Regulation of NF-E2 Activity in Erythroleukemia Cell Differentiation

Abstract

The erythroid transcription factor NF-E2 is an obligate heterodimer composed of two different subunits (p45 and p18), each containing a basic region-leucine zipper DNA binding domain, and it plays a critical role in erythroid differentiation as an enhancer-binding protein for expression of the ␤-globin gene. We show here that dimethyl sulfoxide treatment of wild-type murine erythroleukemia cells, but not a mutant clone of dimethyl sulfoxide-resistant cells, increases NF-E2 activity significantly, which involves both up-regulation of DNA binding and transactivation activities. Both activities were reduced markedly by treatment of cells with 2-aminopurine but not by genistein. Activation of the Ras-Raf-MAP kinase signaling cascade increased NF-E2 activity significantly, but this was suppressed when MafK was overexpressed. Domain analysis revealed an activation domain in the NH 2 -terminal region of p45 and a suppression domain in the basic region-leucine zipper of MafK. These findings indicate that induction of NF-E2 activity is essential for erythroid differentiation of murine erythroleukemia cells, and serine/threonine phosphorylation may be involved in this process. In addition, they also suggest that a MafK homodimer can suppress transcription, not only by competition for the DNA binding site, but also by direct inhibition of transcription. Hence, MafK may function as an active transcription repressor.The erythroid transcription factor NF-E2 is present in extracts of erythroid cell lines (1, 2) and has been shown to be a heterodimer formed between the two basic region-leucine zipper (b-zip) 1 proteins, i.e. the p45 and the p18 subunits. p45, which together with the Drosophila cap'n-collar (CNC) protein defines a b-zip subfamily, is expressed in hematopoietic cells of the erythroid, megakaryocytic, and mast cell lineages (1), whereas p18 is one of the small Maf family proteins including MafK 2 (1-4), and its expression is not restricted to hematopoietic cells (5). Involvement of NF-E2 in erythroid cell differentiation of mouse erythroleukemia (MEL) cells has been suggested by its role as an enhancer-binding protein for expression of the ␤-globin gene, by the lack of ␤-globin mRNA expression in a MEL cell line (CB3) devoid of NF-E2 protein as a result of integration of Friend viral sequences within the p45 NF-E2 gene locus (6), and by the restoration of ␤-globin mRNA by forced expression of p45 cDNA in these cells (6, 7). It has also been found that NF-E2⅐DNA binding increases during erythroid differentiation of MEL cells (8 -10). These results suggest NF-E2 to be one of the key transcription factors that regulate erythroid differentiation of MEL cells. Unexpectedly, targeted disruption of the p45 gene in mice showed essentially no abnormality in erythropoiesis (11,12). It is possible in these conditions, however, that p45-related b-zip factors, such as Nrf1 (13), or Nrf2 (14), may compensate for deficient NF-E2 activity.Previous studies showed that p45 can be phosphorylated by a cAMP-dependent protein kinase in...

Molecular Profiling of Anemia in Acute Renal Allograft Rejection Using DNA Microarrays

“…By regulating heme formation, ALAS indirectly also determines the expression of many other proteins essential for erythroid development, such as other heme pathway enzymes, beta-globin, and the large subunit of nuclear factor (erythroid-derived 2) (NF-E2) (19). NF-E2 is itself an erythroid transcription factor that plays a critical role in erythroid differentiation as an enhancer-binding protein for the expression of the beta-globin gene (20); it additionally plays a role in heme synthesis and the procurement of iron (21). The down-regulation of ALAS and NF-E2 American Journal of Transplantation 2003; 3: 17-22 (mean 2.1-and 1.3-fold, respectively) in our patients with acute rejection will expectedly reduce hemoglobin synthesis, and this could account for the low hematocrit commonly observed in these anemic patients.…”

MafG Sumoylation Is Required for Active Transcriptional Repression

“…In summary, we previously found that small Maf proteins are regulated quite specifically at the transcriptional level (12,13,19) and that their abundance is a critical determinant of positive and negative gene regulation through MARE (20,22). This study revealed that the sumoylation of MafG is an indispensable posttranslational modification that controls the negative regulatory capacity of MafG.…”

“…In the absence of PIASy, the reporter gene was activated and then repressed depending on the abundance of cotransfected MafG (Fig. 4A, left panel) (22). In the presence of PIASy, MafG was unable to activate the reporter gene (Fig.…”

“…The repressor activity of small Mafs is observed when its relative abundance surpasses that of p45 (20,22). Megakaryocytes recovered from MafK-or MafG-overexpressing transgenic mice exhibited reduced PPF and lowered TXS levels, demonstrating that an excess of small Maf proteins represses MARE-dependent transcription, presumably by competing with the small Maf-p45 heterodimer for DNA binding.…”

“…Small Maf proteins lack any canonical transcriptional effector domains and are obligatory partners for CNC and Bach proteins, which contain recognizable functional motifs but cannot bind to DNA as monomers or homodimers. Small Maf homodimers compete with these activating heterodimers for DNA binding and block their activities, a feature characteristic of passive repression (20,22).…”