Nuclear Condensation during Mouse Erythropoiesis Requires Caspase-3 Mediated Nuclear Opening Formation

Mammalian erythropoiesis is a dynamic process in which erythroid progenitors proliferate and differentiate into mature enucleated red blood cells. In the late stage of terminal erythropoiesis, erythroblasts undergo cell-cycle exit, chromatin condensation, and extrusion of the pycnotic nucleus via an asymmetric cell division. Recent genetic and biochemical studies illustrated that various signaling pathways, including histone deacetylation and other chromatin modifications, are involved in chromatin condensation and enucleation. However, it is unclear the global dynamic changes of the nucleosome and how different histones are regulated during chromatin condensation. We proposed to directly characterize the expression levels and localization of different histones and their variants during erythropoiesis. Using a mouse fetal liver erythroblast in vitro culture system and immunofluorescence analysis, we found an unexpected, gradually enlarged nuclear opening through which most histones, except H2AZ, were released out of the nucleus. The same phenotype was observed in freshly purified fetal liver and bone marrow erythroblasts (Figure 1). These openings lack nuclear lamina, nuclear pore complexes, and nuclear membrane but are distinct from nuclear envelope changes during apoptosis and mitosis. Western blot analysis also demonstrated the nuclear release of a fraction of the major histones, however, many well-known nuclear proteins remained in the nucleus in this process. We also demonstrated that the histone release was cell cycle regulated and independent of nuclear exportin.

Using micrococcal nuclease digestion of chromatin followed by next generation sequencing (MNase-seq) technique, we demonstrated that histone release from the nuclear opening is associated with dynamic nucleosomal changes during mouse terminal erythropoiesis. Mechanistically, caspase-3 is involved in the regulation of nuclear openings during erythropoiesis. Inhibition or knockdown of caspase-3 completely blocked nuclear opening formation and histone release, which led to inhibition of chromatin condensation, cell differentiation, and ultimate cell death.

In summary, our study revealed a caspase-3 mediated nuclear opening formation with histone release in mouse erythroblasts that is unique in mammalian cells. The dynamic nuclear opening formation is required for fast release of major histones into cytoplasm to facilitate chromatin condensation throughout erythropoiesis. This “prokaryotic phase” of erythroblast is also associated with genome wide nucleosome localization changes that correlate with the size of the nuclear opening and histone release, which may provide clues for the pathogenesis of erythroid related diseases with unknown etiology.

Figure 1

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Lamin B opening with H2 release in mouse erythropoiesis. (A) E13.5 TER119 negative mouse fetal liver erythroblasts were purified and cultured in vitro in erythropoietin containing medium. Immunofluorescence stains for lamin B, H2A and DNA (DAPI) from erythroblasts cultured on different days were performed. Arrows indicate lamin B openings. Scale bar: 5mm. (B) Same as A except the cells were from fresh total fetal liver cells (left) and bone marrow erythroblasts. Scale bar: 5mm.

Figure 1

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Lamin B opening with H2 release in mouse erythropoiesis. (A) E13.5 TER119 negative mouse fetal liver erythroblasts were purified and cultured in vitro in erythropoietin containing medium. Immunofluorescence stains for lamin B, H2A and DNA (DAPI) from erythroblasts cultured on different days were performed. Arrows indicate lamin B openings. Scale bar: 5mm. (B) Same as A except the cells were from fresh total fetal liver cells (left) and bone marrow erythroblasts. Scale bar: 5mm.

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