The small Maf proteins (MafG, MafK, and MafF), which serve as heterodimeric partner molecules of CNC family proteins for binding in vitro to MARE sites, have been implicated in the regulation of both transcription and chromatin structure, but there is no current evidence that the proteins fulfill these functions in vivo. To elucidate possible contributions of the small Maf proteins to gene regulation, we have ablated the mafG and mafK genes in mice by replacing their entire coding sequences with the Escherichia coli lacZ gene. mafG homozygous mutant animals exhibit impaired platelet formation accompanied by megakaryocyte proliferation, as well as behavioral abnormalities, whereas mafK-null mutant mice are phenotypically normal. Characterization of the mafG and mafK embryonic expression patterns show that their developmental programs are distinct and intersecting, but not entirely overlapping. These results provide direct evidence that the small Maf transcription factors are vital participants in embryonic development and cellular differentiation.

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...

Small Maf proteins are obligatory heterodimeric partner molecules of mammalian Cap'n'Collar proteins that together control a wide variety of eukaryotic genes. Although both MafK and MafG are expressed in overlapping but distinct tissue distribution patterns during embryonic development, the physiological consequences of loss-of-function mutations in either gene are modest. This suggested that compensation by the third small Maf protein, MafF, might be a major reason for such mild phenotypes and that further analysis of MafF might therefore provide important insights for understanding small Maf regulatory function(s). We therefore cloned, mapped, transcriptionally and developmentally characterized, and finally disrupted the mafF gene. We show that murine mafF is transcriptionally regulated by three different promoters and is most abundantly expressed in the lung. The lacZ gene inserted into the mafF locus revealed prominent expression sites in the gut, lung, liver, outflow tract of the heart, cartilage, bone membrane, and skin but not in hematopoietic cells at any developmental stage. Homozygous mafF null mutant mice were born in a normal Mendelian ratio and displayed no obvious functional deficiencies, indicating that MafF activity may be dispensable even in tissues where the expression of other small Maf proteins is quite low.

Coproporphyrinogen oxidase (CPO) catalyzes the sixth step of the heme biosynthetic pathway. To assess the tissue-specific regulation of the CPO gene promoter, mouse genomic DNA clones for CPO were isolated. Structural analysis demonstrated that the mouse CPO gene spans approximately 11 kb and consists of seven exons, just like its human counterpart. Functional analysis of the promoter by transient transfection assays indicated that synergistic action between an SP-1–like element at −21/−12, a GATA site at −59/−54, and a novel regulatory element, CPRE (-GGACTACAG-) at −49/−41, is essential for the promoter activity in murine erythroleukemia (MEL) cells. In nonerythroid NIH3T3 cells, however, the GATA site is not required. Gel mobility shift assays demonstrated that specific DNA-protein complexes can be formed with each element, and that there are cell-specific differences in factors, which bind to the SP-1–like element between MEL and NIH3T3 cells. These results provide evidence for differential regulation of the promoter function of CPO gene between erythroid and nonerythroid cells. © 1998 by The American Society of Hematology.