Transcriptional Regulation of Chromatin Motion Drives Nuclear Blebbing, Defined by Decreased DNA Density

Transcription of DNA into RNA is fundamental to cellular function, influencing not only gene expression but also the biophysical properties of the nucleus. Transcriptional activity has been linked to micron-scale chromatin motion, nuclear blebbing, and, in some cases, nuclear rupture. Nuclear blebbing is a hallmark of human diseases, including various cancers and age-related disorders, which also exhibit significant changes in transcriptional activity. Inhibition of RNA polymerase II has been shown to suppress nuclear blebbing, while modeling suggests that chromatin motion can drive nuclear deformations. However, the precise relationship between transcription, chromatin dynamics, and nuclear blebbing remains unclear. Chapter 1 of this thesis, an already published manuscript primarily written by my PI, examines the definition and composition of nuclear blebs. My contributions to this work included data collection, analysis, and manuscript editing. A key finding from this study is that decreased DNA density provides a more consistent indicator of nuclear blebs than the absence of lamin B1, which varies across conditions and cell types. Building on these findings, Chapter 2 presents my independent research investigating the role of transcriptional regulation in nuclear blebbing and chromatin motion. To disentangle the effects of transcriptional activity from its biochemical output, I modulated transcription through serum starvation and stimulation and live-cell imaged nuclear morphology using NLS-GFP and randomly incorporated Cy3-dNTPs. Serum starvation, which reduces but does not eliminate transcription, led to a drastic suppression of nuclear blebbing and chromatin motion. Conversely, serum stimulation of starved cells rapidly restored transcription, nuclear blebbing, and chromatin motion within a few hours. Further, live imaging of randomly labeled dNTPs revealed that chromatin motion is necessary for nuclear bleb stability. Overall, this work demonstrates that transcription-driven chromatin motion provides a mechanistic basis for nuclear blebbing. By establishing decreased DNA density as a reliable indicator of nuclear blebs, these findings contribute to a clearer framework for studying nuclear architecture and its links to transcriptional regulation and disease-associated nuclear abnormalities.