Kathryn Calame

A single VH gene segment encodes the immune response to phosphorylcholine: Somatic mutation is correlated with the class of the antibody

Abstract The immune response in BALB/c mice to phosphorylcholine is highly restricted in its heterogeneity. Of the 19 immunoglobulins binding phosphorylcholine for which complete V H -segment amino acid sequences have been determined, 10 employ a single sequence, denoted T15 after the prototype V H sequence of this group of antibodies. The remaining 9 of these V H segments are variants differing by 1 to 8 residues from the T15 sequence. Using a cloned V H cDNA probe complementary to the T15 sequence, we isolated from a mouse sperm genomic library clones corresponding to four V H gene segments that by DNA sequence analysis are >85% homologous to one another. These four V H gene segments have been denoted the T15 V H gene family. These V H gene segments are most, if not all, of the germline V H gene segments that could encode the V H sequences of antibodies that bind phosphorylcholine. One of these four genes contains the T15-V H -coding sequence. When the T15-family V H gene segments were compared with the complete V H protein sequences of 19 hybridoma and myeloma immunoglobulins that bind phosphorylcholine, several striking conclusions could be drawn. First, all of these V H regions must have arisen from the germline T15 V H gene segment. Thus virtually the entire immune response to phosphorylcholine is derived from a single V H -coding sequence. Nine of the 19 V H regions were variants differing from the T15-V H -coding sequence and, accordingly, must have arisen by a mechanism of somatic diversification. Second, the variants appear to be generated by a somatic mutation mechanism. They cannot be explained by recombination or gene conversion among members of the T15 gene family. Third, somatic mutation is correlated with the class of the antibody. All of the somatic variation is found in the V H regions derived from antibodies of the IgA and IgG classes. The IgM molecules express the germline T15 V H gene segment exclusively.

Transcriptional Controlling Elements in the Immunoglobulin and T Cell Receptor Loci

Publisher Summary The proteins that fulfill the function of identifying foreign molecular structures for the humoral response are immunoglobulins (Igs) and for the cellular response are T cell receptors (TCRs). Because both Igs and TCRs carry out antigen recognition, it is not surprising that the structure of Ig and TCR proteins and the structure, organization, and rearrangement of the genes that encode them are quite similar. Ig molecules are secreted and recognize soluble antigens, while TCR molecules remain on the T cell surface in close association with other membrane proteins and recognize cellular antigens, many more Ig molecules are synthesized by terminally differentiated B cells than TCR molecules are synthesized by mature T cells. The chapter presents overviews of the various ways in which Ig and TCR genes are regulated during development. However, the detailed portions of the review in the chapter is limited to transcriptional regulation, and it focuses on cis-acting DNA elements and trans-acting cellular proteins, which are required to regulate transcription of Ig and TCR genes. Transcriptional enhancers, which were first discovered in animal viruses, are defined functionally as cis-acting DNA sequences, which activate transcription-initiation in an orientation- and distance-independent manner. In cases in which the difference in transcriptional activity has been quantitated, it seems that the preference is not absolute, because the low levels of transcription are observed in some non-B cell lines. The chapter discusses several models that may explain its B cell-specific activity in immunoglobulin genes.

Common factor 1 is a transcriptional activator which binds in the c-myc promoter, the skeletal alpha-actin promoter, and the immunoglobulin heavy-chain enhancer.

Ubiquitously expressed transcription factors play an integral role in establishing and regulating patterns of gene transcription. Common factor 1 (CF1) is a ubiquitously expressed DNA-binding protein previously identified in our laboratory. We show here that CF1 recognizes sites in several diverse transcription elements, and we demonstrate the ability of the c-myc CF1 site to activate transcription of a basal promoter in both B cells and fibroblasts.

The endogenous immunoglobulin heavy chain enhancer can activate tandem VH promoters separated by a large distance

The availability of a clone containing two linked immunoglobulin heavy chain variable region genes located 15.8 kb apart has allowed us to study the functional capabilities of the immunoglobulin heavy chain transcriptional enhancer element in its normal chromosomal context. In plasmacytoma J606 the 3 VH gene is joined to D and J gene segments, located within 1.7 kb of the heavy chain enhancer, and expressed; the 5 VH gene is 17.5 kb from the enhancer in J606 DNA. Run-on transcription in isolated nuclei demonstrated specific transcription of the 5 VH gene in J606 that was 60% that of the expressed 3 VH gene. No other enhancer elements are detectable closer to the 5 VH gene than the known heavy chain enhancer. Thus, the heavy chain enhancer appears to be capable of activating transcription of a VH promoter located 17.5 kb away and of activating two tandem VH promoters.

mTFE3, an X-linked transcriptional activator containing basic helix-loop-helix and zipper domains, utilizes the zipper to stabilize both DNA binding and multimerization.

Southwestern (DNA-protein) screening of a murine L-cell cDNA library by using a probe for the microE3 site in the immunoglobulin heavy-chain enhancer yielded a clone, mTFE3, which is a member of the subset of basic helix-loop-helix (BHLH) proteins that also contain a leucine zipper (ZIP). Since the individual contribution of these domains is not well understood for proteins which contain them both, mutational analyses were performed to assess the functional roles of the HLH and ZIP regions for DNA binding and multimerization. The HLH region is stringently required for DNA binding but not for multimerization. The ZIP region is not stringently required for binding or multimerization, but stabilizes both multimer formation and DNA binding. A high degree of conservation at both the amino acid and nucleotide levels between the human transcription factor TFE3 and mTFE3 suggests that mTFE3 is the murine homolog of human TFE3. By using fluorescent in situ hybridization, mTFE3 was mapped to mouse chromosome X in band A2, which is just below the centromere. We show that in addition to the immunoglobulin heavy-chain microE3 site, mTFE3 binds to transcriptional elements important for lymphoid-specific, muscle-specific, and ubiquitously expressed genes. Binding of mTFE3 to DNA induces DNA bending.

SV40 enhancer-binding factors are required at the establishment but not the maintenance step of enhancer-dependent transcriptional activation

We have used temperature-sensitive COS cells to design delayed competition experiments in which competition for simian virus 40 (SV40) enhancer factors occurs after enhancer-dependent transcription has been established. The results demonstrate that competition for SV40 enhancer-binding factors has no effect on enhancer-dependent transcription after transcription has been established at the SV40 early promoter. These data show that the enhancer and factors that bind to it are involved in the establishment of stable transcription complexes, although they do not show whether enhancer factors are an integral part of the transcription complexes. Furthermore, the results of delayed competition experiments with a replicating test plasmid are consistent with the possibility that enhancer-dependent stable transcription complexes could be maintained after DNA replication.

Proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer exist in multiple oligomeric forms.

We describe the purification to near homogeneity of proteins binding to site C2 (muE3) in the immunoglobulin heavy-chain enhancer. Proteins binding to this site produce four protein-DNA complexes which are distinguished by their mobility in gel retardation assays and their elution properties in an anion exchange column. DNA affinity-purified preparations of three chromatographically separated pools, containing different subsets of the four complexes, each contained three polypeptides of 42.5, 44, and 45 kilodaltons (kDa). UV crosslinking of protein to enhancer DNA demonstrated that site C2-binding activities in the three different pools bound DNA through proteins of similar sizes (about 45 kDa), even though the protein-DNA complexes formed by these binding activities were quite distinct. Gel exclusion chromatography and equilibrium binding analyses indicated that the distinct protein-DNA complexes were due to different oligomeric forms of the individual subunits and that a larger multimeric form bound with high affinity to the heavy-chain enhancer site C2, while a smaller species had a much lower affinity for heavy-chain enhancer sequences. Purified protein has been used to map high-affinity binding sites for site C2-binding proteins within an immunoglobulin heavy-chain promoter and at site KE3 in the kappa light-chain enhancer.

Strong transcriptional activation of translocated c-myc genes occurs without a strong nearby enhancer or promoter

We have studied the transcriptional activation of translocated c-myc genes in murine plasmacytomas in which the translocation juncture occurs within the first intron of c-myc and juxtaposes c-myc with the immunoglobulin C alpha gene segment. It has been widely suggested that a novel transcriptional enhancer element located near the C alpha gene segment might activate the translocated c-myc gene. We have carried out an extensive search for such an element and find no significant transcriptional enhancer activity in a 22 kb region encompassing the translocation junction, C alpha gene segment and regions 3 of C alpha. We also find that the cryptic promoter region of the translocated c-myc gene is a very weak promoter of transcription. Despite this evidence against the presence of strong transcriptional regulatory elements, the translocated c-myc gene locus is transcribed at high rates that are 25-greater than 100% of that measured for the highly active immunoglobulin genes in murine plasmacytomas. These data suggest the presence of a novel type of strong activator of transcription in the murine heavy chain locus.

Purified mu EBP-E binds to immunoglobulin enhancers and promoters.

We describe the purification to apparent homogeneity of the murine immunoglobulin heavy-chain (IgH) enhancer-binding protein mu EBP-E from murine plasmacytoma cells by ion exchange and affinity chromatography. Glycerol gradient sedimentation, UV cross-linking, and sodium dodecyl sulfate-polyacrylamide gel electrophoresis confirm that mu EBP-E is a 45-kilodalton molecular mass protein. Orthophenanthroline-copper chemical nuclease footprinting with purified protein has identified high-affinity binding sites for mu EBP-E within the IgH enhancer at the previously identified site E and at sites within IgH promoters and in the kappa light-chain enhancer. Equilibrium binding studies indicate that the dissociation constants for mu EBP-E binding to site E within the enhancer and to a binding site within the V1 heavy-chain promoter are quite low, about 2 x 10(-11) M. Comparison of four mu EBP-E recognition sequences detects only limited sequence similarity among binding sites.

An active chromatin structure acquired by translocated c-myc genes.

We used general sensitivity to DNase I digestion to analyze the chromatin structure of c-myc genes in seven murine plasmacytomas. In every case, the 3 portion of c-myc juxtaposed with C alpha displayed a much more DNase I-sensitive chromatin structure than untranslocated c-myc or, in one case analyzed, the reciprocally translocated 5 portion. Our data suggest the presence of regulatory sequences near the C alpha gene segment.