Dissecting the Mechanisms of Transcriptional Synergy in Response to Proinflammatory Cytokines in Endothelial Cells
Combinatorial cytokine signaling is implicated in the pathogenesis of diseases of the cardiovascular system ranging from acute combinatorial cytokine signaling observed in complications from SARS-COVID19 infection to chronic combinatorial cytokine signaling observed in atherosclerosis. There remains a large gap of our understanding how combinatorial cytokine signaling interface to drive proinflammatory programs. We hypothesize that stimulation of HAECs with IFNg and TNFa promotes synergistic gene transcription through the coordinated interplay between p65, STAT1, p300/CBP, and BRD4. Our work demonstrates through the generation of novel statistical framework that IFNg and TNFa dual stimulation for one hour results in the synergistic induction of a small subset of proinflammatory genes. Prolonged dual cytokine stimulation results in synergistic levels of protein expression and cell death. Treatment with A-485 and/or JQ1 reduces levels of synergistic gene and protein expression along with improvements in cell viability. Defining synergistic responses and identifying their unique transcriptional dependencies opens the door to understanding how disparate cytokine signaling drives proinflammatory responses and how these responses can be targeted. Overall design: teloHAECs were treated for one hour with either vehicle, IFNg, TNFa, or both IFNg and TNFa before cells were harvested for RNA. We then performed compartive gene expression profile analysis between all four conditions. teloHAECs were treated for one hour with either vehicle, IFNg, TNFa, or both IFNg and TNFa before cells were harvested for to perform ATAC-seq. We then performed comparative anlysis to determine differerntial peaks that were specific to the dual cytokine condition. Inhibitor treated ATAC: teloHAECs were treated for one hour with vehicle or both IFNg and TNFa (SYN) then either DMSO or an inhibitor (A485 or JQ1) before cells were harvested for to perform ATAC-seq. We then performed comparative anlysis to determine differential peaks. CUTandRUN: teloHAECs were treated for one hour with vehicle or both IFNg and TNFa (SYN) then either DMSO or an inhibitor (A485 or JQ1) before cells were harvested for to perform ATAC-seq. We then performed comparative anlysis to determine differential peaks. CUT&RUN: TELO HAECs were treated for one hour with vehicle or both IFNg and TNFa (SYN) in the presence or absence of DMSO or inhibitors (A485 or JQ1). Cells were fixed in 1% formaldhyde for 1 min before being quenched with glycine. Cells were bound to Concavalin MyOne beads and incubated with antibody for 24 hours. Samples were washed and then pAG-MNase bound for 1 hour at 4C, after which CaCL2 was added and pAG-MNase cleavage went for 30 minutes with samples submerged in ice. DNA was purified via a phenol-chloroform isolation after which the purified DNA was utilized to generate sequencing libraries. DNA libraries were generated using the NEBNext Ultra II DNA Library Prep Kit with the following PCR cycling adjustmentsfor the PCR Enrichment of adaptor ligated DNA  a. 45 sec at 98ºC  b. 15 sec at 98ºC  c. 10 sec at 60ºC  d. Repeat Steps b-c for a total of 14 cycles e. 1 min at 72ºC  final extension. We then performed compartative analysis to determine differential peaks.