Transcriptional regulation and chromatin reorganization in mechanically induced cellular reprogramming and rejuvenation

Abstract

This doctoral thesis explores the remarkable domain of mechanobiology, transcriptional regulation, and its relevance in regenerative medicine. Cells are known to be plastic, capable of transitioning between different states based on their microenvironment. Mechanical cues, in particular, have emerged as a potent trigger for cell-state transitions, such as reprogramming and differentiation, offering exciting prospects for regenerative medicine. However, the molecular mechanisms underlying these mechanically induced transitions remained poorly understood. The first chapter discusses recent work on exploring the role of mechanical environment and nucleus mechanisms in transcriptional regulation in various cell state transitions. The second chapter unveils the power of laterally confined growth of fibroblasts in inducing dedifferentiation programs. The molecular mechanisms underlying mechanically induced cell-state transitions are illuminated, with a focus on the critical role of the somatic transcription factor, Lef1. Lef1 has been implicated in various cellular contexts, but its central role in mechanically induced fibroblast dedifferentiation emerges as a key finding. Network optimization methods applied to time-lapse RNA-seq data identify Lef1-dependent signaling as potential regulators of these transitions, shedding light on Lef1's interaction with downstream reprogramming factors. Moreover, it identifies Smad4 and Atf2 as potential critical activators of Lef1, establishing an important mechanotransduction pathway for fibroblast dedifferentiation. Building on this foundation, the thesis dives into the rejuvenation of fibroblasts - a key focus for functional tissue regeneration. Traditional methods of cellular rejuvenation can be limited, often necessitating complete reprogramming and carrying risks like genomic mutations and low efficiency. However, the mechanically induced partially reprogrammed spheroids offer an innovative solution. When embedded in 3D collagen matrices with optimized stiffness, these spheroids regain fibroblastic properties and transform into 3D connective tissue networks. These redifferentiated fibroblasts exhibit reduced DNA damage, enhanced cytoskeletal gene expression, and acto-myosin contractility. Besides, increased deposition of matrix protein and enhanced collagen remodeling indicate that these redifferentiated fibroblasts are rejuvenation. This work highlights efficient fibroblast rejuvenation through mechanical reprogramming, promising novel approaches in regenerative medicine. We next investigate mechanical reprogramming and redifferentiation with the goal of presenting a cellular rejuvenation approach for aged dermal fibroblasts. Aging is characterized by cellular functional decline and epigenetic changes, necessitating innovative rejuvenation strategies. Mechanically rejuvenated aged fibroblasts reset their global transcription profile, upregulating genes related to cell proliferation and extracellular matrix secretion. Innovative Hi-C analysis reveals genes associated with aging and rejuvenation within reorganized interchromatin contact regions. Imaging experiments unveil chromatin compaction changes, and novel measurements based on Lamina-associated domains (LAD) interactions offer insights into 3D chromatin organization. The findings introduce a multi-omics analysis approach and contribute to the understanding of chromatin-mediated cellular rejuvenation, with potential therapeutic implications. Finally, we consider the context of tissue regeneration and wound healing, where cell-based therapies play a pivotal role. Traditional autologous transplantation is hampered by cellular senescence and reduced remodeling capabilities. The thesis presents an alternative approach, implanting partially reprogrammed aged human dermal fibroblasts into in vitro aged skin models for tissue regeneration and wound healing. The implanted cells exhibit enhanced extracellular matrix protein expression and synthesis, leading to improved tissue regeneration at wound sites. Transcriptome analysis and chromatin biomarkers unveil the upregulation of tissue regeneration and wound healing pathways, offering a novel, non-genetic approach for cell-based therapies in regenerative medicine.