Selective chemical modulation of gene transcription favors oligodendrocyte lineage progression

Article abstract of DOI:10.1016/j.chembiol.2014.05.009

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 extraterminal domain) prote

Full text of DOI:10.1016/j.chembiol.2014.05.009

Chemistry & Biology
Article
Selective Chemical Modulation of Gene Transcription
Favors Oligodendrocyte Lineage Progression
Mar Gacias,1,5 Guillermo Gerona-Navarro,2,3,5 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
2,
Patrizia Casaccia,1, and Ming-Ming Zhou
*
*
1Departments of Neuroscience and Genetics and Genomic Sciences
2Department of Structural and Chemical Biology
Icahn School of Medicine at Mount Sinai, 1425 Madison Avenue, New York, NY 10029, USA
3Department of Chemistry, Brooklyn College, 2900 Bedford Avenue, Room 351 NE, Brooklyn, NY 11210, USA
4Department of Natural Sciences, Hostos Community College of CUNY, Bronx, NY 10451, USA
5Co-first author
*Correspondence: patrizia.casaccia@mssm.edu (P.C.), ming-ming.zhou@mssm.edu (M.-M.Z.)
http://dx.doi.org/10.1016/j.chembiol.2014.05.009
SUMMARY
tors to target genes, assembly of the mediator complex at
enhancer sites, and activation of paused RNA polymerase II
Lysine acetylation regulates gene expression complexes for productive transcriptional elongation (Chiang,
2009). Numerous studies have reported that broad chemical
inhibition of both BET BrDs effectively blocked genome-wide
through modulating protein-protein interactions in
chromatin. Chemical inhibition of acetyl-lysine bind-
ing bromodomains of the major chromatin regulators
BET (bromodomain and extraterminal domain) pro-
teins has been shown to effectively block cell prolif-
eration in cancer and inflammation. However,
whether selective inhibition of individual BET bromo-
domains has distinctive functional consequences
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 (Nico-
deme et al., 2010; Zhang et al., 2012a). However, the use of
selective inhibitors of single BrDs could have distinctive func-
remains only partially understood. In this study, we
show that selective chemical inhibition of the first
bromodomain of BET proteins using our small-mole- tional features.
We addressed this question in oligodendrocyte lineage cells,
cule inhibitor, Olinone, accelerated the progression
of mouse primary oligodendrocyte progenitors
toward differentiation, whereas inhibition of both
bromodomains of BET proteins hindered differentia-
tion. This effect was target specific, as it was not
detected in cells treated with inactive analogs and
independent of any effect on proliferation. Therefore,
selective chemical modulation of individual bromo-
domains, rather than use of broad-based inhibitors,
may enhance regenerative strategies in disorders
the myelin-forming cells of the central nervous system whose dif-
ferentiation has been previously shown to require cell-cycle exit
(Casaccia-Bonnefil and Liu, 2003; He et al., 2007; Magri et al.,
2014a, 2014b) and histone deacetylase activity (Marin-Husstege
et al., 2002; He et al., 2007; Shen et al., 2008). In this lineage, the
early progenitor stage is characterized by global protein lysine
acetylation and decreased global histone acetylation, previously
identified as critical for the proper onset of oligodendrocyte dif-
ferentiation (Shen et al., 2008; Wu et al., 2012; Ye et al., 2009).
Therefore, we reasoned that oligodendrocyte lineage cells would
characterized by myelin loss such as aging and neu- be a suitable biological system to test the functional conse-
quences of BET protein BrD inhibition using chemical inhibitors
selective for only one or both BrDs of BET proteins.
rodegeneration.
Notably, previous studies have reported distinctive functions
of the two BrDs of BET proteins, possibly consequent to the
interaction with lysine-acetylated histones or with transcriptional
INTRODUCTION
Lysine acetylation plays an essential role in gene transcriptional proteins (Gamsjaeger et al., 2011; Huang et al., 2009; Jang et al.,
¨
regulation. The evolutionarily conserved bromodomain (BrD) 2005; Lamonica et al., 2011; Schroder et al., 2012; Shi et al.,
functions as the acetyl-lysine binding domain (Dhalluin et al., 2014; Yang et al., 2005; Zhang et al., 2012a). In the case of hu-
1999) for acetylated histones and transcription factors, which man BRD4, the first BrD appears dedicated to anchoring this
is required for ordered gene transcription in chromatin (Sanchez molecule and its associated proteins to target gene promoter
and Zhou, 2009). BRD4 is a representative member of the BET and enhancer sites in chromatin, through binding to diacetylated
(bromodomain and extraterminal domain) family of proteins, H4K5ac/K8ac (a mark for gene transcriptional activation), while
characterized by two tandem BrDs (BrD1 and BrD2) followed the second BrD was associated with the recruitment of nonhis-
by an extraterminal domain. Through its BrD/acetyl-lysine bind- tone proteins (i.e., transcription factors and the pTEFb complex)
ing, BRD4 functions to facilitate recruitment of transcription fac- to target genes. In the case of BRD3, however, it is the first BrD
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that binds to the hematopoietic transcription factor GATA1 indole synthesis from treatment of phenylhydrazone 1 with
(Gamsjaeger et al., 2011; Lamonica et al., 2011), thereby sug- sulfuric acid (70%) at 0ꢀC (Rodriguez and Temprano, 1989).
gesting context-dependent different functions of the two BrDs The resultant 2,3,4,5-tetrahydro-1H-pyrido-[4,3-b]indol-1-one 2
of the BET proteins in the regulation of ordered gene transcrip- was N-alkylated with a corresponding phthalimido protected
tion in chromatin.
alkyl bromide in the presence of lithium bis(trimethylsilyl) amide
This distinctive and unique ligand-binding selectivity of the two as a base (Coldham et al., 2007). Finally, treatment of N-phthali-
BrDs has been attributed to a few amino acid residues that mido protected substituted pyrido-indoles with hydrazine hy-
distinguish the first and second BrDs within each BET protein, drate and subsequent acetylation afforded the final compounds
while they all share nearly identical residues at the corresponding 4a to 4d with good yields. Similar procedures were used to
acetyl-lysine binding pocket. In an effort to understand specific generate compounds 5 and 6 (Figure 1B).
molecular functions of the individual BrDs of BET proteins, we
We assessed binding affinity of 4a to 4d, 5, and 6 to BrDs of
developed small-molecule chemical inhibitors that are capable BET proteins by using isothermal titration calorimetry (ITC) (Fig-
of selectively modulating acetyl-lysine binding activity of the first ure 1C; Figure S2A). The structure-activity relationship data
and/or second BrDs of BET proteins and evaluated their effects revealed that the length of the N-alkylated chain plays an impor-
on the progression of oligodendrocyte progenitor cells (OPCs) tant role in binding to BRD4-BrD1. An N-alkylated chain of four
toward differentiation.
methylene units as in 4b seems to be optimal in binding to
BRD4-BrD1, with a binding dissociation constant Kd of 3.4 mM.
This represents a greater than 15-fold improvement in binding
affinity compared with those analogs that comprise three, five,
or six methylene groups (i.e., 4a, 4c, and 4d, respectively) (Fig-
ure 1B). Furthermore, elimination of the acetyl group (5) or the
RESULTS AND DISCUSSION
Structure-Guided Development of the Selective BET
BrD Inhibitor Olinone
We used a structure-guided design strategy to develop selective entire N-acetylated group (6) resulted in a dramatic loss in bind-
small-molecule inhibitors for the BET BrDs (Figure 1A). Our ing affinity. We named the best compound of the series 4b Oli-
rational design of BET-specific BrD inhibitors (BrDis) started none for its cellular activity as described below. Consistently,
with a chemical scaffold of tetrahydro-pyrido indole that was we observed that Olinone exhibits preferred binding for the
present in a nuclear magnetic resonance (NMR)-based screen BrD1 over the BrD2 from all three BET proteins BRD4, BRD3,
hit (MS7972) and showed modest activity as an inhibitor of the and BRD2, as assessed by ITC measurements (Figure 1C).
CBP BrD (Figure 1B) (Sachchidanand et al., 2006). This chemical
scaffold is amendable to varying chemical modifications that can using 2D 1H-15
We next profiled Olinone binding selectivity to different BrDs
heteronuclear single-quantum coherence
N
be synthetically added on to optimize its interactions with a (HSQC) NMR spectroscopy (Figure 1D; Figure S2B). The NMR
target protein. Guided by the structural insights of MS7972 method offers a reliable assessment for ligands that bind to target
bound to the CBP BrD, we designed a series of 1-substituted- proteins with tens to hundreds micromolar affinity, which is com-
2,3,4,5-tetrahydro-pyrido-[4,3-b]indol-1-ones as inhibitors for plementary to ITC, which allows quantitative measurements of
the BET BrDs. These molecules extend from the key features ligand binding in low-micromolar affinity. Notably, Olinone
present on MS7972 for the binding to the BET BrDs to contain showed modest binding to the BrDs from CBP and PHIP, with
a longer acetamidoalkyl substituent at the N-indole as well as Kd values of 33 and 103.4 mM, respectively; very weak binding
an endocyclic peptide bond to enhance interactions with the to BRD4-BrD2, with Kd > 300 mM; and almost no detectable bind-
BrDs (Figure 1B).
ing to the BrDs from many other proteins, including BRD7, PCAF,
The synthesis of these compounds was achieved in four steps ASH1L, TAF1L, SMARCA4, BAZ1B, BAZ2B, ATAD2, BPTF, and
(see Figure S1 available online). Phenylhydrazone 1 was pre- TRIM24, which represent different subgroups of the human BrD
pared by reaction of phenylhydrazine with 2,4-piperidinedione family (Figure 1E; Figure S2C). The selectivity for the BrD1 over
in water (with 10% acetic acid) under nitrogen atmosphere. the BrD2 of the BET proteins was further confirmed using a fluo-
Next, the pyrido-indole scaffold was constructed by Fisher rescence anisotropy competition binding assay (Figure S2D).
Figure 1. Structure-Guided Design of Olinone, a Selective Inhibitor for the BET BrD1
(A) Schematic illustration of domain organization of the members of the BET protein family. ET, extraterminal domain.
(B) Structure and binding affinity of Olinone and its analogs for the BRD4-BrD1, determined by using ITC. Results are representative of three independent
experiments, and the error is the 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 versus BRD4-BrD2, as illustrated by 2D 1H-15N HSQC spectra of a given BrD in the free form (black) and in the
presence of Olinone (red), as well as by ITC.
(E) Phylogenetic tree of the human BrD family. Dots indicate the BrDs for which Olinone binding affinity was evaluated in the assay described in (B) and (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 s). Upper right: Structural comparison of BRD4-BrD1 and BRD4-BrD2 (PDB accession number 2YEM), showing steric clash between Olinone
with His437 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 Figures S1 and S2 and Table S1.
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Moreover, through characterization of Olinone binding to a set of selective for the first BrD of the BET proteins over other BrDs
wild-type or acetyl-lysine binding-deficient mutants of the tan- from different transcriptional proteins.
dem BrDs of BRD4 proteins, we observed that the two BET
It is important to note that Olinone has superior selectivity
BrDs function almost independently of each other, consistent between the two BrDs of BET proteins, even as compared with
with a long linker sequence that connects them (Figure S2D). the recently developed MS436, a diazobenzene compound
Collectively, these results demonstrate that Olinone is a highly (Zhang et al., 2013), or the BET-specific BrDi MS417, which is
selective inhibitor against the first BrD of the BET proteins.
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 BrD
Molecular Basis of Selective Recognition of BRD4 BrD1
by Olinone
The molecular basis of Olinone recognition of the BrD1 of BET (Kd = 20–100 nM) but do not discriminate between the first and
proteins was revealed by our X-ray crystal structure of the second BrDis of the BET proteins, because of greater than
˚
BRD4 BrD1/Olinone complex, determined at 0.94 A resolution 95% sequence identity at the acetyl-lysine binding pocket.
(Table S1). Olinone forms a chairlike conformation in the bound
state (Figure 1F, upper left), its triheterocyclic moiety as the Selective BET-BrD1 Inhibition Favors, whereas Broad
seat packs against the side chains of Trp81 and Pro82 in the BET BrD Inhibition Prevents the Differentiation
one-turn helix Z0, and Ile146 in helix C, and interacts with of Oligodendrocyte Progenitors
Asp144 at the opening of the acetyl-lysine binding pocket To begin evaluating the differential effects of selective inhibitors
formed between the ZA and BC loops. Asp144 is one of a few of the first BrD (such as Olinone) and compare them with those
residues that are different between BRD4-BrD1 and BrD2 in of broad BET inhibitors (i.e., MS417 and JQ1), we measured
the acetyl-lysine binding site; its corresponding residue in BrD2 the levels of expression of the BET protein-encoding genes in
is His437, which would clash with the indole moiety of Olinone the developing white matter tracts of the mouse brain. Of the
(Figure 1F, upper right).
analyzed molecules, Brd2 was the most abundant BET transcript
This major contribution to the overall ligand binding by Asp144 in the neonatal brain (Figure 2A). However, it is worth noting that
was confirmed in our detailed structural analysis of the the levels of all the BETs (i.e., Brd3, Brd4 long isoform, and Brd4
BRD4-BrD1/Olinone complex. The acetyl chain of Olinone, as short isoform) progressively declined in developing white matter
its back, intercalates into a hydrophobic pocket lined with tracts during a temporal window that was coincident with the
Val87, Leu92, Leu94, and Tyr139 of the ZA loop. The carbonyl differentiation of progenitors into myelin-forming oligodendro-
oxygen of the acetyl group of Olinone forms a hydrogen bond cytes, as detected by the expression of the myelin gene Mbp
˚
(2.9 A) to the amide nitrogen of the highly conserved Asn140 in (Figure 2A).
BRD4-BrD1. Notably, the acetyl chain of Olinone adopted an
To assess the ability of Olinone to modulate oligodendrocyte
almost identical conformation and position as that of the acety- progenitor differentiation in mouse cultures, we first determined
lated-K5 side chain of histone H4K5ac/K8ac peptide when the effect of Olinone and MS417 on cell viability after 72 hr
bound in BRD4-BrD1, whereas the piperidone ring of Olinone of treatment with increasing concentrations of BrDi, using a
filled the site for K8ac binding in the H4 peptide (Figure 1F, lower 1-methyl-1 H-tetrazole-5-thiol (MTT) assay (Figure 2B). No sig-
left and right). This explains the optimal length of the acetyl side nificant cytotoxicity was observed for Olinone or its inactive
chain of Olinone in its chemical series and Olinone’s effective in- analog compound 5, up to a concentration of 10 mM. In contrast,
hibition of BRD4-BrD1 binding to the diacetylated H4K5ac/K8ac treatment with MS417 was toxic at doses of 500 nM and above
peptide. The binding affinity of the latter interaction, which is (Figure 2B). Therefore, we opted to use Olinone at 1 mM and
important for BRD4 functions in gene transcriptional regulation MS417 at 0.05 mM for most of the cellular studies.
(Chiang, 2009; Filippakopoulos et al., 2012), is estimated to be
We had previously reported that global histone acetylation
a Ki of 26 mM. Given that a few ligand-binding residues in characterizes proliferating progenitors and that deacetylation is
BRD4-BrD1, such as Ile146 and Asp144, are not conserved necessary to initiate a differentiation program (Marin-Husstege
outside the BET proteins in the human BrD family proteins (San- et al., 2002; Shen et al., 2005). We therefore asked whether
chez and Zhou, 2009), this structure explains how Olinone is treatment with the BrD1 specific inhibitor Olinone or with the
Figure 2. Developmental Expression of BET Family Members in Corpus Callosum and Characterization of BrD Inhibition Effect in Oligoden-
droglial Cells
(A) Quantitative RT-PCR showing the transcript levels of Brd2, Brd3, and Brd4-long and Brd4-short isoforms in developing corpus callosum relative to 18S
expression (n = 3 animals per age group; one-way ANOVA with Bonferroni posttest, ***p < 0.01 versus P0).
(B) MTT assay to measure cell viability in primary cultured mOPCs, primary cultured mouse astrocytes, and Olineu cells after 72 hr of treatment with BrDi at
indicated doses (mM). Red lines indicate the dose range matching with the Kd values of the inhibitors, showing no toxicity at the indicated concentrations. Right:
Olinone and MS417 Kd values for the first and second BrDs of BRD4 (n = 3; one-way ANOVA with Dunnett’s posttest, *p < 0.05, ***p < 0.001).
(C) Selective BET-BrD inhibition decreases histone acetylation. Top: experimental timeline. Bottom: automated high-throughput confocal screening for histone
acetylation. The graph shows the quantification of the average nuclear residue-specific histone acetylation staining intensity in reference to DMSO (n = 20
sections; n = 3) (two-tailed t test, **p < 0.01, ***p < 0.001). Error bars show SEM.
(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 hr of BrDi treatment (Olinone at 1 mM and MS417 at 0.05 mM). 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 posttest, ***p < 0.001).
See also Figure S3, and Table S2.
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BrD1/BrD2 inhibitor MS417 would similarly affect histone acety- individual BrDs of BET proteins. This effect was clearly detect-
lation and gene transcription in immortalized oligodendrocyte able as early as the first 24 hr of differentiation (Figure S3).
progenitors (Olineu). The first experiment was conducted in the
To further determine the window of effectiveness of BrDi treat-
presence of mitogens, which represent a strong stimulus toward ment, we treated cells with either Olinone or MS417 for different
the persistence of global acetylation in these cells (Wu et al., amount of time in the presence of mitogens or in their absence. A
2012). Interestingly, in these cells, Olinone treatment resulted 24 hr treatment in the presence of mitogens was not sufficient to
in an average reduction of histone H3 global acetylation, de- detect the effects of MS417 or Olinone (Figure 4A). In contrast,
tected primarily at residues H3K14ac and H3K36ac, with little treatment for 72 hr starting after the removal of mitogens was
changes at H4K5ac and H4K12ac (Figure 2C). Treatment with sufficient to alter the expression of the late differentiation genes
MS417, in contrast, was less effective at reducing the histone (Figure 4B). Thus, inhibition of BrD binding was not sufficient to
acetylation levels at H3K14, although it decreased the levels of remove the inhibitory block of differentiation caused by mitogen
H3K36ac (Figure 2C). Despite the similar effect on histone de- stimulation and was detected only after the cells exited the cell
acetylation and on the expression of oligodendrocyte specific cycle. The first 24 hr following cell-cycle exit represents an
genes (Figure 2D), treatment of cells with Olinone consistently important critical window for the onset of a complex transcrip-
increased the transcript levels of myelin genes associated with tional program of differentiation. We therefore asked whether
the differentiated phenotype, whereas treatment with MS417 BrD inhibition during this critical period was sufficient to induce
decreased the levels of these genes while increasing transcripts long-lasting changes. For this reason, after the first 24 hr of treat-
characteristic of the progenitor stage (Figure 2D). These results ment, we washed out the drugs and allowed the cells to recover
suggested that BrD1-specific inhibition promoted differentiation, in differentiation medium (DM) prior to measuring the transcript
whereas inhibition of both BrD1 and BrD2 prevented it and re- levels of differentiation genes (Figure 4B). In these conditions,
tained the cells at a progenitor stage.
the levels of gene transcripts measured after washout did not
Because an obligatory relationship exists between cell-cycle reveal any change, which indicated that the effect of blocking
exit and differentiation in oligodendrocyte lineage cells (Swiss BrDs was not stable but reversible over time.
and Casaccia, 2010), and BrDis have been reported to inhibit
The next question was to define whether inhibition of one or
proliferation of cancer cells, we asked whether the differential both BrDis would also be detected if cells were treated after
effect on differentiation observed after treatment with Olinone the program of oligodendrocyte differentiation had started
or MS417 could be due to a distinct modulation of the cell (i.e., after at least 48 hr in mitogen-free DM) (Figure 4C). In this
cycle. To address this question, we adopted three different ap- case, the broad BET inhibition by MS417 still significantly
proaches: immunohistochemistry with antibodies for the cell-cy- decreased the expression of late differentiation genes, though
cle marker Ki67 (Figure 3A), expression levels of cell-cycle genes to a lesser extent than continuous treatment, while the prodiffer-
(Figure 3B), and fluorescence-activated cell sorting (FACS) anal- entiation effect of blocking only the first BrD1 by Olinone was
ysis (Figure 3C). A detailed quantification of Olinone- or MS417- dramatically reduced, with the exception of Mog levels (Fig-
treated cells using immunoreactivity for Ki67 in order to identify ure 4C). Collectively, these data suggest that BET activity in
proliferating cells did not reveal any difference among treatment oligodendrocyte lineage cells is differentially affected by inhibi-
groups (Figure 3A). Gene expression data analysis revealed an tors of the first BrD1 or of both BrDs of BET proteins.
overall interesting effect of MS417, but not Olinone, on the tran-
script levels of cell-cycle genes (Figure 3B). Consistent with Specificity and Off-Target Effects
the inhibitory effect of MS 417, treatment with this broad BET in- We then examined the specificity of the effect of BrDi on oligo-
hibitor decreased the expression of the gene encoding for dendrocyte differentiation by treating progenitors with structur-
p21Waf1, which had been previously shown to positively affect ally related but inactive compounds and conducted a detailed
oligodendrocyte differentiation (Zezula et al., 2001) while immunophenotypic (Figure 5A) and transcriptional (Figure 5B)
increasing the levels of cell-cycle genes preventing differentia- characterization of mouse primary oligodendrocyte progenitor
tion, such as E2F1 (Magri et al., 2014b) and c-Myc (Magri differentiation. Immunoreactivity for the proteoglycan NG2
et al., 2014a). Finally, a measure of the DNA content by FACS was used to identify early progenitors, O4 to label cells express-
analysis did not reveal significant differences in the relative pro- ing lipid sulfatides at an intermediate stage of maturation, and
portion of cells in the different stages of the cell cycle (Figure 3C). myelin basic protein (MBP) to identify differentiated oligodendro-
Taken together, these data suggest that the prodifferentiation cytes. In untreated cultures, the progression of progenitors to
effect of the BrD1 inhibitor Olinone could not be interpreted as mature oligodendrocytes was characterized by the progressive
the consequence of precocious cell-cycle exit.
decrease of NG2 and the gradual acquisition of O4 immunoreac-
tivity, followed by the acquisition of a myelinating phenotype,
characterized by the extension of myelin membranes recognized
by both O4 and MBP antibodies.
Temporal Control of BrDi Effectiveness on
Oligodendrocyte Progenitor Differentiation
To gain a better understanding of the effect of the BrD1-specific
Treatment of primary oligodendrocyte progenitors with the
or broad BET BrDis on differentiation, we conducted a dose- BrD1-specific inhibitor Olinone promoted a shift toward the
response experiment and measured the transcript levels of differentiated phenotype, as documented by the increased per-
late differentiation genes. In both cases, we detected a dose- centage of O4+ cells (Figure 5B), compared with DMSO-treated
dependent response, corresponding to either MS417-mediated cells. This effect was not detected in cells that were treated with
decrease or Olinone-induced increase of myelin gene transcripts the inactive Olinone analog compound 5. Treatment of primary
at a dose comparable with the in vitro binding affinity to the oligodendrocyte progenitors in the exact same conditions with
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Figure 3. Treatment with BrDi Does Not Affect the Proliferation of Primary Cultured mOPCs and Cell-Cycle Exit upon Differentiation Stimuli
(A) Representative confocal images of proliferating mOPCs stained with Ki67 antibody (green) and DAPI (blue). The scale bar represents 50 mm. The graph rep-
resents the percentage of mean pixel intensity. Results shown are relative DMSO-treated control cells (set to 100%) (n = 3; one-way ANOVA with Dunnett’s posttest).
(B) qRT-PCR showing the expression levels of cell-cycle genes after 24 hr of BrDi (Olinone at 1 mM and MS417 at 0.05 mM). 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 posttest, ***p < 0.001). Error bars show SEM.
(C) Cell-cycle analysis of BrDi-treated mOPCs for 24 hr as indicated. Color bars represent percentage of cells in each cell-cycle phase (n = 2; two-tailed t test).
Error bars show SEM.
the broader BET inhibitor MS417, in contrast, generated consis- entiation markers in a dose-dependent fashion while increasing
tently opposite effects to those induced by Olinone. MS417 the expression of the transcriptional inhibitor of myelin genes,
antagonized the progression toward a mature phenotype and Hes5 (Figure 5C).
reduced the number of NG2+/O4+ cells (Figure 5B). The effect
was highly specific as the inactive enantiomer of MS417 (Zhang Selective Inhibition of the BrD1 Enhances, whereas
et al., 2012a), called MS566, was incapable of eliciting the same Inhibition of Both BET BrDs Prevents the Differentiation
biological effect (Figure 5B). At a transcriptional level, the prodif- of Oligodendrocyte Progenitors
ferentiation effect of Olinone, but not compound 5, was also To begin to understand whether the inhibitory effect of MS 417
detected by the increased transcript levels for the late-differen- was consequent to the inhibition of both BrDs of BET proteins,
tiation markers Mag, Mog, Plp, and Mbp (Figure 5C), whereas we used two distinct approaches. First, we asked whether
MS417 treatment decreased the expression of these late-differ- Brd2 in cultured progenitors was also the most prominent BET
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Figure 5. Olinone Specifically Promotes the
Expression of the Oligodendrocyte Interme-
diate Differentiation Marker O4
(A) Top: experimental timeline. Bottom: confocal
image of mOPCs treated for 24 hr with DMSO,
Olinone (1 mM), compound
5 (1 mM), MS417
(0.05 mM), and MS566 (0.05 mM) in DM and stained
with NG2 (green), O4 (red) antibodies, and DAPI
(blue). The scale bar represents 50 mm.
(B) Graph showing the quantification of the per-
centage 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 hr of BrDi (Olinone
at 1 mM, compound 5 at 1 mM, MS417 at 0.05 mM,
MS566 at 0.05 mM). 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 posttest, ***p < 0.001).
whereas treatment of MS417 induced a
punctate distribution of the fusion pro-
tein, which resembled that observed
in eGFP-Brd2mut-transfected cells (Fig-
ure 6B). Taken together, these data sug-
gest that the second BrD of Brd2 is
important for its subcellular localization,
given that Olinone, which inhibits only
the first BrD, did not have any effect on
eGFP-Brd2wt, whereas MS417 (inhibit-
ing both the first and second BrDs of
Brd2) as well as a mutant Brd2 in both
BrDs showed altered nuclear distribution.
The second approach entailed the use of additional chemical
protein to be detected in the oligodendrocyte lineage cells (Fig-
ure 6A), which was consistent with our in vivo studies in devel- probes selective for the inhibition of only the first (MS611) or only
oping myelinating tracts (Figure 2A). We then expressed either of the second (MS765) BrD of BET proteins (Figure S2D; see also
a wild-type EGFP-tagged Brd2 (EGFP-Brd2wt) molecule or a the chemical synthesis in the Supplemental Information and Fig-
mutant form of Brd2 (EGFP-Brd2mut) lacking the acetyl-lysine ure S4). Notably, MS765, which is also known as RVX-208, is a
binding domains in the first and second BrD of the protein mole- compound that was developed by Resverlogix and has been
´
cule (Hnilicova et al., 2013) in Olineu cells and asked whether shown to have 15- to 30-fold selectivity for the second BrDs
the two molecules were differentially distributed within the nuclei over the first of BET proteins (McLure et al., 2013; Picaud
of transfected cells. Although the EGFP-Brd2wt transfected et al., 2013). To investigate whether inhibitors of only the first
cells were characterized by a nuclear diffuse distribution, those or second BrD of BET modulated the progression of oligoden-
transfected with the mutant eGFP-Brd2mut, lacking the ability drocyte progenitors toward a differentiated phenotype, we char-
to bind acetyl-lysine, showed a very distinctive punctate acterized the effect of 72 hr treatment with Olinone and MS611
distribution (Figure 6B). Treatment with the BrD1-specific inhib- (as inhibitors of the first BrD), MS765/RVX-208 (as an inhibitor
itor Olinone did not alter the distribution of eGFP-Brd2wt, of the second BrD), and MS417 (as an inhibitor of both BrDs).
Figure 4. Temporal Control of BrD Inhibition on OPC Differentiation
(A) BrDis do not have broad effects on the expression of oligodendrocytic lineage markers in proliferating conditions. Top: experimental time line. Bottom: qRT-
PCR showing the expression levels of differentiation markers after 24 hr of BrDi treatment (Olinone at 1 mM and MS417 at 0.05 mM) 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 posttest, ***p < 0.001).
(B) Top: different experimental paradigms of BrDi treatment during mOPC differentiation. Bottom: qRT-PCR results showing the transcript levels of late dif-
ferentiation markers (Mag, Mog, Mbp, and Plp) in mOPCs differentiated for 72 hr and treated with BrDi as indicated: (I) during 72 hr or (II) during the first 24 hr of
differentiation and then kept in DM with no BrDi. Doses used were 1 mM Olinone and 0.05 mM MS417 (n = 3; one-way ANOVA with Dunnett’s posttest, *p < 0.05,
**p < 0.01, ***p < 0.001).
(C) Top: different experimental paradigms of BrDi treatment. Bottom: qRT-PCR results showing the transcript levels of late differentiation markers (Mag, Mog,
Mbp, and Plp) in mOPCs differentiated for 72 hr and treated with BrDi as indicated: (I) during 72 hr or (III) during the last 36 hr of differentiation. Doses used were
1 mM Olinone and 0.05 mM MS417 (n = 3; one-way ANOVA with Dunnett’s post test, *p < 0.05, **p < 0.01, ***p < 0.001). Error bars show SEM.
Chemistry & Biology 21, 841–854, July 17, 2014 ª2014 Elsevier Ltd All rights reserved 849

Chemistry & Biology
Selective BrD Modulation in Gene Transcription
Figure 6. Selective BrDi Inhibition Differentially Affects Brd2 Nuclear Localization and the Progression of Primary OPCs toward a Fully
Differentiated Phenotype
(A) qRT-PCR showing the relative expression levels of Brd2, Brd3, and the long and short isoforms of Brd4 in primary cultured mOPCs. Brd2 is the highly ex-
pressed isoform in mOPCs. Olig2 (as a marker of oligodendrocyte lineage), Mbp (as a marker of differentiated oligodendrocytes), and Gfap (as a marker of
astrocytes) levels were also determined as controls. Results were normalized to 18S (n = 3). Error bars show SEM.
(B) Changes in Brd2 nuclear localization after BrDi treatment trichostatin A (TSA). Olineu cells were transfected with Brd2-eGFP or mutBrd2-eGFP (with two
BrDs mutated) in the presence or absence of BrDi (Olinone 1 mM, MS417 0.05 mM); DMSO is used as control. TSA (30 mM) was added after 24 hr of BrDi treatment
(legend continued on next page)
850 Chemistry & Biology 21, 841–854, July 17, 2014 ª2014 Elsevier Ltd All rights reserved

Chemistry & Biology
Selective BrD Modulation in Gene Transcription
Differentiation of progenitors into oligodendrocyte was moni- on BrDs in gene activation, including BET BrDs (Ciceri et al.,
tored by measuring transcripts for late differentiation genes 2014; Ember et al., 2014). Although the reverse may also be
(Mag, Mog, Plp, and Mbp), assessing immunoreactivity for late possible, this is less likely for Olinone because of its distinct
differentiation markers (MBP+/O4+) and quantifying the number chemical structure from those dual kinase-bromodomain inhib-
of membrane-bearing cells. We observed that treatment of Oli- itors. Moreover, similar results obtained with MS611, which
none and with the other inhibitor of the first BrD, MS611, shares the same selectivity for the first BrD over the second
increased expression of late differentiation markers (Figure 6C), BrD of BET proteins as that of Olinone, further supports the
whereas the inhibitor of the second BrD, MS765/RVX-208, had findings of this study.
no effect, and the broad MS417 inhibitor inhibited the expression
of these genes (Figure 6C). The number of MBP+ membrane- SIGNIFICANCE
bearing cells (Figure 6D; Figure S5) was similarly increased
by Olinone or MS611, unchanged by MS765/RVX-208, and BET family proteins, through their BrD/acetyl-lysine binding
decreased by MS417 treatment (Figure 6E).
capability, regulate gene transcription in chromatin in multi-
Collectively, our results suggest that selective inhibition of the ple steps, including the recruitment of transcription factors
first BrD (such as by Olinone or MS611) facilitates the progres- to target genes, assembly of the mediator complex at
sion of primary oligodendrocyte progenitors toward a differenti- enhancer sites, and activation of paused RNA polymerase
ated phenotype, inhibition of the second BrD (such as by MS765/ II machinery complexes for productive transcriptional elon-
RVX-208) has no effect, and inhibition of both BrDs of BET gation (Chiang, 2009). The two BrDs of BET proteins, as
proteins (such as MS417) negatively affects the differentiation shown in BRD3 and BRD4, have context dependent different
process.
functions in regulation of ordered gene transcription (Gams-
Because of the dynamic nature of lysine acetylation, chemi- jaeger et al., 2011; Huang et al., 2009; Jang et al., 2005;
¨
cal inhibition of these BrDs may result in lysine deacetylation Lamonica et al., 2011; Schroder et al., 2012; Shi et al., 2014;
and subsequent ubiquitination-mediated degradation of the Yang et al., 2005; Zhang et al., 2012a).
BrD-binding partners. Therefore, chemical inhibition of the
In this study, we highlight the functional importance of
two different BrDs is likely to yield distinct functional outcomes. chemical modulation of acetylation-mediated protein-pro-
In this study, we compared the effect of a selective inhibitor tein interactions in transcriptional control of OPC differen-
(Olinone) for the first BrD of BET proteins with that of an inhib- tiation. We identify Brd2 as the prominent BET expressed
itor (MS417) that is equally effective for both BrDs and in the oligodendrocyte lineage cells. In contrast to what
observed dramatic differences in the biological response of has been reported for other cell types, treatment of oligo-
oligodendrocyte progenitors to these treatments. Whereas Oli- dendrocyte progenitors with BrDis did not modulate the
none promoted differentiation, MS417 inhibited it. This effect acetylation of histone H4 and did not affect proliferation.
was associated with the upregulation of genes such as E2f1 These cells were quite responsive to treatment with selec-
and Myc, which we have shown to be inhibitory for differentia- tive chemical probes specific for the first, second, or both
tion (Magri et al., 2014a, 2014b) and the downregulation of pro- BrDs of BET proteins. In this functional context, we demon-
differentiation genes such as Cdkn1b (Zezula et al., 2001). One strate that a small-molecule BrDi, designed to selectively
explanation is that under MS417 treatment, a key transcription modulate the master chromatin regulators BET proteins,
factor that is recruited by BET proteins could be degraded in could positively or negatively influence the differentiation
cells, whereas Olinone treatment would leave such a transcrip- of oligodendrocyte progenitors, depending on the targeted
tion factor intact because of its association with the second BrD BrD. Our data further indicate that a complete chemical inhi-
of BET proteins. Given the complex nature of gene transcription bition of BET BrD/acetyl-lysine binding activity may possess
progression of oligodendrocyte progenitor differentiation, the adverse effects on oligodendrocyte progenitor differentia-
identification of such a transcription factor(s), and the detailed tion, an important step for myelination of axons. Overall,
mechanism of action of chemical inhibition of BET proteins by this study opens opportunities for the future development
Olinone and/or MS417 will have to await future investigation. of new therapeutic treatments of demyelinating disorders
Finally, recent studies show that some kinase inhibitors, and also novel chemical agents that could improve the effi-
such as BI-2536 and TG-101348, developed against PLK1 ciency of the lineage progression of adult stem cells into
and JAK2-FLT3 kinases, respectively, have off-target effects specialized cell types.
in order to reduce rapid histone acetylation. Cells were fixed at the indicated time points (1 hr of TSA treatment and 6 hr of TSA treatment) (n = 3). The scale bar
represents 50 mm. Error bars show SEM.
(C) Top: experimental timeline. Transcript levels of late differentiation markers (Mag, Mog, Mbp, Plp, and Sirt2) and the myelin gene transcriptional inhibitor (Hes5)
in response to 72 hr of BrDi treatment (Olinone at 1 mM, compound 5 at 1 mM, MS417 at 0.05 mM, MS566 at 0.05 mM, MS611 and MS765/RVX-208 at 0.5 mM).
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 posttest, *p < 0.05,
**p < 0.01, ***p < 0.001). Error bars show SEM.
(D) Representative confocal images of mOPCs treated with DMSO, Olinone (1 mM), and MS417 (0.05 mM) during 72 hr in DM and stained with MBP antibody (gray)
and DAPI (blue). The scale bar represents 100 mm.
(E) Graph showing the quantification of the percentage of mature MBP+ membrane-bearing cells relative to DMSO (n = 3 with 7 or 8 sections per condition; one-
way ANOVA with Dunnett’s post test, *p < 0.05).
See also Figures S4 and S5.
Chemistry & Biology 21, 841–854, July 17, 2014 ª2014 Elsevier Ltd All rights reserved 851

Chemistry & Biology
Selective BrD Modulation in Gene Transcription
´
(with both BrDs mutated), previously described by Hnilicova et al. (2013). After
EXPERIMENTAL PROCEDURES
24 hr of transfection, cells were treated for 24 hr with either BrDis (Olinone,
MS417) at indicted concentrations or with DMSO as negative control. At this
time point, cells were fixed and stained with DAPI (1:10000; Molecular Probes)
for immunochemistry.
ITC
Experiments were carried out on a GE MicroCal auto-ITC200 instrument at
15ꢀC while stirring at 1,000 rpm in the ITC buffer at pH 7.4, consisting of
50 mM sodium phosphate and 150 mM sodium chloride, as described previ-
ously (Zhang et al., 2012a). Compound concentration was determined by
NMR and protein concentrations by A280 measurements. The protein sample
(ꢁ75 mM) was placed in the cell, whereas the microsyringe (130 ml) was loaded
with a solution of the correspondent compound (0.8 mM) in the ITC buffer. All
titrations were conducted using 20 identical injections of 2 ml, with a duration
of 4 s per injection and a spacing of 150 s 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 impli-
cated in the MicroCal Origin software supplied with the instrument to yield
enthalpies of binding (DH) and binding constants (KB). Thermodynamic
parameters were calculated (DG = DH ꢂ TDS = ꢂRTlnKB, where DG, DH,
and DS are the changes in free energy, enthalpy, and entropy of binding,
respectively). In all cases, a single binding site model was used.
Proliferation Assays
Mouse primarily cultured oligodendrocytes were cultured in CC2-coated
eight-well chambers (Lab-Tek) in Sato medium with growth factors (bFGF
and PDGFaa). Cells were treated with either BrDis at indicated concentrations
or DMSO as control for 24 hr. To determine changes in proliferation, cells
were fixed and immunolabeled with antibodies against the cell proliferation
marker Ki67 (ab14298, 1:1000) for 1 hr at room temperature (RT).
Immunohistochemistry
Immunohistochemistry of cultured cells with O4 antibodies was performed
live. Cells were grown on CC2-coated eight-well chambers (Lab-Tek) for all
immunocytochemistry. For staining oligodendrocyte lineage markers, cells
were rinsed gently with PBS and incubated live with O4 hybridoma superna-
tant (1:10, gift from Dr. Bansak, University of Connecticut) for 30 min at
37ꢀC. Cells were then fixed with 4% paraformaldehyde (PFA) for 15 min at
RT and first incubated with blocking/permeabilization solution (phosphate
buffer with gelatin bovine albumin [0.1 M phosphate buffer, 0.1% gelatin,
1% BSA, azide] PGBA plus 10% normal goat serum and 0.5% Triton X-100)
for 1 hr at RT. For costaining experiments, cells were incubated with additional
primary antibodies against NG2 (Chemicon AB5320, 1:300) and MBP (Cova-
nce SMI99, 1:500) overnight at 4ꢀC. A 1 hr incubation with secondary fluores-
cent antibodies (Alexa Fluor 488 or 546) was performed the following day with
counterstaining for DAPI (1:10000; Molecular Probes) to visualize cell nuclei.
Protein Crystallization, X-Ray Diffraction Data Collection,
and Structure Determination
Purified BRD4-BrD1 protein (14 mg/ml) was mixed with Olinone 4b at a 1:10
molar ratio of protein to ligand. The complex was crystallized using the sitting
drop vapor diffusion method at 20ꢀC by mixing 1 ml of protein solution with 1 ml
of the reservoir solution, which contains 25% tert-Butanol and 0.1 M Tris-HCl
(pH 8.5). Crystals were soaked in the corresponding mother liquor supple-
mented with 20% ethylene glycerol as a cryoprotectant before freezing in
liquid nitrogen. X-ray diffraction data were collected at 100 K at beamline
X6A 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 replace-
ment using the program MOLREP (Vagin and Teplyakov, 1997), and the struc-
ture refinement was done using the program Refmac (Murshudov et al., 1997).
The 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 Table S1 (see Supplemental Information).
Image Acquisition and Quantification
Images were captured at 203 and oil immersion 633 objective using LSM 710
Metaconfocal laser scanning microscope (Carl Zeiss MicroImaging). 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 ImageJ software (NIH). To characterize the 24 hr culture,
the proportion of NG2;O4 double positive was calculated by referring to the
total number of cells (DAPI+). To characterize the 72 hr 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 prolif-
eration 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 differ-
ences between control and experimental conditions after combing the total
number of cells in each group, with a significance threshold of p < 0.05.
Primary Oligodendrocytes
OPCs were prepared by sequential immunopanning and kept under undiffer-
entiating conditions, as described previously (Watkins et al., 2008), until the
onset of experiments. Briefly, OPCs were isolated from one P6 mouse pup
brain using an immunopanning system enabling purity of 95%. The dissected
cortex was chopped in papain buffer, incubated for 20 min at 37ꢀC, and
titrated in ovomucoid solution (CellSystems). 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 simpli-
ficonia lectin (Vector Labs, L-1100), for negative selection for 15 min, followed
by a positive selection step with rat antimouse CD140a (Research Diagnostics,
10R-CD140AMS) as a primary antibody and AffiniPure goat antirat immuno-
globulin G (H+L) (Dianova, 112-005-003) as a secondary antibody for 45 min.
The supernatant was aspirated, and the positive selection plate was washed
with Dulbecco’s PBS (DPBS). The adherent OPCs were removed using trypsin,
centrifuged for 10 min at 1,000 rpm, resuspended in mouse OPC (mOPC) Sato
(Watkins et al., 2008), and plated in a T75 culture flask coated with polylysine.
The OPCs were cultured in a humidified incubator at 5% CO2 and 37ꢀC, with
media changes every 2 days. OPC were maintained proliferating in the pres-
ence of basic fibroblast growth factor (bFGF) (20 ng/ml) and platelet-derived
growth factor (PDGF) (10 ng/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.
Acetyl Histone Detection
Olineu cells were plated on 0.1 mg/ml poly-d-lysine (Sigma P7886) coated
96-well plates at a density of 5,000 cells/well. Oligodendrocyte differentiation
medium was supplemented with 1% horse serum (Invitrogen 16050). Twenty-
four hours 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 hr. Cells were gently
washed with PBS after completion of the treatment and fixed with 4% PFA for
20 min at RT. Cells were then washed and permeabilized with 0.1% triton
for 30 min at RT followed by incubation in PGBA plus 10% normal goat serum
for 1 hr. 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 4ꢀC. The following day, samples were incubated for 1 hr
with secondary fluorescent antibody Alexa Fluor 488 (Invitrogen A11034)
and counterstained for DAPI (1:10,000, Molecular Probes). Measurement of
the pixel intensity of each primary antibody listed above was 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.
Olineu Transient Transfection
Olineu cells (15,000 cells/well) were grown on CC2-coated eight-well cham-
bers (Lab-Tek) for all transfection experiments. These cells were transiently
transfected using X-tremeGENE 9 DNA transfection reagent (Roche Diagnos-
tics) following the manufacturer’s protocol and using equal amounts of the
eukaryotic expression vectors pEGFP-C1-Brd2 and pEGFP-C1-Brd2mut
852 Chemistry & Biology 21, 841–854, July 17, 2014 ª2014 Elsevier Ltd All rights reserved

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Selective BrD Modulation in Gene Transcription
Cell-Cycle Assay
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treated with BrDi as indicated. After 24 to 30 hr of treatment, cells were rinsed
twice with DPBS, trypsinized, and pelleted with PBS. One million cells 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.
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significance at p < 0.05.
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SUPPLEMENTAL INFORMATION
Supplemental Information includes Supplemental Experimental Procedures,
five figures, and two tables and can be found with this article online at http://
dx.doi.org/10.1016/j.chembiol.2014.05.009.
Gamsjaeger, R., Webb, S.R., Lamonica, J.M., Billin, A., Blobel, G.A., and
Mackay, J.P. (2011). Structural basis and specificity of acetylated transcription
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AUTHOR CONTRIBUTIONS
He, Y., Dupree, J., Wang, J., Sandoval, J., Li, J., Liu, H., Shi, Y., Nave, K.A., and
Casaccia-Bonnefil, P. (2007). The transcription factor Yin Yang 1 is essential
for oligodendrocyte progenitor differentiation. Neuron 55, 217–230.
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 struc-
ture. 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.
ꢀ
Hnilicova, J., Hozeifi, S., Stejskalova, E., Duskova, E., Poser, I., Humpolıckova,
ꢀ
´
´
´
´
´
ꢀ
J., Hof, M., and Stanek, D. (2013). The C-terminal domain of Brd2 is important
for chromatin interaction and regulation of transcription and alternative
splicing. Mol. Biol. Cell 24, 3557–3568.
Huang, B., Yang, X.D., Zhou, M.M., Ozato, K., and Chen, L.F. (2009). Brd4
coactivates transcriptional activation of NF-kappaB via specific binding to
acetylated RelA. Mol. Cell. Biol. 29, 1375–1387.
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 Cen-
ter at Rockefeller University. We also wish to thank the staff at the X6A beam-
line of the NSLS at 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.), Clinical and Translational Science Awards grant
UL1RR029887 to the Icahn School of Medicine at Mount Sinai, and research
grants from the NIH (R03NS072578 to G.G.-N., R37NS42925 and R01-
NS52738 to P.C., and R01HG004508 and R01CA87658 to M.-M.Z.).
Jang, M.K., Mochizuki, K., Zhou, M., Jeong, H.S., Brady, J.N., and Ozato, K.
(2005). The bromodomain protein Brd4 is a positive regulatory component of
P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol.
Cell 19, 523–534.
Lamonica, J.M., Deng, W., Kadauke, S., Campbell, A.E., Gamsjaeger, R.,
Wang, H., Cheng, Y., Billin, A.N., Hardison, R.C., Mackay, J.P., and Blobel,
G.A. (2011). Bromodomain protein Brd3 associates with acetylated GATA1
to promote its chromatin occupancy at erythroid target genes. Proc. Natl.
Acad. Sci. USA 108, E159–E168.
Magri, L., Gacias, M., Wu, M., Swiss, V.A., Janssen, W.G., and Casaccia, P.
(2014a). c-Myc-dependent transcriptional regulation of cell cycle and nucleo-
somal histones during oligodendrocyte differentiation. Neuroscience.
Published online February 4, 2014. http://dx.doi.org/10.1016/j.neuroscience.
2014.01.051.
Received: August 29, 2013
Revised: April 28, 2014
Accepted: May 1, 2014
Published: June 19, 2014
Magri, L., Swiss, V.A., Jablonska, B., Lei, L., Pedre, X., Walsh, M., Zhang, W.,
Gallo, V., Canoll, P., and Casaccia, P. (2014b). E2F1 coregulates cell cycle
genes and chromatin components during the transition of oligodendrocyte
progenitors from proliferation to differentiation. J. Neurosci. 34, 1481–1493.
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854 Chemistry & Biology 21, 841–854, July 17, 2014 ª2014 Elsevier Ltd All rights reserved