Sumoylation of p45/NF-E2: Nuclear Positioning and Transcriptional Activation of the Mammalian β-Like Globin Gene Locus

Abstract

NF-E2 is a transcription activator for the regulation of a number of erythroid-and megakaryocytic lineagespecific genes. Here we present evidence that the large subunit of mammalian NF-E2, p45, is sumoylated in vivo in human erythroid K562 cells and in mouse fetal liver. By in vitro sumoylation reaction and DNA transfection experiments, we show that the sumoylation occurs at lysine 368 (K368) of human p45/NF-E2. Furthermore, p45 sumoylation enhances the transactivation capability of NF-E2, and this is accompanied by an increase of the NF-E2 DNA binding affinity. More interestingly, we have found that in K562 cells, the ␤-globin gene loci in the euchromatin regions are predominantly colocalized with the nuclear bodies promyelocytic leukemia protein (PML) oncogenic domains that are enriched with the PML, SUMO-1, RNA polymerase II, and sumoylatable p45/NF-E2. Chromatin immunoprecipitation assays further showed that the intact sumoylation site of p45/ NF-E2 is required for its binding to the DNase I-hypersensitive sites of the ␤-globin locus control region. Finally, we demonstrated by stable transfection assay that only the wild-type p45, but not its mutant form p45 (K368R), could efficiently rescue ␤-globin gene expression in the p45-null, erythroid cell line CB3. These data together point to a model of mammalian ␤-like globin gene activation by sumoylated p45/NF-E2 in erythroid cells.

JNK-mediated turnover and stabilization of the transcription factor p45/NF-E2 during differentiation of murine erythroleukemia cells

“…S5). This sumoylation of p45 is required for NF-E2 to transactivate erythroid genes in erythroid cells, including induced MEL, because of enhanced DNA binding by the sumoylated p45/NF-E2 and its ability to bring in/ maintain the active genes loci, such as the β globin, within the nuclear body POD (28). Thus, similar to other studies of the antagonistic effects of ubiquitination and sumoylation (38)(39)(40), K368 of p45 becomes accessible to sumoylation on inactivation of JNK in the induced MEL cells.…”

“…On one hand, the p45 was known to be modified by phosphorylation and sumoylation (27,28), in particular, the cAMPdependent PKA phosphorylates p45, but it had no effect on the DNA-binding or transactivation ability of NF-E2 (27). On the other hand, sumoylation of p45 enhances the transactivation ability of NF-E2, and this is modulated through the association between NF-E2 and the β-globin loci within the nuclear body PML oncogenic domains (POD) in erythroid cells including induced MEL (28).…”

“…On the other hand, sumoylation of p45 enhances the transactivation ability of NF-E2, and this is modulated through the association between NF-E2 and the β-globin loci within the nuclear body PML oncogenic domains (POD) in erythroid cells including induced MEL (28). In contrast to sumoylation, very little, if any, is known about the processes of phosphorylation and ubiquitination of p45 in vivo and their biological roles in gene regulation and erythroid differentiation.…”

Transcriptional control of erythropoiesis: emerging mechanisms and principles

“…Furthermore, lysine 368 and serine 169 of p45/NF-E2 are sumoylated and phosphorylated, respectively, and both modifications are important for bmajor transcriptional activation in CB3 cells (Casteel et al, 1998;Shyu et al, 2005).…”

Chromatin-binding in vivo of the erythroid kruppel-like factor, EKLF, in the murine globin loci

“…CB3, a MEL derivative lacking the expression of p45 [28], was grown in RPMI 1640 medium (Invitrogen) containing the same ingredients as above. The procedures for the establishment of CB3 pools stably expressing human p45/NF-E2 followed those described in ref [29].…”