Characterization Of Chromatin Dynamics In Mammalian Erythropoiesis

Mammalian erythropoiesis is a dynamic process in which erythroid progenitors proliferate and differentiate into mature enucleated red blood cells. In the late stage of erythropoiesis, erythroblasts undergo terminal 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 deacetylations 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. We found that histone H2A and its variants are differentially expressed during erythropoieisis, while the levels of other histones including H2B, histone H3 and histone H4 remained unchanged. To determine their localization, we performed immunofluorescence experiments of these histones together with nuclear lamins. Strikingly, lamin B staining revealed a gradually enlarged nuclear opening through which most histones, except histone H2AZ, were released out of the nucleus. This nuclear opening was confirmed by electron microscopy. The same phenotype was observed in vivo in freshly purified late stage erythroblasts. Western blot analysis confirmed this release, however, many well known nuclear proteins remained in the nucleus in this process. Mechanistically, we demonstrated that caspase 3 was involved in the regulation of nuclear openings during erythropoieiss. Inhibition or knockdown of caspase 3 completely blocked lamin B opening and histone release, which led to inhibition of chromatin condensation and cell differentiation, and ultimate cell death.

In summary, our study demonstrated that the dynamic changes of various histones, with their selective and gradual release out of the nucleus, is required for chromatin condensation and differentiation during erythropoieisis. Studies of the detailed mechanisms are ongoing. Particularly, we are using micrococcal nuclease digestion of chromatin followed by next generation sequencing technology (MNase-seq) to determine genome-wide nucleosome location during the differention. Chromatin- immunoprecipitation (ChIP) will also be performed to determine H2A family protein locations during erythropoiesis.