HEB, a helix-loop-helix protein related to E2A and ITF2 that can modulate the DNA-binding ability of myogenic regulatory factors.

Proteins containing the basic-helix-loop-helix (B-HLH) domain have been shown to be important in regulating cellular differentiation. We have isolated a cDNA for a human B-HLH factor, denoted HEB, that shares nearly complete identity in the B-HLH domain with the immunoglobulin enhancer binding proteins encoded by the E2A and ITF2 genes (E proteins). Functional characterization of the protein expressed from this cDNA indicates that HEB is a third member of the E-protein class of B-HLH factors. HEB mRNA was found to be expressed in several tissues and cell types, including skeletal muscle, thymus, and a B-cell line. HEB, ITF2, and the E12 product of the E2A gene all bound to a similar spectrum of E-box sequences as homo-oligomers. All three factors also formed hetero-oligomers with myogenin, and the DNA-binding specificity and binding off-rates (dissociation rates) were modulated after hetero-oligomerization. Both homo- and hetero-oligomers of these proteins were able to distinguish between very closely related E-box sequences. In addition, HEB was shown to form hetero-oligomers with the E12 and ITF2 proteins. Finally, HEB was able to activate gene expression. These data demonstrate that HEB shares characteristics with other E proteins and show that HEB can interact with members of both the myogenic regulatory class and the E-protein class of B-HLH factors. HEB is therefore likely to play an important role in regulating lineage-specific gene expression.     

E2A and E2-2 are subunits of B-cell-specific E2-box DNA-binding proteins.

A class of helix-loop-helix (HLH) proteins, including E2A (E12 and E47), E2-2, and HEB, that bind in vitro to DNA sequences present in the immunoglobulin (Ig) enhancers has recently been identified. E12, E47, E2-2, and HEB are each present in B cells. The presence of many different HLH proteins raises the question of which of the HLH proteins actually binds the Ig enhancer elements in B cells. Using monoclonal antibodies specific for both E2A and E2-2, we show that both E2-2 and E2A polypeptides are present in B-cell-specific Ig enhancer-binding complexes. E2-box-binding complexes in pre-B cells contain both E2-2 and E2A HLH subunits, whereas in mature B cells only E2A gene products are present. We show that the difference in E2-box-binding complexes in pre-B and mature B cells may be caused by differential expression of E2A and E2-2.     

Defective gene expression,S phase progression,and maturation during hematopoiesis in E2F1/E2F2 mutant mice

E2F plays critical roles in cell cycle progression by regulating the expression of genes involved in nucleotide synthesis, DNA replication, and cell cycle control. We show that the combined loss of E2F1 and E2F2 in mice leads to profound cell-autonomous defects in the hematopoietic development of multiple cell lineages. E2F2 mutant mice show erythroid maturation defects that are comparable with those observed in patients with megaloblastic anemia. Importantly, hematopoietic defects observed in E2F1/E2F2 double-knockout (DKO) mice appear to result from impeded S phase progression in hematopoietic progenitor cells. During DKO B-cell maturation, differentiation beyond the large pre-BII-cell stage is defective, presumably due to failed cell cycle exit, and the cells undergo apoptosis. However, apoptosis appears to be the consequence of failed maturation, not the cause. Despite the accumulation of hematopoietic progenitor cells in S phase, the combined loss of E2F1 and E2F2 results in significantly decreased expression and activities of several E2F target genes including cyclin A2. Our results indicate specific roles for E2F1 and E2F2 in the induction of E2F target genes, which contribute to efficient expansion and maturation of hematopoietic progenitor cells. Thus, E2F1 and E2F2 play essential and redundant roles in the proper coordination of cell cycle progression with differentiation which is necessary for efficient hematopoiesis.     

Generation and Analysis of Partially Haploid Cells with Cre-mediated Chromosome Deletion in the Lymphoid System

The fast accumulation of mutant mouse strains in recent years has provided an invaluable resource for phenotype-based genetic screens. However, study of lymphoid phenotypes can be obscured or impractical if homozygous mutations cause early embryonic defects. To aid phenotype screening of germ line mutations in the lymphoid system, we developed a method to induce loss of heterozygosity (LOH) in developing lymphocytes through chromosome deletion. Chromosome deletion was triggered by Cre/loxP-mediated inverse sister chromatid recombination in the G₂/M phase of the cell cycle, leading to the generation of daughter cells missing part of or the entire recombinant chromosome. We show that the resulting cells were viable and capable of additional rounds of cell division, thus providing raw materials for subsequent phenotypic assessment. We used the recombination system to induce LOH at the E2A locus in developing B cells. A significant loss of pro-B and pre-B cells was observed when the wild-type allele was removed by chromosome deletion from the E2A heterozygous mice, a result consistent with the required role for E2A in B cell development. We also demonstrated the effectiveness of Cre-mediated chromosome deletion in the LOH assay for HEB function in T cell development. Thus, the Cre-mediated chromosome deletion provides a new and effective method for genome-wide assessment of germ line mutations in the lymphoid system.     

Murine pro-B cells require IL-7 and its receptor complex to up-regulate IL-7R alpha, terminal deoxynucleotidyltransferase, and c mu expression

Phenotypic analysis of bone marrow cells from IL-7 knockout (KO) mice revealed that B cell development is blocked precisely at the transition between pro-B cells and pre-B cells. In contrast, the generation of pre-pro-B cells and pro-B cells appeared to be normal, as judged by total cell numbers, proliferative indexes, D-JH and V-DJH gene rearrangements, and mRNA for recombinase-activating gene-1 (RAG-1), RAG-2, TdT, Ig mu, lambda 5, and VpreB. However, upon closer inspection, several abnormalities in pro-B cell development were identified that could be corrected by injection of rIL-7 in vivo. These included the absence of the subset of late pro-B cells that initiates cmu expression for pre-B cell Ag receptor (BCR) formation, and the failure of pro-B cells to up-regulate TdT and the IL-7R alpha (but not the common gamma-chain) chain. Similar defects were present in common gamma-chain and Jak3 KO mice, but not in lambda 5 or (excluding cytoplasmic Ig mu heavy chain (c mu)) RAG-1 KO mice, all of which also arrest at the late pro-B cell stage. Consequently, up-regulation of TdT and IL-7R alpha expression requires signaling through the high affinity IL-7R, but does not require cmu expression or a functional pre-BCR. Taken together, these results suggest that IL-7 and its receptor complex are essential for 1) up-regulating the expression of TdT and IL-7R alpha, 2) initiating the production of cmu and 3) promoting the formation of a functional pre-BCR in/on pro-B cells. These key events, in turn, appear to be prerequisite both for differentiation of pro-B cells to pre-B cells and for proliferation of these cell subsets upon continued stimulation with IL-7.     

Generation of an Adenovirus Vector Lacking E1, E2a, E3, and All of E4 except Open Reading Frame 3

Toxicity and immunity associated with adenovirus backbone gene expression is an important hurdle to overcome for successful gene therapy. Recent efforts to improve adenovirus vectors for in vivo use have focused on the sequential deletion of essential early genes. Adenovirus vectors have been constructed with the E1 gene deleted and with this deletion in combination with an E2a, E2b, or E4 deletion. We report here a novel vector (Av4orf3nBg) lacking E1, E2a, and all of E4 except open reading frame 3 (ORF3) and expressing a beta-galactosidase reporter gene. This vector was generated by transfection of a plasmid carrying the full-length vector sequence into A30.S8 cells that express E1 and E2a but not E4. Production was subsequently performed in an E1-, E2a-, and E4-complementing cell line. We demonstrated with C57BL/6 mice that the Av4orf3nBg vector effected gene transfer with an efficiency comparable to that of the Av3nBg (wild-type E4) vector but that the former exhibited a higher level of beta-galactosidase expression. This observation suggests that E4 ORF3 alone is able to enhance RNA levels from the beta-galactosidase gene when the Rous sarcoma virus promoter is used to drive transgene expression in the mouse liver. In addition, we observed less liver toxicity in mice injected with the Av4orf3nBg vector than those injected with the Av3nBg vector at a comparable DNA copy number per cell. This study suggests that the additional deletion of E4 in an E1 and E2a deletion background may be beneficial in decreasing immunogenicity and improving safety and toxicity profiles, as well as increasing transgene capacity and expression for liver-directed gene therapy.