<0.01; ***P<0.001). Error bars, ± s.e.m.

(D) Representative confocal images of mOPCs treated with DMSO, Olinone (1 μM) and MS417 (0.05 μM) during 72 hours in differentiation medium (DM) and stained with MBP antibody (grey) and DAPI (Blue). Scale bar: 100 mm.

(E) The graph shows the quantification of the percentage of mature MBP+ membrane bearing cells relative to DMSO (n=3 with 7-8 sections per condition; One-Way ANOVA with Dunnett’s post test, *P<0.05).

See also Figure S4, and Figure S5.

The second approach entailed the use of additional chemical probes selective for the inhibition of only the first (MS611) or only of the second (MS765) bromodomain of BET proteins (Figure S2D; and see the chemical synthesis in Supplemental Information and Figure S4). Notably, MS765, which is also known as RVX-208, is a compound that was developed by Resverlogix Corp. and shown to have a 15-30-fold selectivity for the second bromodomains over the first of BET proteins (McLure et al., 2013; Picaud et al., 2013). To investigate whether inhibitors of only the first or second bromodomain of BET modulated the progression of oligodendrocyte progenitors towards a differentiated phenotype, we characterized the effect of 72 hours treatment with: Olinone and MS611 (as inhibitors of the first bromodomain), MS765/RVS-208 (as inhibitor of the second bromodomain) and MS417 (as inhibitor of both bromodomains). Differentiation of progenitors into oligodendrocyte was monitored by measuring transcripts for late differentiation genes (Mag, Mog, Plp and Mbp), assessing immunoreactivity for late differentiation markers (MBP+/O4+) and quantifying the number of membrane-bearing cells. We observed that treatment of Olinone and with the other inhibitor of the first bromodomain, MS611 increased expression of late differentiation markers (Figure 6C) while the inhibitor of the second bromodomain, MS765/RVS-208 had no effect and the broad MS417 inhibitor inhibited the expression of these genes (Figure 6C). The number of MBP+ membrane bearing cells (Figure 6D and Figure S5) was similarly increased by Olinone or MS611, unchanged by MS765/RVS208 and decreased by MS417 treatment (Figure 6E).

Collectively, our results suggest that selective inhibition of the first bromodomain (such as by Olinone or MS611) facilitates the progression of primary oligodendrocyte progenitors towards a differentiated phenotype; inhibition of the second bromodomain (such as by MS765/RVS208) has no effect while inhibition of both bromodomains of BET proteins (such as MS417) negatively impacts the differentiation process.

Due to the dynamic nature of lysine acetylation, chemical inhibition of these bromodomains may result in lysine de-acetylation and subsequent ubiquitination-mediated degradation of the bromodomain-binding partners. Therefore, chemical inhibition of the two different bromodomains is likely to yield distinct functional outcomes. In this study we compared the effect of a selective inhibitor (Olinone) for the first bromodomain of BET proteins with that of an inhibitor (MS417) that is equally effective for both bromodomains, and observed dramatic differences in the biological response of oligodendrocyte progenitors to these treatments. While Olinone promoted differentiation, MS417 inhibited it. This effect was associated with the up-regulation of genes such as E2f1 and Myc, which we have shown to be inhibitory for differentiation (Magri et al., 2014a; Magri et al., 2014b) and the down-regulation of pro-differentiation genes such as Cdkn1b (Zezula et al., 2001) One explanation is that under MS417 treatment, a key transcription factor that is recruited by BET proteins could be degraded in cells, whereas Olinone treatment would leave such a transcription factor intact, owing to its association with the second bromodomain of BET proteins. Given the complex nature of gene transcription progression of oligodendrocyte progenitor differentiation, the identification of such a transcription factor(s), and the detailed mechanism of action of chemical inhibition of BET proteins by Olinone and/or MS417 will have to await future investigation. Finally, recent studies show that some kinase inhibitors such as BI-2536 and TG-101348 developed against PLK1 and JAK2-FLT3 kinases, respectively, have off-target effects on bromodomains in gene activation, including BET bromodomains (Ciceri et al., 2014; Ember et al., 2014). While the reverse may also be possible, this is less likely for Olinone because of its distinct chemical structure from those dual kinase-bromodomain inhibitors. Moreover, similar results obtained with MS611 that shares the same selectivity for the first bromodomain over the second bromodomain of BET proteins as that of Olinone further supports the findings of this study.

SIGNIFICANCE

BET family proteins through their bromodomain/acetyl-lysine binding capability, regulate gene transcription in chromatin in multiple steps, including the recruitment of transcription factors to target genes, assembly of the mediator complex at enhancer sites, and activation of paused RNA polymerase II machinery complexes for productive transcriptional elongation (Chiang, 2009). The two bromodomains of BET proteins, as shown in BRD3 and BRD4, have context dependent different functions in regulation of ordered gene transcription (Gamsjaeger et al., 2011; Huang et al., 2009; Jang et al., 2005; Lamonica et al., 2011; Schroder et al., 2012; Shi et al., 2014; Yang et al., 2005; Zhang et al., 2012a).

In this study, we highlight the functional importance of chemical modulation of acetylation-mediated protein-protein interactions in transcriptional control of the oligodendrocyte progenitor cell differentiation. We identify Brd2 as the prominent BET expressed in the oligodendrocyte lineage cells. In contrast to what reported in other cell types, treatment of oligodendrocyte progenitors with bromodomain inhibitors did not modulate the acetylation of histone H4 and did not affect proliferation. These cells were quite responsive to treatment with selective chemical probes specific for the first, second or both bromodomains of BET proteins. In this functional context, we demonstrate, for the first time, that a small molecule bromodomain inhibitor, designed to selectively modulate the master chromatin regulators BET proteins, could positively or negatively influence the differentiation of oligodendrocyte progenitors, depending on the targeted bromodomain. Our data further indicate that a complete chemical inhibition of BET bromodomain/acetyl-lysine binding activity may possess adverse effects on oligodendrocyte progenitor differentiation, an important step for myelination of axons. Overall, this study opens opportunities for the future development of new therapeutic treatments of demyelinating disorders, and also novel chemical agents that could improve the efficiency of the lineage progression of adult stem cells into specialized cell types.

EXPERIMENTAL PROCEDURES

Isothermal Titration Calorimetry

Experiments were carried out on a GE MicroCal auto-ITC200 instrument at 15°C while stirring at 1,000 rpm in the ITC buffer of pH 7.4, consisting of 50 mM sodium phosphate and 150 mM sodium chloride as described previously (Zhang et al., 2012a). Compound concentration was determined by NMR and protein concentrations by A280 measurements. The protein sample (~75 μM) was placed in the cell, whereas the micro-syringe (130 μL) was loaded with a solution of the correspondent compound (0.8 μM) in the ITC buffer. All titrations were conducted using 20 identical injections of 2 μL with a duration of 4 sec per injection and a spacing of 150 sec between injections. The heat of dilution was determined by independent titrations (protein into buffer) and was subtracted from the experimental data. The collected data were implicated in the MicroCal™ Origin software supplied with the instrument to yield enthalpies of binding (H) and binding constants (KB). Thermodynamic parameters were calculated (ΔG = ΔH − TΔS = −RTlnKB, where ΔG, ΔH and ΔS are the changes in free energy, enthalpy and entropy of binding respectively). In all cases a single binding site model was employed.

Protein Crystallization, X-ray Diffraction Data Collection and Structure Determination

Purified BRD4-BrD1 protein (14 mg/mL) was mixed with Olinone 4b at 1:10 molar ratio of protein:ligand. The complex was crystallized using the sitting drop vapor diffusion method at 20°C by mixing 1 μL of protein solution with 1 μL of the reservoir solution that contains 25% tert-Butanol, 0.1M Tris-HCl pH 8.5. Crystals were soaked in the corresponding mother liquor supplemented with 20% ethylene glycerol as cryoprotectant before freezing in liquid nitrogen. X-ray diffraction data were collected at 100K at beamline ×6A of the National Synchrotron Light Source (NSLS) at Brookhaven National Laboratory. Data were processed using the HKL-2000 suite (Otwinowski and Minor, 1997). The BRD4-BRD1 structure was solved by molecular replacement using the program MOLREP (Vagin and Teplyakov, 1997), and the structure refinement was done using the program Refmac (Murshudov et al., 1997). Graphics program COOT (Emsley and Cowtan, 2004) was used for model building and visualization. Crystal diffraction data and refinement statistics for the structure are displayed in Supplemental Table 1 (see Supplemental Information).

Primary Oligodendrocytes

OPCs were prepared by sequential immunopanning and kept under undifferentiating conditions as described earlier (Watkins et al., 2008) until the onset of experiments. Briefly, oligodendrocyte progenitor cells (OPCs) were isolated from one P6 mouse pup brain using an immunopanning system enabling a purity of 95%. The dissected cortex was chopped in papain buffer, incubated for 20 min at 37°C and titrated in ovomucoid solution (CellSystems GmbH, Troisdorf, Germany). The single cell solution was centrifuged at 1,000 rpm for 10 min and resuspended in panning buffer and transferred to a bacterial culture plate coated with Anti-BSL1 Griffonia simplificonia lectin (Vector Labs, L-1100), for negative selection for 15 min, followed by a positive selection step with rat anti-mouse CD140a (Research Diagnostics, 10R-CD140AMS) as primary antibody and AffiniPure goat anti-rat IgG (H+L) (Dianova, 112-005-003) as secondary antibody for 45 min. The supernatant was aspirated, and the positive selection plate was washed with DPBS. The adherent OPCs were removed using trypsin, centrifuged for 10 min at 1,000 rpm, resuspended in mouse OPC Sato (Watkins et al., 2008) and plated in a T75 culture flask coated with poly-lysine. The OPCs were cultured in a humidified incubator at 5% CO2 and 37°C with media changes every 2 days. OPC were maintained proliferating in the presence of bFGF (20ng/ml) and PDGF (10ng/ml), while oligodendrocyte differentiation was induced by culturing the cells in the absence of mitogens and adding T3 60 nM (Sigma,T5516) to Sato medium.

Olineu transient transfection

Olineu (15.000 cells/well) were grown on CC2-coated 8 well chambers (Lab-TekTM, USA) for all transfection experiments. These cells were transiently transfected using X-tremeGENE 9 DNA transfection reagent (Roche Diagnostics) following the manufacturer’s protocol and using equal amounts of the eukaryotic expression vectors pEGFP-C1-Brd2 and pEGFP-C1-Brd2mut (with both bromodomains mutated) previously described in Hnilicova and coworkers (Hnilicova et al., 2013). After 24 hours of transfection, cells were treated for 24 hours with either BrD inhibitors (Olinone, MS417) at indicted concentrations or with DMSO as negative control. At this time point, one subset of cells were fixed and stained with DAPI (1:10000; Molecular Probes, Eugene, USA) for immunochemistry.

Proliferation assays

Mouse primarily cultured oligodendrocytes were cultured in CC2-coated 8 well chambers (Lab-TekTM, USA) in SATO medium with growth factors (bFGF and PDGFaa). Cells were treated with either BrD inhibitors at indicated concentrations or DMSO as control for 24 hours. To determine changes in proliferation, cells were fixed and immunolabeled with antibodies against the cell proliferation marker Ki67 (ab14298, 1:1000) 1 hour at room temperature.

Immunohistochemistry

Immunohistochemistry of cultured cells with O4 antibodies was performed live. Cells were grown on CC2-coated 8 well chambers (Lab-TekTM, USA) for all immunocytochemistry. For staining oligodendrocyte lineage markers, cells were rinsed gently with PBS and incubated live with O4 hybridoma supernatant (1:10, gift from Dr Bansak, University of Farmington, Farmington, CT) for 30 minutes at 37°C. Cells were then fixed with 4% PFA for 15 minutes at room temperature and first incubated with blocking/permeabilization solution (PGBA plus 10 % normal goat serum, and 0.5% TritonX-100) for 1 hour at room temperature. For co-staining experiments cells were incubated with additional primary antibodies against NG2 (Chemicon AB5320, 1:300) and MBP (Covance SMI99, 1:500) overnight at 4 °C. A 1-h incubation with secondary fluorescent antibodies (Alexa Fluor 488 or 546) was performed the following day with counterstaining for DAPI (1:10000; Molecular Probes, Eugene, USA) to visualize cell nuclei.

Image acquisition and quantification

Images were captured at 20× and oil inmension-63× objective using LSM 710 Metaconfocal laser scanning microscope (Carl Zeiss MicroImaging, Inc.). For the quantification of the cells at different stages, three fields of each well and four wells of each condition were analyzed and the immunopositive cells were counted using NIH ImageJ software. To characterize the 24 hours culture, the proportion of NG2;O4 double positive was calculated by referring to the total number of cells (DAPI+). To characterize the 72 hours culture, the percentage of MBP+ membrane bearing cells was calculated by dividing the number of each type of cells by the number of total number of cells (DAPI+). OPC proliferation was determined by measuring the mean pixel intensity of DAPI/Ki67+ cells (>99% in each group) relative to DMSO values. One-Way ANOVA with Dunnett’s multiple comparison test was performed to assess statistical differences between control and experimental conditions after combing the total number of cells in each group, with a significance threshold of P<0.05.

Acetyl histone detection

Olineu cells were plated on 0.1mg/ml poly-d-lysine (Sigma P7886) coated 96-well plates at a density of 5000 cells per well. ODM medium was supplemented with 1% horse serum (Invitrogen 16050). 24hours after plating, cells were treated with either Olinone at 1 mM or MS417 at 0.05 mM as well as DMSO as a control for 48 hours. Cells were gently washed with PBS after completion of the treatment and fixed with 4% paraformaldehyde for 20min at RT. Cells were then washed and permeabilized with 0.1% triton for 30min at RT followed by incubation in PGBA plus 10 % normal goat serum for 1h. Cells were incubated with primary antibodies (H3-Acteyl 1:500 Upstate 06-599; H3AcK14 1:500 Active Motif 39599; H3AcK36 1:1000 Active Motif 39380; H4AcK5 1:2000 Active Motif 39584 and H4AcK12 1:1000 Active Motif 39928) overnight at 4C. The following day, samples were incubated for 1h with secondary fluorescent antibody AlexaFluor488 (Invitrogen A11034) and counterstained for DAPI (1:10,000, Molecular Probes). Measurement of pixel intensity of each primary antibody listed above were accomplished by automated high throughput screening using plate readers (Perkin Elmer EnVision; TECAN Sapphire and Genios) and high-content microscopes (Molecular Devices ImageXpress Ultra) at the Integrated Screening Core Facility at the Icahn School of Medicine at Mount Sinai. Two tailed Student’s t-tests were performed to assess statistical differences between the average values in each condition, with a significance threshold of P<0.05.

Cell cycle assay

mOPCs cells were grown in proliferating conditions until the reached 75% of confluence. At this point differentiation was induced and the cells were treated with BrDi as indicated. After 24-30 hours of treatment cells were rinsed twice with DPBS, trypsinized and pelleted with PBS. 1x106 per condition were fixed with cold 70% ethanol overnight. Cell cycle analysis was performed using the Muse Cell Cycle Analyzer (Millipore, MCH100106) following the manufacturer’s protocol.

Statistical analysis

Unless otherwise stated, all of the experiments were performed a minimum of three times. The results represent the mean ± S.E.M.. We compared the data using one-way ANOVA or two tailed t-test as appropriate and defined statistical significance at P < 0.05.

Supplementary Material

01
02

Highlights.

  • Oligodendrocyte progenitor cell (OPC) differentiation involves histone acetylation.

  • BET proteins regulate gene transcription during OPC differentiation.

  • Chemical inhibition of the first BrDs of BET proteins promotes OPC differentiation.

  • Chemical inhibition of both BrDs of BET proteins hinders OPC differentiation.

ACKNOWLEDGMENTS

We acknowledge the use of the NMR facility at the New York Structural Biology Center and the ITC instrument at the High-Throughout Screen Resource Center (HTSRC) at the Rockefeller University. We also wish to thank the staff at the X6A beamline of the National Synchrotron Light Sources at the Brookhaven National Laboratory for facilitating X-ray data collection. This work was supported in part by the Research Supplements to Promote Diversity in Health-Related Research Program from the National Cancer Institute (G.G.-N.), CTSA grant (UL1RR029887) to Icahn School of Medicine at Mount Sinai and by research grants from the National Institutes of Health (R03NS072578-01 to GGN, R01-NS52738 and R01NS42925 to PCB, R01HG004508 and R01CA87658 to MMZ).

Footnotes

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ACCESSION CODES

Structure factors and coordinates for the BRD4 bromodomain 1 in complex with Olinone are deposited at the RCSB Protein Data Bank under the RCSB ID code rcsb085830 and PDB ID code 4QB3.

SUPPLEMENTAL INFORMATION

Supplemental Information includes chemical synthesis, three figures and two tables and can be found with this article online.

AUTHOR CONTRIBUTIONS

G.G.-N., M.G., P.C., and M.-M. Z. conceived and designed the experiments for this study. G.G.-N., G.Z., and Y.R. designed the compounds. G.G.-N. and G.Z. performed the chemical synthesis. A.N.P. and J.J. determine the crystal structure. G.G.-N., L.Z., E.R., and A.V. conducted the biochemical study. M.G. J.K., G.M., and B.M. carried out the molecular and cellular biology experiments. G.G.-N. M.G., P.C., and M.-M.Z. wrote the manuscript.

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

01
NIHMS602357-supplement-01.pdf (17.8MB, pdf)
02
NIHMS602357-supplement-02.xlsx (49.2KB, xlsx)

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