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.

Identification of a matrix-associated region 5' of an immunoglobulin heavy chain variable region gene.

In the accompanying report (C. F. Webb, C. Das, S. Eaton, K. Calame, and P. Tucker, Mol. Cell. Biol. 11:5197-5205, 1991), we characterize B-cell-specific protein-DNA interactions at -500 and -200 bp upstream of the mu immunoglobulin heavy chain promoter whose abundances were increased by interleukin-5 plus antigen. Because of the high A + T/G + C ratio of these sequences and the consistent findings by others that enhancer- and promoterlike regions are often located near matrix-associated regions, we asked whether these sequences might also be involved in binding to the nuclear matrix. Indeed, DNA fragments containing the -500 binding site were bound by nuclear matrix proteins. Furthermore, UV cross-linking studies showed that the DNA binding site for interleukin-5-plus-antigen-inducible proteins could also bind to proteins solubilized from the nuclear matrix. Nuclear matrix-associated sequences have also been demonstrated on either side of the intronic immunoglobulin heavy chain enhancer. Our data suggest a topological model by which interactions among proteins bound to the promoter and distal enhancer sequences might occur.

Reassessment of germline heavy chain transcripts from two murine VH families.

While expression of functional heavy chain immunoglobulin mRNA requires rearrangement of variable (VH), diversity (D) and (JH) gene segments, these individual gene segments can be transcribed prior to their rearrangement. It has been proposed that the resulting germline, or sterile, transcripts play an important role in the rearrangement process because strong correlations between rearrangement frequency and sterile transcript levels have been observed in some studies. Murine VH genes have been grouped into families on the basis of coding sequence homology. VH families rearrange in a developmentally regulated manner, so that rearrangements of genes from several VH families are detected earlier than rearrangements of J558 family genes. Paradoxically, the only VH family for which sterile transcripts have been documented is the J558 family. We used RT-PCR analyses to ask whether sterile transcripts from other VH families could be detected in fetal liver samples prior to their rearrangement. While J558 family germline transcripts were easily detected, no sterile transcripts were observed from the S107 family. Our studies also revealed the ability of small quantities of degraded genomic DNA to nonspecifically prime cDNA synthesis, emphasizing the need for caution in interpreting RT-PCR data in which family-specific oligos are used for cDNA production. These results cast doubt on the idea that sterile transcripts are required for V(H)DJ(H) rearrangement.

Isolation and characterization of the promoter of the human 5'-nucleotidase (CD73)-encoding gene.

Ecto-5'-nucleotidase (NT, CD73) is a purine salvage-pathway enzyme located on the surface of various cell types, including subsets of human lymphocytes and certain leukemias and lymphomas. In addition to purine salvage, NT has proposed roles in lymphocyte maturation and activation, and its expression has been associated with the resistance of some tumor cell lines to chemotherapeutic agents. To better understand the regulation of NT gene expression in normal lymphocyte development and the elevated expression seen in some drug-resistant tumor cell lines, we isolated NT genomic clones containing the promoter region. The genomic DNA upstream from the NT start codon is high in G+C content, with one cAMP-responsive element and five consensus Sp-1 binding sites, but no TATAA box. RNase protection assays identified a cluster of potential transcription start points (tsp). One tsp, at -63 bp relative to the start codon, was confirmed as authentic by 5'-RACE (rapid amplification of cDNA ends) cloning. Transient transfection experiments utilizing luc as a reporter gene have demonstrated that a 155-bp NT genomic DNA segment inclusive of the tsp functions as a promoter in both NT+ (WI-L2 and MG) and NT- (Jurkat, Hela and Raji) cell lines. The addition of 5'-flanking sequences extending as far as -1.9 kb did not confer cell-type-specific expression to the core promoter. However, nuclear run-on analysis of nascent NT transcripts suggested that differential transcription initiation is at least partially responsible for the regulation of NT expression. Thus, additional information is necessary, either at the chromatin level, or within elements outside of the promoter region, to direct tissue-specific expression of NT.

The transcription factor, Bright, and immunoglobulin heavy chain expression.

Bright, or B cell regulator of immunoglobulin heavy chain transcription, is a B lymphocyte-specific protein first discovered for its ability to increase immunoglobulin transcription three- to sevenfold in antigen-activated B cells. It interacts with DNA through an ARID, or A/T-rich interaction domain, and is the only member of a previously undescribed family of DNA-binding proteins for which target genes have been identified. The mechanism(s) by which Bright facilitates transcription are unknown. Several proteins that associate with Bright may shed light upon its function. These include the nuclear matrix proteins sp100 and LYSp100B, and suggest that Bright may affect chromatin configuration and nuclear sublocalization. Furthermore, Bruton's tyrosine kinase is required for Bright binding activity, suggesting links between Bright, cell signaling cascades, and X-linked immunodeficiency disease.

Binding characteristics of fluoresceinated Lotus tetragonolobus fucolectin to macrophage populations which are either responsive or refractory to activation by lymphokine.

Fucose binding protein (FBP) from Lotus tetragonolobus seed was studied by fluorescence microscopy for its binding characteristics to various guinea pig peritoneal macrophage populations. Fluoresceinated FBP (FITC-FBP) was bound optimally at 22 degrees C in a punctate distribution and was internalized at 37 degrees C. Binding of FBP to macrophages was reversed specifically by the competitive sugar L-fucose, and not by D-fucose, L-rhamnose, or D-galactose. FBP was bound with greater frequency and intensity to 3-day oil-elicited peritoneal macrophages which are responsive to migration inhibition by FBP and migration inhibitory factor (MIF) than to resident or 7-day inflammatory macrophages which are unresponsive to activation by the same effectors. Competition for visual binding of FITC-FBP to macrophages was demonstrated by preincubation of cells with unlabeled FBP or MIF. Competition of FITC-FBP binding by MIF was reversed by L-fucose. These results indicate that FBP binds preferentially, with greater frequency and intensity, to macrophage subpopulations which are responsive to MIF than to MIF-refractory macrophages. The data further supports the existence of a common receptor site for MIF and FBP on the macrophage membrane which involves fucosyl determinants.

Mechanisms of radiosensitization in bromodeoxyuridine-substituted cells.

The radiosensitization of exponentially-growing V79-171 cells whose DNA has been substituted by bromodeoxyuridine (BrdU) in place of thymidine is decreased if acetone is present during irradiation. Acetone, at a concentration of 1 mol dm-3, removes the majority of the increase in double-strand breaks (dsbs) caused by BrdU substitution, but only removes approximately half of the increase in cell killing. The decrease in cell radiosensitization coincides with the removal of the additional dsbs. The protection afforded by acetone against dsbs is assumed to be due to its ability to scavenge hydrated electrons, thought to be the active species causing the increased DNA damage in the presence of BrdU. The residual component of BrdU radiosensitization which could not be removed by treatment with acetone may be due to either a subset of nonscavengable, lethal dsbs or the influence of BrdU on the fixation of potentially-lethal damage (Iliakis et al. 1992). Cells substituted with BrdU are not sensitized to hydroxyl radicals (from hydrogen peroxide). Also, the enhanced levels of single-strand break (ssb) and dsb production in cells substituted with BrdU arise from analogous events (i.e. increases in the yield of ssbs). These studies support the locally multiply damaged site theory of lesion (dsb) production (Ward 1981) and, in the case of BrdU-substituted cells, the increase in dsbs appears to be due to the production of additional ssbs by hydrated electrons at sites of multiple damage.

A comparison of lactogenic receptors from rat liver and Nb2 rat lymphoma cells by using cross-linking techniques.

Lactogenic receptors were analysed with the use of the cross-linking agent disuccinimidyl suberate to attach covalently 125I-labelled ovine prolactin or human growth hormone to binding sites from (1) liver from pregnant rats and (2) the rat-derived Nb2 lymphoma cell line. Analysis by SDS/polyacrylamide-gel electrophoresis of the proteins cross-linked to labelled hormone in rat liver indicated a major specifically-labelled complex with an Mr of 68,000-72,000, when run under reducing or non-reducing conditions. With Nb2 cells a major specifically-labelled complex with an Mr of 97,000-110,000 was identified, but only when electrophoresis was run using reducing conditions. Assuming one hormone molecule (Mr 22,000-24,000) per hormone-receptor complex, then the receptor proteins have an Mr of 44,000-50,000 for rat liver and 73,000-88,000 for the Nb2 cells. For both cell types the receptors were of lactogenic specificity; lactogenic hormones competed for binding whereas somatogenic hormones did not. These studies suggest that the lactogenic receptors in rat liver membranes and Nb2 cells differ in two respects. Firstly, the Mr of the labelled receptor protein in Nb2 cells is greater than that of the corresponding receptor protein in rat liver membranes; secondly, the Nb2 cell receptor appears to exist as a disulphide-linked oligomer whereas the receptor in rat liver membranes does not.

Immunoglobulin receptor signaling depends on the carboxyl terminus but not the heavy-chain class.

To examine the isotypic and structural requirements involved in signaling through the immunoglobulin (Ig) receptor on B lymphocytes, we established a panel of T15 idiotype-positive transfectants that expressed wild-type IgM, wild-type IgD, or hybrid IgM molecules. Growth inhibition of the transfected lymphoma cells in response to anti-idiotype antibodies was used to measure signaling. Hybrid IgM molecules were constructed so that the membrane-spanning region of the mu heavy chain was replaced by that of delta, gamma 2b, or alpha heavy chains or that of the I-Ab class II (Ia) alpha chain. All transfectants that expressed IgM or hybrid IgM molecules with membrane-spanning regions from another Ig isotype underwent signaling in response to anti-idiotype antibodies, whereas the IgM-Ia hybrid transfectants did not. Transfectants that expressed wild-type IgD molecules also underwent signaling, although this response was particularly sensitive to serum concentrations. These results imply that signaling occurs in a similar manner through heavy-chain receptors of any isotype and suggest that conserved amino acid sequences in the transmembrane regions are important in this process.

The specificity of binding of growth hormone and prolactin to purified plasma membranes from pregnant-rabbit liver.

The binding of 125I-labelled human growth hormone (hGH) to a purified plasma membrane preparation from the liver of pregnant rabbit, and to receptors solubilized from this fraction with Triton X-100, was dependent on time, temperature, the cations used and the receptor concentration. Solubilization did not affect the binding properties of the receptors at low concentrations of Triton X-100. Some somatogenic hormones, such as bovine GH, and some lactogenic hormones, such as ovine prolactin, displaced 125I-labelled hGH from purified plasma membranes and solubilized receptor preparations, but GHs and prolactins from various other species were rather ineffective. The results indicate that although there are binding sites for hGH in these pregnant rabbit liver membranes, few of these are specifically somatogenic or lactogenic. The binding properties of the purified plasma membranes are similar to those of a microsomal preparation studied previously, suggesting that the complex nature of the binding of hGH is not due to the heterogeneity of cellular membranes used to study binding, but is a property of the receptors associated with plasma membranes.

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.

Effect of anti-gamma 3 antibodies on immunoglobulin isotype expression in lipopolysaccharide-stimulated cultures of mouse spleen cells.

To test the hypothesis that gamma 3 is the pivotal isotype for sequential heavy chain switching from mu to each of the gamma isotypes, we have compared the effects of anti-gamma 3 and anti-mu antibodies on the expression of immunoglobulin isotypes in lipopolysaccharide (LPS)-stimulated cultures of mouse spleen cells. IgM-, IgG1-, IgG2b- and IgG2a-containing plasma cells were enumerated by immunofluorescence and secreted immunoglobulins were measured by radioimmunoassay. Although anti-gamma 3 and anti-mu were equally effective in inhibiting the LPS-induced differentiation of IgG3 plasma cells, anti-gamma 3 had no effect on the differentiation of IgM, IgG1, IgG2b, or IgG2a plasma cells. These results support a direct mechanism of heavy chain immunoglobulin switching.

Expression and sequence analyses of serum amyloid A in the Syrian hamster.

Reactive amyloidosis occurs during chronic inflammation and involves deposition of amyloid A (AA) fibrils in many organs. Amyloid A is derived by proteolysis from serum amyloid A component (SAA), a major acute-phase reactant in many species. Since spontaneous amyloidosis occurs commonly in Syrian hamsters, we have studied the structure and expression of SAA genes during inflammation in these animals. Two cDNA clones and one genomic clone were sequenced, suggesting that Syrian hamster SAA is encoded by at least two genes. Hepatic mRNA analyses showed that SAA was inducible in many hamster organs during acute inflammation. These studies also demonstrated that SAA mRNA for one isotype is maximally expressed at a site of local tissue damage.

Differential transcription efficiency of two Ig VH promoters in vitro.

Each Ig variable region gene segment is transcribed from its own unique promoter. While all of these promoters share common consensus elements that contribute to the B cell-specific expression of these genes, the DNA sequence of each promoter is distinct. In this study, we have directly compared the transcription efficiencies of two murine heavy chain (VH) promoters in a murine B cell in vitro transcription system. We found that the promoters differed in both transcription efficiency and the ability to bind specific protein complexes. While some of the transcription differences may be attributed to differences in basal promoter elements, the spacing between the octamer and the heptamer consensus elements was found to be important. Others have reported a direct correlation between transcription efficiency and the probability that individual variable region gene segments will rearrange. Our studies may be of direct importance to those interested in identifying B cell-specific transcription factors and may ultimately help to explain differences in the expression of some VH gene segments.

Induction of immunoglobulin mu mRNA in a B cell transfectant stimulated with interleukin-5 and a T-dependent antigen.

We have established an Ag-specific in vitro system for studying the roles of Ag and IL-5 in B cell differentiation and Ig production. The murine B cell leukemia, BCL1B1, was transfected with mu and kappa genomic sequences and VS107 V regions that conferred a T15 Id and phosphocholine-binding specificity upon the cells. Transfected cells were treated with both the T-dependent Ag phosphorylcholine-key-hole limpet hemocyanin (PC-KLH), and 0.5 ng/ml purified IL-5. After 3.5 days steady state mu-mRNA levels increased three- to fourfold over mu-mRNA levels obtained from untreated cultures, or cultures treated with IL-5 or PC-KLH alone. IL-5 alone caused increased Ig secretion and a nearly twofold increase in proliferation. Additionally, cells treated with PC-KLH alone were able to process the Ag and present it to T cells as shown by subsequent lymphokine production. Thus, both Ag and IL-5 used singly appear to interact with their respective receptors, although neither Ag nor IL-5 increased mu-mRNA levels when used singly. The production of increased mu-mRNA levels obtained with Ag plus IL-5 required polymerase II transcription, suggesting that they may act to increase Ig transcription directly.

Family-specific differences in transcription efficiency of Ig heavy chain promoters.

Murine Ig variable region heavy chain genes (V(H)) are grouped into families based on coding sequence homology. We observed that the accompanying promoter sequences were also conserved in a family-specific manner. Remarkably, no one has directly compared the transcription efficiencies of V(H) genes from different families. Using an in vitro transcription system, we found that transcription efficiencies of different V(H) promoters differed by as much as 70-fold. These differences could be attributed to variation in the octamer-heptamer and TATA sequences, as well as to the presence or absence of initiator elements. The J558 family promoter exhibited the highest level of transcription and specifically interacted with an Oct-1 dimer not bound by other V(H) promoters. These data suggest that differential transcription and regulation of V(H) promoters could occur in vivo. The increased transcription efficiency of the J558 promoter relative to other V(H) promoters also presents a possible explanation for the abundance of J558 sterile transcripts observed before V(H)DJ(H) rearrangement.

An upstream Oct-1- and Oct-2-binding silencer governs B29 (Ig beta) gene expression.

The B cell-specific B29 (Igbeta) gene is activated in the earliest B cell precursors and is expressed throughout B cell development. Tissue-specific expression of the murine B29 gene is controlled by a B cell-specific promoter whose activity is governed by a cassette of upstream transcriptional silencers. This study describes a potent new silencer that is located 5' of the previously identified B29 silencer elements, FROG and TOAD. Like these known elements, the new B29 silencer is not restricted to the B29 promoter. Nuclear proteins from all cell lines tested interacted with this A+T-rich sequence, which closely resembled a noncanonical octamer binding motif and also conformed to the consensus sequence for nuclear matrix attachment regions. Interaction of Oct-1 and Oct-2 with the B29 A+T-rich sequence was confirmed using octamer-specific Abs. Oct-1/Oct-2 binding was required for the inhibitory activity of this sequence because mutations that blocked Oct-1/Oct-2 binding also eliminated inhibition of the B29 promoter. This B29 A+T-rich sequence specifically interacted with isolated nuclear matrix proteins in vitro, suggesting that it may also function as a matrix attachment region element. Maintenance of the level of B29 gene expression through the interaction of the minimal promoter and the upstream silencer elements FROG, TOAD, and the A+T-rich Oct-1/Oct-2 binding motif may be essential for normal B cell development and/or function.

Immunoglobulin gene rearrangements and deletions in human Epstein-Barr virus-transformed cell lines producing different IgG and IgA subclasses.

During differentiation B lymphocytes may switch from the expression of surface IgM to the synthesis of IgG, IgA, or IgE isotypes by using a different heavy chain constant region (CH) gene. The molecular mechanisms by which switching occurs remain controversial. Rearrangements and deletions of CH genes 5' to the expressed gene have often been observed in the mouse and, more recently, in human cells that have switched isotypes. We have used human JH, C micro, C gamma, and C alpha probes to examine the extent of the deletions and rearrangements in clones of Epstein-Barr virus-transformed human cells that produce IgG1, IgG3, IgG4, or IgA1. Though deletions of CH genes 5' to the expressed CH gene were consistently observed, the rearrangement process appeared to be highly variable for the nonproductive CH gene locus: deletion or persistence of 5' CH genes, combinations of deletion and duplication of 5' genes, and deletions extending to 3' CH genes. Our results reveal an unexpected lack of specificity in the DNA deletions in cells that have undergone isotype switching.