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Biomedical subjects

M E Koshland

Publications and source records attributed to M E Koshland.

At least 19 recordsLinked to original sources
PubMed2000

Dynamic chromatin remodeling in the vicinity of J chain gene for the regulation of two stage-specific genes during B cell differentiation.

Dynamic chromatin remodeling during B cell differentiation was identified in the vicinity of J chain gene. In pre-B cells, the enhancer-containing DNase I hypersensitive sites (HSSs) 3-4 were open. However, these HSSs 3-4 turned out to be unassociated with J chain gene expression, as the J chain promoter-containing HSS1 remained in a closed state. The open enhancer HSSs 3-4 in the pre-B cells might be related to the expression of a pre-B cell-specific gene upstream of the HSSs 3-4, which was identified in our Northern blot analyses. The HSSs 3-4 are then closed in the next immature and mature B cell stages until the IL-2 opens the HSSs 3-4 again as well as HSS1 to express J chain gene in the primary immune responses. The dynamic regulation of chromatin structure during B cell differentiation for the expression of two stage-specific genes will provide a good model system for the study of B cell differentiation and gene expression.

Animals↗
PubMed1999

B cell-specific activator protein prevents two activator factors from binding to the immunoglobulin J chain promoter until the antigen-driven stages of B cell development.

The immunoglobulin J chain gene is inducibly transcribed in mature B cells upon antigen recognition and a signal from interleukin-2 (IL-2). B cell-specific activator protein (BSAP), a transcription factor that silences J chain transcription, has been identified as a nuclear target of the IL-2 signal. The levels of BSAP progressively decrease in response to IL-2 and this change correlates with the differentiation of B cells into antibody secreting plasma cells. Here we report the binding of the upstream stimulatory factor (USF) to an E-box motif immediately upstream from the BSAP site on the J chain promoter. Mutations in the USF binding motif significantly decrease J chain promoter activity in J chain expressing B cell lines. We also show that a functional relationship exists between USF and a second J chain positive-regulating factor, B-MEF2, using co-immunoprecipitation assays and transfections. Finally, we provide evidence that the binding of BSAP prevents USF and B-MEF2 from interacting with the J chain promoter during the antigen-independent stages of B cell development. It is not until the levels of BSAP decrease during the antigen-driven stages of B cell development that both USF and B-MEF2 are able to bind to their respective promoter elements and activate J chain transcription.

Animals↗
PubMed1998

Myocyte enhancer factor-related B-MEF2 is developmentally expressed in B cells and regulates the immunoglobulin J chain promoter.

Immunoglobulin J chain gene expression is induced by the delivery of a lymphokine signal to antigen-activated B cells in a primary immune response. A major interleukin 2 (IL-2)-responsive region that contains two adjacent control elements (JA and JB) exists within the J chain promoter. Transcription factor PU.1 positively regulates J chain gene expression by binding to one of the control elements (JB) in the J chain promoter. In the present study we have determined that a myocyte enhancer factor 2 (MEF2)-related nuclear factor, named B-MEF2, positively regulates the J chain gene promoter activity via the second control element (JA). An in vitro translated MEF2 family member, MEF2C, was found to bind the JA site with identical properties as endogenously expressed B-MEF2 in B cell lines. Moreover, in vivo experiments showed that a dominant negative mutant of MEF2C blocked B-MEF2 regulation of the J chain promoter. Consistent with its role as positive regulator of J chain gene expression, B-MEF2 levels were enhanced in highly differentiated B cells. In addition, induction of an IL-2-responsive presecretor cell line BCL1 with IL-2 or IL-5 (which up-regulates J chain gene expression) resulted in an increased expression of B-MEF2. We conclude that a MEF2-related transcriptional factor, B-MEF2, acts as a stage-specific positive regulator of J chain gene expression in the B cell lineage.

Animals↗
PubMed1998

The transcription factor NF-kappaB/p50 interacts with the blk gene during B cell activation.

The B cell-specific transcription factor Pax-5 has been shown previously to interact with the promoter of the blk gene in vitro. blk encodes a tyrosine kinase associated with the B cell receptor, which is expressed during the early but not the final stages of B cell development. To investigate whether Pax-5 regulates expression of the blk gene in vivo during B cell development and/or activation, Pax-5a was overexpressed in B cell lines. Increases in blk promoter activity using a chloramphenicol acetyltransferase reporter gene system suggested a role for Pax-5a as a transcriptional activator. Subsequent site-specific mutagenesis studies showed that mutations of the Pax-5 binding site on blk significantly alter promoter activity, although results suggested that other factors could bind to this region as well. Using mobility shift assays, we detected an inducible transcription factor that interacts strongly with a sequence overlapping the Pax-5 site on the blk promoter and identified this as a homodimer of NF-kappaB/p50, a member of the NF-kappaB/Rel family of transcription factors. This factor was present at high levels in lipopolysaccharide-activated normal B cells and in plasma cell lines but either at low levels or undetectable levels in resting normal B cells or pre-B or mature B cell lines. In contrast, lipopolysaccharide induction of a pre-B cell line (703/Z) induced a complex that contained both NF-kappaB/p50 and p65. These studies suggest that different NF-kappaB complexes are able to interact with a sequence overlapping the Pax-5 site on the blk promoter and that the relative levels of "bound" factor influence levels of blk expression. Since p50 homodimers and p50/p65 heterodimers of the NF-kappaB complex should have opposing effects on blk transcription, this could provide a mechanism to differentially regulate blk expression during B cell development and activation.

Adaptor Proteins, Signal Transducing↗
PubMed1998

Dependence of BSAP repressor and activator functions on BSAP concentration.

During a B cell immune response, the transcription factor BSAP maintains its activator functions but is relieved of its repressor functions. This selective targeting of BSAP activities was shown to be regulated by a concentration-dependent mechanism whereby activator motifs for BSAP had a 20-fold higher binding affinity than repressor motifs. An exchange of activator and repressor motifs, however, showed that the context of the motif, rather than the affinity, determined whether BSAP operated as an activator or repressor.

Animals↗
PubMed1998

A stage-specific enhancer of immunoglobulin J chain gene is induced by interleukin-2 in a presecretor B cell stage.

Interleukin-2 (IL-2)-induced transcription of the J chain gene was used as a model for analyzing cytokine regulation during B cell development. To determine whether IL-2 signals are targeted to a J chain gene enhancer as well as to its promoter, the sequences flanking the J chain gene were first examined for DNase I hypersensitivity. Of six sites identified, two strong ones, 7.5 kb upstream of the J chain gene, were found to be associated with an enhancer that is active only during the antigen-driven stages of B cell development. Further analyses of the enhancer in the IL-2-responsive presecretor BCL1 cells showed that the enhancer is activated at this stage by an IL-2 signal that functions by opening the enhancer chromatin and stimulating STAT5 to bind to a STAT5 element critical for the enhancer induction. Moreover, after this early induction stage, the enhancer was shown to be constitutively open and active in terminally differentiated plasma cells.

Base Sequence↗
PubMed1996

Expression of the immunoglobulin J chain in a murine B lymphoma is driven by autocrine production of interleukin 2.

Expression of the immunoglobulin J chain is initiated by lymphokine signals delivered to activated B cells during a primary immune response. In the mature murine B cell line, CH12.LX, IL-5 and LPS but not IL-2 were found to greatly enhance basal levels of J chain gene expression. Analysis of the IL-2 receptor (IL-2R) showed two defects: an unusually low expression of the IL-2R alpha chain and little or no IL-2R beta chain. Treatment with IL-5 strongly amplified IL-2R alpha chain expression in CH12.LX cells, yet failed to confer IL-2 responsiveness. However, when the IL-2R beta chain was introduced by stable transfection, the cells expressed 400-500 high affinity IL-2R and responded to IL-2 with increased J chain expression. Surprisingly, in the absence of exogenous lymphokine stimulation, the basal levels of J chain and IL-2R alpha in all IL-2R beta transfectants became significantly elevated over time. Analysis showed that CH12.LX cells constitutively synthesized IL-2 and, given a functional IL-2R, responded to the lymphokine in an autocrine fashion to upregulate both J chain and IL-2R alpha. Thus, CH12.LX cells provide a model cell line in which the role of the IL-2R beta chain in differentiative events such as J chain upregulation can be examined.

Animals↗
PubMed1996

An interleukin-2 signal relieves BSAP (Pax5)-mediated repression of the immunoglobulin J chain gene.

Cytokine regulation of B cell development was analyzed using interleukin-2 (IL-2)-induced transcription of the J chain gene as a model system. A nuclear target of the IL-2 signal was identified as the Pax5 transcription factor, BSAP, which recognizes a negative regulatory motif in the J chain promoter. Functional assays showed that BSAP mediates the silencing of the J chain gene during the early stages of B cell development, but repression is relieved during the antigen-driven stages in a concentration-dependent manner by an IL-2-induced down-regulation of BSAP RNA expression. At the low levels present in J chain-expressing plasma cells, BSAP repression could be overridden by positive-acting factors binding to down-stream J chain promoter elements. Overexpression of BSAP in these cells reversed the positive regulation and inhibited J chain gene transcription. Thus, IL-2 regulation of BSAP concentration may provide a mechanism for controlling both repressor and activator functions of BSAP during a B cell immune response.

Animals↗
PubMed1995

Polymer IgM assembly and secretion in lymphoid and nonlymphoid cell lines: evidence that J chain is required for pentamer IgM synthesis.

The requirements for IgM assembly and secretion were evaluated by introducing a constitutively expressed J-chain cDNA into lymphoid and nonlymphoid cell lines expressing the secretory form of monomer IgM. Assays of cell lysates and supernatants showed that only secretory monomer IgM is required for the synthesis and secretion of hexamer IgM, whereas J chain, as well as the secreted form of monomer, is required for the synthesis and secretion of pentamer IgM. Moreover, J chain facilitates the polymerization process so that pentamer IgM is preferentially synthesized. Other components of the polymerization process were found to be shared by all the cell lines examined, whether the cells were of lymphoid or nonlymphoid origin and had a rudimentary or developed secretory apparatus. These results identify monomer IgM and J chain as the two components that determine the B-cell-specific expression of IgM antibodies and, thus, as the appropriate targets for therapeutic regulation of IgM responses.

Animals↗
PubMed1993

Ets-related protein PU.1 regulates expression of the immunoglobulin J-chain gene through a novel Ets-binding element.

In a primary immune response, a signal from interleukin-2 (IL-2) activates transcription of the gene encoding the pentamer IgM joining component, the J chain. Recently, a bifunctional control element (JB) in the J-chain promoter has been identified. This finding was pursued in the present study by purifying and characterizing the nuclear protein (NF-JB) that mediates the positive regulatory activity of the JB element. The analyses revealed that NF-JB is identical to the Ets-related B-cell- and macrophage-specific transcriptional factor, PU.1, despite the fact that the JB site lacks the GGA core reported to be essential for binding by members of the Ets oncoprotein family. The two factors were found to be indistinguishable with respect to their DNA-binding characteristics, size, and peptide structure. Moreover, in transient transfection assays, PU.1 alone activated reporter constructs containing the JB cis-element, and the activation was shown to be dependent on a glutamine-rich sequence in the amino-terminal portion of PU.1. Finally, a dominant negative mutant of PU.1 was capable of suppressing the transcriptional activity of a 1.2-kb J-chain promoter sequence. These results establish an important role for PU.1 in the regulation of immunoglobulin J-chain gene expression and provide new insights into the function(s) of the Ets transcription factors in lymphoid cells.

Amino Acid Sequence↗
PubMed1992

A promoter element that exerts positive and negative control of the interleukin 2-responsive J-chain gene.

In a primary immune response a signal from interleukin 2 (IL-2) induces B lymphocytes to express the gene for the IgM joining component, the J chain. The signaling mechanism was pursued in this study by examining the J-chain gene 5' flanking region for regulatory sequences and interacting nuclear factors. The analyses identified a major control region located between -75 and -45 that encodes two adjacent elements: a T-rich sequence (JA) containing a single positive regulatory motif and an A+G-rich sequence (JB) containing overlapping positive and negative regulatory motifs. Dissection of the two elements indicated that the bifunctional JB sequence is the likely target of the IL-2 signal. The evidence was based on findings that (i) JB activity correlated with J-chain gene transcription--i.e., JB acts as a repressor in J-chain-silent B cells and as an activator in J-chain-expressing cells, and (ii) JB activator function is mediated by a B-cell-specific nuclear protein, NF-JB, that exhibits an IL-2-responsive binding pattern.

Animals↗
PubMed1991

Interleukin 2- and interleukin 5-induced changes in the binding of regulatory factors to the J-chain gene promoter.

In a primary immune response, B cells require signals from the T-cell lymphokines interleukins 2 and 5 (IL-2 and IL-5) to develop into IgM-secreting cells. One role of IL-2 and IL-5 is to activate transcription of the gene encoding the IgM joining component, the J chain. In this study the activation mechanism was investigated by using an inducible beta-lymphoma cell line to examine J-chain RNA expression and factor binding to the J-chain promoter. The analyses revealed that both IL-2 and IL-5 trigger a decrease in the binding of two promoter-specific nuclear proteins that precedes the appearance of J-chain RNA. In combination the two lymphokines effected nearly additive changes in factor binding and J-chain RNA abundance. Both effects were reversed upon withdrawal of the lymphokine stimulus and both were inhibited in the presence of the T-cell lymphokine IL-4. These findings indicate that the IL-2 and IL-5 signal pathways converge to deliver a common signal that regulates the repressor activities of two lymphokine-responsive promoter elements.

Animals↗
PubMed1991

Glycosylphosphatidylinositol: a candidate system for interleukin-2 signal transduction.

The mechanism of interleukin-2 (IL-2) signal transduction was analyzed by use of an inducible B lymphoma. Like normal antigen-activated B lymphocytes, the lymphoma cells respond to IL-2 by proliferating and differentiating into antibody-secreting cells; both responses are blocked by a second interleukin, IL-4. Analyses of the signaling pathway showed that IL-2 stimulated the rapid hydrolysis of an inositol-containing glycolipid to yield two possible second messengers, a myristylated diacylglycerol and an inositol phosphate-glycan. The myristylated diacylglycerol response exhibited the same IL-2 dose dependence as the growth and differentiative responses, and the generation of both hydrolysis products was inhibited by IL-4. These correlations implicate the glycosyl-phosphatidylinositol system in the intracellular relay of the IL-2 signal.

Arachidonic Acid↗
PubMed1989

Mechanism of interleukin-2 signaling: mediation of different outcomes by a single receptor and transduction pathway.

The T cell lymphokine, interleukin-2 (IL-2), plays a pivotal role in an immune response by stimulating antigen-activated B lymphocytes to progress through the cell cycle and to differentiate into antibody-secreting cells. An IL-2 inducible B lymphoma line, in which the growth and differentiation responses are uncoupled, provides a model system for dissecting the signaling mechanisms operating in each response. This system was used to show that both signals are initiated by IL-2 binding to a single, unifunctional receptor complex. Moreover, both signals are transduced by a pathway that does not involve any known second messenger system and that can be blocked by a second T cell lymphokine, interleukin 4. These findings suggest that the pleiotrophic effects of IL-2 are determined by different translations of the signal in the nucleus.

Animals↗
PubMed1986

A model system for peptide hormone action in differentiation: interleukin 2 induces a B lymphoma to transcribe the J chain gene.

Physiological levels of a purified T cell hormone, interleukin 2 (IL-2), were found to stimulate a cloned murine B cell line (BCL1) to secrete pentamer IgM antibody. The peptide hormone acts at the cell surface via specific IL-2 receptors and induces changes in the 5' chromatin of the J chain gene that correlate with its transcription and with the production of the J chain protein required for pentamer IgM assembly. There was no effect of IL-2 on cell proliferation nor on mu heavy chain gene transcription. These results define a specific function for IL-2 in B cell differentiation. In addition, the IL-2/BCL1 system provides a model for examining the mechanism by which signals generated by hormone-receptor interaction are transmitted to the nucleus and regulate gene expression.

Animals↗
PubMed1986

Immunoglobulin J chain gene from the mouse.

During a primary immune response, murine B lymphocytes are induced to express the gene for the immunoglobulin J chain. As a first step in determining the mechanism of induction, genomic DNA clones encoding the murine J chain were obtained from cell lines representative of B lymphocytes before and after J chain expression. Analysis of the coding regions showed that the J chain gene has a different structure from the other immunoglobulin genes. It consists of four exons organized in a simple 7.3-kilobase transcription unit that does not require DNA rearrangement or alternative processing for expression. These structural properties indicate that transcription of the J chain gene is initiated by changes in chromatin conformation, probably involving a J chain-specific DNA-binding factor. Analysis of the 5' flanking sequences of the J chain gene, on the other hand, showed that the promoter region contains two conserved elements that have been implicated in the lymphocyte-specific expression of the light chain genes. The sharing of these elements suggests that, once the J chain gene is activated, its transcription is regulated by mechanisms similar to those controlling the light chain genes.

Amino Acid Sequence↗
PubMed1986

Accessibility of the promoter sequence in the J-chain gene is regulated by chromatin changes during B-cell differentiation.

The gene for the immunoglobulin M (IgM)-polymerizing protein, the J chain, is activated when the mature B cell is triggered to secrete pentamer IgM. Activation of the gene was found to be associated with chromatin changes in a 240-base-pair region at the 5' end of the gene. Analyses of lymphoid lines showed that the 5' region was resistant to nuclease digestion at the immature B-cell stage; it became slightly more accessible in mature B cells and cells at an early stage in the IgM response and then displayed an open, hypersensitive structure in IgM-secreting cells. In addition, analyses of normal, mitogen-stimulated lymphocytes showed that the open hypersensitive structure was coinducible with J-chain gene expression. These results suggest that the 5' chromatin changes precede transcription, making control sequences within the site accessible to regulatory factors.

B-Lymphocytes↗