Nuclear actin structure regulates chromatin accessibility [RNA-seq]
Polymerized b-actin may provide a structural basis for chromatin accessibility. Nuclear actin transport into the nucleus can determine mesenchymal stem cell (MSC) differentiative outcomes through regulated control of gene expression. Using MSC, we show that inhibiting Arp2/3 directed secondary actin branching with CK666, which results in decreased nuclear actin structure, significantly alters chromatin access measured with ATAC-seq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which increases nuclear actin structure. Nuclear visualization shows Arp2/3 inhibition is associated with decreased pericentric H3K9me3 marks. CytoD, in contrast, induces relocation of H3K27me3 marks away from the inner membrane. Treatment induced alterations in the chromatin landscape at 24 hours prompts differential gene expression associated with decreased proliferation and increased differentiation. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest change in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions. Overall design: Mouse marrow-derived mesenchymal stem cells (MSCs) were harvested from C57BL/6J murine. In the first set of experiments, MSCs were treated with either CK666, an Arp2/3 inhibitor, or cytochalasin D (CytoD), and ATAC-seq and RNA-seq were performed on three replicates each of control and stimulated cells. In the second set of experiments, Arp4 was knocked down using an siRNA, and ATAC-seq and RNA-seq were performed on siRNA mock transfected control MSCs, siRNA Arp4 transfected MSCs, and siRNA Arp4 transfected MSCs also stimulated with CK666. bone marrow