Nuclear actin structure regulates chromatin accessibility [ATAC-seq]

Polymerized B-actin may provide a structural basis for chromatin accessibility and actin transport into the nucleus can guide mesenchymal stem cell (MSC) differentiation. Using MSC, we show that using CK666 to inhibit Arp2/3 directed secondary actin branching results in decreased nuclear actin structure, and significantly alters chromatin access measured with ATACseq at 24 h. The ATAC-seq results due to CK666 are distinct from those caused by cytochalasin D (CytoD), which enhances nuclear actin structure. In addition, nuclear visualization shows Arp2/3 inhibition decreases pericentric H3K9me3 marks. CytoD, alternatively, induces redistribution of H3K27me3 marks centrally. Such alterations in chromatin landscape are consistent with differential gene expression associated with distinctive differentiation patterns. Further, knockdown of the non-enzymatic monomeric actin binding protein, Arp4, leads to extensive chromatin unpacking, but only a modest increase in transcription, indicating an active role for actin-Arp4 in transcription. These data indicate that dynamic actin remodeling can regulate chromatin interactions. 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) for 24 hours, 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 for 24 hours. In a third set of experiments, MSCs and NIH3T3 cells were stimulated with CK666 for 4 hours, and ATAC-se qwas performed on three replicates each of stimulated and control cells for both cell types.