Enucleation.

Abstract SCI-18

Late erythroblasts undergo terminal cell cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division, but the signaling pathways and genes involved in these last steps of erythropoiesis are not known. Last year we showed that enucleation of mouse fetal erythroblasts requires both Rac GTPases and mDia2, a downstream effector of Rho GTPases and a formin protein required for nucleation of unbranched actin filaments. Rac and mDia2 are required for formation of the cortical actin ring that apparently powers the separation between the membrane- enveloped nucleus and forming reticulocyte. In collaboration with Drs. Tzutzuy Ramirez Hernandez and Maki Murata-Hori of the Temasek Life Sciences Laboratory in Singapore we showed by time-lapse microscopy that extrusion of the nucleus begins with microtubule- dependent “pushing” of the nucleus to one pole of the cell. Final budding off of the nucleus happens quickly – requiring only about 5 minutes. By analyzing fractionated erythroblasts at all stages of differentiation we confirmed that the nucleus undergoes gradual condensation before being extruded out of the cytoplasm. To investigate how chromatin condensation affects condensation and enucleation, we treated cultured erythroblasts with trichostatin A, a pan-histone deacetylase inhibitor, and discovered that enucleation is completely blocked. We further demonstrated that HDAC1, HDAC2 and HDAC3 are highly expressed in erythroid cells. Using a retroviral system to express shRNAs in developing erythroblasts, we found that downregulation of HDAC2 partially blocked enucleation. We also focused on the role of HDAC6 on enucleation since previous reports showed that HDAC6 forms a complex with mDia2. We confirmed the interaction between HDAC6 and mDia2 and demonstrated that HDAC6 can deacetylate mDia2. This may activate mDia2 and eventually promote enucleation. Our hypothesis is that HDACs play two distinct roles in enucleation: HDAC1, HDAC2 and HDAC3 (class I HDACs) are necessary for the condensation of the chromatin which is required for enucleation; on the other hand, HDAC6 (class II HDAC) interacts with and deacetylates mDia2 to promote enucleation.