Histone modification plays an important role in various biological processes, including gene expression regulation. Bromodomain, as one of histone readers, recognizes specifically the ε-N-lysine acetylation (KAc) of histone. Although the bromodomains and histone acetylation sites of Trypanosoma brucei (T. brucei), a lethal parasite responsible for sleeping sickness in human and nagana in cattle, have been identified, how acetylated histones are recognized by bromodomains is still unknown. Here, the bromodomain factor 2 (TbBDF2) from T. brucei was identified to be located in the nucleolus and bind to the hyperacetylated N-terminus of H2AZ which dimerizes with H2BV. The bromodomain of TbBDF2 (TbBDF2-BD) displays a conserved fold that comprises a left-handed bundle of four α-helices (αZ, αA, αB, αC), linked by loop regions of variable length (ZA and BC loops), which form the KAc-binding pocket. NMR chemical shift perturbation further revealed that TbBDF2-BD binds to the hyperacetylated N-terminus of H2AZ through its KAc-binding pocket. By structure-based virtual screening combining with the ITC experiment, a small molecule compound, GSK2801, was shown to have high affinity to TbBDF2-BD. GSK2801 and the hyperacetylated N-terminus of H2AZ have similar binding sites on TbBDF2-BD. In addition, GSK2801 competitively inhibits the hyperacetylated N-terminus of H2AZ binding to TbBDF2-BD. After treatment of GSK2801, cell growth was inhibited and localization of TbBDF2 was disrupted. Our results report a novel bromodomain-histone recognition by TbBDF2-BD and imply that TbBDF2 may serve as a potential chemotherapeutic target for the treatment of trypanosomiasis.
Trypanosoma brucei is one of the most popular parasites causing African trypanosomiasis which is known as sleeping sickness in humans and nagana in other animals. The African trypanosomiasis is eventually fatal if it is not treated, and there is lack of drugs for this disease . Treatment of African trypanosomiasis is facing lots of problems such as early-stage diagnosis, strong side effect of drugs and drug resistance [1–3]. It is urgent to develop new drugs to overcome these difficulties.
Epigenetics plays important roles in different physiological mechanisms [4–6]. Epigenetic regulation is also very important in virulence gene expression of parasitic protozoa . Bromodomain-containing protein family which naturally binds to acetyl-lysine is a class of important epigenetic factor . Many bromodomain-containing proteins in higher eukaryotes have been identified to be essential for chromatin remodeling and gene transcription [9–12]. For example, Brd4 is an important bromodomain and extra-terminal (BET) family protein. It is involved in the regulation of NF-κB turnover  and also takes part in the repression of the Tat-mediated transactivation of the HIV promoter . BET family proteins also participate in the regulation of inflammation . Some bromodomain-containing proteins of trypanosoma have been explored. TcBDF1 is able to enhance the trypomastigote cell infection . TcBDF2 binds to H4K10Ac and H4K14Ac and is accumulated after UV irradiation, suggesting that TcBDF2 may be involved in the regulation of chromatin remodeling of Trypanosomacruzi . TcBDF3 binds to the acetylated α-tubulin and overexpression of TcBDF3 affects differentiation of T. cruzi . Bromodomain proteins of T. brucei are necessary for maintenance of activated expression site (ES) and expression of VSG genes [19,20], which indicates that bromodomains are essential for transcription regulation and cell differentiation of T. brucei. A more detailed research suggested that TbBDF3 co-localizes with H4K10Ac, H2AZ and H2BV at probable TSSs (transcription start sites) and may play an essential role in transcription initiation . All these previous studies revealed that bromodomain-containing proteins are essential in the physiological regulation of trypanosome and are potential targets for therapy of trypanosomiasis.
The important roles of bromodomain proteins indicate that they are ideal drug targets [22,23]. Many effective small molecule inhibitors have been developed such as (+)-JQ1, I-BET151, SGC-CBP30, GSK2801, and RVX-208 [24–28]. Bromodomain inhibitors have been well studied in cancer, inflammation and viral infection . Recent evidence suggests that bloodstream-form T. brucei treated by I-BET151 would develop into insect-stage features. Meanwhile, the mice infected with I-BET151-treated trypanosomes survived significantly longer than those infected with untreated trypanosomes . These suggest that I-BET 151 inhibits bromodomain binding to chromatin and causes T. brucei to lose the ability to infect mice. Thus, screening inhibitors for bromodomain-containing proteins of T. brucei is urgent to develop effective drugs for research and therapy.
In the present study, the bromodomain factor 2 of T. brucei (TbBDF2) was identified to bind to the histone variant H2AZ/H2BV dimer specifically. Binding assay showed that TbBDF2-BD specifically recognizes the hyperacetylated N-terminus of H2AZ. We further characterized a small molecule compound GSK2801 that binds to TbBDF2 with high affinity. These results provide novel understanding of the bromodomain binding to histone and add future insights into the treatment of trypanosomiasis.
Genes were amplified from the genome of T. brucei by PCR. Genes encoding TbBDF1-BD (fragment 27–129), TbBDF2-BD (fragment 1–111) and TbBDF5-BD1 (fragment 1–123) were cloned into the pET22b (+) vector, respectively. Genes encoding TbBDF5-BD2 (fragment 172–330) and TbBDF3 (fragment 30–154) were cloned into the pET28a (+) vector, respectively. For in situ tagging of TbBDF2, 3′- 371 bp fragment except the stop codon of cDNA was cloned into the pC-PTP-NEO  and pC-EYFP-NEO, respectively. For in situ tagging of H2AZ and H2BV, cDNA fragment of H2AZ (1–582) and H2BV (1–790) were cloned into the pN-PURO-PTP , respectively.
Protein expression and purification
Expression vectors were transformed into Escherichia coli BL21 (DE3), respectively. The transformed cells were cultured in Luria-Bertani (LB) at 37°C until OD600 to 0.8, then induced with 0.8 mM IPTG at 16°C for 20 h. The induced cells were harvested and resuspended in lysis buffer (20 mM Tris, 500 mM NaCl, pH 7.8), then lysed by sonication. Lysate was centrifuged at 14 000 g at 4°C for 20 min to remove precipitant. Protein was purified by Ni2+-NTA column. Eluted protein was further purified by size exclusion chromatography using a Superdex 75 column on an AKTA purification system. [13C, 15N]-TbBDF2-BD was purified in the same way except that LB medium was replaced by M9 medium containing 0.5 g/l 15N-labeled ammonium chloride and 2.5 g/l 13C-labeled glucose. The final NMR samples contained 0.8 mM TbBDF2-BD, 25 mM sodium phosphate (pH 6.8), 150 mM sodium chloride, 1 mM EDTA and 1 mM dl-dithiothreitol in either 90% H2O/10% D2O or 100% D2O.
NMR spectroscopy, data processing, and structure calculation
All the NMR spectra were acquired at 293 K on a Bruker DMX500 spectrometer. 1H, 15N-HSQC, HNCACB, CACB(CO)NH, HNHA, HCC(CO)NH, three-dimensional 15N-NOESY and 13C-NOESY spectra were recorded. The NMR data were processed with NMRPipe  and Sparky . Distance restrains were acquired from 15N-NOESY and 13C-NOESY. Dihedral angle restraints were calculated by TALOS program . Structures were calculated by CYANA 3.0 . The final 20 lowest target function structures were analyzed with MOLMOL . The structure was analyzed with PROCHECK online .
Isothermal titration calorimetry
Experiments were performed on an ITC200 (MicroCal) at 20°C. Proteins were dialyzed into buffer (25 mM sodium phosphate with pH 6.8, 150 mM sodium chloride and 1 mM EDTA). For titration of small molecule compounds, all solutions in the cell and the syringe were diluted into the same final DMSO concentration (no more than 1%). TbBDF2-BD protein solution or protein–GSK2801 mixture in the calorimetric cell was titrated with the peptide solution in the syringe. Data were analyzed using the ORIGIN software (MicroCal) with a single-binding site model.
NMR chemical shift perturbation
15N-labeled TbBDF2-BD was titrated with GSK2801 and acetylated peptides to different molar ratio, respectively. 1H, 15N-HSQC spectra were recorded at each concentration of small molecule compound or peptides for analysis. Chemical shift changes were normalized using function .
Trypanosome cell culture and transfection
The wild-type T. brucei 427 procyclic-form cells were cultivated at 26°C in Cunningham's medium  supplemented with 10% fetal bovine serum (Hyclone). For in situ tagging of TbBDF2, vectors were linearized with XhoΙ and plasmids were transfected to 427 cells by electroporation. Electroporation was carried out in a 2-mm cuvette (BTX) using the BTX ECM630 with parameters set as follows: 2 kV voltages, 200 Ω resistance, and 25 μF capacitance. The transfectants were selected under 2.5 μg/ml G418. For the construction of cell lines that co-express TbBDF2-EYFP and PTP-H2AZ or PTP-H2BV, vectors were linearized with MfeI and BspMI, respectively. The linearized vectors were transfected to cell lines expressing TbBDF2-EYFP by electroporation. The transfectants were selected under 2.5 μg/ml G418 and 2 μg/ml puromycin. Cells lines were validated by the western blot.
Affinity purification of TbBDF2-PTP
Affinity purification was performed as described before with some modifications [29,38]. Briefly, 500 ml of cells expressing TbBDF2-PTP from one of its endogenous loci were harvested and lysed by sonication. Lysate was incubated with 200 μl IgG sepharose 6 fast flow beads (GE) at 4°C for 2 h. IgG beads were washed three times with lysis buffer [20 mM HEPES with pH 7.4, 100 mM NaCl, 1 mM MgCl2, 2% glycerol, 1 mM EDTA, 0.1% Triton X-100, 0.2% NP-40, and protease inhibitor cocktail (Roche)], and proteins were eluted with 0.1 M glycine (PH 2.5). Proteins were then concentrated and separated by ExpressPlu PAGE Gel (Genscript). Gels are stained with Coomassie Brilliant Blue and enriched TbBDF2 was analyzed by mass spectrometry.
In-gel digestion and LC-MS/MS
In-gel digestion of proteins was carried out according to our published procedures . Sample was loaded onto Acclaim PepMap 100 (Thermo Scientific) and analyzed by Q Exactive hybrid quadrupole-Orbitrap Plus mass spectrometer (Thermo Fisher Scientific). The peptides were subjected to NSI source followed by tandem mass spectrometry (MS/MS) in Q Exactive (Thermo) coupled online to the UPLC. Raw data files were searched using the Mascot search engine (version 2.3) against H2AZ sequence and H2BZ sequence. Mass error was set to 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on Cys was specified as fixed modification, oxidation on Met, acetylation on Lys and acetylation on protein N-terminus was specified as variable modification. Peptide ion score was set to ≥20.
Cells expressing TbBDF2-EYFP were harvested and washed twice with PBS. Cells were then resuspended in PBS containing 2% paraformaldehyde and incubated for 10 min in room temperature. After washed twice with PBS, cells were adhered to coverslips. The fixed cells were washed and blocked with blocking buffer (PBS with 1% BSA and 0.1% Triton X-100 PBS) at room temperature for 60 min. After that, cells were incubated with primary antibody (diluted in PBS with 1% BSA) for 60 min, washed with PBS for 5 min for three times, and then incubated with secondary antibody (diluted in PBS with 1% BSA) for 60–90 min. Slides were mounted in mounting medium (Vector Labs) containing DAPI and examined by FV1200MPE-share system (Olympus). Images were analyzed by a FV10-ASW 4.2 viewer. The following primary antibody was used: anti-fibrillarin for detecting nucleolus (16021-1-AP, Proteintech, rabbit anti-fibrillarin polyclonal antibody, used at 1 : 100 dilution). Cy3 conjugated anti- rabbit IgG (BA1032, Boster, used at 1 : 100 dilution) was used as a secondary antibody.
Cells co-expressing TbBDF2-EYFP and H2AZ or H2BV were harvested and lysed by sonication. Lysate was incubated with 30 μl IgG sepharose 6 fast flow beads (GE) at 4°C for 1 h. IgG beads were washed three times with lysis buffer [20 mM HEPES with pH 7.4, 100 mM NaCl, 1 mM MgCl2, 2% glycerol, 1 mM EDTA, 0.5% NP-40 and protease inhibitor cocktail (Roche)], and proteins were eluted with 1% SDS. Proteins were then detected by the western blot. Cells expressing TbBDF2-EYFP were analyzed as a control.
Virtual screening using the PYRX software
Virtual screening was carried out using PyRx 0.8  from http://pyrx.sourceforge.net/. SDF files were obtained from PubChem database and Protein Data Bank. Software was used as described before . After energy minimization by the PyRx, all compounds with reasonable energy function were analyzed manually. Finally, five small molecule compounds (including one control) were selected and purchased from APExBIO Technology, Houston, U.S.A.
In vitro compound sensitive analysis
Four-hundred and twenty-seven cells were treated with different concentrations of GSK2801 and counted daily. IC50 was determined as described  with some modifications. In brief, GSK2801 was serially diluted in DMSO and added into 96-wellplate. 200 μl medium containing 2 × 105 cells was added into each well. After cells were incubated for 72 h, 20 μl resazurin (22.88 mg/ml) was added into each well. After 4, 6 and 8 h, plate was detected by spectra MAX gemini EM, respectively. Curves were fitted using Boltzmann function by the OriginPro 8.0 software and the IC50 value was determined.
TbBDF2 is predominantly located in the nucleolus of T. brucei
Depletion of TbBDF2 inhibited cell growth , suggesting that TbBDF2 is essential for T. brucei. To determine the subcellular localization of TbBDF2, TbBDF2 was tagged with EYFP at the C terminus of one of its endogenous loci in T. brucei. Fluorescence microscopy showed that TbBDF2-EYFP was colocalized with fibrillarin which is a well-known nucleolar protein (Figure 1). This demonstrates that TbBDF2 is predominantly located in the nucleolus.
Subcellular localization of TbBDF2 in the T. brucei.
TbBDF2 binds to the variant histone H2AZ/H2BV dimer
The homologue of TbBDF2 in T. cruzi (TcBDF2) was reported to recognize H4K10Ac and H4K14Ac . However, ITC experiment showed that the bromodomain of TbBDF2 displayed low affinity to acetylated H4 peptide (Supplementary Figure S1), which indicates that TbBDF2 has other preference. In order to identify the TbBDF2 binding proteins, we constructed a cell line stably expressing TbBDF2 with a C-terminal PTP tag (described in the Method). One-step affinity purification of TbBDF2-PTP was performed and TbBDF2-associated proteins were co-purified and identified by mass spectrometry. In addition to the TbBDF2 itself, two histone variants, H2AZ and H2BV were identified by mass spectrometry (Figure 2A). In order to confirm the interactions between TbBDF2 and H2AZ/H2BV, co-precipitation experiments were performed. TbBDF2-EYFP both co-precipitated with PTP-H2AZ and PTP-H2BV (Supplementary Figure S2). Given that H2AZ always dimerizes with H2BV , these results suggest that TbBDF2 is able to bind to the histone variant H2AZ/H2BV dimer functionally.
TbBDF2 binds to the hyperacetylated N-terminus of H2AZ.
TbBDF2 binds to the hyperacetylated N-terminus of H2AZ
As TbBDF2 contains a bromodomain, we wondered whether H2AZ/H2BV are acetylated. To explore the acetylated sites of the H2AZ/H2BV dimer, high-precision LC-MS/MS was performed against the TbBDF2-associated H2AZ and H2BV. The mass spectrometry revealed 13 acetylation sites on H2AZ and 3 acetylation sites on the H2BV. The sequence coverage of the samples is 49.44% for H2AZ and 75.00% for H2BV, respectively (Figure 2B, Supplementary Figure S3 and Table S1). Both N and C termini of H2AZ are hyperacetylated, while N terminus of H2BV is triacetylated. The TbBDF2-associated H2AZ and H2BV were both acetylated at GK motif , which was also observed in H2AZ of chicken and yeast [43–45] (Supplementary Figure S4A,B). Besides, H2AZ of T. brucei has a longer N-terminus than that of higher eukaryotes (Supplementary Figure S4C), which is a unique feature of H2AZ of T. brucei.
To explore the acetylation site recognized by TbBDF2-BD, ITC experiments were performed. Peptides carrying all identified acetylated lysine residues were synthesized (Supplementary Table S2). Additionally, although lysine residues were not identified to be acetylated by mass spectrometry, peptides containing acetylated K31 of H2AZ and acetylated K33, K34, K41 and K42 of H2BV were also synthesized for the ITC experiments. Firstly, TbBDF2-BD was titrated with those hyperacetylated peptides, respectively. Results showed that TbBDF2-BD preferentially recognized the hyperacetylated N-terminus of H2AZ (H2AZ27-63 hKAc) with a Kd of 0.46 mM (Figure 2C–E, Supplementary Figure S5). Thus, we wondered whether TbBDF2-BD recognizes mono-acetylated peptides with the same affinity. Next, mono-acetylated N-terminus of H2AZ peptides were synthesized (Supplementary Table S2) and titrated with TbBDF2-BD, respectively. However, all these peptides interact with the TbBDF2-BD with lower affinity than H2AZ27-63 hKAc (Figure 2E), which suggests that interactions between H2AZ and TbBDF2 may depend on hyperacetylated N-terminus of H2AZ.
The solution structure of TbBDF2-BD
The solution structure of TbBDF2-BD was solved by a series of two- and three-dimensional NMR spectra. In total, 1465 NOE distance restraints and 161 dihedral angle restraints were included in the structure calculation of TbBDF2-BD. The chemical shifts of atoms have been deposited into the Biological Magnetic Resonance Data Bank (25905), and the structures were deposited in Protein Data Bank (2N9G). A summary of the structural statistics for these 20 structures is given in Supplementary Table S3. The ensemble of 20 lowest target function structures and a representative structure are shown in Supplementary Figure S6A,B. TbBDF2-BD displays a conserved overall fold comprising four α helices (αZ, αA, αB, αC) linked by highly variable loop regions (ZA and BC loops). The four helices form a deep cavity that is extended by the two loop regions (ZA and BC loops), creating a hydrophobic pocket which is usually a KAc-binding pocket. By comparing the solution structure of TbBDF2-BD and crystal structure of TbBDF2-BD in complex with I-BET151, some differences were observed: the αA and αC helix exhibit different orientations and the ZA-loop displays varying degrees of twist (Figure 3A). These differences may be partly due to the dynamic nature of NMR, nevertheless, it is possible that conformational changes were induced by the binding of I-BET151.
The H2AZ27-63 hKAc binds to KAc-binding pocket of TbBDF2-BD.
TbBDF2-BD binds to the hyperacetylated N-terminus of H2AZ through its KAc-binding pocket
To determine hyperacetylated H2AZ-binding surface on TbBDF2-BD, NMR chemical perturbation was performed by titrating the hyperacetylated N-terminus of H2AZ peptide into 15N-labeled TbBDF2-BD to different molar ratio. 1H, 15N-HSQC spectra recorded from samples of different protein/peptide molar ratio were overlaid and analyzed (Figure 3B). Many amino acids with obvious chemical shift changes were observed, including A37, E38, E39, L40, D42, Y43, H44, D52, Y85, N86 and W92. These residues are dispersed in ZA-loop and BC-loop (Figure 3C,D, Supplementary Table S4), which form a KAc-binding pocket, revealing that TbBDF2-BD binds to the hyperacetylated N-terminus of H2AZ mainly through its KAc-binding pocket. Besides, the residues in αA, αC helix and ZA-loop also have significant chemical shift changes (Supplementary Table S4) when titrated with peptides, indicating that these residues may also contribute to the interactions between TbBDF2-BD and hyperacetylated H2AZ. Perturbation with unmodified H2AZ peptide indicated no interactions between mock H2AZ and TBBDF2-BD (Supplementary Figure S6C), which further confirmed the hyperacetylation of N-terminus of H2AZ is essential for their interactions.
The small molecule GSK2801 binds to TbBDF2-BD with high affinity
Many small molecule inhibitors have been developed for bromodomains of human, we tried to find some commonly used inhibitors that can inhibit TbBDF2-BD. Twenty-seven commonly used small molecule inhibitors of bromodomain (Supplementary Table S5) were selected to be docked with the solution structure of TbBDF2-BD (Figure 4A, Supplementary Table S6). Molecules with relatively high-binding affinity to TbBDF2-BD, and are located into the KAc pocket of TbBDF2-BD are ideal candidates for subsequent analysis. After virtual screening, four small molecule inhibitors (Supplementary Table S5) were selected for further screening. UNC669, which could not be docked into the KAc-binding pocket of TbBDF2-BD, was selected as a control. ITC experiments were performed, and all five inhibitors were titrated with TbBDF2-BD, respectively. Results showed that GSK2801 binds to TbBDF2-BD with the highest affinity (15 μM) (Figure 4B,C). The affinity of GSK2801 to TbBDF2-BD is 30 times higher than that between the hyperacetylated N-terminus of H2AZ and TbBDF2-BD, which implies its potential to be inhibitor of TbBDF2. Other bromodomains of T. brucei were also titrated with GSK2801 to measure their binding ability with GSK2801 (the bromodomain of TbBDF4 failed to be expressed, therefore was not tested in this study) (Supplementary Figure S7A–D). TbBDF3-BD and TbBDF5-BD1 do not bind to GSK2801. TbBDF1-BD has extremely low binding affinity to GSK2801. TbBDF5-BD2 binds to GSK2801 with the affinity of 83 μM. All results show that GSK2801 prefers to bind to TbBDF2-BD.
GSK2801 binds to the TbBDF2-BD.
GSK2801 competes the KAc-binding pocket of TbBDF2-BD with the hyperacetylated N-terminus of H2AZ
Given that inhibitors are usually located in the KAc-binding pocket of bromodomain , we examined whether GSK2801 binds to the KAc-binding pocket of TbBDF2-BD. NMR chemical shift perturbation was performed to analyze the interface between GSK2801 and TbBDF2-BD. TbBDF2-BD was titrated with increasing concentrations of GSK2801 (Figure 4D). Many the residues displayed obvious chemical shift changes when the molar ratio of TbBDF2 and GSK2801 reached 1 : 0.3. When the molar ratio increased to 1 : 1, the chemical shift perturbation was almost saturated, indicating that the TbBDF2 is sensitive to the small molecule compound GSK2801. Titration of DMSO was performed as a control. Perturbation result of 15N-TbBDF2-BD with DMSO suggested that DMSO does not affect the chemical shift (Supplementary Figure S6D). Similar to the perturbation of the hyperacetylated N-terminus of H2AZ, most residues involved in GSK2801 recognition are dispersed in ZA-loop and BC-loop (Figure 4E, Supplementary Table S4), and are identical with those residues involved in recognizing hyperacetylated H2AZ. It suggests that the GSK2801 and hyperacetylated H2AZ may compete KAc-binding pocket on TbBDF2-BD (Figure 5A,B). A GSK2801-TbBDF2-BD docking model revealed that the GSK2801 binds to the KAc-binding pocket directly. Unlike flat structure of IBET-151 and bromosporine, aromatic rings of GSK2801 form an L-shaped structure to occupy the KAc-binding site and is closer to BC-loop structurally than IBET-151 and bromosporine (Figure 5C,D).
GSK2801 binds to the KAc-binding pocket of TbBDF2-BD.
Given that GSK2801 and H2AZ share a similar binding interface on TbBDF2-BD and GSK2801 has a higher affinity relative to H2AZ, we supposed that GSK2801 competitively inhibits TbBDF2 binding to the hyperacetylated N-terminus of H2AZ. To confirm this hypothesis, we analyzed acetylated H2AZ peptide-binding ability to TbBDF2-BD and TbBDF2-BD/GSK2801 complex by ITC, respectively (Figure 6A,B). There was obvious exothermic phenomenon when the TbBDF2-BD was titrated with the peptide. However, after TbBDF2-BD was saturated with three-fold molar concentration of GSK2801, the exothermic phenomenon disappeared, which suggests that acetylated lysine residues on peptide were unable to compete the KAc-binding pocket of TbBDF2-BD in the presence of GSK2801. Thus, GSK2801 is able to competitively inhibit TbBDF2 binding to the hyperacetylated N-terminus of H2AZ.
The effect of GSK2801 on the procyclic-form T. brucei.
GSK2801 is lethal to T. brucei and affects the cellular location of TbBDF2
Due to high affinity of GSK2801 binding to TbBDF2, we supposed that GSK2801 should strongly affect cell growth of T. brucei. To investigate the effect of GSK2801 on T. brucei, cells were treated with different concentrations of GSK2801 and were counted daily (Figure 6C). After treated with 20 and 30 μM GSK2801 for 2 days, the cell growth was significantly inhibited. Resazurin-based cell viability assays were performed . Fluorescence was detected at three-time points. The calculated IC50 at each time point were almost identical (Figure 6D, Supplementary Figure S7E–G). The average value of IC50 is 1.368 μM. Small molecules often have side effects in vivo [46,47]. Affinity of GSK2801 to TbBDF2-BD is not comparable to the IC50 of GSK2801, implying GSK2801 has other effects beyond the inhibition of TbBDF2-BD. Indeed, we found that GSK2801 also binds to other bromodomains of T. brucei, such as TbBDF5-BD2 (Supplementary Figure S7D).
To explore the effect of GSK2801 on TbBDF2 in vivo, the cellular location of TbBDF2 was monitored by a fluorescence microscopy after the treatment of 10 μM GSK2801 for 2 days (Figure 6E). In the presence of 10 μM GSK2801, the dominant nucleolus localization of TbBDF2 disappeared, and TbBDF2 diffused in the whole nucleus. This phenomenon was also observed in cells treated by 20 μM and 30 μM GSK2801 (data not shown). From these results, we concluded that GSK2801 should be an effective inhibitor of TbBDF2.
Trypanosoma brucei is an early divergent eukaryote, which displays unique cellular processes during its life cycle. Histone modifications of T. brucei have been reported to be quite different from those of higher eukaryotes . All those differences make the roles of histone modifications and biological effects caused by recognition of these modifications special to T. brucei. However, little progress has been made to explore the roles of histone readers in T. brucei. For that purpose, we initiate our research by studying the structure and function relationship of bromodomain of T. brucei, which is a kind of histone reader responsible for recognition of acetylated histones. TbBDF2 is one of the five bromodomain-containing proteins of T. brucei  and was identified previously to be essential for the cell growth of T. brucei . In the present study, TbBDF2 was revealed as a reader that binds to the hyperacetylated N-terminus of variant histone H2AZ through its bromodomain. To the best of our knowledge, it is the first report of bromodomain binding to the hyperacetylated variant histones in T. brucei.
According to our results, we observed that acetylation occurs in a GK motif of H2AZ and H2BV of T. brucei, which has been reported as a modification motif of CBP/p300 . Acetylated GK motif was also found in H2AZ of chicken and yeast, which implies a common future of hyperacetylated histone variants and a conserved physiological function. Acetylated H2AZ of chicken and yeast has a significant role in gene activation [43–45]. The K14Ac of H2AZ associates with activated genes in yeast, and is enriched at the promotor of active gene . The hyperacetylated H2AZ is only distributed in the active gene of chicken , which indicates the importance of hyperacetylated H2AZ in maintaining the active state of gene expression. Moreover, H2AZ acetylation is also important for gene deregulation in cancer and for histone methylation and DNA methylation . H2AZ and H2BV have been reported to be abundant in TSSs . In our study, we found that TbBDF2 associates with hyperacetylated H2AZ and H2BV. However, previous ChIP-sequencing results showed that TbBDF2 distributed overall the genome . It seems that hyper-acetylation state of H2AZ and H2BV may be extremely low and displayed different genome localization. We have tried to obtain a poly-antibody against hyperacetylated N-terminus of H2AZ to investigate the chromatin localization of hyperacetylated H2AZ. However, it is difficult to obtain an ideal antibody. The hyperacetylation of H2AZ may only happen at specific times or during specific cellular events, which may explain why there is no colocalization signal between TbBDF2 and H2AZ in ChIP results.
TbBDF2 is mainly localized in the nucleolus where ribosome assembly occurs . The transcription of pre-rRNAs is extremely active, which requires chromatin continued open to transcription machines. H2AZ and H2BV have been found in the genome region of 5S DNA and rDNA spacer in T. brucei . Hyperacetylation of H2AZ and H2BV may be necessary for maintaining the open state in these areas. It has also been reported that H2A.Z and its acetylated form of mouse were found primarily 500–600 bp upstream of the spacer promoter and to a lesser extent at the rRNA gene terminator . All these evidences imply that the binding of TbBDF2 to hyperacetylated H2AZ/H2BV may play an important role in rRNA biogenesis.
Recent work has demonstrated that bromodomain proteins of T. brucei, including TbBDF2, are necessary for monoallelic expression of VSG genes . Depletion of TbBDF2 results in significantly increased transcription of VSGs located at silent bloodstream expression sites. In addition, ChIP followed by Q-PCR showed that TbBDF2 is localized to the promoter region of both the active and silent ES . These results are consistent with the nucleolus localization of TbBDF2, which indicates the importance of TbBDF2 in the regulation of VSGs expression in T. brucei.
Bromodomain is usually responsible for recognizing mono-acetylated mark. Here, we show that TbBDF2-BD is able to interact with the hyperacetylated N-terminus of H2AZ. We tried to get crystal of TbBDF2-BD/hyperacetylated H2AZ; however, we failed. Though the exact binding mode is not clear, our study shows that the hyperacetylated H2AZ binds to the classic KAc-binding pocket of TbBDF2-BD and the binding of H2AZ is able to induce conformational change of αA, αC helix and ZA-loop of TbBDF2-BD. In previous studies, Brdt-BD1 was found to recognize H4K5AcK8Ac in one pocket . It revealed a novel-binding mode that one bromodomain binds to two acetylated sites synergistically. Brdt-BD1 possesses a wide pocket, providing a large contact surface, which is specifically attached by H4K5AcK8Ac. Here, TbBDF2-BD displays a similar structure, which enables it to bind to two or multi-acetylated sites (Figure 7).
Comparison of KAc-binding pockets from bromodomain homologies.
To date, many small molecule inhibitors have been developed for bromodomains, some of which are in clinic trial. Whether these inhibitors are suitable for the treatment of trypanosomiasis promotes us to screen inhibitors for TbBDF2. Here, we found that GSK2801 displays the highest affinity to TbBDF2-BD and strongly inhibits TbBDF2 binding to hyperacetylated H2AZ. Model of GSK2801/TbBDF2-BD complex shows that GSK2801 lies in the KAc-binding pocket of TbBDF2-BD perfectly (Figure 5C), which may explain the observation that GSK2801 prevents TbBDF2 from binding to hyperacetylated H2AZ. Given that the binding of TbBDF2 to H2AZ may play an important role in gene regulation of T. brucei, GSK2801 provides a theoretical basis for drug design for African trypanosomiasis. GSK2801 shows reasonable metabolic process  and has a higher affinity for TbBDF2-BD compared with I-BET151 in vitro , which suggests that GSK2801 is an ideal lead compound for designing high-specificity TbBDF2-BD inhibitors or drugs for trypanosomiasis.
In summary, we identified that TbBDF2 is predominantly located in the nucleolus and binds to the hyperacetylated N-terminus of H2AZ, implying a role of TbBDF2 in the gene expression regulation. Structural study revealed that TbBDF2-BD binds to the hyperacetylated N-terminus of H2AZ through its KAc-binding pocket. A small molecule compound GSK2801 was found to have high affinity to TbBDF2-BD, therefore may serve as a framework for further drug design.
S.L., X.T., and X.Y. designed the research. X.Y., S.L., J.Z. performed the experiments. S.L., X.Y., X.W., X.T. analyzed the data. X.Y., S.L., and X.T., wrote the paper. X.Z. and C.X. discussed and gave advises on the manuscript.
This work was supported by National Natural Science Foundation of China. Grant numbers are [U1332137] (to X. T.),  (to S. L.) and  (to C. X.).
We are grateful to Ziyin Li of University of Texas Health Science Center at Houston for providing the wild-type T. brucei 427 procyclic-form cells and plasmids (pC-PTP-NEO and pC-EYFP-NEO) used for in situ tagging.
The Authors declare that there are no competing interests associated with the manuscript.