BRD4 interacts with PML/RARα in acute promyelocytic leukemia

doi:10.1007/s11684-017-0604-x

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 Zhang^1,²(

)

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

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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 Author(s): 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:

https://academic.hep.com.cn/fmd/EN/10.1007/s11684-017-0604-x
https://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.

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