Nuclear myosin VI cooperates with actin to promote transcriptional cluster formation at androgen receptors

Steroid hormone receptors are ligand-binding transcription factors essential for mammalian physiology. The androgen receptor (AR) binds testosterone mediating gene expression for sexual, somatic, and behavioral functions and is involved in various conditions, including androgen insensitivity syndrome and prostate cancer. Our previous work revealed the actin-dependent formation of transcriptional hubs consisting of the AR, the mammalian formin disheveled-associated activator of morphogenesis 2 (DAAM2) and active RNA Polymerase II (RNA Pol-II). Of note, highly dynamic nuclear F-actin polymerization by DAAM2, directly at the AR is essential for androgen signaling. To better understand actin-driven AR transcriptional activity, we turned our interest to the unconventional myosin VI, which was previously proposed to be involved in RNA Pol-II transcription. Indeed, dihydrotestosterone-dependent mass spectrometry of immunoprecipitated eGFP-myosin VI identified the AR as a prominent associator. Consistent with this, structured illumination microscopy in prostate cancer cells revealed signal-dependent nuclear enrichment of myosin VI, which localized in close proximity to AR as well as RNA Pol-II clusters and the actin nucleator DAAM2. Using live-cell structured illumination microscopy imaging, we directly visualized a ligand-dependent dynamic association between AR, myosin VI, and nuclear actin, revealing their spatially coordinated reorganization at AR clusters. Pharmacological inhibition of actin polymerization or inhibition of the myosin VI motor domain disrupted the formation of AR-related transcriptional clusters. Furthermore, reporter gene analysis and proliferation assays supported a critical role for myosin VI in AR signaling. Our findings thus uncover myosin VI as an essential regulator for the spatial organization of androgen-dependent transcription.