Histone Deacetylation Makes an Important Contribution to Chromatin Condensation and Enucleation during Murine Erythroblast Terminal Differentiation.

Mammalian erythroid differentiation is characterized by progressive chromatin condensation and culminates in nuclear extrusion. Core histone modifications are known to play a significant role in formation of heterochromatin in several other differentiated cell types. To elucidate molecular mechanisms governing changes in mammalian erythroblast chromatin preceding enucleation, we used a model system in which Friend virus-infected murine spleen erythroblasts undergo terminal differentiation and enucleation in vitro during 0–44 hours. At the conclusion of terminal differentiation of murine erythroblasts, we found that histone H3(K9) dimethylation markedly increased while acetylation of histone H3(K9) and histone H4(K12) dramatically decreased, strongly correlating with nuclear chromatin condensation. To test the effects of altering histone deacetylation on chromatin condensation and enucleation, we added either 1 or 2 mM sodium butyrate, an inhibitor of histone deacetylase, to cultures of mouse erythroblasts and compared specific parameters of differentiation with untreated control cells, which normally terminally differentiate at 44 hrs. Western blotting revealed that control cells had the predicted dramatic decrease in histone H4(K12) acetylation while 1 mM or 2mM butyrate-treated erythroblasts did not show diminished levels of acetylation. Numbers of erythroblasts were 5- and 8-fold higher at 44 hrs in cultures treated with 1 and 2mM butyrate, respectively, compared to untreated control cells and there was a correlated marked reduction in expelled nuclei and young reticulocytes. We also found that nuclei in the butyrate-treated erythroblast populations were 1.5 fold larger relative to nuclei with condensed chromatin in terminally differentiated erythroblasts in control cultures. Evaluation of hemoglobinization of erythroblasts by benzidine/hemotoxin staining showed that hemoglobin synthesis continued after butyrate treatment, indicating cell viability and the absence of significant butyrate-induced apoptosis. In sum, these data show that butyrate exposure maintains erythroblasts in a nucleated state with relatively uncondensed chromatin and inhibits them from normally differentiating into young reticulocytes. Thus widespread deacetylation of histones appears to be necessary to achieve extensive chromatin condensation during mammalian erythroid maturation. Our data add support to the attractive idea that during normal mammalian erythropoiesis chromatin condensation may be essential for the transition from nucleated erythroblasts to enucleated reticulocytes.