BRD4 Interacted With PML/RARɑ And They Co-regulated A Subset Of Target Genes In Acute Promyelocytic Leukemia

Acute Promyelocytic Leukemia(APL)has been researched most deeply and has the best effects of prognosis.It is a subset of acute myeloblastic leukemia(AML)which is the type M3 of the FAB classification.A typical t(15;17)(q22,q21)translocation results in the promyelocytic leukemia retinoic acid receptorα(PML/RARɑ)fusion protein which plays a crucial role in leukemogenesis of APL.PML/RARɑ onco-fusion protein contains the protein-protein interaction domain of PML and also includes the DNA bingding domain and the ligand binding domain of RARɑ in APL.Pharmacological concentrations of all-trans retinoic acid(ATRA)caneffectively relieve the inhibition of PML/RARɑ and degrade PML/RAR fusion protein,consequently inducing the differentiation of APL cells.BRD4 is a kind of epigenetic reader and transcriptional regulator.It is reported that BRD4 interacts with multiple factors including recognization of histone acetylation,activation of a transcriptional complex and RNAPII.Furthermore,BRD4 can interact with sequence-specific DNA-binding transcription proteins in a gene-specific manner.Recent researches have shown that bromodomains of BRD4 can be specifically targeted by a selective small molecule inhibitor JQ1 which has great anti-proliferative effect on a range of cancers,especially hematological malignancies.JQ1 has already been proven to effectively treat certain cancers.As different tumor types have different genetic and epigenetic landscape,the action of BET inhibitors might be selectively influenced by distinct transcriptional regulators associated with BRD4.Here,we found that BRD4 interacted with PML/RARɑ in APL.Furthermore,PML/RARɑ and BRD4 co-regulated a subset of target genes,such as CDKN2 D,InG1,CDK13,MYC,CSF3 R,IRF1,EGR4,DAP,RUNX1,JUNB,SPI1 and FTL which are responsible for proliferation,cell cycle and apoptosis.By RT-qPCR analysis,the levels of messenger RNA of these target genes were influenced by the inhibition of BRD4 or PML/RARɑ.ByChIP-qPCR analysis,BRD4 and PML/RARɑ occupancy at these target genes regulatory regions,such as MYC promoter,CDK13 promoter,EGR4 enhancer,CDKN2 D promoter,InG1 promoter,IRF1 enhancer,FTL enhancer,CEBPE promoter and RUNX1 promoter,were all lost after BRD4 or PML/RARɑinhibition.By ChIP-re-ChIP assay,we found that BRD4 and PML/RARɑwere localized in the same regions of their target genes.Hence,BRD4 was invovled in APL and associated with PML/RARɑ.