The DNA binding protein BCL6 regulates NFκB-controlled endothelial inflammatory gene expression

Abstract

BackgroundNF{kappa}B drives acute vascular inflammation by activating gene expression programs in endothelial cells to promote leukocyte recruitment. Numerous negative feedback regulators of NF{kappa}B activation have been defined that promote resolution of inflammation. However, the identities of endogenous suppressors of NF{kappa}B transcription are less clear. In macrophages, the transcriptional repressor BCL6 was shown to substantially overlap with NF{kappa}B-driven genes and influence the response to LPS. We identified that the DNA binding protein BCL6 was expressed in endothelial cells. Although the role of BCL6 in adaptive immune cells has been characterized, how BCL6 modifies transcription in endothelial cells has not been studied. ObjectiveBased on prior knowledge that BCL6 represses part of the LPS-induced transcriptome in macrophages, we asked whether BCL6 regulated endothelial pro-inflammatory state by direct interaction with NF{kappa}B. MethodsWe analyzed public datasets of RNA and ChIP-Seq, probed BCL6 expression in human tissue, and tested BCL6 knockdown, overexpression and pharmacological manipulation on TNF induced gene expression in vitro using human primary endothelium isolated from the heart. ResultsWe demonstrate that the DNA binding protein BCL6 is basally expressed in the endothelium, with chromatin marks reflective of a superenhancer, and is particularly enriched in aortic endothelial cells (ECs) compared with ECs from other organs. Although basal expression was relatively low, BCL6 was rapidly upregulated in cardiac endothelium stimulated with TNF, through direct action of NF{kappa}B. The BCL6 consensus DNA binding motif overlaps with that of NF{kappa}B. BCL6 target genes included endothelial pro-inflammatory chemokines and adhesion molecules, as well as NF{kappa}B-related genes themselves. BCL6 knockdown and the degrading BCL6 inhibitor BI-3802 augmented the endothelial cell response to TNF. Surprisingly, antagonism of the BTB domain of BCL6 with small molecules 79-6, FX1 or BI-3812, blocked leukocyte adherence and accordingly suppressed both NF{kappa}B transcriptional activity as well as the expression of many genes in response to TNF. Lastly, we show that HDAC activity is increased by TNF, and can be reduced in the presence of BTB domain inhibitors. ConclusionsOur results demonstrate that BCL6 is a repressor of NF{kappa}B-driven gene expression and inflammation in cardiac endothelial cells. These findings indicate that targeting of BCL6 may enhance vascular inflammation resolution.