BRD4 interacts with PML/RARα in acute promyelocytic leukemia

Abstract

Bromodomain-containing 4 (BRD4) has been considered as an important requirement for disease maintenance and an attractive therapeutic target for cancer therapy. This protein can be targeted by JQ1, a selective small-molecule inhibitor. However, few studies have investigated whether BRD4 influenced acute promyelocytic leukemia (APL), and whether BRD4 had interaction with promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein to some extent. Results from cell viability assay, cell cycle analysis, and Annexin-V/PI analysis indicated that JQ1 inhibited the growth of NB4 cells, an APL-derived cell line, and induced NB4 cell cycle arrest at G1 and apoptosis. Then, we used co-immunoprecipitation (co-IP) assay and immunoblot to demonstrate the endogenous interaction of BRD4 and PML/RARα in NB4 cells. Moreover, downregulation of PML/RARα at the mRNA and protein levels was observed upon JQ1 treatment. Furthermore, results from the RT-qPCR, ChIP-qPCR, and re-ChIP-qPCR assays showed that BRD4 and PML/RARα co-existed on the same regulatory regions of their target genes. Hence, we showed a new discovery of the interaction of BRD4 and PML/RARα, as well as the decline of PML/RARα expression, under JQ1 treatment.

Keywords

BRD4 / PML/RARα / APL / interaction

Introduction

Acute promyelocytic leukemia (APL) is the M3 type of acute myeloblastic leukemia (AML) and defined by a reciprocal chromosomal translocation t(15;17) generating promyelocytic leukemia-retinoic acid receptor α (PML/RARα) fusion protein in the majority of patients [¹]. This fusion protein has a crucial role in the leukemogenesis of APL [²]. Pharmacological concentrations of all-trans retinoic acid (ATRA) treatment can exert terminal differentiation on APL cells and achieve over 90% complete remission in patients [¹–³]. This approach is considered to be the first example of target therapy in cancer, providing a unique model for mechanistic studies in APL and probably in other types of AML.

Chromatin regulators have been explored as candidate drug targets, because they are deregulated in numerous cancers and sensitive to small-molecule inhibitors [⁴–¹¹]. BRD4 belongs to the human bromodomain and extra-terminal (BET) protein family [¹²] and is known as a chromatin reader, which is critical for transcriptional elongation. The roles of BRD4 in various epigenetic and chromatin activities have also been well documented [¹³,¹⁴]. JQ1, which is a recently developed small-molecule bromodomain inhibitor [¹⁵–¹⁷], competitively binds to the bromodomain pocket of BRD4. JQ1 displaces BRD4 from the active chromatin and separates RNA Pol II from targeted genes [¹³]. Previous studies indicated that JQ1 can exert treatment effects on hematological malignancies [¹⁸,¹⁹], such as different types of AML [²⁰–²²], multiple myeloma [²³], Burkitt’s lymphoma [²⁴], diffuse large B cell lymphoma [²⁵], and T cell acute lymphoblastic leukemia [²⁶]. Various solid tumors, such as neuroblastoma [²⁷,²⁸] and medulloblastoma [²⁸], were also shown to be responsive to JQ1. Although BET family proteins are expressed in most cancer cells, the reason why only some of these proteins appear to be sensitive to JQ1 treatment remains unknown. However, recent data demonstrated that BRD4 selectively interacts with DNA binding proteins and transcriptional factors [²⁹], and this phenomenon suggests the importance of local genetic and epigenetic environment [³⁰]. Thus, we tested whether APL cells were responsive to JQ1 treatment, and whether the interaction between PML/RARα and BRD4 existed on chromatin of NB4 cells.

Materials and methods

Cell lines, cell culture, and reagents

NB4 cell, an APL-derived cell line, carries the t(15;17) translocation and expresses the PML/RARα fusion protein. PR9 cell is a PML/RARα-inducible model under Zn²⁺ treatment. NB4 and PR9 cells were grown in culture media including 90% RPMI-1640 and 10% fetal bovine serum (Giboco® Life Technologies) at 37 °C under 5% CO₂/95% air. JQ1, ATRA, ZnSO₄, and dimethyl sulfoxide (DMSO) were purchased from Sigma-Aldrich. JQ1 was dissolved by DMSO as a stock solution at 2 mg/mL. ATRA was dissolved in ethanol as a stock solution at 1 mmol/L. ZnSO₄ was dissolved in sterile water as a stock solution at 100 mmol/L.

Cell viability assay

Experiments were performed in 96-well plates. Approximately 10 000 cells were cultivated in each well with 100 µL of culture media containing 10% FBS supplement. The fresh media contained serially diluted JQ1 (5, 10, 50, 100, and 500 nmol/L or 1, 2, 2.5, and 10 µmol/L) or vehicle (DMSO, 0.05%). We added 10 µL of the solution Counting Kit-8 (Dojindo) to each well after 48 h or 72 h. After additional 4 h of incubation, absorbance was measured at 450 nm. We used GraphPad Prism software to normalize and calculate the values.

Cell cycle analysis

NB4 cells were treated with 0.5 µmol/L JQ1 for 24, 48, or 72 h. We collected the treated or control cells, washed them with PBS and fixed them with 70% ice-cold ethanol at –20 °C overnight. After one night, we rewashed the fixed cells with PBS, added RNase and PI to them, and finally used flow cytometry (Coulter) to analyze the cells.

Annexin-V/PI analysis

We used Annexin V-PI Apoptosis Detection Kit (BD Biosciences) to measure the percentage of apoptotic cells. We collected 5×10⁵ cells and utilized binding buffer to wash the cells. Then, we added 5 mL of Annexin-V and 10 mL of PI to the cells. After 15 min of incubation in the dark at room temperature, we detected the fluorescent intensities using flow cytometry (Coulter).

Immunoblot analysis

A total of 3×10⁶ cells were collected, washed with PBS, and lysed using RIPA buffer (Sigma-Aldrich). We used SDS-PAGE gel to separate equal amounts (40 µg) of proteins, which were extracted from control or treated cells. These cells were transferred into PVDF membranes, incubated with properly diluted primary antibodies at 4 °C for one night followed by horseradish peroxidase (HRP)-conjugated secondary antibodies, and visualized with enhanced Chemiluminescence Reagent Plus (Millipore).

Antibodies used in the experiments were anti-PML+RARα fusion protein (Abcam Catalog No. ab43152), anti-BRD4 (H-250) (Santa Cruz Biotech Catalog No. sc-48772), and β-actin (Cell Signaling Catalog No. 5125).

Co-immunoprecipitation (Co-IP) assay

We used Univeral Magnetic Co-IP Kit (Active Motif) to obtain the NB4 cells nuclear extracts and lysates. All steps were operated according to the manufacturer’s instructions.

Antibodies used in the experiments were anti-PML+ RARα fusion protein (Abcam Catalog No. ab43152), anti-BRD4 (H-250) (Santa Cruz Biotech Catalog No. sc-48772), anti-BRD4 (Bethyl Laboratories, Inc. Catalog No. A301-985A), and normal rabbit IgG (Santa Cruz Biotech Catalog No. sc-2027).

RNA isolation and quantitative real-time PCR

We used RNeasy Mini Kit (Qiagen) to isolate total RNA from NB4 cells and Reverse Transcription System (Promega) to synthesize cDNA from 1000 ng of RNA. We utilized standard SYBR green reagents and protocols (TOYOBO) to perform qPCRs in triplicate. We performed the Ct method to quantify and normalize the target mRNA expression to GAPDH expression. The following are the primers for the quantitative reverse transcription-PCR (qRT-PCR): PML/RARα (forward: 5′-AAGTGAGGTCTTCCTGCCCAA-3′; reverse: 5′-GGTGGGCACTATCTCTTCAGA-3′), CTCF (forward: 5′-TCAGTGCAGTTTGTGCA GTTA-3′; reverse: 5′-TTCCCCTGAATGGGTTCTCAT-3′), CDK13 (forward: 5′-CCCCTAGTCCCTACAGCAG-3′; reverse: 5′-GCCTAGATGAATACGGGCTTCTG-3′), EGR4 (forward: 5′-TCCTCGTCAAGTCCACTGAAG-3′; reverse: 5′-CAGGAGTCGGCTAAGTCCC-3′), CSF3R (forward: 5′-GAGCTGAGAACTACCGAACGG-3′; reverse: 5′-GGCCTGAGGGTCTCCAAGA-3′), JUNB (forward: 5′-ACAAACTCCTGAAACCGAGCC-3′; reverse: 5′-CGAGCCCTGACCAGAAAAGTA-3′), MYC (forward: 5′-GGCTCCTGGCAAAAGGTCA-3′; reverse: 5′-CTGCTAGTTGTGCTGATGT-3′), SPI1 (forward: 5′-GTGCCCTATGACACGGATCTA-3′; reverse: 5′-AGTCCCAGTAATGGTCGCTAT-3′), InG1 (forward: 5′-AACAACGAGAACCGTGAGAAC-3′; reverse: 5′-TGGTTGCACAGACAGTACGTG-3′), DAP (forward: 5′-CTCCCGAAGGGAAACTAGAGA-3′; reverse: 5′-TTCTGCACAATTCGCATTCCA-3′), RUNX1 (forward: 5′-CTGCCCATCGCTTTCAAGGT-3′; reverse: 5′-GCCGAGTAGTTTTCATCATTGCC-3′), IRF1 (forward: 5′-CTGTGCGAGTGTACCGGATG-3′; reverse: 5′-ATCCCCACATGACTTCCTCTT-3′), CDKN2D (forward: 5′-AGTCCAGTCCATGACGCAG-3′; reverse: 5′-ATCAGGCACGTTGACATCAGC-3′), FTL (forward: 5′-CAGCCTGGTCAATTTGTACCT-3′; reverse: 5′-GCCAATTCGCGGAAGAAGTG-3′), and GAPDH (forward: 5′-GAAGGTGAAGGTCGGAGTC-3′; reverse: 5′-GAAGATGGTGATGGGATTTC-3′).

ChIP-qPCR and re-ChIP-qPCR assays

The chromatin immunoprecipitation (ChIP) and re-ChIP assays for BRD4 and PML/RARα were performed using ChIP-IT ® High Sensitivity Kit and Magnetic Chromatin Re-Immunoprecipitation Kit (Active Motif) according to the manufacturer’s protocol. The antibodies used for the ChIP assay were PML (PG-M3) (Santa Cruz Biotech), RARα (C-20x) (Santa Cruz Biotech), BRD4 (Bethyl Laboratories, Inc. Catalog No. A301-985A), and normal IgG (Santa Cruz Biotech). The purified ChIP DNA samples were used as templates by qPCR for target genes. All primers for ChIP-qPCR and re-ChIP-qPCR were as follows: CDK13 (forward: 5′-GAGGCGTGTTGTTGTCTCG-3′; reverse: 5′-AGTCCCGAGTCCTCCAAGT-3′), MYC (forward: 5′-AGAGGCTTGGCGGGAAAA-3′; reverse: 5′- GCCTCTCGCTGGAATTACTAC-3′), EGR4 (forward: 5′-TCCGGATTCACAGAACCACC-3′; reverse: 5′-CTTCTGTTGCCCACCTTGAA-3′), CDKN2D (forward: 5′-CCTTTCTCCCTGGGTCAGTT-3′; reverse: 5′-AACGCGTCGCTCCTGATT-3′), InG1 (forward: 5′-GAGGACGAGGGCTTTTCTCT-3′; reverse: 5′-CACAGTCCTCTCCAAGCCA-3′), IRF1 (forward: 5′-TGAGGTCACACAGCCTGT-3′; reverse: 5′-TCCCTACCTATTCCTCCCCA-3′), FTL (forward: 5′-CAGAGGCCAGTGACCTTAG-3′; reverse: 5′-GGACAGAGACCCAGAGATGG-3′), CEBPE (forward: 5′-AGTAGACCCAAGAGACACGC-3′; reverse: 5′-CTCTGCCTCTGGTTTCCTGT-3′), and RUNX1 (forward: 5′- AATCTCCTTGCCACCCTCAG-3′; reverse: 5′-CACTCTCCACTTCCTCACCA-3′).

Accession numbers

We used Accession No. GSE19202 at NCBI Gene Expression Omnibus to obtain ChIP-on-chip data. We also downloaded ChIP-seq data used in this study from NCBI SRA (http://www.ncbi.nlm.nih.gov/sra) through Accession Nos. SRR617752, SRR1106515, SRR1143134, SRR846906, SRR617757, and SRR747901.

Statistical analysis

Quantitative data are shown as the mean±standard deviation (SD) of three independent experiments. We used GraphPad Prism software to assess statistical analyses and two-tailed t-tests to validate the significance of all data. Statistical significance was considered as P-values of less than 0.05.

Results

APL cells were responsive to JQ1 treatment, indicating G1 phase arrest and apoptosis

We first conducted a series of cellular assays to test APL cells under JQ1 treatment. NB4 cells were treated with serially diluted JQ1 for 48 h or 72 h and assayed through a CCK-8 kit. The growth of NB4 cells was obviously inhibited by JQ1, indicating dose- and time-dependence (Fig. 1A). To detect whether the cellular effect of JQ1 had association with NB4 cell cycle progression, NB4 cells were sustained over a 3-day time course after 0.5 µmol/L JQ1 treatment. JQ1 induced significant G1 phase arrest when treated cells were compared with control cells (Fig. 1B). Interestingly, the population of a sub-G1 also increased after JQ1 treatment for 72 h (Fig. 1B). Then, we performed apoptotic assays on NB4 cells. The percentage of apoptotic cells increased after treatment with 0.5 µmol/L or 1.0 µmol/L JQ1 for 24, 48, or 72 h (Fig. 1C). These results showed that JQ1 inhibited NB4 cell growth and induced NB4 cell apoptosis. Thus, the response of NB4 cells to JQ1 suggested the importance of BRD4 in APL cells.

BRD4 interacted with PML/RARα and was functionally associated with the stability of PML/RARα in NB4 cells

To determine whether BRD4 interacted with PML/RARα, we first evaluated BRD4 expression in NB4 cells (Fig. 2A). The DMSO treatment was regarded as control. The level of BRD4 protein was not influenced by independent treatment of JQ1 or ATRA or by combined ATRA and JQ1 (Fig. 2A). Then, we conducted co-immunoprecipitation experiments using specific antibodies of PML/RARα and BRD4 in NB4 cells. Endogenous interaction of BRD4 and PML/RARα was observed (Fig. 2B). However, we also discovered that BRD4 did not interact with wild-type PML and RARA. Moreover, we respectively treated the cells with JQ1 and ATRA and examined the protein expression levels of BRD4 and PML/RARα in the NB4 cells. The results showed that the expression of BRD4 was barely affected either by JQ1 or ATRA treatment (Fig. 2A), but the expression of PML/RARα was downregulated by independent treatments with ATRA or JQ1 (Fig. 2C). To further test this observation, we conducted a similar experiment in PR9 cells which can express PML/RARα under the induction of Zn²⁺ treatment. After incubation with Zn²⁺ for 4 h, PR9 cells were treated with ATRA or JQ1 for 48 h. Western blots were applied to evaluate changes in the expression of PML/RARα before and after treatment. The level of PML/RARα protein decreased in the JQ1-treated PR9 cells, similar to that of the ATRA-treated PR9 cells (Fig. 2D). This reduction in PML/RARα not only appeared at the protein level but also at the mRNA level (Fig. 2E). Hence, these results indicate that PML/RARα and BRD4 are probably in a complex loop, in which BRD4 may participate in retaining the stability of PML/RARα in APL cells.

BRD4 exhibited substantial impact on PML/RARα-regulated genes, particularly those associated with cell growth and apoptosis

Then, we evaluated whether BRD4 participated in the PML/RARα-involved gene regulation. We first conducted an electronic analysis of ChIP-on-chip/seq data published by our group and another study. The results show a total of 2424 candidate genes containing binding regions of both PML/RARα and BRD4 (Fig. 3A). Among these genes, we tested a series of genes responsible for cell proliferation and apoptosis through RT-qPCR in NB4 cells treated with JQ1 or ATRA. After 24 h of treatment with 0.5 µmol/L JQ1, downregulated genes included EGR4, CSF3R, MYC, and SPI1 (Fig. 3B), while treatment with 1 µmol/L JQ1 for 24 h also resulted in reduced mRNA levels of CTCF, CDK13, and JUNB (Fig. 3B). Treatment with 0.5 µmol/L or 1 µmol/L JQ1 for 24 h upregulated InG1, DAP, RUNX1, IRF1, CDKN2D, and FTL (Fig. 3C). However, ATRA treatment downregulated MYC and JUNB (Fig. 3B) but upregulated InG1, EGR4, CDK13, RUNX1, DAP, CTCF, CDKN2D, SPI1, CSF3R, FTL, CEBPE, PIM1, and IRF1 (Fig. 3C). BRD4 and PML/RARα-involved gene regulation is markedly more complex than previously inferred. Nevertheless, BRD4 clearly functioned as an important factor coordinating PML/RARα regulatory complexes in APL.

BRD4 and PML/RARα bound to the same regulatory regions on chromatin

Based on the above observations, we further examined whether BRD4 and PML/RARα existed in the same regulatory regions of the tested genes through ChIP-qPCR assay in the cells before and after treatment with JQ1 or ATRA. In the untreated NB4 cells, enrichment levels of PML/RARα and BRD4 were markedly high in most of the tested regions (Fig. 4A). However, enrichment levels of both BRD4 and PML/RARα were significantly reduced in the treated cells (Fig. 4B and 4C). In addition, we performed sequential ChIP (re-ChIP) assays. ChIP regions immunoprecipitated with the first indicated antibody (anti-BRD4/ anti-PML/RARα) were re-immunoprecipitated with the second indicated antibody (anti-PML/RARα/ anti-BRD4) (Fig. 4D and 4E). These results provided evidence that BRD4 and PML/RARα bound to the same narrow regions on chromatin, and their bindings were reduced under JQ1 or ATRA treatment in NB4 cells. These phenomena implicate a functional interplay between these two factors for the transcription regulation of their target genes.

Discussion

This study demonstrates that BRD4 is an important chromatin regulator in APL. First, the results revealed that suppression of BRD4 exhibited growth inhibitory effects on NB4 cells. Second, the experimental results showed that BRD4 interacted with PML/RARα, and JQ1-mediated BRD4 inhibition decreased the protein and mRNA levels of PML/RARα. Third, BRD4 exerted substantial impact on PML/RARα-regulated genes. Fourth, BRD4 and PML/RARα co-occupied the same regions on chromatin in NB4 cells. Our results may provide a new direction of mechanistic and therapeutic studies in APL and probably in other types of AML.

BRD4 has recently attracted attention, because this chromatin binding factor plays a crucial role in various types of cancer. This molecule also exhibits promising potential in cancer therapy, once targeted by its inhibitors. BRD4 influences mitotic progression [³¹,³²]. BRD4 binds to the transcription start sites of genes expressed during the M/G1 transition, functionally recruits P-TEFb to mitotic chromosomes, and consequently promotes the expression of proliferation-related genes [³¹,³²]. P-TEFb is known to include a core cyclin-dependent kinase-9 (CDK9) [³³–³⁵] and phosphorylates the RNA polymerase II (RNA Pol II) CTD, leading to transcription [³⁶]. Although further clarification is warranted, our data show the G1 phase suppression in JQ1-treated APL cells, implicating the presence of aforementioned BRD4-associated mechanisms in APL cells. At the transcriptomic level, those genes related to promote proliferation and targeted by PML/RARα were also markedly downregulated in JQ1-treated NB4 cells. This phenomenon also provides another evidence that BRD4 is an important part in the cell cycle of APL. Moreover, the interaction between BRD4 and PML/RARα suggests that the transcription regulation of proliferation-promoting genes by PML/RARα was possibly mediated through BRD4. This observation is also supported by the dynamic changes in PML/RARα and BRD4 bindings under ATRA or JQ1 treatment and by changes in the expression of the proliferation-promoting genes.

The pharmacological concentration of ATRA (10⁻⁷–10⁻⁶ mol/L) can influence the expression of PML/RARα [¹]. Here, PML/RARα was also affected by JQ1 at mRNA and protein levels. JQ1 targeted the N-terminal bromodomains of BRD4 and resulted in BRD4 displaced from active chromatin. We inferred that changes in the expression of these target genes upon JQ1 treatment might influence associative pathways and cause the activation of proteasome, ultimately leading to reduction of PML/RARα fusion protein. The attenuation of PML/RARα fusion protein affected its target genes, consequently impacting cell functions. Nevertheless, the PML/RARα fusion gene, as a target gene, may be regulated by BRD4. In addition, ATRA could induce NB4 cell differentiation [¹–³]. We conducted cell differentiation experiments on the NB4 cells. However, our data presented that JQ1 did not induce granulocytic differentiation of NB4 cells. Therefore, the different function mechanisms between ATRA and JQ1 need to be further elucidated.

Thus, in this work, we revealed that BRD4 exerted impact on PML/RARα-regulated genes. We selected several candidate target genes, such as CDKN2D, InG1, CDK13, MYC, CSF3R, IRF1, EGR4, DAP, RUNX1, JUNB, SPI1, FTL, and CTCF, from these overlapping genes. The data showed that the expression of these genes was altered in JQ1-treated NB4 cells. Upregulated genes included InG1, DAP, RUNX1, IRF1, CDKN2D, and FTL. By contrast, CTCF, CDK13, EGR4, CSF3R, JUNB, MYC, and SPI1 were downregulated. We provided an explanation of the downregulated genes according to previous reports, that is, JQ1 competitive binding to the bromodomain pocket resulted in the displacement of BRD4 from active chromatin and subsequent removal of RNA Pol II from target genes, consequently affecting the expression of target genes. Moreover, under JQ1 treatment, the downregulation of target genes could be partially explained by the observed loss of BRD4 recruitment to their regulatory regions. For the upregulated genes, we inferred that BRD4 may have interacted with co-repressors or co-activators, directly influencing its target genes or indirectly affecting downstream target genes regulated by target genes of BRD4. However, upon JQ1 treatment, co-repressor release and co-activator recruitment could upregulate several target genes. ChIP-qPCR analysis showed that treating NB4 cells with JQ1 led to reduced levels of BRD4 at candidate target genes regulatory regions. However, whether such JQ1-BET bromodomain inhibition caused a general reduction in BRD4 occupancy associated with the genome in NB4 cells remains ambiguous.

BRD4 is a widely expressed protein in various types of cancer, whereas PML/RARα is a distinct oncoprotein only occurring in APL. Thus, physical and functional interplay between these two proteins may provide an additional link between APL and other types of AML. For instance, we previously reported that a master transcription factor of myeloid differentiation, PU.1, interacted with PML/RARα in APL. Thus, we provided the first evidence why the formation of this distinct oncoprotein blocks myeloid differentiation in APL. Accordingly, benefits of this investigation may go beyond mechanistic and therapeutic studies in APL to include those in other types of AML.

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