Unraveling Higher-Order Chromatin Architecture and its Role in Cell Engineering for Improved Regenerative Outcomes


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The physical structure of chromatin has recently emerged as a key contributor to genome regulation and cellular function. Chromatin packing dictates the conformation of the 2-meter-long DNA polymer within the ~10 μm cell nucleus. This conformation can influence gene function by regulating the accessibility of molecular regulators to DNA, altering the transcriptional rates for multiple genes simultaneously. Remarkably, even though stem cells and differentiated or specialized cells possess the same DNA sequence, they exhibit strikingly distinct functions. This raises a fascinating question: what molecular mechanisms determine the cell fate if the underlying genetic blueprint or the DNA sequence itself remains unchanged? This can be partly attributed to differences in their epigenome, including molecular and structural modifications to chromatin that can influence the likelihood of expression of multiple genes, thereby regulating cell phenotype or function without changing the DNA sequence itself. Chapter 1 of this thesis introduces the importance of determining the physical structure of the genome to understand its role in regulating transcription and modulating cellular functions. Furthermore, this section discusses methodologies to study chromatin architecture in the context of transcription. Next, Chapter 2 discusses high-resolution electron microscopy and polymer physics-based analysis techniques to identify and characterize the functional properties of higher-order chromatin packing domains, the building blocks of genome organization. This work demonstrates that chromatin fibers fold into packing domains with an average radius of ~80 nm (~200 kbp in genomic size) and have heterogeneous morphological properties that are transcriptionally relevant. These spatially well-defined higher-order domains can be potentially engineered to achieve specific global transcriptional patterns. This can be particularly critical to efficiently manipulating cell fate for regenerative engineering applications, such as maximizing differentiation towards a target cell type. Chapter 3 explores this idea of chromatin engineering through contact guidance-induced nuclear deformation as a model to efficiently control differentiation outcomes in human mesenchymal stem cells (hMSCs). Specifically, microtopography-induced nuclear deformation elicits cellular transcriptional reprogramming, possibly through the regulation of 3D chromatin conformation, and consequently maximizes lineage-specific differentiation towards osteoblasts, and facilitates in-vivo bone regeneration. Finally, Chapter 4 summarizes this thesis work, and provides an outlook for future directions. Additionally, the chapter outlines the potential chromatin engineering avenues in improving regenerative outcomes. Overall, the presented work combines nanoimaging and molecular approaches to investigate the complex relationship between chromatin structure and function, while offering a promising method for cell engineering in regenerative applications.

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