Project Summary/Abstract It is well known that stem cell differentiation and cell fate are regulated by mechanical forces and mechanics. We have reported earlier that cell mechanical properties dictate stress-induced spreading and differentiation in embryonic stem cells (ESCs). Other reports have also demonstrated the effect of forces on pluripotent stem cells. However, how force regulates (pluripotent) stem cell differentiation and cell fate at the level of chromatin remains elusive. This is an important question since the chromatin is the hub of gene transcription and DNA replication and DNA damage repair, critical for pluripotent stem cell self-renewal and differentiation and cancer stem cell self-renewal. Our preliminary results chromatin domain stretching but not compression rapidly and nuclear protein LAP2β mediates force transmission from nuclear lamina to chromatin. In addition, RNA polymerase II (Pol II) is recruited and elongated in response to stretching but not compression and demethylation of H3K9me3 is necessary for force-induced gene upregulation. Built on these results, we propose 3 specific aims to elucidate nuclear mechanobiology mechanisms in the living cells. Aim 1: To determine if chromatin stretching is necessary for force-induced stem cell differentiation; Aim 2: To test the hypothesis that H3K9me3 and H3K9ac regulate force-induced pluripotent stem cell gene transcription; Aim 3: To determine if large-strain induced telomere attrition contributes to transition from normal stem cells to cancer stem cells. Our experimental designs are rigorous and the likelihood of generating insightful discovery is high. The long-term goal is to develop novel approaches and strategies to intervene pathological processes like malignant tumors.