Unique function for carboxyl-terminal domain of Oct-2 in Ig-secreting cells.

The activity of Ig gene promoters and enhancers is regulated by two related transcription factors, Oct-1 (ubiquitous) and Oct-2 (B lineage specific), which bind the octamer motif (ATTTGCAT) present in these elements. As Ig promoter-binding factors, Oct-1 and Oct-2 each work together with a B lymphocyte-specific cofactor OCA-B/OBF-1/Bob-1 that interacts with them through their POU (DNA-binding) domains. Because both can mediate Ig promoter activity in B cells, there has been some question as to whether these two octamer-binding factors serve distinct functions in lymphocytes. We have shown previously that the silencing of B lymphocyte-specific genes in plasmacytoma x T lymphoma hybrids can be prevented by preserving Oct-2 expression. The pronounced effect of this transcription factor on the phenotype of plasmacytoma x T lymphoma hybrids established a critical role for Oct-2 not only in maintaining Ig gene expression, but in maintaining the overall genetic program of Ig-secreting cells. In the present study, we have explored the functional differences between Oct-1 and Oct-2 using chimeric Oct-1/Oct-2 proteins in cell fusion assays. Our results provide further evidence for an essential role for Oct-2 in Ig-secreting cells and identify the C-terminal domain of Oct-2 as responsible for its unique function in these cells.

Deletional analyses reveal an essential role for the hs3b/hs4 IgH 3' enhancer pair in an Ig-secreting but not an earlier-stage B cell line.

The Ig heavy chain (IgH) locus is controlled by multiple regulatory sequences mapping both within the IgH transcription unit (E mu) and downstream (3') of IgH coding sequences (hs3a, hs1,2, hs3b and hs4). Enhancer knockout studies in mice have implicated E mu in the control of IgH variable region gene assembly, but single-enhancer knockouts involving the 3' IgH enhancers have yet to shed light on their function. Transfection studies in mice and cell lines have suggested that the 3' enhancers behave similarly to a locus control region as first identified in the beta-globin locus. We have exploited this property to form mini-loci in a surface Ig(+) and an Ig-secreting cell line as a means for studying the functions of the 3' IgH enhancers. Importantly, this experimental system allows for the analysis of enhancer function within the context of chromatin. The mini-loci consisted of an Ig gamma 2b transcription unit linked to the four murine 3' IgH enhancers. Using targeted deletions of enhancer pairs within these mini-loci, we have discovered a critical and apparently developmentally regulated role for the hs3b/hs4 enhancer pair in IgH transgene expression.

Role of OCA-B in 3'-IgH enhancer function.

OCA-B (alternately called Bob1 and OBF-1) is a B cell-specific coactivator that interacts with the ubiquitously expressed Oct-1 and the B cell-restricted Oct-2 to activate transcription via the octamer site (5'-ATGCAAAT-3'). OCA-B-/- mice appear to undergo normal Ag-independent B cell maturation. However, Ag-dependent B cell differentiation, including germinal center formation, production of secondary Ig isotypes, and proliferation in response to surface Ig cross-linking, is greatly affected. We demonstrate that the observed reductions in expression of class-switched isotypes in OCA-B-/- mice may be due in part to deficiencies in the function of the 3'-IgH enhancer elements. Furthermore, we find that surface Ig cross-linking represses all the Ig enhancers and that this repression is absent in OCA-B-/- B cells. These results suggest an important role for OCA-B in Ig enhancer function in vivo.

3' IgH enhancer elements shift synergistic interactions during B cell development.

IgH gene expression is tightly controlled over the course of B cell development, B cell activation, and the subsequent differentiation of these cells into Ig-secreting plasmacytes. There are several transcriptional enhancers that map within and downstream of the IgH locus, and some of these have been clearly implicated in the developmental regulation of IgH gene assembly and expression. While some of the individual enhancers from this locus have been studied extensively, the functional interactions possible among this group of enhancers have been largely unexplored. In the present study, we have measured the transcriptional activities of combinations of enhancers introduced into B-lineage cell lines at several different developmental stages. We detected a developmental progression in which the 3' enhancers are initially inactive, then become strongly active through synergistic interactions, and finally achieve a strong level of activity with little interdependency. The relative contributions of E mu (the intron enhancer) and of the 3' enhancers also change as a function of developmental stage. We discuss these results in light of parallel studies of developmental changes in transcription factor requirements.

Transcription factor effects on chromosome constitution of cell hybrids.

When immunoglobulin (Ig)-secreting plasmacytomas are fused to a T-cell lymphoma, Ig gene expression ceases in greater than 95% of the resulting hybrids. In the rare hybrids that continue to express Ig, all other tested B lymphocyte-specific genes also remain active. The low frequency with which these Ig-expressing hybrids are recovered, along with the fact that cell fusions can lead to chromosome loss, led us to propose that this rare phenotype was due to loss of a T-cell-derived chromosome encoding a factor or factors with gene silencing activity. To identify the relevant chromosome, we have used a polymerase chain reaction (PCR)-assisted method of chromosome mapping to analyze both Ig-silenced (common) and Ig-expressing (rare) hybrids. Although no single chromosome was found to correlate with Ig gene silencing, we discovered that the two types of hybrids had undergone distinct patterns of chromosome loss. Moreover, we found that ectopic expression of a B-cell-specific transcription factor (Oct-2) dramatically altered both the phenotype and chromosome constitution of hybrids arising in these cell fusions.

E47 activates the Ig-heavy chain and TdT loci in non-B cells.

The E2A proteins, E12 and E47, are basic helix-loop-helix (bHLH) proteins essential for the B-cell lineage. Initially identified as immunoglobulin enhancer-binding proteins, they have also been shown to activate immunoglobulin enhancer-based reporters in transient transfection assays. Here, we examine the relationship between E2A DNA binding activity and activation of the endogenous, chromosomal immunoglobulin heavy chain (IgH) locus. Using sterile I(mu) transcription as an indicator of IgH enhancer activity, we see a direct correlation between E2A DNA binding activity and I(mu) transcription in stable BxT hybrids. We also observe a 1000-fold stimulation of endogenous I(mu) transcription in fibroblasts that express high levels of E47 and less stimulation in cells that express E12. By contrast, none of the other IgH enhancer-binding proteins tested (E2-2, Pu.1, Oct-2, OCA-B, TFE3 and USF) were able to activate I(mu) transcription. E47 overexpression also resulted in transcriptional activation of the endogenous gene encoding TdT, indicating that it, too, is a target of E2A proteins early in the B-cell lineage. Our results indicate that E2A proteins have the distinctive property of activating silent, chromatin-embedded B-cell-specific genes, underscoring their crucial role in B-cell development.

Immunoglobulin gene transcription ceases upon deletion of a distant enhancer.

The tissue-specific E mu enhancer within the immunoglobulin heavy chain (IgH) locus has recently been shown to be essential for efficient V region gene assembly in early B lineage cells. However, we and others have shown that late stage, Ig-secreting cells can produce IgH in the absence of E mu. In the present study we have explored the notion that another enhancer found in the far 3' region of the IgH locus (3' alpha E) takes on an important regulatory role in cells that have reached this terminal stage in B cell development. The technique of homologous recombination was used to disrupt the 3' alpha E region in an E mu-deficient, Ig gamma 2a-secreting cell line. Loss of 3' alpha E completely abolished Ig heavy chain gene expression, demonstrating that transcription of this gene was dependent upon sequences that reside over 70 kb downstream. The ability of these sequences to function efficiently in the absence of E mu may also provide an explanation for deregulated c-myc expression in many Ig-secreting tumors.

Mammalian cell fusion in an electroporation device.

We and others have been interested in the phenomenon of gene 'extinction' in somatic cell hybrids, reasoning that the study of this process is likely to reveal underlying mechanisms responsible for limiting the expression of specialized genes only to appropriate cell types. In the course of our studies in this area, we have developed a simple and economical method of fusing mammalian cells, using an electroporation device. In fusions between murine myeloma and T lymphoma lines, hybrid cell recoveries were typically one per 10(5) [corrected] input myeloma cells. Because of our interest in the regulation of immunoglobulin heavy chain (IgH) gene expression, we analyzed the hybrids for both IgH gene composition and expression. The hybrid lines were phenotypically indistinguishable from those generated by the more conventional, polyethylene glycol (PEG)-induced fusion protocol. There was a notable increase, however, in the number of hybrids that retained IgH-encoding chromosomes from both parental lines.

Constitutively expressed Oct-2 prevents immunoglobulin gene silencing in myeloma x T cell hybrids.

Recent experiments involving disruption of the Oct-2 gene have shown that this largely B cell-restricted transcription factor is not required in the early stages of B cell development. However, B cells that lack Oct-2 may be blocked from differentiation past the surface immunoglobulin-positive stage. To identify a possible function for Oct-2 in the late stage immunoglobulin-secreting cell, we have used the method of somatic cell fusion. When the immunoglobulin-producing myeloma MPC11 is fused to a T lymphoma, Oct-2 production ceases, as does the expression of immunoglobulin, J chain, and several other B cell-specific gene products. In the present study, we show that by preventing the loss of Oct-2 in the hybrid cells, we can preserve expression of all other tested B cell-specific genes. These results establish a central role for Oct-2 in maintaining the genetic program of the immunoglobulin-secreting plasmacyte.

Coordinate silencing of myeloma-specific genes in myeloma x T lymphoma hybrids.

It has been well-established that Ig genes are transcriptionally silenced when Ig-producing myeloma lines are fused to non-B cells. In the present study, we analyzed the expression of several other myeloma-specific genes in fusions of myelomas with the T lymphoma, BW5147. Seven of the eight genes analyzed behaved coordinately with the Ig loci; they were silent in most myeloma x T hybrids but active in the rare hybrid that retained Ig gene expression. Cloned IgH genes introduced into the two types of hybrids behaved as their endogenous counterparts. The coordinate behavior of these several genes in the panel of "exceptional" and "extinguished" hybrids suggests a central and bimodal switch for alternately activating and de-activating the genetic program of the Ig-secreting plasmacyte. The switch between an active and an inactive transcriptional state involves, at some level, a change in the methylation status of the IgH genes. Methylation and transcriptional activity were inversely correlated. In Ig-extinguished hybrids the myeloma-derived locus was methylated de novo, whereas in the rare Ig-expressing hybrid, the T cell-derived locus was demethylated de novo.

The octamer/mu E4 region of the immunoglobulin heavy-chain enhancer mediates gene repression in myeloma x T-lymphoma hybrids.

We have shown previously that the immunoglobulin heavy-chain enhancer acts as a repressor of gene transcription in hybrids between immunoglobulin-producing myelomas and a T-lymphoma line. We have now mapped this repressive activity to a 51-bp enhancer subfragment which contains the octamer and mu E4 protein-binding motifs. Even a single copy of this subfragment will repress gene expression in hybrid cells. Mutational analyses of the repressor fragment suggest that in non-B cells, a strong transcriptional repressor(s) functions through the same motifs important for gene activation in B cells. Changes in chromatin structure that accompany reporter gene repression suggest a general mechanism for prohibiting immunoglobulin heavy-chain locus activation in inappropriate cell types.

Immunoglobulin gene expression only in the right cells at the right time.

Study of immunoglobulin gene transcriptional control has disclosed a complexity that rivals that of the antibody repertoire itself. Although some DNA sequences that regulate transcription of these genes were identified almost a decade ago, additional regulatory sequences continue to be discovered, some in regions far removed (as much as 200 kb) from the actual site of transcription. This has invoked models of regulation in which the higher-order structure of chromatin must be taken into account. Within the regulatory DNA sequences themselves, there is a bewildering array of sites that bind proteins and a growing catalog of proteins that can bind each of those sites. The proteins are believed to be the physical link between the regulatory DNA sequences and the protein machinery actually carrying out transcription. Specific interactions among proteins that can bind the same site and among proteins that bind different sites have been described. A challenge remaining is to identify those interactions that occur in vivo and that lead to progressively more efficient Ig production in the developing B cell, but which disallow Ig production in non-B cells.

An enhancer at the 3' end of the mouse immunoglobulin heavy chain locus.

A tissue-specific enhancer (E mu) lies between the joining (JH) and mu constant region (C mu) gene segments of the immunoglobulin heavy chain (IgH) locus. Since mouse endogenous IgH genes are efficiently transcribed in its absence, the normal function of this enhancer remains ill-defined. Recently, another lymphoid-specific enhancer of equal strength has been identified 3' of the rat IgH locus. We have isolated an analogous sequence from mouse and have mapped it 12.5 kb 3' of the 3'-most constant region gene (C alpha-membrane) of the BALB/c mouse locus. The mouse and rat sequences are 82% homologous and share with other enhancers several DNA sequence motifs capable of binding protein. However, in transient transfection assays, the mouse sequence behaves as a weaker enhancer. The role of this distant element in the expression of endogenous IgH genes, both in E mu-deficient, Ig-producing cell lines and during normal B cell development, is discussed.

Immunoglobulin heavy-chain enhancer is required to maintain transfected gamma 2A gene expression in a pre-B-cell line.

The immunoglobulin heavy-chain (IgH) enhancer serves to activate efficient and accurate transcription of cloned IgH genes when introduced into B lymphomas or myelomas. The role of this enhancer after gene activation, however, is unclear. The endogenous IgH genes in several cell lines, for example, have lost the IgH enhancer by deletion and yet continue to be expressed. This might be explained if the role of the enhancer were to establish high-level gene transcription but not to maintain it. Alternatively, other enhancers might lie adjacent to endogenous IgH genes, substituting their activity for that of the lost IgH enhancer. To address both of these alternatives, we searched for enhancer activity within the flanking regions of one of these IgH enhancer-independent genes and designed an experiment that allowed us to consider separately the establishment and maintenance of expression of a transfected gene. For the latter experiment we generated numerous pre-B cell lines stably transformed with a gamma 2a gene. In this gene, the IgH enhancer lay at a site outside the heavy-chain transcription unit, between DH and JH gene segments. After expression of the transfected gene was established, selective conditions were chosen for the outgrowth of subclones that had undergone D-J joining and thus IgH enhancer deletion. Measurements of gamma 2a expression before and after enhancer deletion revealed that the enhancer was required for maintenance of expression of the transfected gene. The implication of this finding for models of enhancer function in endogenous genes is discussed.

Presentation of IgG2a antigens to class II-restricted T cells by stably transfected B lymphoma cells.

Here we describe a panel of BALB/c T cells specific for IgG2a of the b allotype in association with I-Ad. We used DNA-mediated gene transfer techniques to localize antigenic determinants recognized by responding T cells. Initially a truncated IgG2aa gene comprising a variable domain and the CH3 domain (not including the membrane exons) from the BALB/c IgG2aa heavy chain was introduced into myeloma cells. The V-CH3 protein was expressed at high levels under control of the Ig heavy chain enhancer. Secretion of the V-CH3 protein did not require assembly of H-H dimers or an association with light chains. To generate stably transfected B cell lines that would stimulate our class II-restricted T cells, we replaced most of the BALB/c IgG2aa CH3 exon with CH3 coding sequences from a C57BL/6 IgG2ab cDNA clone and introduced these constructs into Ia+ B lymphoma cells. The IgG2ab CH3-transfected B cells were recognized by BALB/c Igh-1b-specific T cell hybrids in the absence of exogenous antigen. Experiments using glutaraldehyde-fixed cells as stimulators indicate that presentation of the secreted form of V-IgG2ab CH3 requires processing. We found that a significant fraction of the endogenously synthesized V-IgG2ab CH3 protein was, however, present as already processed antigen.

Role of the octamer motif in hybrid cell extinction of immunoglobulin gene expression: extinction is dominant in a two enhancer system.

We have shown previously that genes activated by the immunoglobulin heavy chain (IgH) enhancer or promoter in mouse myeloma cells are extinguished upon fusion of the myeloma with a mouse T cell lymphoma. Here we show that the conserved octamer sequence shared by the IgH enhancer and promoter, when multimerized to form a tissue-specific enhancer, can also render a gene extinguishable under the same experimental conditions. Extinction, however, is not correlated with either absence of the tissue-specific transcription factor OTF-2 or loss of its ability to bind the octamer sequence. It was also found that extinction mediated by the IgH enhancer is dominant to transcriptional activation by the SV40 enhancer. We propose, therefore, that the T cell-negative regulator responsible for IgH gene extinction does not simply prevent IgH enhancer activation but interferes with gene expression more directly, perhaps by disrupting the transcription complex established as a result of tissue-specific enhancer activation.

Genes activated in the presence of an immunoglobulin enhancer or promoter are negatively regulated by a T-lymphoma cell line.

The tissue-specific expression of immunoglobulin genes can be partially explained by a requirement for activating factors found only in B lymphocytes and their derivatives. However, loss of immunoglobulin expression upon fusion of an immunoglobulin-producing myeloma cell with a T lymphoma cell (BW5147) or fibroblast (L cell) suggests that negatively acting factors also play a role in the tissue specificity of immunoglobulin genes. Expression of a cloned immunoglobulin heavy-chain gene introduced into myeloma cells was suppressed after fusion of the myeloma transformants with BW5147. The presence of either the immunoglobulin heavy-chain enhancer or promoter conferred suppression, under similar conditions, upon a heterologous gene that is normally expressed in both B and T lymphocytes. These immunoglobulin heavy-chain gene control regions, or gene modifications induced by them, are subject to negative control by T-lymphocyte-derived factors.

T cell recognition of endogenous IgG2a expressed in B lymphoma cells.

We recently characterized a panel of C57BL/6J T cell clones specific for IgG2a of the a allotype in association with I-Ab. Several of the clones gave surprisingly strong responses in the presence of normal spleen cells from several H-2b strains, including C3H.SW, A.BY, D1.LP and BALB.B, without the addition of exogenous antigen. Experiments using Igh-congenic mouse strains demonstrated that this response was directed towards shared allotypic determinants expressed on endogenously synthesized IgG2a molecules in various strains. Here we present evidence that the cell(s) responsible for this stimulation are conventional low density splenic accessory cells. Presentation of endogenously synthesized IgG2a by this population of dendritic cells, macrophages, and/or activated B cells was chloroquine sensitive. Thus, we conclude that this response is probably directed towards secreted IgG2a molecules that are internalized, processed and re-expressed at the cell surface in association with class II molecules. We also tested the ability of T cell clone B61-34 to respond in the presence of the B lymphoma cell line M12.C3.A2. A strong response was again observed in the absence of exogenous antigen. Northern gel analysis of M12.C3.A2 messenger RNA provided evidence that these cells synthesize IgG2a in both a secreted and a membrane form. The response directed towards endogenous IgG2a expressed in M12.C3.A2 lymphoma cells is chloroquine resistant and considerably more efficient than that stimulated by IgG2a added as exogenous soluble antigen. These results demonstrate for the first time that B cells have the capacity to present antigenic determinants expressed on endogenously synthesized immunoglobulins to class II-restricted T cells. The implications of these findings with respect to network models of immune regulation are discussed.

DNA rearrangement causes a high rate of spontaneous mutation at the immunoglobulin heavy-chain locus of a mouse myeloma cell line.

The spontaneous mutation rate of immunoglobulin genes expressed in myeloma cells is well above that of other genes expressed in these or in other cell types. The nature of such mutations in one myeloma cell line, MPC11, was explored at the molecular level. Included in this study were MPC11 variants representing 24 independent and spontaneous mutations affecting immunoglobulin secretion. Of the mutants studied, 19 had ceased immunoglobulin heavy chain (IgH) production (nonproducers), and 5 produced from as little as 1/1,000 to as much as 1/10 the amount of immunoglobulin produced by MPC11 (low producers). Only one of the MPC11 mutants (a nonproducer) showed no evidence of DNA rearrangement in or near the expressed IgH gene. The formerly expressed gamma 2b gene had been deleted in 18 of the 19 nonproducers. All of the low producers had undergone DNA rearrangement in or near the expressed IgH gene, and three of them produced immunoglobulin of a new heavy chain class. The cause for reduced heavy-chain synthesis in the low producers is not yet known. However, in several of these mutants, the defect appeared to be posttranscriptional. In these cell lines, steady-state IgH mRNA levels were much lower than in the parent cell line, while the heavy-chain gene transcription rate remained unchanged.