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.     

Baculovirus-expressed myogenic determination factors require E12 complex formation for binding to the myosin-light-chain enhancer.

Two recombinant baculoviruses BcV-myf4 and BcV-myf5 have been constructed to synthesize the human myogenic determination factors myogenin (myf4) and myf5 in eucaryotic cells. Both recombinant proteins are localized to the nucleus of virus-infected Spodoroptera frugiperda (sf) insect cells and can be recovered as soluble factors. The virus-produced proteins exhibit high-affinity binding to a muscle-specific DNA sequence in the presence of the ubiquitous helix-loop-helix (HLH) protein E12, but only marginal binding in unsupplemented sf nuclear extracts. Both baculovirus-encoded myogenic factors are able to heterooligomerize with E12 in the absence of DNA-binding sites. We conclude from our results that these muscle-specific HLH proteins produced in eucaryotic cells largely depend on dimerization with E12 or similar HLH proteins to recognize the myosin-light-chain-enhancer-MEF-1-binding site. We have no evidence for intracellular protein modifications exerting major effects on the interaction between these factors and DNA.     

Constitutive and inducible factors bind to regulatory element 3 in the promoter of the gene encoding mouse granulocyte colony-stimulating factor.

The expression of the mouse gene (G-CSF) encoding granulocyte colony-stimulating factor is controlled by at least three regulatory elements, GPE1, GPE2 and GPE3 (G-CSF promoter elements). A set of 30-mer oligodeoxyribonucleotides (oligos) scanning the GPE3 region (-104 to -51) of the G-CSF promoter was synthesized, and the tetramer of each oligo was inserted upstream from the cat gene with the simian virus 40 enhancer element. By introducing these hybrid genes into human squamous carcinoma CHU-2 and mouse macrophage BAM3 cells, the enhancer core element of the GPE3 was localized to the region from -98 to -79 in the promoter. A nuclear factor which specifically binds to the core element of the GPE3 was constitutively detected in human CHU-2 cells, whereas the expression of a similar, but distinctly different, factor was significantly induced in BAM3 cells by lipopolysaccharide. The results suggest that these nuclear factors play important roles in the constitutive expression of G-CSF in CHU-2 cells and its inducible expression in macrophages.     

An upstream regulatory element of the NCAM promoter contains a binding site for homeodomains.

In the present study, we have analyzed an upstream regulatory element of the neural cell adhesion molecule (NCAM) promoter which is required for full promoter activity. It contains an ATTATTA motif that resembles the core recognition sequence of homeodomain (HD) proteins of the Antennapedia (Antp) and related types. Electrophoretic mobility shift (EMSA) and DNase I footprinting analyses revealed that the Drosophila HDs coded by the Antp and the zerknüllt (zen) genes bind this site in vitro. In contrast, the engrailed (en) protein did not produce a detectable footprint. The functional relevance of the ATTATTA motif was demonstrated by showing that a two-nucleotide exchange curtailed stimulation of an heterologous promoter. An oligonucleotide known to be recognized with high affinity by Antp-like HDs efficiently competed for endogenous factor binding. These results suggest that the NCAM gene may be a target for HD proteins.     

Evidence of a tissue-restricting DNA regulatory element in the mouse IRBP promoter

The expression of interphotoreceptor retinoid binding protein (IRBP) is limited to photoreceptor cells of the retina and pinealocytes of the pineal gland. We sought to define cis-elements of the mouse IRBP 5' flanking region that are required for this restricted activity. In vitro transient transfections of retinoblastoma and neuroblastoma cells and in vivo experiments with transgenic Xenopus laevis indicate that -1783/+101 and -156/+101 IRBP gene fragments directed expression predominantly to the retina and pineal, with minor neuronal expression elsewhere. In contrast, a -70/+101 fragment was less restrictive in controlling expression, exhibiting activity not only in retina, but also in forebrain, hindbrain, spinal cord, and motor neurons innervating gills.     

Two tissue-specific factors bind the erythroid promoter of the human porphobilinogen deaminase gene.

We have studied the erythroid-specific promoter of the human gene coding for Porphobilinogen Deaminase (PBGD) by DNaseI footprinting, gel retardation and methylation interference assays. We show that this promoter, which is inducible during MEL cell differentiation, contains three binding sites for the erythroid-specific factor NF-E1 and one site for a second erythroid-specific factor, which we name NF-E2. NF-E1 is a factor that also binds the promoter and the enhancer (present in the 3' flanking region) of the human beta-globin gene. NF-E2 has not yet been described and although it binds to a sequence containing the Ap1 consensus, it appears to be different from Ap1.