Mechanoenhancers in DNA let cells sense tissue stiffness and may offer a way to tame fibrosis
Researchers mapped DNA switches that respond to tissue stiffness and used epigenetic editing to dial down stiffness-driven gene activity, including in IPF lung fibroblasts.
Summary
Mechanical cues such as tissue stiffness influence cell behavior and are implicated in fibrosis and aging. This study asked how cells turn those cues into changes in gene activity. Researchers grew human cells on soft and stiff gels and found widespread changes in gene expression and chromatin accessibility. They identified and validated mechanically responsive regulatory DNA elements called mechanoenhancers, which can be preferentially active on either soft or stiff materials. Using epigenetic editing, they shifted gene expression on rigid materials toward levels seen on softer ones. According to the abstract, editing mechanoenhancers in lung fibroblasts from healthy donors and donors with idiopathic pulmonary fibrosis (IPF) reduced stiffness-driven transcriptional responses and prevented activation of genes associated with the fibroblast-to-myofibroblast transition. These cell-based findings suggest a potential route to targeting mechanically driven disease, not an established treatment.
Detailed Summary
Mechanical cues such as extracellular matrix stiffness influence cell behavior and play roles in aging, fibrosis, tumor formation, and atherosclerosis. How these cues reshape regulatory DNA activity remains incompletely understood. This study, published in Science, combined epigenome profiling, epigenetic editing, and CRISPR screening to identify mechanically responsive regulatory elements called mechanoenhancers.
The researchers cultured primary human neonatal foreskin fibroblasts (HFFs) and A549 tumor-derived lung epithelial cells for 20 hours on soft (1 kPa) or stiff (50 kPa) hydrogels, as well as tissue culture plastic. RNA-seq identified 4,009 differentially expressed genes in HFFs and 221 in A549 cells, with 76 shared. Shared genes included canonical YAP/TAZ-associated genes such as CYR61, CTGF, and ANKRD1, which were upregulated on stiffer materials. About 23% of accessible chromatin peaks in HFFs and 15% in A549 cells differed between soft and stiff hydrogels, with an equal distribution favoring each stiffness condition.
Motif and footprinting analyses implicated stiffness-dependent transcription factor engagement. TEAD motifs showed greater protein engagement on stiff hydrogels in both cell types; CEBP family motifs were preferentially bound on soft hydrogels in HFFs. HiCAR analysis in HFFs suggested that long-range chromatin contacts may contribute to stiffness-dependent transcriptional changes. Differential accessibility alone, however, does not establish that every responsive region is a functional enhancer.
Functional testing identified a mechanically responsive enhancer approximately 14 kb upstream of FZD2 that becomes activated on stiffer materials. The researchers also investigated regulation of MYH9, which encodes non-muscle myosin IIA and showed stiffness-dependent expression in HFFs. A CRISPR interference screen targeted 114 accessible regions around MYH9 using 5,192 guide RNAs. The supplied excerpt ends during reporting of this screen, so its subsequent validation results cannot be assessed here.
The abstract reports that mechanoenhancers regulate functions including apoptosis, mechanotransduction, proliferation, and migration. Editing these elements on rigid materials tuned gene expression toward levels observed on softer materials. In lung fibroblasts from healthy and IPF donors, editing reduced stiffness-mediated transcriptional responses and prevented activation of genes associated with the fibroblast-to-myofibroblast transition. These findings support a potential strategy for targeting mechanically driven disease, but the supplied material does not establish clinical efficacy, long-term effects in tissue, or superiority over pathway inhibitors.
Key Findings
- After 20 hours on 1 versus 50 kPa hydrogels, 4,009 genes in HFFs and 221 in A549 cells were differentially expressed, with 76 shared
- About 23% of accessible chromatin peaks in HFFs and 15% in A549 cells differed between soft and stiff hydrogels
- TEAD motifs showed greater protein engagement on stiff hydrogels in both cell types; CEBP family motifs were preferentially bound on soft hydrogels in HFFs
- Functional testing identified a stiffness-responsive enhancer approximately 14 kb upstream of FZD2 that becomes activated on stiffer materials
- A MYH9-focused CRISPRi screen targeted 114 accessible regions using 5,192 guide RNAs; the excerpt ends before its detailed validation results
- The abstract reports that mechanoenhancer editing on rigid materials tuned gene expression toward levels observed on softer materials
- According to the abstract, editing in healthy and IPF donor lung fibroblasts reduced stiffness-driven transcription and prevented activation of genes associated with the fibroblast-to-myofibroblast transition
Methodology
The researchers used RNA-seq and ATAC-seq in HFFs and A549 cells cultured for 20 hours on 1 kPa and 50 kPa polyacrylamide hydrogels and on tissue culture plastic. Sequencing experiments were performed in at least duplicate per condition. Differential expression was defined using FDR<0.05 and |log2 fold change|>0.5. Analyses included transcription factor motif enrichment and TOBIAS footprinting; HiCAR chromatin interaction mapping was performed in HFFs on soft and stiff hydrogels. Functional testing included enhancer reporter constructs and dCas9-KRAB CRISPRi. The described MYH9 screen used 5,192 guides across 114 regions, with cells stained and sorted into MYH9-high and MYH9-low bins. The abstract additionally reports phenotypic and single-cell RNA-seq CRISPR screening and experiments in healthy and IPF donor lung fibroblasts, but their detailed methods and results are not included in the supplied excerpt.
Study Limitations
The detailed experiments provided use cultured neonatal foreskin fibroblasts and the A549 tumor-derived cell line, which may not reflect aged human tissue. The authors suggest that primary and immortalized cells may differ in mechanical memory. The excerpt ends during the MYH9 screen results; detailed later screening results and IPF fibroblast experiments are unavailable, with the latter supported here only by the abstract. The supplied material does not establish long-term in vivo durability, off-target safety, or delivery feasibility. No conflict-of-interest statement is included in the supplied text.
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