Role of force regulated nuclear structure in expression of osteogenesis

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Abstract: Role of force regulated nuclear structure in expression of osteogenesis Bone marrow mesenchymal stem cells (MSC) exist in a multipotential state, where osteogenic and adipogenic genomes are silenced in heterochromatin at the inner nuclear leaflet. Activating the osteogenic differentiation program involves multiple regulatory factors, including physical force, which is generated in the marrow space during dynamic exercise. MSC experience mechanical force through their cytoskeletal attachments to substrate, inducing signaling that alters gene expression. We showed that intranuclear actin structures are affected by changes in the cytoplasmic cytoskeleton, and direct changes in intranuclear actin due to knock down of nuclear restricted mDia2 (preventing intranuclear actin polymerization) exert profound regulatory control on gene expression. Further, although both dynamic and static mechanical force activate RhoA to control formation of the actin cytoskeleton, the nature of these forces appear to have widely variant effects on gene expression – dynamic force inhibits adipogenesis and promotes multipotentiality, while static force is associated with osteogenesis. Dynamic versus static applications may affect gene expression through generating different forces on the nucleus, affecting nuclear structure and nuclear access of the mechanoresponders, Yap and β-catenin. We here hypothesize that nuclear structure, modified by force activated actin polymerization, contributes to selective MSC differentiation and fate. To address this hypothesis, we propose to define how cellular actin structure resulting from dynamic or static mechanical force differentially regulate nuclear architecture and gene expression. In SA1 we will find if dynamic and static strain differentially modify nuclear architecture (F-actin and lamin structure, nucleoli size and spacing, cell and nuclear stiffness and FISH localization of Runx2). Our data shows that loss of intranuclear actin polymerization decreases lamin B1 at the inner nuclear leaflet, thus we will find if force alters osteogenic gene silencing through lamins, and if alterations in formin mDia2 or laminB1 modulate heterochromatization of the osteogenic genome. In SA2 we will ask if dynamic and static force differentially regulate nuclear entry of Yap or β-catenin, and relate cytoskeletal stress transfer to the nuclear membrane to changes in nuclear access of these molecules. Unbiased RNAseq will allow us to compare gene expression after dynamic vs static force and ask if nuclear Yap and β- catenin are critical. Lastly in SA3 we determine if intranuclear actin structure directly controls access to the osteogenic genome. We will define nuclear structure after mDia2 knock down (decrease intranuclear F-actin, induces osteogenesis), mDia1 knock down (decreasing cytoplasmic F-actin) and altering secondary actin branching (induces adipogenesis). In these conditions we will relate nuclear structure to activation of Runx2 and PPARγ cistromes. Upon completion of our objectives we will be able to establish how mechanical forces and nuclear actin polymerization control epigenetic induction of osteogenesis and regulate nuclear transfer of YAP and β-catenin.