Immunoglobulin Heavy Chain Enhancer Research Articles

Evaluation of antisense inhibition in a B cell model.

An in vitro model has been developed in an attempt to optimise antisense inhibition in B cells as a prelude to transgenic studies. The hypotheses tested were that i) the 3'-untranslated region would be an appropriate target for antisense inhibition; 2) the immunoglobulin heavy chain intronic enhancer could be used to enhance antisense inhibition via increased production of antisense transcripts; and 3) the mouse metallothionein-1 promoter would allow induction of antisense inhibition in B cells. Secreted IgM protein and mRNA were monitored following the stable transfection of a B cell line, HO-2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA under the control of either the mouse metallothionein-1 promoter or homologous (ie same promoter as target sequence) immunoglobulin heavy chain promoter. The 3'-untranslated region proved to be an appropriate target resulting in 70% inhibition of IgM secretion. Compared with untransfected and sense controls, significant decreases in IgM secretion (and RNA levels) were detected in clones transfected with antisense constructs utilising the mouse metallothionein-1 promoter and the immunoglobulin heavy chain intronic enhancer elements. These clones exhibited a further significant reduction in secreted IgM production upon zinc induction. Hybridisation studies demonstrated that decreased protein production was most likely attributable to reduction in RNA levels. In contrast, transfection with antisense constructs had no effect on membrane IgM protein levels which not only confirmed the specificity of antisense action but meant that the B cell remained sensitive to receptor ligation. We conclude that reasonable antisense inhibition of gene product expression can be achieved in B cells by targeting the 3'-untranslated region and using both an inducible promoter (mouse metallothionein-1) and the IgH enhancer to aid antisense RNA production.

Evaluation of novel control elements by construction of eukaryotic expression vectors

A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3′ enhancer (3′E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3′E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3′ enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.

Involvement of Stat3 in interleukin-6-induced IgM production in a human B-cell line.

Interleukin-6 (IL-6) is an important B-cell growth and differentiation factor. IL-6 treatment of the human lymphoblastoid cell line, SKW6.4, leads to increased IgM production. We have previously shown that IL-6 induces activation of JAK1 and JAK2 in human B cell lines. A chimeric IL-6 receptor, comprised of the intracellular tail of the IL-6 receptor subunit gp130 fused to the extracellular domain of the epidermal growth factor (EGF) receptor, was stably transfected into SKW6.4 cells. EGF treatment induced IgM production in cells transfected with an intact gp130 cytoplasmic tail, but not in untransfected cells or cells transfected with a cytoplasmic tail lacking all four signal transducers and activators of transcription (Stat) binding sites. Moreover, EGF treatment induced Stat3 phosphorylation in cells transfected with the intact chimeric EGF-gp130 receptor along with induction of DNA-mobility shift of a classical interferon-gamma-activated site. To define further the relation between Stat3 activation and enhanced IgM production, we determined the effect of chimeric gp130 on the transcriptional activation of a genetic element linked to immunoglobulin production, namely the immunoglobulin heavy chain enhancer (IgH-enhancer). Parental as well as transfected SKW6.4 cells were transiently transfected with an IgH-enhancer-luciferase construct. The transcriptional activity of the IgH-luciferase construct was induced upon ligation of the full-length chimeric receptor but not by truncated gp130 receptors. Moreover, the gp130-induced activity of this reporter gene was abrogated by Stat3EE, a mutant Stat3 incapable of binding DNA. These results indicate that IL-6-induced B-cell differentiation, as measured by IgM production, may be controlled by Stat3 proteins.

Constitutive expression of terminal deoxynucleotidyl transferase in transgenic mice is sufficient for N region diversity to occur at any Ig locus throughout B cell differentiation.

N region diversity in Ag receptors is a developmentally regulated process in B and T cells that correlates with the differential expression of terminal deoxynucleotidyl transferase (TdT). Absent in fetal and newborn mice, TdT expression is restricted to early T and pro-B cells in adults. To extend the TdT expression pattern throughout B cell ontogenesis, we generated transgenic mice carrying a TdT cDNA under the regulatory elements of the N-myc gene and the IgH enhancer. High expression was observed in secondary lymphoid organs consistent with TdT activity beyond the pre-B cell stage. This suggests that TdT transgene expression is not down-regulated as is the endogenous gene. Unlike normal mice, extensive N region diversity was found in rearranged lambda light chain genes of adult transgenic animals. Therefore, expression of TdT appears sufficient for N region diversity to occur at any Ig locus. More importantly, expression of the transgene takes place during fetal development. As a consequence, the potential fetal B cell repertoire is modified as both rearranged heavy and light chain genes now show N region additions. Constitutive expression of TdT throughout B cell differentiation does not therefore appear deleterious and suggests that TdT is recruited only to participate in the V(D)J recombination process.

Selective utilization of basic helix-loop-helix-leucine zipper proteins at the immunoglobulin heavy-chain enhancer.

The microE3 E box within the immunoglobulin heavy-chain (IgH) enhancer binds several proteins of the basic helix-loop-helix-leucine zipper (bHLHzip) class, including TFE3, USF1, and Max. Both TFE3 and USF have been described as transcriptional activators, and so we investigated their possible roles in activating the IgH enhancer in vivo. Although TFE3 activated various enhancer-based reporters, both USF1 and Max effectively inhibited transcription. Inhibition by USF correlated with the lack of a strong activation domain and was the result of the protein neutralizing the microE3 site. The effects of dominant-negative derivatives of TFE3 and USF1 confirmed that TFE3, or a TFE3-like protein, is the primary cellular bHLHzip protein that activates the IgH enhancer. In addition to providing a physiological role for TFE3, our results call into question the traditional view of USF1 as an obligate transcriptional activator.

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

Bent DNA in the Human Adenovirus Type 2 E1A Enhancer Is an Architectural Element for Transcription Stimulation

ELF-1 Interacts with and Transactivates the IgH Enhancer π Site

We previously identified a B-cell-specific regulatory element in the immunoglobulin heavy chain (IgH) enhancer, pi, with striking similarity to binding sites for ets-related transcription factors. Whereas the ability of ets-related factors to bind to and transactivate the pi site has been substantiated, the identification of the particular member of the ets family responsible for B-cell-specific regulation of the pi site has remained controversial. We have used antibodies specific for individual members of the ets family to evaluate which ets-related factor in B-cell nuclear extracts interacts with the IgH pi site. We present strong evidence that ELF-1 is highly expressed in B-cells and is one of two major factors specifically interacting with the murine IgH enhancer pi site in B-cell nuclear extracts. Binding of ELF-1 correlates with activity of the pi site, since mutations abolishing function of pi also inhibit binding of ELF-1. Furthermore, we demonstrate that ELF-1 can transactivate the IgH enhancer in HeLa cells, suggesting a role for ELF-1 in B-cell-specific IgH gene expression.

CREB Proteins Function as Positive Regulators of the Translocated bcl-2 Allele in t(14;18) Lymphomas

The translocated and normal bcl-2 alleles in the DHL-4 cell line with the t(14;18) translocation were separated by pulsed field electrophoresis. An in vivo footprint over a cAMP response element (CRE) in the bcl-2 5'-flanking sequence was identified on the translocated allele. Electrophoretic mobility shift assays with the bcl-2 CRE demonstrated complexes with mobilities identical to those with a consensus CRE. UV cross-linking experiments revealed that proteins with molecular masses of 34, 43, and 67 kDa bound to the bcl-2 CRE site. Electrophoretic mobility shift assay with an antibody specific to the phosphorylated cAMP response-binding protein (CREB) demonstrated that phosphorylated CREB was present in DHL-4 cells. Treatment with phorbol 12-myristate 13-acetate (PMA) led to an increase in both the amount of phosphorylated CREB and the bcl-2 promoter activity. The response to PMA was dependent on an intact CRE site. The activity of the bcl-2 promoter was increased 20-fold in a construct with the immunoglobulin heavy chain enhancers, and mutation of the CRE site abolished most of the induction. The addition of PMA increased the activity of the bcl-2-immunoglobulin enhancer construct by 3.5-fold. Access to the CRE site is blocked in the silent normal bcl-2 allele, while CREB proteins bind to the site on the translocated allele. We conclude that the CRE site functions as a positive regulatory site for the translocated bcl-2 allele in t(14;18) lymphomas.

Establishment and characterization of choroid plexus carcinoma cell lines: connection between choroid plexus and immune systems.

Murine choroid plexus cell lines were produced from choroid plexus carcinoma generated in transgenic mice harboring the viral oncogene simian virus 40 large tumor antigen under transcriptional control of an intronic enhancer region from the human immunoglobulin heavy chain (IgH) gene. Two morphologically distinct cell lines have been cloned. These established cell lines retained the characteristics of choroid plexus cells in that they expressed such choroid plexus cell marker or related proteins as transthyretin and α2‐macroglobulin. They were tumorigenic in nude mice. In the cell lines, the μA and μB (HE2) motifs within the IgH intronic enhancer were active and we also demonstrated the existence of the proteins binding to these motifs, suggesting a potential link between the choroid plexus and immune systems. It is considered that these binding proteins act as trans‐activators for the enhancer and may belong to the class of ETS‐related proteins. These cell lines and xenografts should be useful materials for analyses of choroid plexus functions.

E47 activates the Ig-heavy chain and TdT loci in non-B cells.

The E2A proteins, E12 and E47, are basic helix-loop-helix (bHLH) proteins essential for the B-cell lineage. Initially identified as immunoglobulin enhancer-binding proteins, they have also been shown to activate immunoglobulin enhancer-based reporters in transient transfection assays. Here, we examine the relationship between E2A DNA binding activity and activation of the endogenous, chromosomal immunoglobulin heavy chain (IgH) locus. Using sterile I(mu) transcription as an indicator of IgH enhancer activity, we see a direct correlation between E2A DNA binding activity and I(mu) transcription in stable BxT hybrids. We also observe a 1000-fold stimulation of endogenous I(mu) transcription in fibroblasts that express high levels of E47 and less stimulation in cells that express E12. By contrast, none of the other IgH enhancer-binding proteins tested (E2-2, Pu.1, Oct-2, OCA-B, TFE3 and USF) were able to activate I(mu) transcription. E47 overexpression also resulted in transcriptional activation of the endogenous gene encoding TdT, indicating that it, too, is a target of E2A proteins early in the B-cell lineage. Our results indicate that E2A proteins have the distinctive property of activating silent, chromatin-embedded B-cell-specific genes, underscoring their crucial role in B-cell development.

Concerted repression of an immunoglobulin heavy-chain enhancer, 3' alpha E(hs1,2).

The transcription factor, B-cell-specific activator protein (BSAP), represses the murine immunoglobulin heavy-chain 3' enhancer 3' alpha E(hs1,2) in B cells. Analysis of various 3'alpha E deletional constructs indicates that sequences flanking a and b BSAP-binding sites are essential for appropriate regulation of the enhancer. An octamer motif 5' of the a site and a specific G-rich motif 3' of the b site were identified by competition in electrophoretic mobility-shift assays and methylation-interference foot-printing analysis. Site-directed mutagenesis of either the octamer or G-rich sites resulted in the complete release of repression of 3' alpha E(hs1,2), implicating these two motifs in the repression of this enhancer in B cells. However, when both BSAP-binding sites were mutated, the octamer and G-rich motifs functioned as activators. Moreover, in plasma cells, when BSAP is not expressed, 3' alpha E(hs1,2) is active, and its activity depends on the presence of the other two factors. These results suggest that in B cells, 3' alpha E (hs1,2) is down-regulated by the concerted actions of BSAP, octamer, and G-rich DNA-binding proteins. Supporting this notion of concerted repression, a physical interaction between BSAP and octamer-binding proteins was demonstrated using glutathione S-transferase fusion proteins. Thus, concerted repression of 3' alpha E (hs1,2) in B cells provides a sensitive mechanism by which this enhancer, either individually or as part of a locus-controlling region, is highly responsive to any of several participating factors.

Regulation of 3' IgH enhancers by a common set of factors, including kappa B-binding proteins.

Targeted disruption of the p50 subunit of NF-kappa B resulted in isotype class switch defects resembling those observed in mice in which the downstream IgH enhancer 3'alpha E(hs1,2) was deleted. We postulated that kappa B binding proteins may regulate class switching by interacting with 3'alpha E(hs1,2) or with other IgH 3' enhancers with which 3'alpha E(hs1,2) synergizes. kappa B binding sites were identified in 3'alpha E(hs1,2) and 3' alpha-hs4, the distal 3' IgH enhancer. A kappa B binding site within 3'alpha E(hs1,2) contributes to at least half the activity of the enhancer in plasma cells, while the same kappa B binding site participates in the complex repression of the enhancer in B cells. In the case of 3'alpha-hs4, a kappa B binding complex activates the enhancer in pre-B, B cells and plasma cells. Additional binding sites within 3'alpha-hs4 for factors known to regulate 3'alpha E(hs1,2), including Oct-1 and BSAP, were identified, and their contribution to 3'alpha-hs4 regulation during B cell development was assessed. Oct-1 positively regulates the enhancer in pre-B and B cells, while BSAP is a repressor in pre-B cells and an activator at the B cell stage. These studies identify kappa B binding proteins as key modulators of 3'alpha E(hs1,2) and 3'alpha-hs4, and suggest coregulation of the two enhancers by a common set of factors.

B cell lineage-specific activator protein (BSAP). A player at multiple stages of B cell development.

B cell lineage-specific activator protein (BSAP), encoded by the Pax 5 gene, is a critical modulator of early B cell differentiation. Binding sites for BSAP have been identified in the promoters of multiple genes as well as at multiple sites within the IgH locus. While in vivo target sites critical for early B cell differentiation have not yet been identified, candidate sites include a 3 IgH enhancer that is active early in B cell development. BSAP may play an additional role later in development, such as in the regulation of isotype class switching.

Mutually Exclusive Interaction of a Novel Matrix Attachment Region Binding Protein and the NF-μNR Enhancer Repressor: IMPLICATIONS FOR REGULATION OF IMMUNOGLOBULIN HEAVY CHAIN EXPRESSION

The immunoglobulin heavy chain (IgH) intronic enhancer stimulates transcription from functional promoters in B lymphocytes but not other cell types. The observation that binding sites for the nuclear factor-mu negative regulator (NF-muNR) enhancer repressor overlap nuclear matrix attachment regions (MARs) in this enhancer has lead to the hypothesis that the cell type specificity of the enhancer might be controlled by regulating nuclear matrix attachment (Scheuermann, R. H., and Chen, U. (1989) Genes & Dev. 3, 1255-1266). To understand the role of MARs in IgH enhancer regulation, we have identified a novel MAR-binding protein, MAR-BP1, from soluble nuclear matrix preparations based on its ability to bind to the MARs associated with the IgH enhancer. Purified MAR-BP1 migrates as a 33-kDa protein, and it can be found in nuclear matrix preparations from a number of different types of lymphoid cell lines. Although specific binding sites have been difficult to localize by chemical or enzymatic footprinting procedures, NF-muNR binding sites are critical for efficient MAR-BP1 binding. Indeed, binding of the IgH enhancer to either intact nuclear matrix preparations or to MAR-BP1 is mutually exclusive to NF-muNR binding. These results are consistent with a model for cell-type specific regulation in which binding of the NF-muNR repressor to the IgH enhancer prevents nuclear matrix attachment in inappropriate cells by interfering with MAR-BP1/enhancer interaction.

GABP and PU.1 compete for binding, yet cooperate to increase CD18 (beta 2 leukocyte integrin) transcription.

CD18 (beta 2 leukocyte integrin) is a leukocyte-specific adhesion molecule that plays a crucial role in immune and inflammatory responses. A 79-nucleotide fragment of the CD18 promoter is sufficient to direct myeloid transcription. The CD18 promoter is bound by the B lymphocyte- and myeloid-restricted ets factor, PU.1, and disruption of the PU.1-binding sites significantly reduces promoter activity. However, PU.1 alone cannot fully account for the leukocyte-specific and myeloid-inducible transcription of CD18. We identified a ubiquitously expressed nuclear protein complex of extremely low electrophoretic mobility that also binds to this region of the CD18 promoter. This binding complex is a heterotetramer composed of GABP alpha, and ets factor, and GABP beta, a subunit with homology to Drosophila Notch. GABP alpha competes with the lineage restricted factor, PU.1, for the same critical CD18 ets sites. The CD18 promoter is activated in myeloid cells by transfection with both GABP alpha and GABP beta together, but not by either factor alone. Transfection of non-hematopoietic cells with the two GABP subunits together with PU.1 is sufficient to activate CD18 transcription in otherwise non-permissive cells. Thus, GABP and PU.1 compete for the same binding sites but cooperate to activate CD18 transcription.

π1 Binding Sites Are Negative Regulators of bcl-2 Expression in Pre-B Cells

The bcl-2 gene is differentially regulated during B-cell development, with low-level expression in pre-B cells and higher-level expression in mature B cells. These changes correlate with susceptibility to cell death by apoptosis and suggest that the Bcl-2 protein may play a role in the control of cell death during B-cell development. We have identified two negative regulatory regions in the human bcl-2 5' flanking and 5' untranslated regions in pre-B cells; these regions have no significant function in mature B cells. Further investigation of these regions revealed two pre-B-cell-specific enhancer elements (pi 1 sites) in the 5' negative regulatory region and one in the 3' negative regulatory region. Mutational analysis confirmed that these three sites functioned as negative regulators of the bcl-2 promoter in the pre-B-cell line Nalm-6. Electrophoretic mobility shift assays with each of the three sites demonstrated a complex of identical mobility to that formed with the immunoglobulin heavy-chain enhancer pi 1 site. UV cross-linking experiments revealed that a protein with a molecular mass of 58 kDa bound to the three bcl-2 sites and to the immunoglobulin enhancer site. This protein reacted with an antibody against Ets family proteins. Constructs with the isolated pi 1 sites linked to the simian virus 40 promoter were used in transient transfection experiments in the pre-B-cell line. The bcl-2 sites decreased expression of the simian virus 40 promoter, while the immunoglobulin enhancer site increased its expression. The pi 1 sites in the bcl-2 gene may play a role in the developmental regulation of bcl-2 expression during B-cell differentiation.

Random activation of a transgene under the control of a hybrid hCD2 locus control region/Ig enhancer regulatory element.

Locus control regions such as those of human CD2 and beta-globin differ from classical enhancers in that, whereas the former confer high level, copy-dependent, position-independent expression to linked genes in transgenic mice, the latter do not, expression levels being dependent on the site of integration. We report that the position independence of the CD2 locus control region is modified by coupling it to the immunoglobulin heavy chain enhancer. Whilst in the majority of transgenic lines the Ig heavy chain enhancer has little or no effect on T cell expression of the hCD2 transgene, in others transgene expression is non-specifically extinguished in a proportion of lymphoid cells. The transgenic locus chromatin appears inaccessible to DNase I in these cells, which do not express the gene. Furthermore, mice homozygous for the hybrid hCD2-Ig heavy chain enhancer construct contain T cells with both an active and an inactive transgene. The 'decision' to express or repress the gene appears to be a random process which involves each chromosome separately, occurs at early stages in differentiation and is heritable by daughter cells. These data suggest the possibility that stochastic decisions might control a number of biological processes.

Disregulation of leukosialin (CD43, Ly48, sialophorin) expression in the B-cell lineage of transgenic mice increases splenic B-cell number and survival.

Leukosialin (also known as Ly48, CD43, and sialophorin) is a major cell surface sialoglycoprotein found on a variety of hematopoietically derived cells. The precise function of this molecule is poorly understood but it has been implicated in cell proliferation and intercellular adhesion. We developed a transgenic mouse model to assess leukosialin's function in vivo. Our approach was to alter mouse CD43 (mCD43) expression in the B-cell lineage where it is tightly regulated, by expressing it in peripheral B cells where it is normally absent. To drive expression of leukosialin in mature B cells, the immunoglobulin heavy chain enhancer was fused to the mCD43 gene. mCD43-immunoglobulin heavy chain enhancer transgenic mice display splenomegaly due to increased numbers of B cells. Transgenic B cells show a striking increase in their ability to survive in vitro compared to B cells from nontransgenic control mice. This prolonged survival is reflected in a decreased susceptibility to apoptosis. These observations suggest that mCD43 plays an important role in the regulation of B-cell survival. The alteration of the temporal expression, or "disregulation," of a gene in transgenic mice provides a general strategy for elucidating the in vivo role of other molecules involved in cell signaling and adhesion.

Regulated activity of the IgH intron enhancer (E mu) in the T lymphocyte lineage.

The activity of the IgH (E mu) enhancer in the T lymphocyte lineage has been investigated using both transgenic mice and transfection studies. Thymocyte fractionation experiments indicate that a transgene consisting of the bacterial chloramphenicol acetyl transferase (CAT) gene, linked to E mu and the SV40 early promoter (E mu-CAT), is expressed only in thymocytes with a mature medullary phenotype and not in immature cells. Transfection of this same construct into two thymoma cell lines representing different stages of thymocyte development mimics the pattern of activity observed in vivo. Further transfection experiments suggest that this pattern of expression might be attributed to the differential activity of the E2E3 and octanucleotide motifs of E mu during development. In contrast, an Ig lambda transgene (linked to E mu and an Ig V lambda promoter) is expressed in the majority of thymocytes. We envisage that the different patterns of expression of the two transgenes reflect interactions between their respective promoters and the factors which are bound to E mu at different stages of thymocyte development. Although differing in their pattern of expression within the thymus, the two transgenes share the property of extinction in peripheral T lymphocytes. These results indicate that the expression of E mu-linked transgenes in the thymus cannot simply be explained by activation of the enhancer in a lymphoid progenitor cell prior to B/T lineage divergence. Rather, the enhancer (or components of it) must be independently activated (and inactivated) during T lymphocyte development. Furthermore, this activity is consistent with the developmental timing of Ig DH-JH rearrangements in these cells.

Derivation and partial analysis of two highly active myeloma cell transfectants

Vectors have been designed to optimise the expression of heterologous proteins in transfected mouse myeloma cells. The over-ridingly important DNA element contained in these constructs is the classical mouse immunoglobulin heavy chain enhancer. It is shown that even in the absence of a well-known promoter element, the enhancer can drive gene expression in stable cell transfectants and the main transcriptional start site utilized in such situations has been mapped to within the previously defined enhancer region. Using chicken lysozyme as a reporter function in these vectors, two transfected myeloma cell clones have been isolated which secrete this protein at levels 50–100-times as high as those usually obtained with the same vectors and it is shown that in molar terms this is at least as high as endogenous immunoglobulin produced by a related line. Analysis of these lines show that in one case only a single copy, and in the other two to three copies, of the apparently unrearranged vector have integrated at a single locus within the genome. Possible explanations for the high-level expression are discussed.