GSE193505
Depletion of Sun1/2 Induces Heterochromatin Accrual in MSCs
Mesenchyal Stem Cells (MSCs) differentiation into multiple lineages, such as osteocytes and adipocytes, has been shown to be regulated by mechanical signals. The Linker of the Nucleoskeleton and Cytoskeleton (LINC) complex has been shown to be required for mechanical signal transduction, regulation of MSCs differentiation, and nuclear integrity. The LINC complex is made of Nesprins and Sun proteins. Nesprin proteins associate with the cytoskeleton on the outer nuclear membrane and Sun proteins are bound to the inner nuclear membrane where they bind to inner nuclear proteins and chromatin. We investigated the role of the Sun1/2 in regulating the inner nuclear functions of chromatin organization and adipogenic differentiation independently of the LINC complex function.  We show that depletion of Sun1/2 increased nuclear area and perimeter, and decreased circularity. Expression of a dominant-negative KASH (dnKASH) domain targeting the SUN domain on Sun proteins inhibiting Nesprin-SUN association resulting a loss of Nesprin localization to the nuclear envelope decreased nuclear area and circularity. Adipogenesis was inhibited during Sun1/2 depletion while dnKASH expression accelerated adipgoenesis. RNA-seq data showed decreased adipogensis and increased immune response during Sun1/2 depletion. dnKASH responded oppositely with increased adipogenic gene expression and decreased immune response. We also observed increased H3K9me3 levels, increased H3K9me3 foci count, and enrichment on Adipoq during Sun1/2 depletion. No increase of H3K9me3 levels, foci count, or increased H3K9me3 enrichment on Adipoq was found during dnKASH expression. We conclude that physically decoupling of the LINC complex via dnKASH accelerates adipogenesis and that Sun1/2 regulates chromatin organization and adipogenesis independently of the LINC complex function. Overall design: Investigation of the tole of Sun1/2 proteins in regulating adiogenic differentiation independent of LINC complex function. Sample groups are delineated via labeling of "M" followed by a number between 6-14. Such that samples labeled with "M6" are the first sample group and "M15" is the last. siSun depleted cells are labled  with “S” while siCntl treated cells are labeled with “C”. Dominant-negative KASH domain was induced by Doxycycline and is labeled with “D” no doxycycline control is labeled with “C”. Samples labeled with "Ap" are treated with only adiogeneic media. Samples labeled with "Na" have been grown in growth media. siSun and siCntl replication groups are M11-15. Dominant-negative KASH and control replication groups are M6-10.