Regulation of YAP Promotor Accessibility in Endothelial Mechanotransduction

Abstract

BACKGROUND: Endothelial cells are constantly exposed to mechanical forces in the form of fluid shear stress, extracellular stiffness, and cyclic strain. The mechanoresponsive activity of YAP (Yes-associated protein) and its role in vascular development are well described; however, whether changes to transcription or epigenetic regulation of YAP are involved in these processes remains unanswered. Furthermore, how mechanical forces are transduced to the nucleus to drive transcriptional reprogramming in endothelial cells is poorly understood. The YAP target gene, AmotL2 (angiomotin-like 2), is a junctional mechanotransducer that connects cell-cell junctions to the nuclear membrane via the actin cytoskeleton. METHODS: We applied mechanical manipulations including shear flow, stretching, and substrate stiffness to endothelial cells to investigate the role of mechanical forces in modulating YAP transcription. Using in vitro and in vivo endothelial depletion of AmotL2, we assess nuclear morphology, chromatin organization (using transposase-accessible chromatin sequencing), and whole-mount immunofluorescent staining of the aorta to determine the regulation and functionality of YAP. Finally, we use genetic and chemical inhibition to uncouple the nuclear-cytoskeletal connection to investigate the role of this pathway on YAP transcription. RESULTS: Our results reveal that mechanical forces sensed at cell-cell junctions by the YAP target gene AmotL2 are directly involved in changes in global chromatin accessibility and activity of the histone methyltransferase EZH2, leading to modulation of YAP promotor activity. Functionally, shear stress–induced proliferation of endothelial cells in vivo was reliant on AmotL2 and YAP/TAZ expression. Mechanistically, uncoupling of the nuclear-cytoskeletal connection from junctions and focal adhesions led to altered nuclear morphology, chromatin accessibility, and YAP promotor activity. CONCLUSIONS: Our findings reveal a role for AmotL2 and nuclear-cytoskeletal force transmission in modulating the epigenetic and transcriptional regulation of YAP to maintain a mechano-enforced positive feedback loop of vascular homeostasis. These findings may offer an explanation as to the proinflammatory phenotype that leads to aneurysm formation observed in AmotL2 endothelial deletion models.

A phenotypic screening approach to target p60AmotL2-expressing invasive cancer cells

“…Supporting this hypothesis, a recent study from our lab has shown that impairing the AmotL2 signaling axis by using short hairpin RNAs—which is phenocopied by p60AmotL2 via acting as a dominant-negative of the main isoform—leads to reduced chromatin accessibility [ 20 ]. While the mechanism of decreased chromatin accessibility in p60AMOTL2-expressing cells is not explored here, a role for perturbed cytoplasmic contractile actin filaments [ 17 ] cannot be ruled out.…”

“…This complex initiates the formation of specific radial actin filaments connecting cellular junctions to the nuclear lamina [ 15 , 17 ]. AmotL2 has been implicated in various biological processes, including aortic development, blastocyst hatching, epithelial cell polarity, and regulation of the Hippo signaling pathway, which controls organ size and cell growth [ 12 , 17 – 20 ].…”