Control of immunoglobulin heavy chain constant region gene expression.
Explore the source record for details and available documents.
Animals↗
Biomedical subjects
P Rothman
Publications and source records attributed to P Rothman.
Identification of a conserved lipopolysaccharide-plus-interleukin-4-responsive element located at the promoter of germ line epsilon transcripts.
Treatment of splenic B lymphocytes and certain B-lineage cell lines with the mitogen lipopolysaccharide (LPS) and the lymphokine interleukin-4 (IL-4) induces expression of germ line immunoglobulin C epsilon transcripts and class switching to the C epsilon gene. We show that LPS-plus-IL-4 induction of germ line epsilon transcripts (termed I epsilon transcripts) occurs at the transcriptional level in an Abelson murine leukemia virus-transformed pre-B-cell line. A 1.1-kb region of DNA surrounding the I epsilon promoter endows inducible transcription to a heterologous reporter gene stably transfected into these cells; such inducible expression depends on combined treatment with LPS and IL-4. Analyses of constructs transiently introduced into a B-cell lymphoma line demonstrated that LPS-plus-IL-4-inducible expression can be conferred by a 179-bp segment of DNA spanning the I epsilon transcriptional initiation site. Mutational analyses demonstrated that this expression depended on DNA sequences within a conserved region directly upstream from the I epsilon transcriptional initiation region. One nuclear protein that is constitutively expressed in normal B cells binds to the downstream end of the conserved sequence; its binding specificity correlates with the functional effect of several mutations. Two additional proteins, which are induced by IL-4 treatment of splenic B cells, bind to the transcription initiation sites of I epsilon. These proteins are indistinguishable in binding assays from proteins previously shown to bind an enhancer region of the class II major histocompatibility complex gene A alpha.
Abelson murine leukemia virus↗
Isolation of coordinately regulated genes that are expressed in discrete stages of B-cell development.
We have utilized subtractive hybridization to isolate 16 distinct cDNA sequences representing genes expressed in pre-B-cell lines but not myeloma cell or fibroblast lines. These sequences represent RNA transcripts that vary in abundance in pre-B-cell lines from 0.001% to 0.05%. Five of these sequences were not related to any known genes. One was related to but distinct from known myosin regulatory light chain genes and another encoded a protein with lectin domains. Three represented previously identified genes encoding carbonic anhydrase type II, thymosin, and CD2; these genes were not previously known to be specifically expressed in early stages of B-cell development. Other isolated genes corresponded to pre-B-cell-specific or pre-B-cell/B cell-specific genes recently described by others. The isolated cDNA sequences may be divided into two general categories--those representing genes expressed only in the pre-B-cell stage of B-cell development and those expressed in both the pre-B-cell and B-cell stages. The in vivo expression patterns of the identified genes suggest that some function specifically in lymphocytes while others may have roles in additional lineages.
Animals↗
Structure and expression of germline immunoglobulin gamma 3 heavy chain gene transcripts: implications for mitogen and lymphokine directed class-switching.
We have characterized the structure and expression of transcripts synthesized from the murine germline immunoglobulin gamma 3 heavy chain gene in certain B-lineage cells. The transcripts initiate upstream of the switch gamma 3 region, generating a 5' exon that is spliced to C gamma 3 exons. Expression of this germline transcript is induced when splenic B cells or A-MuLV-transformed pre-B cell lines are cultured in the presence of lipopolysaccharide (LPS). Addition of interleukin-4 (IL-4) to these lipopolysaccharide (LPS) cultures dramatically inhibits induction of the germline gamma 3 transcript. Induction of germline gamma 3 transcripts occurs before the increased accumulation of gamma 3-producing cells and VDJ-gamma 3 mRNA in cultures of splenic B cells. These data provide further evidence that germline CH transcriptional units are important components in the regulation of heavy chain class-switching. In addition, the pre-B cell lines that we describe represent the first example of permanent cell lines that regulate expression of the germline gamma locus in response to LPS plus IL-4 treatment in a manner analogous to normal B cells; therefore these lines should represent an excellent model system to further study the molecular mechanisms by which germline expression is regulated by these agents.
Animals↗
Structure and expression of germ line immunoglobulin heavy-chain epsilon transcripts: interleukin-4 plus lipopolysaccharide-directed switching to C epsilon.
We have isolated cDNA clones complementary to a truncated immunoglobulin heavy-chain C epsilon RNA transcript previously found to be induced in B lymphoid cells by treatment with lipopolysaccharide (LPS) combined with interleukin-4 (IL-4). We demonstrate that this transcript initiates from a promoter upstream of the germ line epsilon class-switch recombination region (S epsilon region). The major germ line C epsilon transcript contains a small 5' exon contributed by sequences upstream of the S epsilon region spliced to the normal C epsilon exons. Treatment of splenic B lymphoid cells with LPS plus IL-4 induces the expression of transcripts from the germ line epsilon transcription unit followed by expression of normal immunoglobulin epsilon heavy-chain mRNA. Furthermore, we demonstrate that similar treatment of transformed precursor B cell lines induces the expression of germ line epsilon transcripts followed by class switching to epsilon expression in these lines. This is the first demonstration of switching to epsilon in cells of the pre-B stage. The general structure of the germ line epsilon transcript and transcription unit is similar to that previously characterized for germ line gamma 2b transcripts. However, expression of these two germ line transcription units in B-lineage cells is inversely regulated by IL-4 (plus LPS) treatment, correlating with the effects of these treatments on switching to these loci.
Animals↗
The molecular events in heavy chain class-switching.
Heavy chain class-switching is the process by which B lymphoid cells change the constant region of the immunoglobulin heavy chain they produce. Class-switching is most commonly accomplished by recombinationldeletion between switch recombination regions that lie upstream of each germline heavy chain constant region gene. Recent studies support a model that recombination to specific switch regions is directed by modulation of the accessibility of these regions to a common class-switch recombination system. T cell lymphokines seem to be able to alter the accessibility of different heavy chain constant region loci, and thereby direct the specificity of class-switch recombination in B cells.
Animals↗
Mitogen plus interleukin 4 induction of C epsilon transcripts in B lymphoid cells.
To elucidate the mechanism of IL-4-induced enhancement of IgE and IgG1 production, murine splenic B cells and A-MuLV-transformed cells were cultured with LPS and IL-4 and assayed for epsilon and gamma 1 transcripts. Concomitant treatment with IL-4 and LPS induced expression of C epsilon transcripts in both normal and transformed cells. Expression of these truncated C epsilon transcripts preceded accumulation of normal epsilon mRNA in treated cells. Consistent data were obtained with respect to gamma 1 RNA expression. These results suggest that IL-4 can direct class switching in the context of a mechanism associated with differential expression of germline constant region genes.
Animals↗
Mitogen- and IL-4-regulated expression of germ-line Ig gamma 2b transcripts: evidence for directed heavy chain class switching.
Treatment of murine B cells with bacterial lipopolysaccharide (LPS) in the presence or absence of different lymphokines results in cell populations that differentially express particular immunoglobulin heavy chain constant region (CH) genes. This class switch involves recombination between switch regions located upstream of the germ-line CH genes. We have treated Abelson murine leukemia virus-transformed pre-B cells and normal splenic B cells with LPS or LPS plus the lymphokine IL-4 and examined the effect on the germ-line gamma 2b locus and gamma 2b class switching. In both cell types, LPS induces transcription specifically through the germ-line gamma 2b locus before gamma 2b class switching. Furthermore, IL-4 inhibits LPS induction of germ-line gamma 2b transcripts in spleen cells and correspondingly abrogates switching to this CH gene. Thus treatment with mitogens and lymphokines can alter transcription of germ-line CH genes in B lineage cells and thereby directly regulate class switching in the context of a recombinase accessibility mechanism.
Animals↗
Control of recombination events during lymphocyte differentiation. Heavy chain variable region gene assembly and heavy chain class switching.
Our recent studies have focused on the organization of immunoglobulin genes in mice and humans and the mechanism and control of the recombination events that are involved in their assembly and expression. This report describes our progress in this area with particular focus on elucidating factors that influence the generation of the antibody repertoire in normal and diseased states. We present a detailed analysis of the organization of the human VH locus, studies that help to elucidate the nature of the recombination defect in mice with severe combined immunodeficiency, and studies of transgenic mice that focus on the mechanism that regulates tissue-specific variable region gene assembly. In addition, we also characterize mechanisms that control the heavy chain class-switch process. Although the latter process apparently involve a recombination system distinct from that involved in variable region assembly, we find that the two recombination events appear to be controlled by similar mechanisms.
Animals↗
Further dissection of the functional heterogeneity within the OKT4+ and OKT8+ human T cell subsets.
Previous studies have suggested functional heterogeneity within the OKT4+ and the OKT8+ populations. For example, after activation the OKT4+ population contains not only helper cells but also cells capable of suppressing B cell differentiation. Previous studies also indicate that the reciprocal T cell population, OKT8+, does not provide helper activity but contains cytotoxic effector cells and radiosensitive cells important in the suppression of B cell differentiation. Using a new differentiation antigen, OKT17, which recognizes a surface antigen present on the majority of resting normal peripheral T lymphocytes but is present only on a subset of OKT4+ cells after activation, evidence was obtained that two functionally mature subsets can be distinguished within the OKT4+ population itself: OKT4+17+ radiosensitive suppressor cells and OKT4+17- radiosensitive helper cells. Recently, another monoclonal antibody, OKT20, has been described which is present on a small percentage of resting lymphocytes but is expressed in varying proportions on activated T cells. Functional analysis of normal resting human T lymphocytes demonstrated that the OKT20-depleted T cell subset was able to generate cytotoxic cells and to suppress antibody production to the same extent as did OKT8+ cells. On the other hand, when unselected T lymphocytes were cultured for six days in a mixed lymphocyte reaction and then depleted of OKT20 reactive cells, the cytotoxic effector T cells were eliminated. In contrast, OKT20-depleted T cells after identical activation were still able to suppress antibody production. These data provide evidence that following activation of OKT8+ cells, the OKT20 differentiation antigen becomes selectively expressed on cytotoxic effectors but not on suppressor cells.
Antibodies, Monoclonal↗