Principal Investigator Harvey Lodish
Mammalian erythroid cells undergo enucleation during a late stage of differentiation, a process that does not occur in other vertebrates. This process has critical physiological and evolutional significance for the morphogenesis and hemoglobin enrichment of mature mammalian red blood cells. Although enucleation has been known for decades the mechanisms that regulate the process remain obscure. Using the new in vitro culture system of fetal liver erythroid progenitors Jing developed, Peng Ji is investigating the mechanism of mammalian erythroid cell enucleation. This and many of our other studies on EpoR signal transduction make use of the system Jing Zhang developed; purified fetal liver erythroid progenitors (so-called CFU-Es) are plated on fibronectin- coated dishes and cultured in the presence of Epo; they undergo normal terminal proliferation and differentiation that can be followed on a single cell level by FACS.
Since actin filaments have been shown to be critical for enucleation, Peng determined the role of different Rho GTPases, the master regulators of actin nucleation, on enucleation. Peng showed that deregulation of Rac GTPase during the late stages of erythropoiesis completely blocks enucleation of cultured mouse fetal erythroblasts without affecting their normal proliferation and differentiation. The contractile actin ring formed on the plasma membrane of late-stage erythroblasts at the boundary between the cytoplasm and nucleus of enucleating cells was disrupted when Rac GTPase was inhibited in late stages of erythropoiesis. Peng further demonstrated that the Rac GTPase activity is mediated by a downstream target protein, mDia2, a formin protein required for nucleation of unbranched actin filaments. These results reveal important roles for Rac GTPase and mDia2 in enucleation of mammalian erythroblasts.
Peng, together with Francisco Sanchez-Rivera, is also focusing on the role of histone deacetylases (HDACs) in chromatin and nuclear condensation and enucleation of late erythroid cells. They showed that inhibition of HDAC activities by Trichostatin A completely blocked enucleation, and that specific inhibition of HDAC6 activity partially blocked enucleation. Peng further showed that mDia2 is acetylated in vivo and his current aim is to determine whether HDAC6 can deacetylate mDia2 and in so doing promote red cell enucleation. In parallel they are investigating the roles of HDACs in inactivating gene transcription and condensing chromatin prior to enucleation. In collaboration with Tzutzuy Ramirez, a fellow with Dr. Maki Murata Hori of the Temasek Life Sciences Laboratory, Singapore, they are investigating the roles of many cytoskeletal and other proteins in nuclear migration and enucleation of these cells, in part using video microscopy of cells expressing fluorescent- tagged proteins.
At the same time, Peng is also interested in the roles of mDia2 on hematopoietic stem cell homing and migration. He is currently generating an mDia2 knockout mouse model and he will use these mice to study the roles of mDia2 in hematopoiesis.