The Transcriptional And Epigenetic Study Of VEGF Signaling Pathway

Angiogenesis plays a key role in the development of blood vessels and progression of many diseases like cancer.Vascular endothelial growth factors A(VEGFA)is the most important regulatory factor in angiogenesis.VEGFA binds to its receptor VEGFR2,transducts the signal from extracellular to the nuclei and changes the gene expression.The signal transduct from extracellular to the nucleus has been well demonstrated,but little is known about how the signal reshape epigenetic landscape and change the gene expression.Here,we tackle the question and interrogate the epigenetic and transcriptional response of endothelial cells to VEGFA treatment.To identify the genes regulated by VEGFA in endothelial cells,we measured mRNA expression by RNA-seq in human umbilical vein endothelial cells(HUVECs)after VEGFA stimulation.We found that 874 genes were differentially expressed(DEG).These DEGs were grouped into seven clusters(G1-7)according to their temporal expression pattern.More than half(55%)of the genes in clusters G4-5 were transcription factors(TFs)and rapidly up-regulated at 1 h,suggesting that VEGFA signals impact cellular behavior by rapidly modifying their transcriptional output.Then,we used chromatin immunoprecipitation followed by high-throughput sequencing(ChIP-seq)to profile the genome-wide chromatin occupancy of multiple histone modifications in HUVEC cells stimulated by VEGFA to gain epigenetic chromatin landscape and its relationship to gene expression in angiogenesis.We found that the epigenetic strength at the proximal promoter,not its dynamic variation,contributes to transcriptional response to VEGFA stimulation.To gain greater insight into the epigenetic regulation of the VEGFA transcriptional response,we next interrogated epigenetic marks on distal chromatin regions(±1–100 kb from TSS).The result indicated that VEGFA induced widespread alterations of the epigenetic landscape at distal regulatory regions,which was associated with dynamic gene transcription and critical for regulating VEGFA responses.Dense collections of enhancer,termed super-enhancers(SEs),activate the expression of cell lineage specification genes and functions in development and diseases.However,little is known about the identity and function of SEs in angiogenesis and VEGFA signaling.Here,we found that dynamics of component enhancers within SEs is an important and novel epigenetic feature that regulates transcription in response to VEGFA.Transcription factors(TFs)recognize specific DNA motifs to shape the chromatin landscape and transcriptome.To further elucidate the function of TF of endothelial cells in regulation of enhancer dynamics and transcription responsiveness to VEGF,we set out to map the genome-wide chromatin occupancy of key endothelial TFs.Based on the co-occurrence of the motifs,we were able to identify six main groups of TFs.Many of these motifs contained the recognition sites of ETS factors,which highlights the dominant role of ETS family TFs in endothelial cells.To test the hypothesis that altered TF occupancy contributed to enhancers dynamics,we selected six overrepresented TFs(ETS1,ERG,FLI,GATA2,JUN,and RBPJ)and two histone modification enzymes(EP300 and EZH2)with known functions in angiogenesis and profiled their genomic occupancy in HUVEC.The results demonstrated that dynamics of TF and epigenetic enzyme chromatin occupancy accounted for VEGF-induced changes of chromatin landscape.To test the possibility that changes in TF occupancy and epigenetic state contribute to the VEGFA transcriptional response,we interrogated the relationship between TF binding clusters and DEGs.We found that DEGs were highly enriched in the regions adjacent to dyanmic TFs and EP300(but not EZH2).Hence,this study pinpointed that the dynamic TF binding is one of critical mechanism governing epigenetic and transcriptional response to VEGFA.Finally,to identify master regulators(MRs)that control VEGFA-induced responses of endothelial cells,we integrated DEGs,dynamic TF-AE linkages,motif-dynamic AE linkages,AE-DEG associations,and protein–protein interactions from the STRING to construct a transcriptional network.We identified MAFK,MAFF,and MAFG,the members of small MAF family,as the master regulators of this network.Individual knockdown of MAFF and MAFG,not MAFK,by siRNA disrupted VEGF-induced migration of HUVECs.When all three MAFs were depleted,VEGFA-stimulated cell migration was almost completely abolished.On the other hand,up-regulation of each MAF stimulated cell migration.Proliferation assays showed that MAFF and MAFG,not MAFK,are essential for VEGFA-stimulated endothelial cell proliferation,and their overexpression was sufficient to enhance this process.HUVEC tube formation ability was significantly attenuated by knocking down MAFF or all three MAFs simultaneously,while enhanced by overexpression of MAFF or MAFG.The Matrigel plug assay showed that depletion of MAFF,MAFG,or both in endothelial cells reduced cell proliferation,increased cell death,and dampened vascular tube formation within the transplanted Matrigel.In contrast,overexpression of either MAFF or MAFG dramatically enhanced new vessel formation by promoting cell growth and reducing apoptosis.Moreover,HUVECs overexpressing MAFs also stimulated host endothelial cell recruitment into the Matrigel plug,and HUVECs with knocking down MAF did the opposite,which further affirmed the proangiogenic role of MAFs.Together,the data demonstrated that the small MAFs are novel MRs of VEGFA signaling and augment angiogenesis.Collectively,these results reveal that VEGFA stimuli could rapidly reconfigure the chromatin landscape to orchestrate the angiogenesis.