Nuclear Dynamics and Gene Activation during Erythroid Maturation.

We have investigated the relationships among nuclear positioning, association with RNA polymerase II (PolII) and expression of the murine β-globin locus during erythroid differentiation, as well as the role of the locus control region (LCR) in these processes. Fetal liver cells from wildtype and LCR-deletion mouse strains were stained with a panel of antibodies, and flow cytometry was used to define and isolate cells from four stages of erythropoiesis spanning pro-erythroblasts (stage 1) to orthochromatic normoblasts and nucleated RBC (stage 4). DNA FISH analyses reveal that with increasing erythroid maturation the β-globin locus is less likely to be located in the nuclear periphery. Immuno-FISH demonstrates that PolII speckles initially are spread diffusely throughout the nucleus, with the exception of extreme periphery. With erythroid maturation the number of speckles decline and become more centrally located. Combined DNA FISH /PolII immuno-staining reveals an increase in co-localization of PolII and β-globin alleles during erythroid maturation. These results are consistent with a model in which the locus is more likely to be in the nuclear periphery and away from PolII foci prior to activation, and with maturation, the locus re-locates more centrally and associates with PolII speckles, leading to β-globin activation. To investigate this model, α and β-globin primary transcript FISH were performed and revealed that while β activation lags behind α, both start at stage 2 and increase with maturation. Notably, activation occurs prior to the increased localization of the locus away from the periphery during maturation, suggesting that localization of the locus away from the periphery may be secondary to the redistribution of PolII during erythroid maturation. Analysis of sorted erythroid cells from mice with a targeted deletion of the LCR reveals less co-localization of the β-globin locus and PolII speckles and loss of re-localization away from the periphery during maturation. Moreover, primary transcript FISH analysis of sorted cells from mice lacking the LCR and those carrying a combined deletion of HS 2 and 3 suggests that the LCR affects the likelihood that an allele is expressed, as well as the amount of transcript generated during periods of expression. Taken together our results suggest that the LCR plays a role in locating to, or stabilization of, interactions between the locus and PolII speckles, increasing the probability of β-globin expression, after which the LCR also affects the rate of transcription.