Mechanoresponsive genomic enhancers potentiate the cellular response to matrix stiffness [scRNA-seq]

Epigenetic control of gene regulation and cell phenotype is influenced by changes in the mechanical microenvironment. Yet how mechanical forces precisely influence epigenetic state to regulate transcriptional responses remains largely unmapped. Here, we combine genome-wide epigenome profiling, epigenome editing, and phenotypic and single-cell RNA-seq screening to identify a sub-class of genomic enhancers that is responsive to the mechanical microenvironment. These enhancers regulate genes that act as key drivers for a variety of functional behaviors including apoptosis, mechanotransduction, proliferation, and migration. We find distinct patterns wherein subsets of mechano-enhancers can be preferentially active on either soft or stiff extracellular matrix (ECM) contexts. Epigenetic editing of these mechano-enhancers on rigid materials precisely tunes mechanically-induced gene expression to levels observed on softer materials, and broadly enables reprogramming of cellular response to the mechanical microenvironment. These mechanically-activated enhancers act as key signal transducers and may constitute a new class of targets that can be modulated to alter mechanically-driven disease states. To determine the genes regulated by the pREs identified in the proliferation and migration screens, we made a sub-library of gRNAs targeting 87 of the pREs (10 gRNAs/pRE) with the largest effect sizes in either or both of the previous screens. This library also included positive controls of promoter-targeting and known enhancer-targeting gRNAs, and 100 non-targeting negative control gRNAs, for a total of 1,005 gRNAs in the sub-library. We transduced HFFs expressing dCas9KRAB cultured on TCP with this gRNA library at 0.33 MOI, and eight days post-transduction we profiled 103,440 quality single cell transcriptomes. We recovered a median of 1 gRNA per cell and identified an average of 159 cells containing each gRNA. To identify significant gene linkages to each pRE, we tested cells that had each gRNA versus all cells that did not receive that gRNA, and compared expression for all genes within +/- 1Mb from the targeted pRE.