Intricate network of nuclear morphology, chromatin dynamics and macromolecular crowding facilitates efficient nuclear functioning

The highly crowded nuclear interior impacts molecular motion and assembly, and therefore influence sub-cellular organization and function. However, we have limited understanding of nuclear crowding at the mesoscale (10-100 nanometers), which corresponds to the size of regulatory molecules such as RNA polymerase II. Here, we leveraged nuclear Genetically Encoded Multimeric nanoparticles (nucGEMs) to characterize physical properties of nucleus and to study its impact on transcription. nucGEMs are self-assembling fluorescent tracer particles of 40-nm diameter whose motion can be tracked to infer biophysical properties. By tracking GEMs, we found that nucleus is more crowded than the cytoplasm. Within the nucleus, nucGEMs majorly populated euchromatic compartments, excluded from heterochromatin and the nucleolus. Changing nuclear volume by growing cells to confluency or by exposing them to hypo and hyper osmotic shock analogously altered nuclear crowding. Increasing heterochromatin using HDAC inhibitors or inhibiting transcription using actinomycin D led to an immediate decrease in nucGEM diffusion followed by slow and gradual recovery. Interestingly, transcriptional activity, as monitored by 5EU incorporation, also changed in-correlation with nuclear crowding changes. These findings indicate that nuclear morphology, chromatin dynamics and nuclear processes altogether shape the molecular crowding of the nucleus, which in turn affects transcription. Furthermore, through an osmotic-stress resistance screen, we discovered that MYC confers resistance to increased molecular crowding. We found that overexpression of key transcriptional regulator MYC, increased crowding and elevated transcription rate. We propose that MYC enables transcription even in a hyper-crowded molecular environment ensuring cell proliferation, shedding a new light on the function of this molecule. Our findings indicate a direct link between crowding and transcription suggesting that nuclear crowding may play a crucial role in several biological processes such as stem cell differentiation and oncogenic transformation of normal cells.