HDAC2 is required for chromatin condensation and subsequent enucleation of cultured mouse fetal erythroblasts

Background. Mammalian erythroid cells undergo chromatin condensation and enucleation in their final stages of differentiation. We previously reported that Rac GTPases and their downstream target mDia2 are required for enucleation of in vitro cultured mouse fetal liver erythroblasts. However, it is not clear how chromatin condensation is achieved and whether it is required for enucleation. Design and Methods. Mouse fetal liver erythroblasts were purified from embryonic day 14.5 pregnant mouse and cultured in erythropoietin containing medium. Enucleation was determined by flow-cytometry based analysis after histone deacetylases inhibitor treatment or lentiviral shRNA infection. Results. We show here that histone deacetylases play critical roles in chromatin condensation and enucleation in cultured mouse fetal liver erythroblasts. Enzymatic inhibition of histone deacetylases by trichostatin A or valproic acid prior to the start of enucleation blocks chromatin condensation, contractile actin ring formation and enucleation. We further demonstrate that histone deacetylase 1, 2, 3 and 5 are highly expressed in mouse fetal erythroblasts. ShRNA down-regulation of histone deacetylase 2, but not the other histone deacetylases, phenotypically mimics trichostatin A and valproic acid treated cells with significant inhibition of chromatin condensation and enucleation. Importantly, knockdown of histone deacetylase 2 does not affect erythroblast proliferation, differentiation, or apoptosis. DOI: 10.3324/haematol.2010.029827 Conclusions. These results identify histone deacetylase 2 as an important regulator in mediating chromatin condensation and enucleation in the final stages of mammalian erythropoiesis. Introduction Mammalian erythropoiesis from the Colony Forming Unit Erythroid (CFU-E) progenitor involves sequential erythropoietin dependent and independent stages(1). In the early stages erythropoietin prevents CFU-E apoptosis; it supports rapid cell divisions and induction of hundreds of genes encoding erythrocyte-important proteins. The latter stages are erythropoietin independent and involve chromatin condensation, cell cycle arrest, and enucleation. Whereas in all vertebrates the erythrocyte nucleus becomes highly condensed, enucleation is unique to mammalian erythrocytes. The extruded nucleus is surrounded by a plasma membrane which shows a distinct surface protein expression profile with low levels of glycophorin A and transferrin receptor compared to the incipient reticulocyte; little cytoplasm remains in the extruded nucleus (2). The membrane enveloping the nucleus also contains high levels of phosphatidylserine, which serve as a signal for macrophage engulfment(3). Although our understanding of mammalian erythroid cell enucleation has increased since morphological studies decades ago(4, 5), most of the molecular mechanisms remain unknown. Recent studies suggest that non-apoptotic activities of caspases(6, 7) and of the tumor suppressor protein Rb(8, 9) play important roles in erythroid enucleation. In addition, several studies suggest that enucleation requires that the erythroid cells interact with extracellular matrix proteins(10), macrophages(11, 12), or sinusoidal endothelium(13). These observations, however, are limited and there is little evidence demonstrating that any single regulatory pathway plays a primary role in the enucleation process. DOI: 10.3324/haematol.2010.029827 Using an in vitro cultured mouse fetal liver erythroblast system(14), we previously demonstrated that deregulation of Rac GTPases during the late stages of erythropoiesis completely blocked enucleation of cultured mouse fetal erythroblasts without affecting normal proliferation or differentiation. A contractile actin ring normally forms on the plasma membrane of late-stage erythroblasts at the boundary between the cytoplasm and nucleus of enucleating cells; formation of this cytoskeletal structure was disrupted when Rac GTPase was inhibited in late stages of erythropoiesis. Rac GTPase activity is mediated by the downstream target protein, mDia2, a formin required for nucleation of unbranched actin filaments(2). These results revealed important roles for the Rac GTPase and mDia2 in enucleation of mammalian erythroblasts(15). Chromatin condensation during the late stages of erythropoiesis involves many histone modifications such as deacetylations, presumably catalyzed by histone deacetylases (HDACs). There are 18 known HDACs. HDAC1, 2, 3 and 8 belong to class I; these HDACs are homologous to the yeast RPD3 protein and reside in the nucleus. HDAC4, 5, 7 and 9 belong to class IIa and they shuttle between the nucleus and the cytoplasm. HDAC6 and HDAC10 are class IIb HDACs that contain two catalytic HD domains. All of these HDACs contain a zinc catalytic domain that is inhibited by Trichostatin A (TSA)(16). Histone deacetylases play essential roles in chromatin remodeling, epigenetic regulation, and gene expression, processes that are critical for normal cell differentiation and proliferation (17-19). HDACs also play important roles in determining the fate of hematopoietic cells. In red cells, HDACs negatively regulate the IL-3mediated growth of early erythroid precursors by suppressing their responsiveness to IL3, and play an important role in erythropoietin-mediated differentiation and survival of erythroid precursors (20). DOI: 10.3324/haematol.2010.029827 Histone deacetylases also play critical roles in terminal erythropoiesis. Popova et al recently demonstrated that the activities of histone deacetylases are required for chromatin condensation in Friend-virus infected murine spleen erythroblasts (FVA cells)(21). In this study, HDAC5 expression was shown to be increased during terminal erythropoiesis. However, a role for HDAC5 in mammalian erythroblast chromatin condensation and enucleation needs to be re-examined as no loss of function studies have been done. The use of FVA cells also raises concerns as retrovirus infection often introduces genetic and epigenetic modifications. Here we utilized the in vitro mouse fetal erythroblast culture system and demonstrated that HDAC2, but not other HDACs, plays a critical role in chromatin condensation and subsequent enucleation of primary mouse erythroblasts. Importantly, shRNA knockdown of HDAC2 in the late stages of erythropoiesis did not block normal differentiation or proliferation of primary erythroblasts. These results demonstrated significant roles of HDAC2 during terminal mammalian erythropoiesis. In addition, as various HDAC inhibitors have increasingly been developed and studied in clinical trials for cancer therapy, our study also provided strong evidence that anemia should be assessed in cancer patients with HDAC inhibitor treatment. DOI: 10.3324/haematol.2010.029827