Nuclear relocation of a transactivator subunit precedes target gene activation

Murine erythroleukemia (MEL) cells are a model system to study reorganization of the eukaryotic nucleus during terminal differentiation. Upon chemical induction, MEL cells undergo erythroid differentiation, leading to activation of globin gene expression. Both processes strongly depend on the transcriptional activator NF-E2. Before induction of differentiation, both subunits of the NF-E2 heterodimer are present, but little DNA-binding activity is detectable. Using immunofluorescence microscopy, we show that the two NF-E2 subunits occupy distinct nuclear compartments in uninduced MEL cells; the smaller subunit NF-E2p18 is found primarily in the centromeric heterochromatin compartment, whereas the larger subunit NF-E2p45 occupies the euchromatin compartment. Concomitant with the commitment period of differentiation that precedes globin gene activation, NF-E2p18, along with other transcriptional repressors, relocates to the euchromatin compartment. Thus, relocation of NF-E2 p18 may be a rate-limiting step in formation of an active NF-E2 complex. To understand the mechanisms of NF-E2 localization, we show that centromeric targeting of NF-E2p18 requires dimerization, but not with an erythroid-specific partner, and that the transactivation domain of NF-E2p45 may be necessary and sufficient to prevent its localization in centromeric heterochromatin. Finally, using fluorescence in situ hybridization, we show that, upon differentiation, the beta-globin gene loci relocate away from heterochromatin compartments to euchromatin. This relocation correlates with both transcriptional activation of the globin locus and relocation of NF-E2p18 away from heterochromatin, suggesting that these processes are linked.