MSE PhD Defense: “Chromatin as an Active and Adaptive Material”

The three-dimensional organization of chromatin within the cell nucleus plays a critical role in regulating gene expression, maintaining genome stability, and guiding cellular responses to environmental cues. Despite advances in imaging and sequencing technologies, the fundamental principles governing chromatin architecture and dynamics, particularly the role of associated proteins like HP1α in driving these processes, remain poorly understood. Additionally, although multiple studies have demonstrated the sensitivity of chromatin organization to microenvironmental cues, the underlying physical mechanisms driving this sensitivity and its impact on transcription, especially in response to mechanical cues, remain largely unexplored. Therefore, elucidating the mechanisms that regulate chromatin organization, particularly those shaped by the interplay of molecular interactions and external mechanical forces, is crucial for understanding how genome structure and function are interrelated and how they might be controlled for therapeutic purposes.

To bridge this gap, this thesis integrates theoretical modeling, computational simulations, and experimental approaches to investigate the principles that regulate genome organization. To explore the molecular drivers of chromatin structure, we developed a novel polymer-based model using kinetic data of the chromatin architectural protein HP1α, extracted from FRAP experiments in vivo. This model was designed to predict both the structural organization and dynamic behavior of constitutive heterochromatin, which comprises gene-poor, transcriptionally silent regions essential for genome stability. By incorporating HP1α binding kinetics and its affinity for methylated chromatin, the model accurately predicts heterochromatin domain sizes and sub-diffusive motion. These predictions were validated using Hi-C and high-resolution imaging data, revealing how transient HP1α interactions contribute to heterochromatin structure and mobility. Moreover, the model provides a mechanistic explanation for the maintenance of epigenetic memory within these regions.

Next, to investigate how the mechanical properties of the cellular microenvironment influence genome architecture, we designed experiments that replicate cellular responses to changes in tissue stiffness, guided by predictions from our stiffness-dependent polymer model. Focusing on lung fibrosis, we used IMR90 cells cultured on synthetically engineered hydrogels with defined stiffnesses representing healthy and fibrotic (disease-like) pulmonary environments. As substrate stiffness increased from soft, healthy-like conditions to stiff, fibrotic-like conditions, cells exhibited marked changes in chromatin accessibility, nuclear epigenetic landscape, and gene expression. These effects were characterized using RNA-seq, ATAC-seq, and super-resolution OligoSTORM imaging. Our findings uncovered a mechanosensitive mechanism by which chromatin reorganization mediates both transcriptional responses and epigenetic regulation, offering insights into the gene-level consequences of microenvironmental changes during disease progression.

Together, this thesis presents a multiscale integrative framework that combines computational modeling with biological experimentation to advance our understanding of genome organization. By bridging the gap between molecular-scale interactions and tissue-level mechanical cues, it provides new insights into how chromatin architecture encodes both dynamic responsiveness and long-term cellular identity. Ultimately, this work contributes to a broader understanding of how biophysical principles shape gene regulation, with implications for development, disease progression, and the design of novel therapeutic strategies.