Nuclear relocation of a transactivator subunit precedes target gene activation
Nuclear relocation of a transactivator subunit precedes target gene activation
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DOI：
10.1073/pnas.211444898
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发表时间：
2001-10-09
影响因子：
11.1
通讯作者：
Groudine, M
中科院分区：
文献类型：
--
作者：
Francastel, C;Magis, W;Groudine, M
小鼠红白血病（MEL）细胞是研究终末分化过程中真核细胞核重组的模型系统。在化学诱导后，MEL细胞经历红系分化，导致珠蛋白基因表达的激活。这两个过程强烈依赖于转录激活因子NF-E2。在诱导分化之前，NF-E2异源二聚体的两个亚基都存在，但几乎检测不到DNA结合活性。使用免疫荧光显微镜，我们发现，两个NF-E2亚基占据不同的核隔室在未诱导的MEL细胞;较小的亚基NF-E2 p18被发现主要在着丝粒异染色质隔室，而较大的亚基NF-E2 p45占据常染色质隔室。伴随着珠蛋白基因激活前的分化承诺期，NF-E2 p18沿着与其他转录抑制因子一起重新定位到常染色质区室。因此，NF-E2 p18的重新定位可能是活性NF-E2复合物形成的限速步骤。为了了解NF-E2定位的机制，我们发现NF-E2 p18的着丝粒靶向需要二聚化，但不需要红细胞特异性伴侣，并且NF-E2 p45的反式激活结构域可能是必要的，足以防止其在着丝粒异染色质中的定位。最后，利用荧光原位杂交，我们发现，在分化，β-珠蛋白基因位点搬迁远离异染色质车厢常染色质。这种重新定位与珠蛋白基因座的转录激活和NF-E2 p18远离异染色质的重新定位相关，表明这些过程是相关的。
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.