Depletion of Sun1/2 induces heterochromatin accrual in mesenchymal stem cells during adipogenesis

Abstract

Mechanical signals regulate adipogenic differentiation of mesenchymal stem cells (MSCs). Critical to the mechano-regulation of MSCs, Linker of the Nucleoskeleton and Cytoskeleton (LINC) complexes are integral to both nucleo-cytoskeletal signal transduction and structural integrity of the nucleus. The LINC complex is made of Nesprin proteins that associate with the cytoskeleton on the outer nuclear membrane (ONM) and Sun proteins that bound to nuclear lamina and chromatin at the inner nuclear membrane (INM). In addition to their role in the LINC complex function, depletion of Sun1/2 effects chromosomal tethering to the nuclear envelope, nuclear morphology, and chromatin organization. Suggesting that Sun1/2 proteins may regulate chromatin organization and adipogenic differentiation independent of the LINC complex mediated nucleo-cytoskeletal connectivity. To test this hypothesis Sun1/2 depletion was compared to expression of a dominant-negative KASH (dnKASH) domain to decouple nucleus from cytoskeleton by inhibiting Nesprin-SUN association. Sun1/2 depletion inhibited fat droplet formation and production of adipogenic proteins such as Adipoq, which were supported by RNA-seq showing decreased adipogensis. In contrast dnKASH responded oppositely, increasing fat droplet formation, Adipoq and adipogenic gene expression. At the chromatin level, Sun1/2 depletion increased H3K9me3 levels, increased H3K9me3 foci count, and enrichment on Adipoq. 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 depletion of Sun1/2 increases heterochromatin accrual and inhibits adipogenesis independent of the LINC complex function.

Prrx1-driven LINC complex disruption in vivo reduces osteoid deposition but not bone quality after voluntary wheel running

“…Studies involving LINC complex disruption via KASH overexpression in vitro show decreases osteogenic activity in human bone marrow mesenchymal stem cells by reducing Runx2 via increased histone acetylation [47]. We recently reported that LINC disruption also decreases adipogenic potential in vitro [48]. Despite this evidence, our results indicate no change in adipogenic and osteogenic potential of these cells when extracted from bone marrow.…”

Data‐Driven and Cell‐Specific Determination of Nuclei‐Associated Actin Structure

“…As to effects in MSC, it has been recently reported that depleting LINC function via depletion of SUN proteins can alter heterochromatin states altering lineage selection. [ 41 ] Depleting SUN proteins results in functionally different heterochromatin rearrangements than does dnKASH expression, [ 21 ] suggesting that changes in the heterochromatin state are affected by both nuclear envelope structural composition and F‐actin contractility. To this end, our method can be implemented with both fixed and live cell imaging to detect changes in F‐actin under variety of mechanical forces and thus enable researchers to develop new hypotheses in cell mechanobiology.…”

“…These isolated MSC stocks were stably transduced with a doxycycline‐inducible plasmid expressing an mCherry‐tagged dominant‐negative KASH (dnKASH) domain. [ 21 ] The dnKASH plasmid was lentiviral packaged as a generous gift from Dr. Daniel Conway (Addgene # 125 554). In order to label actin in living MSCs, RFP‐tagged chromobody against actin was used (Chromotek acr‐TagRFP).…”

Data driven and cell specific determination of nuclei-associated actin structure

“…As to effects in MSC, it has been recently reported that depleting LINC function via depletion of Sun proteins can alter heterochromatin states altering lineage selection 41 . Depleting Sun proteins results in functionally different heterochromatin rearrangements than does dnKASH expression 21 , suggesting that changes the heterochromatin state is affected by both nuclear envelope structural composition and F-actin contractility. To this end, our method can be implemented with both fixed and live cell imaging to detect changes in F-actin under variety of mechanical forces and thus enable researchers to develop new hypotheses in cell mechanobiology.…”

“…These isolated MSC stocks were stably transduced with a doxycycline-inducible plasmid expressing an mCherry tagged dominant-negative KASH (dnKASH) domain 21 . The dnKASH plasmid was lentiviral packaged as a generous gift from Dr. Daniel Conway (Addgene # 125554).…”