Immunoglobulin Mu Heavy Chain Research Articles

A Negative Cofactor Containing Dr1/p19 Modulates Transcription with TFIIA in a Promoter-specific Fashion

An activity that modulated the relative levels of transcription from the adenovirus major late promoter (MLP), and the immunoglobulin heavy chain mu promoter (mu) was purified as a 90-kDa factor. This factor is suggested to be a heterotetramer of two subunits: a 20-kDa polypeptide identical to the previously described Dr1/p19 and a novel 30-kDa polypeptide. The Dr1/p19 protein has been characterized as a repressor of transcription, and the 30-kDa protein is related to a recently identified yeast gene proposed to encode a repressor of transcription. The 90-kDa factor forms a complex with TATA-binding protein on DNA and at high concentrations of both factors protects over a 150-base pair region around the promoter from DNase I cleavage. The conformation of this complex as assayed by footprinting analysis is altered by the transcription factor TFIIA on the MLP but not on the mu promoter. Similarly, TFIIA reverses the repression of transcription by the 90-kDa factor on the MLP but not on the mu promoter. Thus, the interactions of TATA-binding protein, TFIIA, and the 90-kDa factor are promoter-specific.

Precise Alignment of Sites Required for μ 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.

IgH (mu-switch and gamma-1) region restriction fragment length polymorphism in human narcolepsy.

Predisposition to narcolepsy involves genetic factors both in humans and in a canine model of the disorder. In humans, narcolepsy is strongly associated with HLA DR15 and DQB1*0602. In Dobermans and Labradors, narcolepsy is transmitted as a single autosomal recessive gene with full penetrance (canarc-1). Canine narcolepsy is not linked with DLA, the canine equivalent of HLA, but co-segregates with a DNA segment with high homology with the mu immunoglobulin heavy-chain (IgH) switch-like region (S mu). To determine if the IgH locus is involved in genetic predisposition to human narcolepsy, restriction fragment length polymorphisms specific for the IgM and IgG cluster within this locus were studied in sporadic cases of the disease, as well as in five families with two or more affected individuals. Comparisons were made between control populations and both familial and sporadic cases and for patients with and without HLA-DR15 and DQB1*0602. RFLP analysis at the S mu and gamma-1 loci, which cover over 200 kb of 14q32.3, indicates that there is no evidence for any association between the IgH region and human narcolepsy.

Identification of an activity in B-cell extracts that selectively impairs the formation of an immunoglobulin mu s poly(A) site processing complex.

The immunoglobulin mu heavy-chain transcription unit is differentially expressed during B-cell development, producing mRNAs that encode secreted (mu s) and membrane-bound (mu m) forms of the heavy-chain polypeptide. Whereas the mu s mRNA and the mu m mRNA are produced in approximately equal abundance in B cells, an increase in the utilization of the mu s poly(A) site contributes to the production of the mu s mRNA as the predominant form in a plasma cell. Previous experiments have demonstrated a correlation between the formation of a stable complex on a poly(A) site and the relative function of the poly(A) site. We have thus investigated the parameters determining the interaction of these factors with the immunoglobulin poly(A) sites. Assays of complex formation involving the two immunoglobulin poly(A) sites by using HeLa cell activities revealed the formation of stable complexes with no apparent difference between the mu s site and the mu m site. In contrast, the mu s-specific complex was markedly less stable when a B-cell extract was used. Fractionation of B-cell extracts has revealed an activity that specifically destabilizes the mu s polyadenylation complex, suggesting that the function of this poly(A) site may be regulated by both positive- and negative-acting factors.

Analysis of a Mouse Gene Encoding Three Steps of Purine Synthesis Reveals Use of an Intronic Polyadenylation Signal without Alternative Exon Usage

A single mouse genomic locus encodes proteins catalyzing three steps of purine synthesis, glycinamide ribonucleotide synthetase (GARS), aminoimidazole ribonucleotide synthetase (AIRS), and glycinamide ribonucleotide formyltransferase (GART). This gene has 22 exons and spans 28 kilobases. The existence of a second genetic locus and closely related pseudogenes was ruled out by Southern analysis. Mouse tissues express two related classes of messages encoded by this single locus: a trifunctional GARS-AIRS-GART mRNA and a monofunctional GARS mRNA. These transcripts used the same set of multiple transcriptional start sites, and both used the same first 10 exons. CCAAT and TATA elements were not found for this locus. Exon 11, which represented the last coding sequence of the GARS domain, was differentially utilized for the two messages. The trifunctional mRNA was generated by splicing exon 11 to exon 12, the first coding sequence for the AIRS domain with subsequent use of a polyadenylation signal at the end of exon 22. Genomic sequence corresponding to the 3'-UTR of the monofunctional GARS mRNA was contiguous with exon 11, so that the smaller message arose from the recognition of one of the multiple polyadenylation signals present within the intron between exons 11 and 12. Hence, polyadenylation of the primary transcript at a position corresponding to an intron of the genomic locus was responsible for the generation of the monofunctional GARS class of mRNAs. This utilization of an intronic polyadenylation site without alternative exon usage is comparable to the mechanism whereby both secreted and membrane-bound forms of the immunoglobulin mu heavy chain are made from a single genetic locus.

On the linkage between RNA processing and RNA translatability.

The immunoglobulin mu heavy chain gene of mouse hybridoma cells is expressed in two forms, microseconds and microns, differing in their use of 3' exons. As for many other mammalian genes, mutations in the mu gene which prematurely terminate translation often have the effect of reducing the amount of these mu RNAs. To test the generality of this relationship, we selected mutant hybridoma cell lines defective in IgM production and searched both for translation termination mutations which do not reduce the amount of mu RNA as well as for mutants which show the more commonly observed reduction in mu RNA. As observed previously, the amount of microseconds RNA is normal in mutants terminating in the C mu 4 exon; by contrast the amount of microns RNA is reduced in these mutants, indicating that the effect of the mutation is influenced by some feature near the 3' end of the RNA. Mutations terminating translation in other C region exons have a graded effect on RNA content, ranging from 10% the normal level for termination in the C mu 3 exon down to 1% for termination in the C mu 2 exon. By contrast, a mutant cell line terminating in the leader exon contained 25% the normal amount of mu RNA, suggesting that translation past some point might be required to fully engage the RNA degradation process.

Dependence of enhancer-mediated transcription of the immunoglobulin mu gene on nuclear matrix attachment regions.

Transcription of the immunoglobulin mu heavy chain locus is regulated by an intronic enhancer that is flanked on both sides by nuclear matrix attachment regions (MARs). These MARs have now been shown to be essential for transcription of a rearranged mu gene in transgenic B lymphocytes, but they were not required in stably transfected tissue culture cells. Normal rates of transcriptional initiation at a variable region promoter and the formation of an extended deoxyribonuclease I (DNase I)--sensitive chromatin domain were dependent on MARs, although DNase I hypersensitivity at the enhancer was detected in the absence of MARs. Thus, transcriptional activation of the mu gene during normal lymphoid development requires a synergistic collaboration between the enhancer and flanking MARs.

Role of the mu immunoglobulin heavy chain transmembrane and cytoplasmic domains in B cell antigen receptor expression and signal transduction.

Membrane immunoglobulins (mIg) serve as the recognition components of B lymphocyte antigen receptors. Binding of antigen to these receptors leads to dramatic effects on B cell growth and viability. We have examined the structural elements of mIgM that are involved in antigen receptor assembly and function. Expression of transfected wild-type mIgM in a B cell line led to assembly with the other two known components of the antigen receptor, Ig-alpha and Ig-beta, expression on the cell surface, and when cross-linked by anti-IgM antibodies, stimulation of signal transductin reactions, including tyrosine protein phosphorylation, inositol phosphate production, and increases in cytoplasmic calcium concentration. Replacement of the highly conserved COOH-terminal 41 amino acids of mIgM heavy chain (mu m) with the transmembrane and cytoplasmic domains of human CD8 alpha resulted in a molecule which was expressed on the B cell surface at levels comparable to wild-type mIgM, but which did not form a complex with Ig-alpha or Ig-beta and did not stimulate any of the signaling reactions mentioned above. Replacement of the basic three-amino-acid cytoplasmic domain of mu m with a different but similarly charged sequence had no effect on cell-surface expression or signaling function. On the other hand, removal of the entire cytoplasmic domain resulted in a molecule which did not bind Ig-alpha and Ig-beta and which did not transduce signals. This effect is probably due to altered post-translational processing of this mutant molecule. Finally, a series of eight single-amino-acid substitutions in the transmembrane domain was constructed. Most of these resulted in the removal of hydroxyl groups from conserved residues postulated to be important for interactions with other components. Each of these mutant molecules was capable of transducing signals when cross-linked by anti-IgM, but one was partially defective. Since alteration of any single residue was not sufficient to disrupt signaling completely, the interactions required for signaling are likely to involve multiple residues, so that elimination of one hydroxyl group does not prevent the interaction. We propose that the cytoplasmic domain of mu m does not play a critical role in receptor function but that the transmembrane domain specifies interactions with other components, probably Ig-alpha and Ig-beta, required for proper antigen receptor signal transduction.

Stimulation of kappa light-chain gene rearrangement by the immunoglobulin mu heavy chain in a pre-B-cell line.

B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin mu heavy chain induces rearrangement activity at the kappa light-chain locus. To examine this issue in more detail, we isolated five matched pairs of mu- and endogenously rearranged mu+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five mu+ cell lines, substantial expression of mu protein on the cell surface was observed, and this correlated with an enhanced frequency of kappa immunoglobulin gene rearrangement compared with that in the matched mu- cell lines. This increased kappa gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the mu+ cells. Consistently, introduction of a functionally rearranged mu gene into one of the mu- pre-B-cell lines resulted in a fivefold increase in kappa gene rearrangements. In three of the four clonally matched pairs with increased kappa gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C kappa locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of mu protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C kappa transcript correlated with the measured frequency of rearranged kappa genes. These results support a regulated model of B-cell development in which mu protein expression in some way targets the V(D)J recombinase to the kappa gene locus.

Stimulation of kappa light-chain gene rearrangement by the immunoglobulin mu heavy chain in a pre-B-cell line.

B-lymphocyte development exhibits a characteristic order of immunoglobulin gene rearrangements. Previous work has led to the hypothesis that expression of the immunoglobulin mu heavy chain induces rearrangement activity at the kappa light-chain locus. To examine this issue in more detail, we isolated five matched pairs of mu- and endogenously rearranged mu+ cell lines from the Abelson murine leukemia virus-transformed pro-B-cell line K.40. In four of the five mu+ cell lines, substantial expression of mu protein on the cell surface was observed, and this correlated with an enhanced frequency of kappa immunoglobulin gene rearrangement compared with that in the matched mu- cell lines. This increased kappa gene rearrangement frequency was not due to a general increase in the amount of V(D)J recombinase activity in the mu+ cells. Consistently, introduction of a functionally rearranged mu gene into one of the mu- pre-B-cell lines resulted in a fivefold increase in kappa gene rearrangements. In three of the four clonally matched pairs with increased kappa gene rearrangements, the increase in rearrangement frequency was not accompanied by a significant increase in germ line transcripts from the C kappa locus. However, in the fourth pair, K.40D, we observed an increase in germ line transcription of the kappa locus after expression of mu protein encoded by either an endogenously rearranged or a transfected functional heavy-chain allele. In these cells, the amount of the germ line C kappa transcript correlated with the measured frequency of rearranged kappa genes. These results support a regulated model of B-cell development in which mu protein expression in some way targets the V(D)J recombinase to the kappa gene locus.

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.

Isolation of new nonsense and frameshift mutants in the immunoglobulin mu heavy-chain gene of hybridoma cells.

In order to expand the experimental material available for genetic and biochemical analyses of the natural immunoglobulin genes, we have isolated a variety of mutant mouse hybridoma cell lines. Some of these mutants have partial or complete deletions of the mu gene. Other mutants have nonsense or frameshift mutations in the exons encoding the variable and the second and third constant region domains of the mu heavy chain. When combined with earlier mutant data, this collection of genotypically and phenotypically tight mutants of known sequence spans most of the 10 kb of the mu gene, providing material for a variety of studies of genetic recombination and mRNA metabolism.

Somatic hypermutation of an immunoglobulin mu heavy chain transgene.

We have analyzed somatic hypermutation of an immunoglobulin (Ig) heavy chain transgene. Hybridomas expressing the transgene were produced from immunized transgenic mice and transgene copies were sequenced to assay for mutation. In two IgM-producing hybridomas, as well as in several IgG-producing hybridomas, mutations were found in the VDJ region of the transgene. In the IgM-producing hybridomas, both mutated and unmutated transgene copies were present and expressed as mRNA. Several mutated transgene copies were present in a single cell and these showed different patterns of mutation. Two IgG-producing hybridomas isolated from a single animal also showed a hierarchical pattern of mutation indicating that transgene mutations can accumulate during B cell proliferation, similar to the mutational process for endogenous antibody genes. Among hybridomas that expressed both IgG and IgM molecules derived from the transgene, the isotype-switched gamma transgene copy exhibited a higher level of mutation than the mu transgene copies. Our results indicate that the 15-kb ARSmu transgene contains all the sequence information required to target the Ig-specific hypermutational machinery, and raise the possibility that sequences associated with the endogenous CH locus might enhance somatic mutation.

A topoisomerase II-like protein is part of an inducible DNA-binding protein complex that binds 5' of an immunoglobulin promoter.

We previously identified a B cell-specific protein-DNA complex 5' of an immunoglobulin mu heavy chain promoter. The sequences to which this protein complex bound were required for induction of immunoglobulin mRNA levels with interleukin-5 + antigen. Further studies identified a second sequence 5' of these regulatory sequences that bound to both the nuclear matrix and to a similar interleukin-5 + antigen inducible protein complex. Therefore, we sought to identify the putative regulatory proteins that comprised this DNA-binding complex. In this study, we have used anti-topoisomerase II antibodies to demonstrate that one of the proteins found in the interleukin-5 + antigen inducible complexes is serologically related to topoisomerase II. To our knowledge, this is the first report where a topoisomerase II related protein participates in an inducible mobility shifted protein-DNA complex. These data suggest a model in which the enhanced immunoglobulin gene transcription observed after treatment with interleukin-5 + antigen might be explained by the induction of a protein complex that acts to relieve torsional stress along the gene.

The B29 and mb‐1 polypeptides are differentially expressed during human B cell differentiation

Surface immunoglobulin on mouse B cells is associated with a heterodimer comprising the products of the mb-1 and B29 genes. Here we report that antibodies raised against a peptide sequence from the intracytoplasmic C terminus of the B29 murine gene product detect the 37-kDa component of the human heterodimer, indicating that this component in man is also encoded by the B29 gene. The immunocytochemical reactivity of these anti-B29 antibodies was compared with those of antibodies to the mb-1 protein. Of 25 cases of acute lymphoblastic leukaemia (ALL), 24 were positive for mb-1 whereas B29 was expressed in only 13 cases. Most of these B29-positive ALL expressed immunoglobulin mu heavy chain in their cytoplasm (pre-B ALL). In lymphoid tissue sections, anti-B29-labeled B cell follicles in a similar fashion to anti-mb-1, with the striking exception that plasma cells were unreactive for B29, but positive for mb-1. These results suggest that the synthesis of B29 begins later in precursor B cells than that of mb-1, and ceases before the terminal plasmacyte phase.

Effects of mutation position on frequency of marker rescue by homologous recombination.

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

Effects of mutation position on frequency of marker rescue by homologous recombination.

Homologous recombination between transferred and chromosomal DNA can be used for mapping mutations by marker rescue, i.e., by identifying which segment of wild-type DNA can recombine with the mutant chromosomal gene and restore normal function. In order to define how much the fragments should overlap each other for reliable mapping, we have measured how the frequency of marker rescue is affected by the position of the chromosomal mutation relative to the ends of the transferred DNA fragments. For this purpose, we used several DNA fragments to effect marker rescue in two mutant hybridomas which bear mutations 673 bp apart in the exons encoding the second and third constant region domains of the immunoglobulin mu heavy chain. The frequency of marker rescue decreased greatly when the mutation was located near one of the ends of the fragments, the results indicating that fragments should be designed to overlap by at least several hundred base pairs. Possible explanations for this "end effect" are considered.

A comparative analysis of cytoplasmic mu (C mu) expression in acute lymphoblastic leukemia by molecular and immunologic techniques. Identification of leukemia cases expressing C mu mRNA transcripts in the absence of detectable C mu proteins.

Among acute lymphoblastic leukemias derived from the B-cell lineage, the subset of cases expressing cytoplasmic mu heavy chain proteins (C mu) in the absence of surface immunoglobulin has been designated pre-B-cell acute lymphoblastic leukemia. This group, traditionally identified using immunologic smear techniques, has been associated with a poor prognosis in some series. In a comparative study, 25 cases of B-lineage acute lymphoblastic leukemia were analyzed for C mu expression using molecular and immunologic techniques. RNA derived from cryopreserved blast cells was hybridized in both Northern and slot-blot analyses using a probe (pBZ311) containing four exons of the human immunoglobulin heavy chain mu constant region gene. Expression of C mu proteins was assessed simultaneously by slide immunofluorescence and flow cytometric techniques in all samples. These studies were correlated with immunoglobulin heavy and light chain gene rearrangements, cell-surface immunophenotype, cytogenetics, and other clinicopathologic features. C mu mRNA transcripts were detected in 14 of 25 cases, whereas C mu proteins were detected in only 9 of these cases using flow cytometric techniques. Only four of these nine cases were positive by slide immunofluorescence techniques. These studies imply that molecular and flow cytometric techniques may be a more sensitive means to assess C mu expression. The identification of five cases that expressed C mu mRNA transcripts in the absence of detectable C mu proteins also suggests that molecular techniques may be valuable in identifying a unique subgroup of pre-B-cell acute lymphoblastic leukemia cases that contain C mu mRNA transcripts, but lack C mu proteins.

A defect in the humoral immune response to protein antigens and haptens in immunoglobulin mu heavy-chain transgenic mice

We have examined the antibody response in mice expressing a functionally rearranged mu Ig heavy chain derived from a hybridoma antibody with specificity for the hapten 4-hydroxy-3-nitrophenyl (NP). Transgenic mice and their normal littermates were immunized with the antigens NP-OVA, the synthetic polypeptide (Tyr,Glu)-Ala-Lys ((T,G)-A-L), or saline. The presence of serum antibodies to NP-BSA, OVA, (T,G)-A-L, and BSA was examined by ELISA. Sera were evaluated prior to immunization and at periods of up to 4 months following immunization. Prior to immunization, transgenic mice had high levels of IgM anti-NP antibody but no detectable antibody to the other antigens. Both the primary and secondary antibody responses of transgenic mice to NP, OVA, and (T,G)-A-L were depressed when compared with the response of nontransgenic mice. Because of reports that these transgenic mice have increased proportions of CD5 + B-cells, a subpopulation associated with the production of autoantibodies, we examined these mice for the production of both IgG and IgM rheumatoid factors and anti-DNA antibodies. Transgenic mice had a modest increase in the spontaneous production of IgM anti-DNA. These data demonstrate a functional defect in the humoral immune response of mu transgenic mice.

The tetrameric structure of NF-mu NR provides a mechanism for cooperative binding to the immunoglobulin heavy chain mu enhancer.

We have analyzed the structure and DNA-binding characteristics of the IgH enhancer-binding regulatory protein NF-mu NR. We estimate that there are at least 5000 molecules of NF-mu NR protein/nucleus of non-B cells, based on the yield of active protein following purification. Purified NF-mu NR exists as a tetramer of 40-kDa subunits, even in the absence of its DNA-binding substrate. The protein complex binds to four binding sites flanking the immunoglobulin heavy chain mu enhancer core. Separately, individual sites bind to the tetrameric complex with affinities varying over a 100-fold range. However, when a low affinity site and a high affinity site are present on the same DNA molecule, both are occupied at the same NF-mu NR concentration; thus, there is a strong cooperative interaction between binding sites. The tetrameric structure provides a mechanism for binding cooperativity in which initial binding is mediated through a high affinity site on the DNA molecule followed by the engagement of the low affinity site juxtaposed to adjacent protein subunits. The presence of multiple binding sites flanking the mu enhancer core may reflect the influence of NF-mu NR binding on enhancer three-dimensional structure, transcription factor binding, and/or nuclear matrix interactions for cell type-specific enhancer function.