Protocol for chromatin accessibility profiling of human endothelial cells cultured under fluid shear stress using ATAC-seq.

Chromatin accessibility influences gene regulation and can be quantified using assay for transposase-accessible chromatin with high-throughput sequencing (ATAC-seq). Recapitulating in vivo fluid shear stress (FSS) mechano-regimes in vitro allows the study of atheroprone and atheroprotective mechanisms. In this protocol, we show how to culture and harvest endothelial cells from microfluidic channels for the preparation of ATAC-seq, highlighting optional growth factor stimulation and different FSS rates. This extends the application of ATAC-seq to the analysis of in vitro mechanically stimulated cells. For complete details on the use and execution of this protocol, please refer to Jatzlau et al.1.

Fluid shear stress-modulated chromatin accessibility reveals the mechano-dependency of endothelial SMAD1/5-mediated gene transcription.

Bone morphogenetic protein (BMP) signaling and fluid shear stress (FSS) mediate complementary functions in vascular homeostasis and disease development. It remains to be shown whether altered chromatin accessibility downstream of BMP and FSS offers a crosstalk level to explain changes in SMAD-dependent transcription. Here, we employed ATAC-seq to analyze arterial endothelial cells stimulated with BMP9 and/or FSS. We found that BMP9-sensitive regions harbor non-palindromic GC-rich SMAD-binding elements (GGCTCC) and 69.7% of these regions become BMP-insensitive in the presence of FSS. While GATA and KLF transcription factor (TF) motifs are unique to BMP9- and FSS-sensitive regions, respectively, SOX motifs are common to both. Finally, we show that both SOX(13/18) and GATA(2/3/6) family members are directly upregulated by SMAD1/5. These findings highlight the mechano-dependency of SMAD-signaling by a sequential mechanism of first elevated pioneer TF expression, allowing subsequent chromatin opening to eventually providing accessibility to novel SMAD binding sites.