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K Calame

Publications and source records attributed to K Calame.

At least 55 records · Page 3Linked to original sources
PubMed1991

A dominant negative form of transcription activator mTFE3 created by differential splicing.

Transcription factor E3 (mTFE3) is a murine transcription activator that binds to the intronic enhancer of the immunoglobulin heavy chain gene. A naturally occurring splice product of mTFE3 messenger RNA (mRNA) lacked 105 nucleotides that encode an activation domain; both absolute and relative amounts of long and truncated mRNAs varied in different tissues. Cells were cotransfected with complementary DNAs that encoded the two mRNA forms in amounts that corresponded to the amounts of each mRNA found in different cells. Small changes in substoichiometric amounts of the truncated form of mRNA effected trans-dominant negative modulation of mTFE3 activity. These findings identify a function for differential splicing in the regulation of transcription factor activity.

Amino Acid Sequence↗
PubMed1991

Novel protein-DNA interactions associated with increased immunoglobulin transcription in response to antigen plus interleukin-5.

Although much has been learned about basal levels of immunoglobulin (Ig) transcription, the regulatory effects of cytokines and antigen (Ag) upon Ig expression in lymphocytes have not been fully characterized. We previously reported that Ag plus interleukin-5 (IL-5) caused increased steady-state Ig mRNA levels in Ag-specific cell lines. In this study, we have identified a region between -250 and -125 bp 5' of the Ig transcription start site that is necessary for the induction of increased mu mRNA levels by Ag plus IL-5. Mobility shift and UV cross-linking studies indicated that IL-5 plus Ag induced increased protein binding to this region. Furthermore, this sequence was found to be closely related to another A + T-rich sequence at -525 bp 5' of the transcription start site. Both sequences exhibited similar B-cell-specific and inducible protein binding. Our data suggest that treatment with IL-5 plus Ag induces several DNA-binding proteins, some of which may participate in increasing Ig transcription above basal levels by binding to sequences 5' of the octamer motif.

Animals↗
PubMed1991

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.

Actins↗
PubMed1990

Ig/EBP-1: a ubiquitously expressed immunoglobulin enhancer binding protein that is similar to C/EBP and heterodimerizes with C/EBP.

We report the isolation and characterization of cDNA clones that encode a protein with the same DNA binding specificity as the immunoglobulin heavy chain enhancer binding protein E (muEBP-E). We call the gene encoding this protein Ig/EBP-1. A fusion protein encoded by the cDNA binds specifically to muEBP-E-binding sites (E sites) in both the IgH enhancer and the VH1 promoter. Sequence analysis reveals that Ig/EBP-1 is a member of the "basic-zipper" family of DNA-binding proteins that are characterized by basic regions and heptad repeats of leucine residues. Among known family members, Ig/EBP-1 demonstrates highest homology to C/EBP throughout the DNA-binding domain and leucine repeat region. Ig/EBP-1 and C/EBP have highly overlapping binding specificities; both cloned proteins bind to the IgH enhancer and the VH1 promoter E sites, and Ig/EBP-1 binds to previously characterized C/EBP binding sites in the Rous sarcoma virus (RSV) LTR and the murine albumin promoter. Consistent with their homology in the leucine repeat region, Ig/EBP-1 and C/EBP form heterodimers; Ig/EBP-1 is the first member of this family that has been found to heterodimerize with the well-characterized C/EBP. Ig/EBP-1 mRNA is present in all tissues and cell lines examined, although its levels vary almost 20-fold from different sources, with highest levels in early B cells. In tissues where Ig/EBP-1 and C/EBP are both present, heterodimers may be functionally important. The presence of Ig/EBP-1 in fibroblasts and other tissues where C/EBP is not expressed suggests that Ig/EBP-1 may be functionally important for the activity of the RSV enhancer in these cell types. Finally, elevated expression of Ig/EBP-1 in early B cells may explain in part the enhancer-independent activity of VH promoters early in B-cell development.

Amino Acid Sequence↗
PubMed1990

Structural characteristics of the variable regions of immunoglobulin genes encoding a pathogenic autoantibody in murine lupus.

We have studied several monoclonal anti-double-stranded (ds) DNA antibodies for their ability to accelerate lupus nephritis in young NZB X NZW F1 female mice and to induce it in BALB/c mice. Two identified as pathogens in both strains have characteristics previously associated with nephritogenicity: expression of IgG2a isotype and IdGN2 idiotype. Both pathogenic antibodies used the combination of genes from the VHJ558 and VK9 subfamilies. Two weak pathogens failed to accelerate nephritis in young BW mice, but induced lupus nephritis in BALB/c mice. They both express IdGN2; one is cationic and an IgG3, the other is an IgG2a. Additional MAbs (some IgG2a, one IdGN2-positive) did not accelerate or induce nephritis. We have cloned and sequenced the variable regions of the immunoglobulin genes of one pathogenic autoantibody. No unique V, D, or J gene segments and no evidence of unusual mechanisms in generating diversity were used to construct this antibody. These data argue against use of unique abnormal Ig genes by systemic lupus erythematosus individuals to construct pathogenic autoantibody subsets. Instead, the major abnormality may be immunoregulatory.

Animals↗
PubMed1990

Activator proteins which regulate immunoglobulin heavy chain gene transcription in B lymphocytes.

While our results do not completely explain the B-cell specificity or other regulated aspects of IgH transcription, they show that IgH transcriptional factors belong to several different DNA-binding families which are capable of dimerization. It may be that formation of heteromultimers provides an additional level of regulation which might confer functional tissue specificity. Our results also suggest that enhancers and promoters may interact through protein-protein interactions, looping out the intervening DNA. Finally, we have shown that the same protein may regulate several genes including IgH and c-myc, indicating that transcriptional regulatory mechanisms for different genes are both complex and interconnected.

Animals↗
PubMed1989

A transcriptional repressor of c-myc.

In murine plasmacytomas there is deregulated transcription of a translocated c-myc allele and undetectable transcription of the normal, unrearranged c-myc allele. Deregulated c-myc transcription probably contributes to the transformed phenotype of the tumour cells, whereas repression of the normal allele probably reflects the normal turn-off of c-myc in non-dividing plasma cells. We previously identified a plasmacytoma-specific protein which binds to the c-myc promoter region 290 base pairs 5' of the P1 transcription start site. This plasmacytoma repressor factor (myc-PRF; formerly myc-PCF) is not found in cell lines representing earlier B-cell stages during which c-myc is transcribed, so it could be a negative regulator of c-myc transcription in terminally differentiated B cells. Here we report that site-directed deletion of the binding site for this protein leads to a 30-fold increase in transcription of a stably transfected c-myc fusion construct in plasmacytoma cells but has no effect in L cells or 18-81 pre-B cells, which lack the protein. Myc-PRF interacts with another widely distributed protein, myc-CF1 (common factor 1), which binds nearby, and this association may be important in myc-PRF repression.

Animals↗
PubMed1989

Transcriptional regulation of immunoglobulin heavy chain and T-cell receptor beta chain genes.

We have identified factors that bind to functionally important regions in IgH chain promoters and enhancer. One promoter factors is identical to u-EBP-E, an enhancer binding protein. Several promoter-binding proteins are present preferentially in either B cells or fibroblasts although most enhancer-binding proteins have a ubiquitous distribution. Additional characterization of these factors will further our understanding of the mechanisms by which IgH promoters and enhancers interact to achieve B-cell restricted and developmental stage-specific expression of IgH genes. The identification of a TCR beta chain enhancer will allow us to pursue similar questions with respect to the regulated expression this locus.

Animals↗
PubMed1989

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.

Animals↗
PubMed1988

A transcriptional enhancer 3' of C beta 2 in the T cell receptor beta locus.

Run-on transcription experiments were used to demonstrate that transcription of T cell receptor beta chain V genes is activated by DNA rearrangement, in a manner similar to immunoglobulin genes. A transcriptional enhancer likely to be involved in this activation has been identified. A 25-kilobase region from J beta 1 to V beta 14 was tested for enhancer activity by transient transfections, and an enhancer was found 7.5 kilobases 3' of C beta 2. The beta enhancer has low activity relative to the simian virus 40 viral enhancer, does not display a preference for V beta promoters, has a T cell-specific activity, and binds two purified immunoglobulin heavy chain enhancer factors.

Animals↗
PubMed1988

In vivo functional analysis of in vitro protein binding sites in the immunoglobulin heavy chain enhancer.

We have systematically investigated the functional role of protein binding sites within the mouse immunoglobulin heavy chain enhancer which we previously identified by in vitro binding studies (1,2). Each binding site was deleted, mutant enhancers were cloned 3' of the chloramphenicol acetyl transferase gene in the vector pA10CAT2 and transfected into plasmacytoma cells. We demonstrate that the newly identified site E, located at 324-338 bp, is important for enhancer function; previously identified sites B(uE1), Cl(uE2), C2(uE3) and C3 were also shown to be important for enhancer activity. Sites A and D are not required for IgH enhancer function, as assayed by our methods. Thus, including the octamer site, six protein binding sites which bind at least six different proteins are important for enhancer function in vivo.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

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.

Animals↗
PubMed1988

Differential stability of c-myc mRNAS in a cell-free system.

We have developed a simple cell-free system for studying the stability of different mRNAs in vitro. We demonstrate that the threefold greater stability in vivo of truncated c-myc mRNA (lacking exon 1) compared with that of full-length c-myc mRNA is maintained in our in vitro system. Chimeric mRNAs in which the first exon of c-myc was fused to immunoglobulin C alpha heavy chain or glyceraldehyde-3-phosphate dehydrogenase mRNAs were not rapidly degraded, demonstrating that c-myc exon 1 alone is not sufficient to tag mRNAs for rapid degradation. Competition experiments show that full-length c-myc mRNA is specifically recognized by a factor(s) responsible for its rapid degradation. This system will allow further characterization and purification of these factors.

Animals↗
PubMed1987

A plasmacytoma-specific factor binds the c-myc promoter region.

We used an electrophoretic mobility-shift assay to study proteins that bind to sequences in the 5' flanking region of the murine c-myc gene. By comparing the DNA-protein complexes formed with extracts from cells representing earlier stages of B-cell development with those from plasmacytomas, we identified a plasmacytoma-specific protein that binds to a region within the c-myc promoter. Five other regions of this promoter show extensive sequence-specific binding, but the binding is not clearly B-cell stage-specific. Methylation-interference and o-phenanthroline/copper-protection experiments identified a single plasmacytoma-specific protein binding site 290 base pairs 5' of the transcription start site P1. Homologues of a core sequence, d(AGAAAGGGAAAGGA), within the 25-base-pair binding site are found at three additional sites in the murine c-myc locus. The plasmacytoma-specific occurrence of this protein suggests that it may play a role in the transcriptional repression of the normal c-myc gene observed in plasmacytomas.

Animals↗
PubMed1987

Multiple DNA sequence elements are necessary for the function of an immunoglobulin heavy chain promoter.

Sequences required for the function of the mouse V1 immunoglobulin heavy chain variable-region (VH) promoter were identified by transient transfection of the normal and mutated promoters into plasmacytoma cells. Our results identify four regions required for normal promoter function: (i) the octamer ATGCAAAT, previously identified by others; (ii) a heptamer, CTAATGA; (iii) a pyrimidine-rich region; and (iv) a region between positions -125 and -251 relative to the transcription start site. Sequence analysis of 19 mouse and human VH 5' flanking regions shows that the heptamer and pyrimidine stretch are strongly conserved. We have also demonstrated that the octamer functions in an orientation independent manner in the VH promoter.

Animals↗