Precise alignment of sites required for mu enhancer activation in B cells.

The lymphocyte-specific immunoglobulin mu heavy-chain gene intronic enhancer is regulated by multiple nuclear factors. The previously defined minimal enhancer containing the muA, muE3, and muB sites is transactivated by a combination of the ETS-domain proteins PU.1 and Ets-1 in nonlymphoid cells. The core GGAAs of the muA and muB sites are separated by 30 nucleotides, suggesting that ETS proteins bind to these sites from these same side of the DNA helix. We tested the necessity for appropriate spatial alignment of these elements by using mutated enhancers with altered spacings. A 4- or 10-bp insertion between muE3 and muB inactivated the mu enhancer in S194 plasma cells but did not affect in vitro binding of Ets-1, PU.1, or the muE3-binding protein TFE3, alone or in pairwise combinations. Circular permutation and phasing analyses demonstrated that PU.1 binding but not TFE3 or Ets-1 bends mu enhancer DNA toward the major groove. We propose that the requirement for precise spacing of the muA and muB elements is due in part to a directed DNA bend induced by PU.1.

Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins.

The enhancer for the immunoglobulin mu heavy chain gene (IgH) activates a heterologous gene at the pre-B cell stage of B lymphocyte differentiation. A lymphoid-specific element, microB, is necessary for enhancer function in pre-B cells. A microB binding protein is encoded by the PU.1/Spi-1 proto-oncogene. Another sequence element, microA, was identified in the mu enhancer that binds the product of the ets-1 proto-oncogene. The microA motif was required for microB-dependent enhancer activity, which suggests that a minimal B cell-specific enhancer is composed of both the PU.1 and Ets-1 binding sites. Co-expression of both PU.1 and Ets-1 in nonlymphoid cells trans-activated reporter plasmids that contained the minimal mu enhancer. These results implicate two members of the Ets family in the activation of IgH gene expression.

Regulation of immunoglobulin gene transcription.

Analysis of the immunoglobulin gene suggests that their expression is controlled through the combinatorial action of tissue- and stage-specific factors (OTF-2, TF-microB, NF-kappa B), as well as more widely expressed E motif-binding factors such as E47/E12. Two basic issues cloud understanding of how these factors are involved in immunoglobulin gene regulation. First, cloning of these factors shows them to be members of families of proteins, all with similar DNA-binding specificities. OTF-2 is a member of the POU domain family, NF-kappa B is a related protein, and the microE5/kappa E2-binding factors are members of the bHLH family. Second, these binding sites and associated factors are involved in the regulation of many genes, not only the immunoglobulin genes, and in fact not only lymphoid-specific genes. These facts complicate understanding which member of a family is in fact responsible for interaction with, and activation of, a particular binding element in an enhancer/promoter. Recently, more detailed analysis of the interactions between such proteins and their related binding sites suggest that a certain level of specificity may in fact be encoded by the DNA element such that one family member of a protein is preferentially bound, or alternatively that the protein-DNA interactions that occur give subtle alterations in protein conformation that unmask an activation or protein-protein interactive domain. An additional level of regulation is imparted by combinatorial mechanisms such as adjacent DNA-binding elements and factors that may alter activity, as well as "cofactors" that, by forming a complex with the bound factor, affect its activation of a gene in a particular cell type. A third level of specificity may be obtained by factors such as NF-kappa B and the bHLH family due to their ability to create heterogeneous complexes, creating unique complexes in a tissue- or stage-specific manner. The multiple functions transcription factors such as NF-kappa B and OTF-2 play in the transcriptional regulation of multiple genes seems complex in contrast to a one factor, one gene regulation model. However, this type of organization may limit the number of factors lymphocytes would require if each lymphoid-specific gene were activated by a unique factor. Thus what appears to be complexity at the molecular level may reflect an economical organization at the cellular level. Investigation of the key factors controlling these genes suggests an ordered cascade of transcription factors becomes available in the cell during B cell differentiation.(ABSTRACT TRUNCATED AT 400 WORDS)

Temperature-sensitive poliovirus mutant fails to cleave VP0 and accumulates provirions.

A temperature-sensitive mutant of poliovirus, VP2-103, was isolated and characterized. A single nucleotide change, resulting in the substitution of glutamine for arginine at amino acid 76 of the capsid protein VP2, prevented the maturation of virions at the nonpermissive temperature. Particles indistinguishable from the previously elusive provirions were observed; these particles have been proposed to be penultimate in virion morphogenesis. Cleavage of VP0 into VP2 and VP4, the products found in mature virions, was not observed in VP2-103-infected cells at the nonpermissive temperature. The cleavage of VP0 in wild-type poliovirus-infected cells is dependent on RNA packaging; this reaction has been postulated to be autocatalytic. The existence of RNA-containing provirionlike particles in VP2-103-infected cells shows that RNA packaging can be uncoupled from VP0 cleavage.

Complex regulation of the immunoglobulin mu heavy-chain gene enhancer: microB, a new determinant of enhancer function.

The B-lymphocyte-specific activity of the immunoglobulin mu heavy-chain gene enhancer has been attributed to the octamer motif (ATTTGCAT) present within the enhancer that binds a B-cell-specific factor designated NF-A2/OTF-2. However, significant residual enhancer activity even after deletion of this element has suggested the presence of a second critical functional determinant. We have used deletion and mutational analyses to define an element, microB (TTTGGGGAA), that is essential for B-cell-specific enhancer activity in S194 myeloma cells in the absence of the octamer. Transfection analysis in a panel of lymphoid cell lines suggests that the presence of either microB or octamer leads to considerable enhancer activity in cell lines representing later stages of B-cell differentiation, whereas both elements are needed for function in cell lines representing earlier stages. Furthermore, in contrast to the results in pre-B-cell lines, both microB and octamer elements function independently in certain T-cell lines in which the mu enhancer is active.

Conditional poliovirus mutants made by random deletion mutagenesis of infectious cDNA.

Small deletions were introduced into DNA plasmids bearing cDNA copies of Mahoney type 1 poliovirus RNA. The procedure used was similar to that of P. Hearing and T. Shenk (J. Mol. Biol. 167:809-822, 1983), with modifications designed to introduce only one lesion randomly into each DNA molecule. Methods to map small deletions in either large DNA or RNA molecules were employed. Two poliovirus mutants, VP1-101 and VP1-102, were selected from mutagenized populations on the basis of their host range phenotype, showing a large reduction in the relative numbers of plaques on CV1 and HeLa cells compared with wild-type virus. The deletions borne by the mutant genomes were mapped to the region encoding the amino terminus of VP1. That these lesions were responsible for the mutant phenotypes was substantiated by reintroduction of the sequenced lesions into a wild-type poliovirus cDNA by deoxyoligonucleotide-directed mutagenesis. The deletion of nucleotides encoding amino acids 8 and 9 of VP1 was responsible for the VP1-101 phenotype; the VP1-102 defect was caused by the deletion of the sequences encoding the first four amino acids of VP1. The peptide sequence at the VP1-VP3 proteolytic cleavage site was altered from glutamine-glycine to glutamine-methionine in VP1-102; this apparently did not alter the proteolytic cleavage pattern. The biochemical defects resulting from these mutations are discussed in the accompanying report.

The NF-kappa B-binding site mediates phorbol ester-inducible transcription in nonlymphoid cells.

The mouse immunoglobulin kappa light-chain enhancer can interact with at least three independent nuclear proteins. One of these proteins, NF-kappa B, is constitutively present only in nuclear extracts derived from B cells and plasma cells. A DNA-binding protein with the same sequence specificity (and therefore presumed to be NF-kappa B itself) can be induced in pre-B cells, T cells, and nonlymphoid cells by phorbol 12-acetate-13-myristate (PMA); however, it is not clear whether the induced factor can activate transcription in nonlymphoid cells as NF-kappa B does in B cells. In this paper we show that multimerization of a fragment of the mouse kappa enhancer that carried only the binding site for NF-kappa B behaved like a B-cell-specific regulatory element. Furthermore, this unit served to activate transcription in nonlymphoid cells after treatment with PMA (but not with cyclic AMP derivatives), and the kinetics of transcription activation correlated well with the kinetics of factor induction. Thus, the induced DNA-binding activity appeared to be functionally indistinguishable from that of NF-kappa B.