Complexity of the erythroid transcription factor NF-E2 as revealed by gene targeting of the mouse p18 NF-E2 locus.

High-level globin expression in erythroid precursor cells depends on the integrity of NF-E2 recognition sites, transcription factor AP-1-like protein-binding motifs, located in the upstream regulatory regions of the alpha- and beta-globin loci. The NF-E2 transcription factor, which recognizes these sites, is a heterodimer consisting of (i) p45 NF-E2 (the larger subunit), a hematopoietic-restricted basic leucine zipper protein, and (ii) a widely expressed basic leucine zipper factor, p18 NF-E2, the smaller subunit. p18 NF-E2 protein shares extensive homology with the maf protooncogene family. To determine an in vivo role for p18 NF-E2 protein we disrupted the p18 NF-E2-encoding gene by homologous recombination in murine embryonic stem cells and generated p18 NF-E2-/- mice. These mice are indistinguishable from littermates throughout all phases of development and remain healthy in adulthood. Despite the absence of expressed p18 NF-E2, DNA-binding activity with the properties of the NF-E2 heterodimer is present in fetal liver erythroid cells of p18 NF-E2-/- mice. We speculate that another member of the maf basic leucine zipper family substitutes for the p18 subunit in a complex with p45 NF-E2. Thus, p18 NF-E2 per se appears to be dispensable in vivo.

Integration of complete transferred DNA units is dependent on the activity of virulence E2 protein of Agrobacterium tumefaciens.

Agrobacterium tumefaciens transfers transferred DNA (T-DNA), a single-stranded segment of its tumor-inducing (Ti) plasmid, to the plant cell nucleus. The Ti-plasmid-encoded virulence E2 (VirE2) protein expressed in the bacterium has single-stranded DNA (ssDNA)-binding properties and has been reported to act in the plant cell. This protein is thought to exert its influence on transfer efficiency by coating and accompanying the single-stranded T-DNA (ss-T-DNA) to the plant cell genome. Here, we analyze different putative roles of the VirE2 protein in the plant cell. In the absence of VirE2 protein, mainly truncated versions of the T-DNA are integrated. We infer that VirE2 protects the ss-T-DNA against nucleolytic attack during the transfer process and that it is interacting with the ss-T-DNA on its way to the plant cell nucleus. Furthermore, the VirE2 protein was found not to be involved in directing the ss-T-DNA to the plant cell nucleus in a manner dependent on a nuclear localization signal, a function which is carried by the NLS of VirD2. In addition, the efficiency of T-DNA integration into the plant genome was found to be VirE2 independent. We conclude that the VirE2 protein of A. tumefaciens is required to preserve the integrity of the T-DNA but does not contribute to the efficiency of the integration step per se.

Erythropoiesis and globin gene expression in mice lacking the transcription factor NF-E2.

Previous studies in transgenic mice and cultured cells have indicated that the major enhancer function for erythroid cell expression of the globin genes is provided by the heterodimeric basic-leucine zipper transcription factor NF-E2. Globin gene expression within cultured mouse erythroleukemia cells is highly dependent on NF-E2. To examine the requirement for this factor in vivo, we used homologous recombination in embryonic stem cells to generate mice lacking the hematopoietic-specific subunit, p45 NF-E2. The most dramatic aspect of the homozygous mutant mice was an absence of circulating platelets, which led to the death of most animals due to hemorrhage. In contrast, the effect of loss of NF-E2 on the erythroid lineage was surprisingly mild. Although neonates exhibited severe anemia and dysmorphic red-cell changes, probably compounded by concomitant bleeding, surviving adults exhibited only mild changes consistent with a small decrease in the hemoglobin content per cell. p45 NF-E2-null mice responded to anemia with compensatory reticulocytosis and splenomegaly. Globin chain synthesis was balanced, and switching from fetal to adult globins progressed normally. Although these findings are consistent with the substitution of NF-E2 function in vivo by one or more compensating proteins, gel shift assays using nuclear extracts from p45 NF-E2-null mice failed to reveal novel complexes formed on an NF-E2 binding site. Thus, regulation of globin gene transcription through NF-E2 binding sites in vivo is more complex than has been previously appreciated.

Pluripotent hematopoietic stem cells contain high levels of mRNA for c-kit, GATA-2, p45 NF-E2, and c-myb and low levels or no mRNA for c-fms and the receptors for granulocyte colony-stimulating factor and interleukins 5 and 7.

Pluripotent hematopoietic stem cells (PHSCs) were highly enriched from mouse bone marrow by counterflow centrifugal elutriation, lineage subtraction, and fluorescence-activated cell sorting based on high c-kit receptor expression (c-kitBR). We used reverse transcriptase polymerase chain reaction to assay the c-kitBR subset and the subsets expressing low (c-kitDULL) and no (c-kitNEG) c-kit receptor for expression of mRNA encoding hematopoietic growth factor receptors and transcription factors. The c-kitBR cells had approximately 3.5-fold more c-kit mRNA than unfractionated bone marrow cells. The c-kitDULL cells had 47-58% of the c-kit mRNA found in c-kitBR cells and the c-kitNEG cells had 4-9% of the c-kit mRNA present in c-kitBR cells. By comparing mRNA levels in c-kitBR cells (enriched for PHSCs) with those of unfractionated bone marrow, we demonstrated that c-kitBR cells contained low or undetectable levels of mRNA for c-fms, granulocyte colony-stimulating factor receptor, interleukin 5 receptor (IL-5R), and IL-7R. These same cells had moderate levels of mRNA for erythropoietin receptor, IL-3R subunits IL-3R alpha (SUT-1), AIC-2A, and AIC-2B, IL-6R and its partner gp-130, and the transcription factor GATA-1 and high levels of mRNA for transcription factors GATA-2, p45 NF-E2, and c-myb. We conclude from these findings that PHSCs are programmed to interact with stem cell factor, IL-3, and IL-6 but not with granulocyte or macrophage colony-stimulating factor. These findings also indicate that GATA-2, p45 NF-E2, and c-myb activities may be involved in PHSC maintenance or proliferation.

Isolation of a differentially regulated splicing isoform of human NF-E2.

The transcription factor NF-E2 (nuclear factor erythroid 2), interacting via DNA motifs within regulatory regions of several hematopoietic genes, is thought to mediate the enhancer activity of the globin locus control regions. By screening a human fetal liver cDNA library with probes derived from mouse NF-E2, we have isolated a splicing variant of the NF-E2 gene (fNF-E2) that differs in the 5' untranslated region from the previously reported cDNA (aNF-E2). The fNF-E2 isoform is transcribed from an alternative promoter located in the 3' end of the first intron and joined by alternative splicing to the second and third exons, which are shared by both RNA isoforms. Although the two forms produce the same protein, they are expressed in different ratios during development. fNF-E2 is more abundant in the fetal liver and less abundant in the adult bone marrow compared to the previously described form. Their distribution apparently follows the differential expression of fetal and adult hemoglobins.

NOR network transduction procedures : "merging of gates" and "substitution of gates" for fan-in and fan-out restricted networks, NETTRA-G3-FIFO and NETTRA-PG1-FIFO /

"December 1974."--T.p.

Program manual : NOR network transduction based on connectable and disconnectable conditions (reference manual of NOR network transduction programs NETTRA-G1 and NETTRA-G2) /

"UIUCDCS-R-75-698"

Network transduction based on permissible functions : general principles of NOR network transduction NETTRA programs /

Includes bibliographical references.

NOR network transduction system : principles of the NETTRA system /

Bibliography: p. 209-212.

Program manual for the NETTRA system /

"UILU-ENG 77 1743."

Program manual: NOR network pruning procedures using permissible functions : (reference manual of NOR network transduction programs NETTRA-PG1, NETTRA-P1, and NETTRA-P2) /

Includes bibliographical references (page 78).

Program manual, NOR network transduction by generalized gate merging and substitution : (reference manual of NOR network transduction programs NETTRA-G3 and NETTRA-G4) /

Includes bibliographical references.

Pruning procedures for NOR networks using permissible functions : principles of NOR network transduction programs NETTRA-PGI, NETTRA-P1, and NETTRA-P2 /

Bibliography: p. 49.

Network transduction programs based on connectable and disconnectable conditions with fan-in and fan-out restrictions : (a description of NETTRA-G1-FIFO and NETTRA-G2-FIFO) /

Includes bibliographical references.

NOR network transduction by generalized gate merging and substitution procedures : (principles of NOR network transduction programs NETTRA-G3 and NETTRA-G4) /

Includes bibliographical references.