Please wait a minute...
Frontiers of Medicine

ISSN 2095-0217

ISSN 2095-0225(Online)

CN 11-5983/R

Postal Subscription Code 80-967

2018 Impact Factor: 1.847

Front. Med.    2018, Vol. 12 Issue (6) : 726-734    https://doi.org/10.1007/s11684-017-0604-x
RESEARCH ARTICLE |
BRD4 interacts with PML/RARα in acute promyelocytic leukemia
Qun Luo1, Wanglong Deng1, Haiwei Wang2, Huiyong Fan1, Ji Zhang1,2()
1. State Key Laboratory of Medical Genomics, Ruijin Hospital Affiliated to Shanghai Jiao Tong University School of Medicine, Shanghai 200025, China
2. Institute of Health Sciences, Shanghai Institutes for Biological Sciences and Graduate School, Chinese Academy of Sciences, Shanghai 200025, China
 Download: PDF(480 KB)   HTML
 Export: BibTeX | EndNote | Reference Manager | ProCite | RefWorks
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     
Corresponding Authors: Ji Zhang   
Just Accepted Date: 02 February 2018   Online First Date: 26 March 2018    Issue Date: 03 December 2018
 Cite this article:   
Qun Luo,Wanglong Deng,Haiwei Wang, et al. BRD4 interacts with PML/RARα in acute promyelocytic leukemia[J]. Front. Med., 2018, 12(6): 726-734.
 URL:  
http://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0604-x
http://academic.hep.com.cn/fmd/EN/Y2018/V12/I6/726
Fig.1  JQ1 exerted effects on NB4 cells. (A) NB4 cells were exposed to various concentrations of JQ1 for 48 h or 72 h. CCk-8 assay displayed the cytotoxic effect of JQ1 after the proportions of viable cells between treated and control cells were compared. (B) NB4 cells were cultured with JQ1 at 0.5 µmol/L for 24, 48, or 72 h. Collected cells were stained with PI. Flow cytometry detected the DNA content and cell cycle distribution. The percentage of cells in the cycle is shown. (C) NB4 cells were treated with JQ1 at 0.5 µmol/L or 1 µmol/L for 24, 48, or 72 h. The percentage of apoptotic NB4 cells (lower and upper right quadrants) in Annexin-V/PI assay was measured by flow cytometry. All results were representative images from three experiments. All data were the mean±SD of three replicates. Two-tailed t-tests were used to validate the significance of all data. P-value<0.05 was considered as statistically significant (*P<0.05; **P<0.01; ***P<0.001).
Fig.2  BRD4 interacted with PML/RARα, and JQ1 treatment affected PML/RARα protein and mRNA levels in NB4 cells. (A) Immunoblot analysis of BRD4 protein in JQ1 or/and ATRA treated-NB4 cells for 24, 48, or 72 h. β-Actin was used as an internal control. (B) Co-IP assay of BRD4 and PML/RARα in NB4 cells. Immunoblots of input lysates or immunoprecipitates were analyzed using the indicated antibodies. (C) In NB4 cells, PML/RARα fusion protein was diminished upon JQ1 or ATRA treatment for 12, 24, 48, or 72 h through immunoblot analysis. The band of PML/RARα fusion protein was at 130 kDa. β-Actin was used as an internal control. (D) Immunoblot experiment was conducted on Zn2+- incubated PR9 cells treated with JQ1 or ATRA for 48 h. PML/RARα fusion protein level was decreased under JQ1 or ATRA treatment. β-Actin was used as an internal control. (E) In NB4 cells, the mRNA level of PML/RARα was also depressed by JQ1 for 24 h as shown by RT-qPCR analysis. Error bars represent SD of triplicate measurements. Two-tailed t-tests were performed to validate the significance of all data. P-value<0.05 was considered as statistically significant (*P<0.05; **P<0.01; ***P<0.001).
Fig.3  BRD4 exerted impacts on PML/RARα target genes. (A) Venn diagram of genes targeted by BRD4 and PML/RARα. RT-qPCR results of (B) downregulated and (C) upregulated genes in NB4 cells incubated with 0.5 µmol/L or 1?µmol/L JQ1 or 1 µmol/L ATRA for 24 h. The data represent the mean of three replicates±SD of three experiments. Two-tailed t-tests were used to validate the significance of all data. P-value<0.05 was considered as statistically significant (*P<0.05; **P<0.01; ***P<0.001).
Fig.4  BRD4 and PML/RARα occupied the same regulatory regions. The PML/RARα and BRD4 occupancy at indicated target genes regulatory regions was validated by ChIP-qPCR assays. ChIP-qPCR was performed on NB4 cells in the (A) absence or (B) presence of 0.5 µmol/L JQ1 treatment for 24 h. (C) ChIP-qPCR analysis of PML/RARα and BRD4 on the chromatin prepared from NB4 cells treated with 1 µmol/L ATRA for 24 h. (D) Re-ChIP using BRD4 for the first round of ChIP. (E) Re-ChIP using PML/RARα for the first round of ChIP. The experiments showed that BRD4 and PML/RARα were localized in the same regions of their target genes. Data are shown as fold enrichment of ChIPed DNA versus IgG DNA. Graphs indicated three independent biological replicates. Error bars represent SD of triplicate measurements.
1 Zhou GB, Zhang J, Wang ZY, Chen SJ, Chen Z. Treatment of acute promyelocytic leukaemia with all-trans retinoic acid and arsenic trioxide: a paradigm of synergistic molecular targeting therapy. Philos Trans R Soc Lond B Biol Sci 2007; 362(1482): 959–971
https://doi.org/10.1098/rstb.2007.2026 pmid: 17317642
2 Wang K, Wang P, Shi J, Zhu X, He M, Jia X, Yang X, Qiu F, Jin W, Qian M, Fang H, Mi J, Yang X, Xiao H, Minden M, Du Y, Chen Z, Zhang J. PML/RARα targets promoter regions containing PU.1 consensus and RARE half sites in acute promyelocytic leukemia. Cancer Cell 2010; 17(2): 186–197
https://doi.org/10.1016/j.ccr.2009.12.045 pmid: 20159610
3 Wang ZY, Chen Z. Acute promyelocytic leukemia: from highly fatal to highly curable. Blood 2008; 111(5): 2505–2515
https://doi.org/10.1182/blood-2007-07-102798 pmid: 18299451
4 Dawson MA, Kouzarides T. Cancer epigenetics: from mechanism to therapy. Cell 2012; 150(1): 12–27
https://doi.org/10.1016/j.cell.2012.06.013 pmid: 22770212
5 Chen SS, Raval A, Johnson AJ, Hertlein E, Liu TH, Jin VX, Sherman MH, Liu SJ, Dawson DW, Williams KE, Lanasa M, Liyanarachchi S, Lin TS, Marcucci G, Pekarsky Y, Davuluri R, Croce CM, Guttridge DC, Teitell MA, Byrd JC, Plass C. Epigenetic changes during disease progression in a murine model of human chronic lymphocytic leukemia. Proc Natl Acad Sci USA 2009; 106(32): 13433–13438
https://doi.org/10.1073/pnas.0906455106 pmid: 19666576
6 Elsässer SJ, Allis CD, Lewis PW. Cancer. New epigenetic drivers of cancers. Science 2011; 331(6021): 1145–1146
https://doi.org/10.1126/science.1203280 pmid: 21385704
7 You JS, Jones PA. Cancer genetics and epigenetics: two sides of the same coin? Cancer Cell 2012; 22(1): 9–20
https://doi.org/10.1016/j.ccr.2012.06.008 pmid: 22789535
8 Cole PA. Chemical probes for histone-modifying enzymes. Nat Chem Biol 2008; 4(10): 590–597
https://doi.org/10.1038/nchembio.111 pmid: 18800048
9 Geutjes EJ, Bajpe PK, Bernards R. Targeting the epigenome for treatment of cancer. Oncogene 2012; 31(34): 3827–3844
https://doi.org/10.1038/onc.2011.552 pmid: 22139071
10 Issa JP, Kantarjian HM. Targeting DNA methylation. Clin Cancer Res 2009; 15(12): 3938–3946
https://doi.org/10.1158/1078-0432.CCR-08-2783 pmid: 19509174
11 Marks PA, Xu WS. Histone deacetylase inhibitors: potential in cancer therapy. J Cell Biochem 2009; 107(4): 600–608
https://doi.org/10.1002/jcb.22185 pmid: 19459166
12 Taniguchi Y. The bromodomain and extra-terminal domain (BET) family: functional anatomy of BET paralogous proteins. Int J Mol Sci 2016; 17(11): 1849
https://doi.org/10.3390/ijms17111849 pmid: 27827996
13 Lovén J, Hoke HA, Lin CY, Lau A, Orlando DA, Vakoc CR, Bradner JE, Lee TI, Young RA. Selective inhibition of tumor oncogenes by disruption of super-enhancers. Cell 2013; 153(2): 320–334
https://doi.org/10.1016/j.cell.2013.03.036 pmid: 23582323
14 Jang MK, Mochizuki K, Zhou M, Jeong HS, Brady JN, Ozato K. The bromodomain protein Brd4 is a positive regulatory component of P-TEFb and stimulates RNA polymerase II-dependent transcription. Mol Cell 2005; 19(4): 523–534
https://doi.org/10.1016/j.molcel.2005.06.027 pmid: 16109376
15 Filippakopoulos P, Qi J, Picaud S, Shen Y, Smith WB, Fedorov O, Morse EM, Keates T, Hickman TT, Felletar I, Philpott M, Munro S, McKeown MR, Wang Y, Christie AL, West N, Cameron MJ, Schwartz B, Heightman TD, La Thangue N, French CA, Wiest O, Kung AL, Knapp S, Bradner JE, Bradner JE. Selective inhibition of BET bromodomains. Nature 2010; 468(7327): 1067–1073
https://doi.org/10.1038/nature09504 pmid: 20871596
16 Nicodeme E, Jeffrey KL, Schaefer U, Beinke S, Dewell S, Chung CW, Chandwani R, Marazzi I, Wilson P, Coste H, White J, Kirilovsky J, Rice CM, Lora JM, Prinjha RK, Lee K, Tarakhovsky A. Suppression of inflammation by a synthetic histone mimic. Nature 2010; 468(7327): 1119–1123
https://doi.org/10.1038/nature09589 pmid: 21068722
17 Decker TM, Kluge M, Krebs S, Shah N, Blum H, Friedel CC, Eick D. Transcriptome analysis of dominant-negative Brd4 mutants identifies Brd4-specific target genes of small molecule inhibitor JQ1. Sci Rep 2017; 7(1): 1684
https://doi.org/10.1038/s41598-017-01943-6 pmid: 28490802
18 Bastien G, Diogo FTV, Jana K, Sami N, Julianne O, André H, Geneviève L, Iman F, Mathieu T, Véronique L, Elizabeth O, Milena K, Dominique G, Joël R, Paul SM, Jalila C, Anne M, Josée H, Guy S, Benjamin HK, Philippe PR, Trang H. High-throughput screening in niche-based assay identifies compounds to target preleukemic stem cells. J Clin Invest 2016; 126(12): 4569–4584
https://doi.org/10.1172/JCI86489 pmid: 27797342
19 Abedin SM, Boddy CS, Munshi HG. BET inhibitors in the treatment of hematologic malignancies: current insights and future prospects. Onco Targets Ther 2016; 9: 5943–5953
https://doi.org/10.2147/OTT.S100515 pmid: 27729803
20 Zuber J, Shi J, Wang E, Rappaport AR, Herrmann H, Sison EA, Magoon D, Qi J, Blatt K, Wunderlich M, Taylor MJ, Johns C, Chicas A, Mulloy JC, Kogan SC, Brown P, Valent P, Bradner JE, Lowe SW, Vakoc CR. RNAi screen identifies Brd4 as a therapeutic target in acute myeloid leukaemia. Nature 2011; 478(7370): 524–528
https://doi.org/10.1038/nature10334 pmid: 21814200
21 Dawson MA, Prinjha RK, Dittmann A, Giotopoulos G, Bantscheff M, Chan WI, Robson SC, Chung CW, Hopf C, Savitski MM, Huthmacher C, Gudgin E, Lugo D, Beinke S, Chapman TD, Roberts EJ, Soden PE, Auger KR, Mirguet O, Doehner K, Delwel R, Burnett AK, Jeffrey P, Drewes G, Lee K, Huntly BJ, Kouzarides T. Inhibition of BET recruitment to chromatin as an effective treatment for MLL-fusion leukaemia. Nature 2011; 478(7370): 529–533
https://doi.org/10.1038/nature10509 pmid: 21964340
22 Saenz DT, Fiskus W, Manshouri T, Rajapakshe K, Krieger S, Sun B, Mill CP, DiNardo C, Pemmaraju N, Kadia T, Parmar S, Sharma S, Coarfa C, Qiu P, Verstovsek S, Bhalla KN. BET protein bromodomain inhibitor-based combinations are highly active against post-myeloproliferative neoplasm secondary AML cells. Leukemia 2017; 31(3): 678–687
https://doi.org/10.1038/leu.2016.260 pmid: 27677740
23 Delmore JE, Issa GC, Lemieux ME, Rahl PB, Shi J, Jacobs HM, Kastritis E, Gilpatrick T, Paranal RM, Qi J, Chesi M, Schinzel AC, McKeown MR, Heffernan TP, Vakoc CR, Bergsagel PL, Ghobrial IM, Richardson PG, Young RA, Hahn WC, Anderson KC, Kung AL, Bradner JE, Mitsiades CS. BET bromodomain inhibition as a therapeutic strategy to target c-Myc. Cell 2011; 146(6): 904–917
https://doi.org/10.1016/j.cell.2011.08.017 pmid: 21889194
24 Mertz JA, Conery AR, Bryant BM, Sandy P, Balasubramanian S, Mele DA, Bergeron L, Sims RJ 3rd, Mele DA. Targeting MYC dependence in cancer by inhibiting BET bromodomains. Proc Natl Acad Sci USA 2011; 108(40): 16669–16674
https://doi.org/10.1073/pnas.1108190108 pmid: 21949397
25 Chapuy B, McKeown MR, Lin CY, Monti S, Roemer MG, Qi J, Rahl PB, Sun HH, Yeda KT, Doench JG, Reichert E, Kung AL, Rodig SJ, Young RA, Shipp MA, Bradner JE. Discovery and characterization of super-enhancer-associated dependencies in diffuse large B cell lymphoma. Cancer Cell 2013; 24(6): 777–790
https://doi.org/10.1016/j.ccr.2013.11.003 pmid: 24332044
26 Knoechel B, Roderick JE, Williamson KE, Zhu J, Lohr JG, Cotton MJ, Gillespie SM, Fernandez D, Ku M, Wang H, Piccioni F, Silver SJ, Jain M, Pearson D, Kluk MJ, Ott CJ, Shultz LD, Brehm MA, Greiner DL, Gutierrez A, Stegmaier K, Kung AL, Root DE, Bradner JE, Aster JC, Kelliher MA, Bernstein BE. An epigenetic mechanism of resistance to targeted therapy in T cell acute lymphoblastic leukemia. Nat Genet 2014; 46(4): 364–370
https://doi.org/10.1038/ng.2913 pmid: 24584072
27 Puissant A, Frumm SM, Alexe G, Bassil CF, Qi J, Chanthery YH, Nekritz EA, Zeid R, Gustafson WC, Greninger P, Garnett MJ, McDermott U, Benes CH, Kung AL, Weiss WA, Bradner JE, Stegmaier K. Targeting MYCN in neuroblastoma by BET bromodomain inhibition. Cancer Discov 2013; 3(3): 308–323
https://doi.org/10.1158/2159-8290.CD-12-0418 pmid: 23430699
28 Bandopadhayay P, Bergthold G, Nguyen B, Schubert S, Gholamin S, Tang Y, Bolin S, Schumacher SE, Zeid R, Masoud S, Yu F, Vue N, Gibson WJ, Paolella BR, Mitra SS, Cheshier SH, Qi J, Liu KW, Wechsler-Reya R, Weiss WA, Swartling FJ, Kieran MW, Bradner JE, Beroukhim R, Cho YJ. BET bromodomain inhibition of MYC-amplified medulloblastoma. Clin Cancer Res 2014; 20(4): 912–925
https://doi.org/10.1158/1078-0432.CCR-13-2281 pmid: 24297863
29 Wu SY, Lee AY, Lai HT, Zhang H, Chiang CM. Phospho switch triggers Brd4 chromatin binding and activator recruitment for gene-specific targeting. Mol Cell 2013; 49(5): 843–857
https://doi.org/10.1016/j.molcel.2012.12.006 pmid: 23317504
30 Asangani IA, Dommeti VL, Wang X, Malik R, Cieslik M, Yang R, Escara-Wilke J, Wilder-Romans K, Dhanireddy S, Engelke C, Iyer MK, Jing X, Wu YM, Cao X, Qin ZS, Wang S, Feng FY, Chinnaiyan AM. Therapeutic targeting of BET bromodomain proteins in castration-resistant prostate cancer. Nature 2014; 510(7504): 278–282
https://doi.org/10.1038/nature13229 pmid: 24759320
31 Yang Z, He N, Zhou Q. Brd4 recruits P-TEFb to chromosomes at late mitosis to promote G1 gene expression and cell cycle progression. Mol Cell Biol 2008; 28(3): 967–976
https://doi.org/10.1128/MCB.01020-07 pmid: 18039861
32 Yang Z, Yik JH, Chen R, He N, Jang MK, Ozato K, Zhou Q. Recruitment of P-TEFb for stimulation of transcriptional elongation by the bromodomain protein Brd4. Mol Cell 2005; 19(4): 535–545
https://doi.org/10.1016/j.molcel.2005.06.029 pmid: 16109377
33 Marshall NF, Price DH. Purification of P-TEFb, a transcription factor required for the transition into productive elongation. J Biol Chem 1995; 270(21): 12335–12338
https://doi.org/10.1074/jbc.270.21.12335 pmid: 7759473
34 Md SJ,Pei WH, Yih J, Edward BS, Andrew P. Short Communication: The broad-spectrum histone deacetylase inhibitors vorinostat and panobinostat activate latent HIV in CD4+ T cells in part through phosphorylation of the T-Loop of the CDK9 subunit of P-TEFb. AIDS Res Hum Retroviruses 2016; 32(2): 169–173
https://doi.org/10.1089/aid.2015.0347 pmid: 26727990
35 Sansó M, Levin RS, Lipp JJ, Wang VY, Greifenberg AK, Quezada EM, Ali A, Ghosh A, Larochelle S, Rana TM, Geyer M, Tong L, Shokat KM, Fisher RP. P-TEFb regulation of transcription termination factor Xrn2 revealed by a chemical genetic screen for Cdk9 substrates. Genes Dev 2016; 30(1): 117–131
https://doi.org/10.1101/gad.269589.115 pmid: 26728557
36 Price DH. P-TEFb, a cyclin-dependent kinase controlling elongation by RNA polymerase II. Mol Cell Biol 2000; 20(8): 2629–2634
https://doi.org/10.1128/MCB.20.8.2629-2634.2000 pmid: 10733565
[1] Tiange Wang, Min Xu, Yufang Bi, Guang Ning. Interplay between diet and genetic susceptibility in obesity and related traits[J]. Front. Med., 2018, 12(6): 601-607.
[2] Dan Huang, Yan Yang, Jian Sun, Xiaorong Dong, Jiao Wang, Hongchen Liu, Chengquan Lu, Xueyu Chen, Jing Shao, Jinsong Yan. Annexin A2-S100A10 heterotetramer is upregulated by PML/RARα fusion protein and promotes plasminogen-dependent fibrinolysis and matrix invasion in acute promyelocytic leukemia[J]. Front. Med., 2017, 11(3): 410-422.
[3] Haiyan He, Ran An, Jian Hou, Weijun Fu. Arsenic trioxide induced rhabdomyolysis, a rare but severe side effect, in an APL patient: a case report[J]. Front. Med., 2017, 11(2): 284-286.
[4] Yonglan Zhu,Fang Zhang,Shanzhen Zhang,Wanglong Deng,Huiyong Fan,Haiwei Wang,Ji Zhang. Regulatory mechanism and functional analysis of S100A9 in acute promyelocytic leukemia cells[J]. Front. Med., 2017, 11(1): 87-96.
[5] Wei Wang,Ming Li,Li Wang,Xueqing Yu. DQB1*060101 may contribute to susceptibility to immunoglobulin A nephropathy in southern Han Chinese[J]. Front. Med., 2016, 10(4): 507-516.
[6] Du Yan, Han Xue, Pu Rui, Xie Jiaxin, Zhang Yuwei, Cao Guangwen. Association of miRNA-122-binding site polymorphism at the interleukin-1 α gene and its interaction with hepatitis B virus mutations with hepatocellular carcinoma risk[J]. Front. Med., 2014, 8(2): 217-226.
[7] Jia-Xin XIE, Jian-Hua YIN, Qi ZHANG, Rui PU, Wen-Ying LU, Hong-Wei ZHANG, Guang-Wen CAO, Jun ZHAO, Hong-Yang WANG, . Association of novel mutations and heplotypes in the preS region of hepatitis B virus with hepatocellular carcinoma[J]. Front. Med., 2010, 4(4): 419-429.
[8] SUN Pin, ZHANG Zhongbin, WU Fen, WAN Junxiang, JIN Xibeng, XIA Zhaolin. Association of the genetic polymorphism of EPHX1 and EPHX2 with the susceptibility to chronic benzene poisoning[J]. Front. Med., 2007, 1(3): 320-326.
[9] WANG Zhonggao. Not asthma, but GERD: case report[J]. Front. Med., 2007, 1(1): 115-119.
Viewed
Full text


Abstract

Cited

  Shared   
  Discussed