Lamin A depletion and RSG influence PPARγ DNA binding and chromatin organization in murine adult fibroblast cells.

Lamin A/C proteins, integral to the nuclear lamina and present throughout the nucleoplasm, contribute to the mechanical stability of the nucleus and influence transcription as seen in laminopathies like certain lipodystrophies and progeria. By using light-sheet fluorescence correlation spectroscopy (FCS), earlier, we proved that lamin A is an important determinant of chromatin viscoelasticity in murine adult fibroblasts (MAFs). Here, by using MAF wild-type and lamin A knockout (KO) cells, we aimed to clarify the role of lamin A in chromatin organization and the DNA binding of PPARγ, a key transcription factor in adipogenesis also implicated in lipodystrophies. We analyzed the distribution of chromatin marks by confocal microscopy and measured PPARγ mobility and DNA binding by FCS and fluorescence recovery after photobleaching (FRAP). Conspicuous overall changes were detected upon lamin A depletion and PPARγ agonist (rosiglitazone) treatment. Confocal imaging showed a remarkable decrease in nuclear volume in the absence of lamin A and an increase upon rosiglitazone treatment indicating chromatin decondensation. Heterochromatin was enriched in a distinct peripheral rim, and the thickness of the constitutive heterochromatin rim diminished in the absence of lamin A. Colocalization analysis showed that the overlap between euchromatin and heterochromatin decreased in lamin A KO cells. PPARγ colocalized with euchromatin, whereas its localization was anti-correlated with constitutive heterochromatin, especially in the absence of lamin A. FCS and FRAP indicated that PPARγ had a fast-diffusing fraction bound with short residence times on DNA and a slow, more stably bound fraction throughout the nucleus including the periphery. Rosiglitazone increased PPARγ's colocalization with the euchromatin and enhanced its DNA binding, which was more pronounced in lamin A KO cells. Our results suggest that lamin A plays a primary role in determining nuclear volume and overall chromatin organization with likely functional consequences exemplified by regulating ligand-induced DNA binding of PPARγ, which may have epigenomic and transcriptional consequences.