Nuclear relocation of a transactivator subunit precedes target gene activation

Abstract

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.

Figure 1

Targeting of NF-E2p18 but not NF-E2p45 to centromeric heterochromatin. Cells were stained with antibodies against proteins indicated on the left followed by FITC-conjugated secondary antibodies (Left) and DAPI (Center). (Right) The merge of both images. (A and C) Staining of undifferentiated MEL interphase cells. (B) Example of metaphase chromosomes from MEL cells. (Magnifications: ×100.)

Figure 2

Relocation of NF-E2p18 after MEL cell commitment to differentiation. (A) Differentiated MEL cells after 4 days of DMSO treatment. Cells were stained as in Fig. 1. (B) Staining with antibodies against NF-E2p18 of uninduced MEL cells or DMSO-treated, alone or in association with inhibitors of erythroid differentiation, for 12 h. DEX, Dexamethasone; IMID, imidazole. (Magnifications: ×100.)

Figure 3

Transcriptional activators and repressors occupy distinct compartments in the nucleus of MEL cells. MEL cells were transiently transfected with expression vectors encoding tagged versions of the wild-type NF-E2 subunits, p18 (red) and p45 (green). The yellow color results from the overlap between red and green fluorescence. Overlap between the two subunits increases with treatment of the cells with DMSO for 24 h. (Magnifications: ×100.)

Figure 4

Identification of protein domains involved in targeting of NF-E2. (A) Staining of NIH/3T3 cells with antibodies against NF-E2p18. (B) Expression of a Myc-tagged NF-E2p18; full-length protein (Upper) and deletion of the C-terminal dimerization domain (NF-E2p18DLZ) (Lower). (C) Expression of a Flag-tagged NF-E2p45; full-length protein (Upper) and deletion of the N-terminal transactivation domain (Lower). (D) Localization of NF-E2p18 containing 80 aa of the NF-E2p45 transactivation domain at its N terminus. (Magnifications: ×100.)

Figure 5

Centromeric targeting of transactivation domain-deficient NF-E2p45 mutants Myc-tagged NF-E2p18, full length or mutant (NF-E2p18DLZ), was cotransfected with deletion mutants of NF-E2p45 lacking the first 80 (NF-E2p45DN80) or 206 (NF-E2p45DN206) N-terminal amino acids of the transactivation domain. (Magnifications: ×100.)

Figure 6

Relocation of the globin loci upon induction of differentiation. (Upper) Examples of fluorescence in situ hybridization detection of the β-globin loci in interphase MEL cells. The β-globin loci are detected with a Texas red-conjugated probe (red); murine centromeres are revealed by their bright DAPI staining (blue). (Magnifications: ×100.) (Lower) Before induction of differentiation, the β-globin loci preferentially associate with centromeric heterochromatin or the nuclear periphery. Upon induction of differentiation, both loci are found dissociated from these heterochromatin compartments.