Immunoglobulin Heavy Chain Intronic Enhancer Research Articles

Role of immunoglobulin heavy chain intronic enhancer Eμ in cell selection at the pre-B to immature B cell transition (P1437)

Abstract We previously described an Eμ-dependent checkpoint for allelic exclusion, operating at the pre-B to immature B cell transition. We now show that Eμ functions at this transition to ensure that Igμ levels, and consequently B cell receptor (BCR) levels, reach a threshold required for positive selection into the immature B cell pool. We find that Ig-mediated signalling is reduced in pre-B cells expressing an Eμ-deficient IgH allele, as is the size of the emerging immature B cell compartment. Some Eμ-deficient B cells break the rules of allelic exclusion as “double-producers” to achieve higher BCR levels. We previously showed that B cells expressing only the Eμ-deficient allele underwent heightened light-chain editing, consistent with the hypothesis that deficient BCR signals were driving further light chain gene rearrangements in search of an IgH/IgL combination with superior signalling properties. In the current study, we tested this by providing Eμ-deficient mice with a pre-assembled VL gene. As predicted, both heavy chain allelic exclusion and the size of the immature B cell pool were affected. In addition, we found that the double-producers in allelic exclusion-defective mice include a subpopulation in which the BCR generated from the Eμ-deficient allele is autoreactive. These results demonstrate that Igμ levels in developing B cells have an important impact on the breadth of the BCR repertoire, the maintenance of allelic exclusion, and the emergence of autoimmune B cells.

Lymphoid Hyperplasia and Lymphoma in Transgenic Mice Expressing the Small Non-Coding RNA, EBER1 of Epstein-Barr Virus

BackgroundNon-coding RNAs have critical functions in diverse biological processes, particularly in gene regulation. Viruses, like their host cells, employ such functional RNAs and the human cancer associated Epstein-Barr virus (EBV) is no exception. Nearly all EBV associated tumours express the EBV small, non-coding RNAs (EBERs) 1 and 2, however their role in viral pathogenesis remains largely obscure.Methodology/Principal FindingsTo investigate the action of EBER1 in vivo, we produced ten transgenic mouse lines expressing EBER1 in the lymphoid compartment using the mouse immunoglobulin heavy chain intronic enhancer Eμ. Mice of several of these EμEBER1 lines developed lymphoid hyperplasia which in some cases proceeded to B cell malignancy. The hallmark of the transgenic phenotype is enlargement of the spleen and mesenteric lymph nodes and in some cases enlargement of the thymus, liver and peripheral lymph nodes. The tumours were found to be of B cell origin and showed clonal IgH rearrangements. In order to explore if EBER1 would cooperate with c-Myc (deregulated in Burkitt's lymphoma) to accelerate lymphomagenesis, a cross-breeding study was undertaken with EμEBER1 and EμMyc mice. While no significant reduction in latency to lymphoma onset was observed in bi-transgenic mice, c-Myc induction was detected in some EμEBER1 single transgenic tumours, indicative of a functional cooperation.Conclusions/SignificanceThis study is the first to describe the in vivo expression of a polymerase III, non-coding viral gene and demonstrate its oncogenic potential. The data suggest that EBER1 plays an oncogenic role in EBV associated malignant disease.

Bright/ARID3A contributes to chromatin accessibility of the immunoglobulin heavy chain enhancer

Bright/ARID3A is a nuclear matrix-associated transcription factor that stimulates immunoglobulin heavy chain (IgH) expression and Cyclin E1/E2F-dependent cell cycle progression. Bright positively activates IgH transcriptional initiation by binding to ATC-rich P sites within nuclear matrix attachment regions (MARs) flanking the IgH intronic enhancer (Eμ). Over-expression of Bright in cultured B cells was shown to correlate with DNase hypersensitivity of Eμ. We report here further efforts to analyze Bright-mediated Eμ enhancer activation within the physiological constraints of chromatin. A system was established in which VH promoter-driven in vitro transcription on chromatin- reconstituted templates was responsive to Eμ. Bright assisted in blocking the general repression caused by nucleosome assembly but was incapable of stimulating transcription from prebound nucleosome arrays. In vitro transcriptional derepression by Bright was enhanced on templates in which Eμ is flanked by MARs and was inhibited by competition with high affinity Bright binding (P2) sites. DNase hypersensitivity of chromatin-reconstituted Eμ was increased when prepackaged with B cell nuclear extract supplemented with Bright. These results identify Bright as a contributor to accessibility of the IgH enhancer.

A Regulated Nucleocytoplasmic Shuttle Contributes to Bright's Function as a Transcriptional Activator of Immunoglobulin Genes

Bright/ARID3a has been implicated in mitogen- and growth factor-induced up-regulation of immunoglobulin heavy-chain (IgH) genes and in E2F1-dependent G1/S cell cycle progression. For IgH transactivation, Bright binds to nuclear matrix association regions upstream of certain variable region promoters and flanking the IgH intronic enhancer. While Bright protein was previously shown to reside within the nuclear matrix, we show here that a significant amount of Bright resides in the cytoplasm of normal and transformed B cells. Leptomycin B, chromosome region maintenance 1 (CRM1) overexpression, and heterokaryon experiments indicate that Bright actively shuttles between the nucleus and the cytoplasm in a CRM1-dependent manner. We mapped the functional nuclear localization signal to the N-terminal region of REKLES, a domain conserved within ARID3 paralogues. Residues within the C terminus of REKLES contain its nuclear export signal, whose regulation is primarily responsible for Bright shuttling. Growth factor depletion and cell synchronization experiments indicated that Bright shuttling during S phase of the cell cycle leads to an increase in its nuclear abundance. Finally, we show that shuttle-incompetent Bright point mutants, even if sequestered within the nucleus, are incapable of transactivating an IgH reporter gene. Therefore, regulation of Bright's cellular localization appears to be required for its function.

Nuclear Matrix Binding Regulates SATB1-mediated Transcriptional Repression

Special AT-rich binding protein 1 (SATB1) originally was identified as a protein that bound to the nuclear matrix attachment regions (MARs) of the immunoglobulin heavy chain intronic enhancer. Subsequently, SATB1 was shown to repress many genes expressed in the thymus, including interleukin-2 receptor alpha, c-myc, and those encoded by mouse mammary tumor virus (MMTV), a glucocorticoid-responsive retrovirus. SATB1 binds to MARs within the MMTV provirus to repress transcription. To address the role of the nuclear matrix in SATB1-mediated repression, a series of SATB1 deletion constructs was used to determine protein localization. Wild-type SATB1 localized to the soluble nuclear, chromatin, and nuclear matrix fractions. Mutants lacking amino acids 224-278 had a greatly diminished localization to the nuclear matrix, suggesting the presence of a nuclear matrix targeting sequence (NMTS). Transient transfection experiments showed that NMTS fusions to green fluorescent protein or LexA relocalized these proteins to the nuclear matrix. Difficulties with previous assay systems prompted us to develop retroviral vectors to assess effects of different SATB1 domains on expression of MMTV proviruses or integrated reporter genes. SATB1 overexpression repressed MMTV transcription in the presence and absence of functional glucocorticoid receptor. Repression was alleviated by deletion of the NMTS, which did not affect DNA binding, or by deletion of the MAR-binding domain. Our studies indicate that both nuclear matrix association and DNA binding are required for optimal SATB1-mediated repression of the integrated MMTV promoter and may allow insulation from cellular regulatory elements.

Regulation of Immunoglobulin Promoter Activity by TFII-I Class Transcription Factors

The restriction of immunoglobulin variable region promoter activity to B lymphocytes is a well known paradigm of promoter specificity. Recently, a cis-element, located downstream of the transcription initiation site of murine heavy chain variable promoters, was shown to be critical for B cell activity and specificity. Here we show that mutation of this element, termed DICE (Downstream Immunoglobulin Control Element), reduces in vivo activity in B cells. Gel mobility shift assays show that DICE forms B cell-specific complexes that were also sensitive to DICE mutation. DICE mutation strongly reduces the ability of a distal immunoglobulin heavy chain intronic enhancer to stimulate transcription. We also identify a DICE-interacting factor: a TFII-I-related protein known as BEN (also termed Mus-TRD1 and WBSCR11). Dominant-negative and RNAi-mediated knockdown experiments indicate that BEN can both positively and negatively regulate IgH promoter activity, depending on the cell line.

Transcriptional activation by LR1 at the Emu enhancer and switch region sites.

LR1 is a B cell-specific, sequence-specific duplex DNA binding activity which is induced in B cells carrying out class switch recombination. Here we identify several properties of LR1 which enable it to function in transcriptional regulation. We show that LR1 contributes to transcriptional activation by the Emu immunoglobulin heavy chain intron enhancer by binding to a site within the enhancer core. We further show that LR1 bends DNA upon binding. In addition, we show that LR1 is itself a bona fide transcriptional activator, as multimerized LR1 sites produce an element which can enhance transcription from a minimal promoter. In order for class switch recombination to occur, an activating signal must be transmitted via the Emu core, and both S regions targeted for recombination must be actively transcribed. The properties of LR1 that we have identified suggest distinct potential functions of LR1 duplex DNA binding activity in class switch recombination. First, LR1 may contribute to recombinational activation by the Emu core. Second, there are multiple potential LR1 duplex binding sites in each of the G-rich switch regions, and LR1 bound at contiguous sites may enhance recombination by stimulating transcription of the S regions.

Detection of aberrant isotype switch recombination in low-grade and high-grade gastric MALT lymphomas

Gastric mucosa-associated lymphoid tissue (MALT) lymphoma originates from reactive lymphocytic infiltrates during chronic gastritis, closely associated with Helicobacter pylori infection. MALT lymphomas may be either "low grade" or "high grade," and transformation from low grade to high grade can occur. To obtain information on the maturational state of MALT lymphoma cells, we investigated their ability to undergo isotype switch recombination, which together with immunoglobulin variable gene somatic mutation, contributes to normal B-cell maturation. Using specific probes for the immunoglobulin heavy-chain (IgH) switch regions, we found by Southern blot that 3 out of 5 low-grade cases and 2 out of 2 high-grade cases showed rearrangements within IgH switch regions, which appeared aberrant in 4 of the 5 cases. The cloning of two rearranged fragments from one low-grade and one high-grade case confirmed the aberrant nature of the rearranged fragments. A deletion from the switch mu region (S mu) to the first constant mu exon (C mu 1) and a second deletion from the second constant mu exon (C mu 2) to the gamma 3 region (gamma 3) was detected in the low-grade case. In the high-grade case, there was a deletion of the IgH intronic enhancer (E mu) and a 336-base pair (bp) insertion into the S mu region of a gene (KIAA0307) normally located at 15q24. These data demonstrate for the first time the ability of MALT lymphoma cells to undergo aberrant isotype switch recombinations, which might be directly involved in the development or progression of malignancy.

Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms

Helix-Loop-Helix Proteins: Regulators of Transcription in Eucaryotic Organisms

Hypomethylation is necessary but not sufficient for V(D)J recombination within a transgenic substrate

Although an inverse correlation between CpG methylation and V(D)J recombination has been demonstrated for both artificial substrates and endogenous genes, it is not known whether all hypomethylated targets are competent to rearrange or if other factors are required. We have created several artificial V(D)J recombination substrate transgenes whose methylation can be controlled by breeding into different genetic backgrounds. A transgene which contains the immunoglobulin heavy chain intronic enhancer rearranges efficiently in B lymphocytes when the transgene loci are unmethylated. When the same loci become methylated, upon breeding into a different mouse strain, no rearrangement can be detected. A similar transgene, but lacking the enhancer, also shows no evidence of V(D)J recombination when it is methylated. Even when this enhancerless transgene is hypomethylated, however, no V(D)J recombination can be detected in B lymphocytes. Thus, hypomethylation is required to permit V(D)J recombination but not all hypomethylated targets are capable of recombination. The results may indicate that the immunoglobulin enhancer is required for the assembly of factors involved in V(D)J recombination.

Cux/CDP homeoprotein is a component of NF-muNR and represses the immunoglobulin heavy chain intronic enhancer by antagonizing the bright transcription activator.

Nuclear matrix attachment regions (MARs) flanking the immunoglobulin heavy chain intronic enhancer (Emu) are the targets of the negative regulator, NF-muNR, found in non-B and early pre-B cells. Expression library screening with NF-muNR binding sites yielded a cDNA clone encoding an alternatively spliced form of the Cux/CDP homeodomain protein. Cux/CDP fulfills criteria required for NF-muNR identity. It is expressed in non-B and early pre-B cells but not mature B cells. It binds to NF-muNR binding sites within Emu with appropriate differential affinities. Antiserum specific for Cux/CDP recognizes a polypeptide of the predicted size in affinity-purified NF-muNR preparations and binds NF-muNR complexed with DNA. Cotransfection with Cux/CDP represses the activity of Emu via the MAR sequences in both B and non-B cells. Cux/CDP antagonizes the effects of the Bright transcription activator at both the DNA binding and functional levels. We propose that Cux/CDP regulates cell-type-restricted, differentiation stage-specific Emu enhancer activity by interfering with the function of nuclear matrix-bound transcription activators.

Evaluation of antisense inhibition in a B cell model.

An in vitro model has been developed in an attempt to optimise antisense inhibition in B cells as a prelude to transgenic studies. The hypotheses tested were that i) the 3'-untranslated region would be an appropriate target for antisense inhibition; 2) the immunoglobulin heavy chain intronic enhancer could be used to enhance antisense inhibition via increased production of antisense transcripts; and 3) the mouse metallothionein-1 promoter would allow induction of antisense inhibition in B cells. Secreted IgM protein and mRNA were monitored following the stable transfection of a B cell line, HO-2.2, with a series of plasmid constructs containing antisense or sense target sequence DNA under the control of either the mouse metallothionein-1 promoter or homologous (ie same promoter as target sequence) immunoglobulin heavy chain promoter. The 3'-untranslated region proved to be an appropriate target resulting in 70% inhibition of IgM secretion. Compared with untransfected and sense controls, significant decreases in IgM secretion (and RNA levels) were detected in clones transfected with antisense constructs utilising the mouse metallothionein-1 promoter and the immunoglobulin heavy chain intronic enhancer elements. These clones exhibited a further significant reduction in secreted IgM production upon zinc induction. Hybridisation studies demonstrated that decreased protein production was most likely attributable to reduction in RNA levels. In contrast, transfection with antisense constructs had no effect on membrane IgM protein levels which not only confirmed the specificity of antisense action but meant that the B cell remained sensitive to receptor ligation. We conclude that reasonable antisense inhibition of gene product expression can be achieved in B cells by targeting the 3'-untranslated region and using both an inducible promoter (mouse metallothionein-1) and the IgH enhancer to aid antisense RNA production.

V(D)J recombination frequency is affected by the sequence interposed between a pair of recombination signals: sequence comparison reveals a putative recombinational enhancer element.

The immunoglobulin heavy chain intron enhancer (Emu) not only stimulates transcription but also V(D)J recombination of chromosomally integrated recombination substrates. We aimed at reproducing this effect in recombination competent cells by transient transfection of extrachromosomal substrates. These we prepared by interposing between the recombination signal sequences (RSS) of the plasmid pBlueRec various fragments, including Emu, possibly affecting V(D)J recombination. Our work shows that sequences inserted between RSS 23 and RSS 12, with distances from their proximal ends of 26 and 284 bp respectively, can markedly affect the frequency of V(D)J recombination. We report that the entire Emu, the Emu core as well as its flanking 5' and 3' matrix associated regions (5' and 3' MARs) upregulate V(D)J recombination while the downstream section of the 3' MAR of Emu does not. Also, prokaryotic sequences markedly suppress V(D)J recombination. This confirms previous results obtained with chromosomally integrated substrates, except for the finding that the full length 3' MAR of Emu stimulates V(D)J recombination in an episomal but not in a chromosomal context. The fact that other MARs do not share this activity suggests that the effect is no mediated through attachment of the recombination substrate to a nuclear matrix-associated recombination complex but through cis-activation. The presence of a 26 bp A-T-rich sequence motif in the 5' and 3' MARs of Emu and in all of the other upregulating fragments investigated, leads us to propose that the motif represents a novel recombinational enhancer element distinct from those constituting the Emu core.

Evaluation of novel control elements by construction of eukaryotic expression vectors

A novel mammalian eukaryotic expression vector for the production of immunoglobulin heavy chain (IgH) genes has been designed. This expression vector contains the variable heavy chain (VH) promoter, the IgH intron enhancer (μE) and the IgH 3′ enhancer (3′E). This construct, designated pTIF-1, was stably transfected into the myeloma cell line J558L. A fivefold increase in the expression level of a rearranged IgH gene was observed when using the pTIF-1 vector containing the 3′E compared to an expression vector lacking this enhancer. Interestingly, this positive effect on the expression level of the 3′ enhancer appears to be position independent. The introduction of two recently identified Ig control elements, HS3 and HS4, to the vector cassette did not further elevate the expression level in the cell line tested. The pTIF-1 vector can be used for expression of any antibody specificity, using PCR amplification of the VDJ region of interest. Furthermore, the constant region can easily be exchanged, which further facilitates studies to dissect different effector functions of IgH constant genes.

Establishment and characterization of choroid plexus carcinoma cell lines: connection between choroid plexus and immune systems.

Murine choroid plexus cell lines were produced from choroid plexus carcinoma generated in transgenic mice harboring the viral oncogene simian virus 40 large tumor antigen under transcriptional control of an intronic enhancer region from the human immunoglobulin heavy chain (IgH) gene. Two morphologically distinct cell lines have been cloned. These established cell lines retained the characteristics of choroid plexus cells in that they expressed such choroid plexus cell marker or related proteins as transthyretin and α2‐macroglobulin. They were tumorigenic in nude mice. In the cell lines, the μA and μB (HE2) motifs within the IgH intronic enhancer were active and we also demonstrated the existence of the proteins binding to these motifs, suggesting a potential link between the choroid plexus and immune systems. It is considered that these binding proteins act as trans‐activators for the enhancer and may belong to the class of ETS‐related proteins. These cell lines and xenografts should be useful materials for analyses of choroid plexus functions.

Gene-targeted deletion and replacement mutations of the T-cell receptor beta-chain enhancer: the role of enhancer elements in controlling V(D)J recombination accessibility.

To assess the role of transcriptional enhancers in regulating accessibility of the T-cell receptor beta-chain (TCRbeta) locus, we generated embryonic stem cell lines in which a single allelic copy of the endogenous TCRbeta enhancer (Ebeta) was either deleted or replaced with the immunoglobulin heavy-chain intronic enhancer. We assayed the effects of these mutations on activation of the TCRbeta locus in normal T- and B-lineage cells by RAG-2 (recombination-activating gene 2)-deficient blastocyst complementation. We found that Ebeta is required for rearrangement and germ-line transcription of the TCRbeta locus in T-lineage cells. In the absence of Ebeta, the heavy-chain intronic enhancer partially supported joining region beta-chain rearrangement in T- but not in B-lineage cells. However, ability of the heavy-chain intronic enhancer to induce rearrangements was blocked by linkage to an expressed neomycin-resistance gene (neo(r)). These results demonstrate a critical role for Ebeta in promoting accessibility of the TCRbeta locus and suggest that additional negative elements may cooperate to further modulate this process.

Early B‐Cell Factor (EBF) Down‐Regulates Immunoglobulin Heavy Chain Intron Enhancer Function in a Plasmacytoma Cell Line

The immunoglobulin heavy chain intron enhancer contains two potential binding sites for early B-cell factor (EBF). To investigate the functional properties of these, EBF was expressed in the EBF non-expressing S194 plasmacytoma cell line and found to down-regulate the activity of a co-transfected immunoglobulin heavy chain intron enhancer reporter construct. The expression of an unrelated reporter construct was unaltered. Dividing the immunoglobulin heavy chain intron enhancer into two subregions showed that the EBF mediated down-regulation of expression was mediated by at least two independent sites. These data indicate a role for EBF in the regulation of immunoglobulin gene expression.

Regulatory regions 3' of the immunoglobulin heavy chain intronic enhancer differentially affect expression of a heavy chain transgene in resting and activated B cells.

We have compared the expression patterns of three Ig heavy chain transgenes. The three constructs differ only by deletion of J-C intron sequences located downstream of the Emu enhancer region. When stably transfected into a myeloma cell line, all three constructs are expressed at comparable levels. However, transgenic mice carrying each construct show dramatic differences in transgene expression. Our results indicate that, in addition to the Emu enhancer, at least two regions, RegA and RegS, within the J-C intron influence transgene expression. RegA, located directly downstream of the core Emu enhancer, is involved in up-regulation of transgene expression after LPS activation of splenocytes. RegS, located within or downstream of the Smu switch region, is important for normal levels of transgene expression in splenocytes of heavy chain transgenic mice.

IgH Intronic Enhancer Element HE2 (μB) Functions as a cis‐Activator in Choroid Plexus Cells at the Cellular Level as well as in Transgenic Mice

Immunoglobulin heavy-chain (IgH) gene expression is regulated largely by the IgH gene intronic enhancer (ENHiH), which is composed of multiple protein-binding motifs. These motifs are DNA elements that are important for the regulation of IgH gene transcription. It has been reported that the HE2 (mu B) and mu A motifs within the ENHiH affect B cell-specific gene expression. To examine the function of the HE2 and mu A elements in vivo, we established transgenic mouse lines. A deletion mutant of the human ENHiH that contains the HE2 and mu A motifs, but lacks the motifs corresponding to murine E5, E3, and octamer, functioned not only in B lymphocytes but also in choroid plexus cells, which secrete CSF. As a result, we obtained choroid plexus tumor-bearing transgenic mice and could establish choroid plexus carcinoma cell lines. In addition, using the luciferase assay, we confirmed at the cellular level that the HE2 motif shows a fair degree of enhancer activity in cultured choroid plexus carcinoma cells. These results suggest the existence of a trans-acting factor for the HE2 motif in choroid plexus cells. Actually, in this cultured cell line, the existence of a protein binding to the HE2 motif was demonstrated by a gel retardation assay. Due to the sequence homology between the HE2 motif and the Ets-binding sites, an Ets-related protein is a highly probable candidate for being the binding factor.

Regulation and targeting of recombination in extrachromosomal substrates carrying immunoglobulin switch region sequences.

We have used extrachromosomal substrates carrying immunoglobulin heavy-chain S mu and S gamma 3 switch region sequences to study activation and targeting of recombination by a transcriptional enhancer element. Substrates are transiently introduced into activated primary murine B cells, in which recombination involving S-region sequences deletes a conditionally lethal marker, and recombination is measured by transformation of Escherichia coli in the second step of the assay. Previously we found that as many as 25% of replicated substrates recombined during 40-h transfection of lipopolysaccharide (LPS)-stimulated primary cells and that efficient recombination was dependent on the presence of S-region sequences as well as a transcriptional activator region in the constructs (H. Leung and N. Maizels, Proc. Natl. Acad. Sci. USA 89:4154-4158, 1992). Here we show that recombination of the switch substrates is threefold more efficient in LPS-cultured primary B cells than in the T-cell line EL4; the activities responsible for switch substrate recombination thus appear to be more abundant or more active in cells which can carry out chromosomal switch recombination. We test the role of the transcriptional activator region and show that the immunoglobulin heavy-chain intron enhancer (E mu) alone stimulates recombination as well as E mu combined with a heavy-chain promoter and that mutations that diminish enhancer-dependent transcription 500-fold diminish recombinational activation less than 2-fold. These observations suggest that the enhancer stimulates recombination by a mechanism that does not depend on transcript production or that is insensitive to the level of transcript production over a very broad range. Furthermore, we find that E mu stimulates recombination when located either upstream or downstream of S mu but that the position of the recombinational activator does affect the targeting of recombination junctions, suggesting that the relatively imprecise targeting of switch junctions in vivo may reflect the availability of many potential activator sites within each switch region.