An atheroprotective transcriptome induced by JQ1 (a BRD4 inhibitor) in human endothelial cells

The bromodomain and extra terminal domain (BET) family of proteins, including BRD2, BRD3, and BRD4, play a key role in many cellular processes, including inflammatory gene expression, mitosis, and viral/host interaction by controlling the assembly of histone acetylation-dependent chromatin complexes. Previous studies have shown that multiple BET inhibitors (BETi), including JQ1, have therapeutic effects in cancer and cardiovascular diseases. Some BETi have entered different phases of clinical trials. Pharmacologically, JQ1 functions by displacing BET proteins from chromatin by competitively binding to the acetyl-lysine recognition pocket of BET bromodomains. JQ1 has been used as a chemical probe to investigate the role of BET bromodomains in the transcriptional regulation of cardiovascular diseases. For example, JQ1 has been shown to attenuates inflammation and experimental atherosclerosis (Mol Cell. 2014 Oct 23; 56(2): 219–231.). JQ1 has also recently been shown to reduce EndoMT and cardiac fibrosis (J Mol Cell Cardiol. 2019 Feb;127:83-96.). However, the molecular targets of JQ1 dependent or independent of BRD4 remains unknown. To depict the transcriptomic signature of JQ1 in human endothelial cells, we observed a vasoprotective and atheroprotective transcriptome by JQ1 treatment using genome-wide RNA-seq based transcriptomic profiling. JQ1 is a magic bullet in cardiovascular disease prevention. Further elucidation of new molecular targets of JQ1 will lead to the identification of potentially new therapeutic targets to treat cardiovascular diseases. Overall design: Human umbilical vein endothelial cells (HUVECs, passage 3, 2 different donors) were seeded in 0.2% gelatin-coated dishes the day before experiment. The next day, cells were treated with BRD4 inhibitor (+)-JQ1 (10 uM, purchased from Cayman Chemicals, #11187) for 48 hours. After treatment, total RNA was isolated using the RNA-Easy Mini Plus kit (QIAGEN). High quality RNA samples (pre-assessed by Nanodrop measurements) were further processed in the Genome Research Center of the University of Rochester Medical Center. The TruSeq RNA Sample Preparation Kit V2 (Illumina, San Diego, CA) was used for next generation sequencing library construction per manufacturer's protocols. Briefly, mRNA was purified from 100ng total RNA with oligo-dT magnetic beads and fragmented. First-strand cDNA synthesis was performed with random hexamer priming followed by second-strand cDNA synthesis. End repair and 3' adenylation was then performed on the double stranded cDNA. Illumina adaptors were ligated to both ends of the cDNA, purified by gel electrophoresis and amplified with PCR primers specific to the adaptor sequences to generate amplicons of approximately 200-500bp in size. The amplified libraries were hybridized to the Illumina single end flow cell and amplified using the cBot (Illumina, San Diego, CA) at a concentration of 8pM per lane. Single end reads of 100nt are generated for each sample and aligned to the organism specific reference genome. Sequenced reads were cleaned using Trimmomatic-0.32 before mapping some of to the human reference genome (GRCh38.p2) with STAR-2.4.2a. Raw read counts were obtained using HTSeq and gencode 23 human gene annotations. DESeq2-1.10.1 was used to perform data normalization and differential expression analysis with an adjusted p-value threshold of 0.05. Then, Cufflinks-2.0.2 Software was used with the gencode 23 human gene annotations to perform differential expression analysis with an FDR cutoff of 0.05.