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Regulation of Germline Promoters by the Two Human Ig Heavy Chain 3' Enhancers¹

Yanzhong Hu^*, Qiang Pan^*, Evangelia Pardali, Frederick C. Mills, Ralph M. Bernstein, Edward E. Max, Paschalis Sideras and Lennart Hammarström^2,*

^* Division of Clinical Immunology, Karolinska Institutet at Huddinge Hospital and Center for Oral Biology, Novum, Huddinge, Sweden; Department for Cell and Molecular Biology, Umeå University, Umeå, Sweden; and Division of Therapeutic Proteins, Center for Biologic Evaluation and Research, Food and Drug Administration, Bethesda, MD 20892

	Abstract

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The human IgH 3' enhancers, located downstream of each of the two C genes, modulate germline (GL) transcription of the IgH genes by influencing the activity of promoter-enhancer complexes upstream of the switch and intervening (I) regions. The regulation of GL 1 and 2 promoters by different human 3' enhancer fragments was investigated in cell lines representing various developmental stages. Both 1HS1,2 and 2HS1,2 fragments show equally strong enhancer activity on the GL 1 and 2 promoters in both orientations when transiently transfected into a number of mature B cell line (DG75, CL-01, and HS Sultan). However, there is no activity in a human pre-B cell line (NALM-6) nor a human T cell line (Jurkat). HS3 shows no enhancer activity by itself in any of the cell lines, whereas a modest effect is noted using HS4 in the three mature B cell lines. However, the combination of the 2HS3-HS1,2-HS4 fragments, which together form a potential locus control region, displays a markedly stronger enhancer activity than the individual fragments with a differential effect on the 1 and 2 promoters as compared with the 3 promoter. Our results suggest that the human GL promoter may be regulated by two independent pathways. One pathway is induced by TGF-ß₁ which directs IgA isotype switch through activation of the GL promoter and no TGF-ß₁-responsive elements are present in the different 3' enhancer fragments. The other route is through the human 3' enhancer regions that cis-up-regulate the GL promoter activity in mature B cells.

	Introduction

Top Abstract Introduction Materials and Methods Results Discussion References

 
The human Ig heavy chain locus is located on the long arm of chromosome 14 and contains a duplicated set of --- genes, giving rise to two IgA subclasses: IgA1 and IgA2 (1). Both participate in the mucosal immune defense (2) but are expressed to varying degrees at different anatomical sites. However, the basic molecular mechanism regulating the production of IgA1 vs IgA2 is still unknown.

IgM-positive B lymphocytes switch to IgG, IgA, or IgE production upon Ag or mitogen stimulation (3, 4). The transition, known as the heavy chain class switch, does not alter the variable region of the Ig molecule produced and therefore it does not change the Ag binding specificity. The genetic basis of the isotype switch is a DNA looping out and deletion recombination event, which replaces the expressed Cµ-C gene complex with one of the seven downstream heavy chain genes and removes the intervening DNA (5, 6). This recombination occurs between regions of repetitive DNA sequences, switch regions (S), which are located 5' of each heavy chain constant region gene, except . The Ab isotype is not utilized randomly and specific stimuli induce B cell responses with characteristic isotype profiles (7, 8, 9, 10, 11).

Cytokine-directed isotype switching is preceded by expression of the corresponding I region and IgH constant region (CH) gene. The cytokine-induced germ line (GL)³ transcripts promote switch recombination by increasing the accessibility of the switch region to a recombinase (12, 13). TGF-ß is a factor that can direct switching from IgM to IgA in humans (14, 15) and mice (16, 17) by inducing GL transcripts (15, 18) through activation of its corresponding promoter/enhancer elements in the I region (19, 20, 21, 22) and it also increases the half life of mRNA (17). The GL promoter regions both contain a putative cAMP-responsive element-binding protein site, Pu.1 and Sp1 binding sites, that are highly homologous to each other and act as basal promoters, and the transcription factor AML1 is able to mediate the TGF-ß₁-induced activation of the GL promoter (23). Upstream of the basal promoter elements there are two silencers which regulate the basal promoter activity (18).

cis-Regulatory elements have been described within the IGHC gene locus and participate in the regulation of IgH gene expression and isotype switch. The intronic Eµ enhancer, located between the J segments and the µ-chain gene (24, 25) regulates the assembly of VDJ and µ-chain genes (26, 27) and may act as a promoter regulating µ GL transcripts (28). A large deletion extending from the J_H segments through Eµ results in a significant block of switch recombination at the µ locus, possibly by shutting down transcription through Sµ (27). More recent data have shown that switching is affected but not abolished by deletion of Eµ and its flanking region (29) or Eµ core sequences alone (30). This phenomenon, taken together with data of Gu et al. (27), suggests retained switching capacity at the locus (27, 31) and implies that there could be another cis-regulatory element within the IGHC gene locus that is involved in the regulation of IgH transcription and gene rearrangement.

A sequence with enhancer activity was previously found about 16 kb downstream of the C gene in the mouse (32) and 25 kb downstream of the C gene in the rat (33). The potential function of these 3' enhancers includes regulation of the IGHC gene locus rearrangements, transcription, isotype switch (33, 34, 35, 36, 37), and somatic mutation (38). The presence of an enhancer in the human IgCH locus was long suspected but the region of interest was difficult to clone due to the presence of a stretch of tandem repeats (39) sequences. However, Mills et al. (40) recently cloned three B cell-specific DNase I-hypersensitive elements (HS1,2, HS3, and HS4) located downstream of the C1 and C2 genes which were homologous to the four murine HS fragments. These fragments were also independently described by two additional research groups (41, 42). The homologous sequences were more conserved in the core sequence rather than in the flanking repeats and showed enhancer activity in transient infections (40, 41, 42).

The mouse 3' enhancers have been shown to play an important role in regulation of the Ig isotype switching through their influence on the activity of the GL region promoters (43). In this paper, we present the first data describing the potential function of the human 3' IgH enhancers in regulation of IgA isotype switching via interaction with the GL promoters. Moreover, since the mouse enhancers have been suggested to function together as a locus control region (LCR) (44), we have studied the synergy of various human 3' enhancer combinations.

	Materials and Methods

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Vector construction

The pGL-2 basal luciferase vector (Promega, Madison, WI) was modified by inserting a SphI-NotI polylinker at bp 5451–5453 to remove the putative TGF-ß-responsive binding site (Fig. 1c). The E fragment, containing the basal promoter element and the D and B fragments containing silencer elements (Fig. 1b) (18), were cut out with the restriction enzymes SalI and XbaI from chloramphenicol acetyltransferase reporter constructs (18) and were subcloned between the XhoI and SphI restriction sites which generated the "backbone" constructs I1-E, I1-D, I1-B, I2-E, I2-D, and I2-B. The WT-228 construct was made by inserting the GL 3 promoter fragment (45) into the pGL-3 basal vector.

View larger version (28K): [in this window] [in a new window]   FIGURE 1. Schematic map of the human IgH 3' enhancer fragments (a), the intervening region (I) upstream of the 1 and 2 switch regions (b), and the luciferase reporter vector pGL-2 (c). a, 3' enhancer fragments located downstream of both C1 and C2 genes are indicated by open circles (38 ). The 1HS3, 1HS1,2, and 1HS4 fragments are located 7, 11, and 20 kb downstream of the C1 gene, respectively, whereas the 2HS3, 2HS1,2, and 2HS4 fragments are located about 7, 12, and 22 kb downstream of the C2 gene. b, The white rectangle represents the I1 and I2 exons, the E represents the basal GL promoter from -248 to +79; the D fragment includes the E element and its upstream silencer from -351 to +79 (the silencer is indicted by a gray rectangle). The B represents a distal GL promoter region which includes two silencer fragments and its downstream E element from -627 to +79. c, A SphI-NotI polylinker was inserted at the bp 5451–5453 of the basal pGL-2 vector. The different GL 1 or GL 2 promoter fragments were inserted between at the XhoI and SphI sites with the same orientation as the luciferase gene. The enhancer fragment inserting site lies at 2004 bp (BamHI site) or 2010 bp (SalI site) downstream of the luciferase reporter gene (indicated by a black arrow).

1HS1,2. A 960-bp fragment was PCR amplified using the construct pGL3-1HS1,2 (40) as a template. The primers SA2.1A and SA2.2B (40) were employed although the original MluI recognition site at the 5' and 3' end of each primer was changed into a BamHI recognition site. The 1HS1,2 fragment was directly subcloned into the backbone constructs at a BamHI site in both orientations. The orientation of the insert in HS1,2-containing constructs was determined by digestion with EcoRI. The constructs containing a single copy of the insert were identified by digestion with HindIII and EcoRI restriction enzymes.

2HS1,2. A 1070-bp 2HS1,2 fragment was derived from the construct pE1.3 (40) using PCR. The primers and the subcloning site in the backbone constructs were the same as in the 1HS1,2.

1HS3. A 982-bp 1HS3 fragment was generated from the construct pGL-1HS3 (40) using PCR employing the primers SA2.5A and SA 2.6A (40). The product was inserted into the intermediate vector pB-S at a HindIII restriction site and then subcloned at a SalI site in the backbone constructs in both orientations (determined by digestion with BamHI).

2HS3. A 700-bp 2HS3 fragment was derived from the construct pSM 0.7 (40) by digestion with SmaI and inserted at a SmaI site in the intermediate vector pB-S. The fragment was then subcloned into the backbone vectors at a SalI site in both orientations (determined by sequencing).

2HS4. A 468-bp fragment was generated from the construct pA2E14 (40) using PCR employing primers SA8A and SA9B (40). The 2HS4 fragment was first cloned at a SacII site of the pB-S vector and then subcloned at a SalI site in the backbone constructs in both orientations (determined by sequencing).

1HS3-HS1,2. The 1HS1,2 fragment was first subcloned at the BamHI site of the intermediate vector pB-S-generating pB-S-HS1,2. The 1HS3 was inserted at the HindIII restriction site of pB-S-HS1,2-generating pB-S-HS3-HS1,2. The 1HS3-HS1,2 was then subcloned into the corresponding backbone vectors at the SalI restriction site. The orientation and copy number of the 1HS3-HS1,2 in the backbone constructs were determined by digestion with EcoRI.

2HS3-HS1,2-HS4. The 2HS1,2 and 2HS4 were inserted into the intermediate vector pB-S at BamHI and SacII sites, respectively, generating pB-S HS1,2-HS4. The 2HS3 was inserted at the SmaI site of construct pB-SHS1,2-HS4-generating pB-S-HS3-HS1,2-HS4. The orientation of each fragment was determined by digestion with BamHI (HS3), EcoRI (HS1, 2), or by sequencing (HS4). The 2HS3-HS1,2-HS4 was subcloned into the corresponding backbone vectors at the SalI restriction site. The orientation of the 2HS3-HS1,2-HS4 in the backbone vectors was determined by digestion with ClaI.

A schematic map of the human 3' enhancer segments (1HS1,2, 1HS3 and 2HS1,2, 2HS3, and 2HS4) presented by Mills et al. (40) is shown in Fig. 1a. A map of the enhancer containing constructs is shown in Fig. 2. The restriction enzymes and T₄ ligase were purchased from Promega.

View larger version (28K): [in this window] [in a new window]   FIGURE 2. Schematic diagram of the test constructs. The distal GL promoter fragments are indicated by the filled rectangles which are inserted upstream of the luciferase gene. The B, D, and E represent the GL 1 (2) promoter fragments indicated in Fig. 1b. The enhancer fragments inserted downstream of the luciferase gene are represented by an open rectangle. The orientation of the enhancers is indicated by the arrow. 1HS1,2 and 2HS1,2 are inserted at the BamHI site directly, whereas HS3, HS4, and HS3-HS1,2-HS4 were first cloned into the modified pB-S vector (see Materials and Methods) and then subcloned at the SalI restriction site.

The DG75 cell line (EBV- negative Burkitt’s lymphoma, sIgM⁺) (37), CL-01 (46) (a germinal center phenotype Burkitt’s lymphoma, a kind gift from Paolo Casali, Cornell University Medical School, New York, NY), HS Sultan (Burkitt’s lymphoma, obtained from the American Type Culture Collection, Manassas, VA), Jurkat (37) (a CD4-positive human T cell line), and NALM-6 (37) (a human pre-B cell line, cyIgM⁺, sIgM^-) were all cultured in RPMI 1640 medium supplemented with 100 µg/ml penicillin, 100 µg/ml streptomycin, 2 mM 2-ME, 2 mM L-glutamine, 10 µM sodium pyruvate, and 10% FCS at 37°C in an atmosphere of 5% CO₂. TGF-ß₁ was purchased from British Biotechnology (Oxford, U.K.) and used at a final concentration of 1 ng/ml.

Transfection

Cells were transiently transfected with the supercoiled plasmids by electroporation. Three to 5 µg of enhancer containing constructs along with 2 µg of human CMV-ß-gal construct were mixed with 10⁷ cells in 500 of µl complete medium. The cells were exposed to a single pulse at 960 µF and at an appropriate voltage for each cell line (300 V for the DG75 cell line, 360 V for the pre-B cell line NALM-6, 180 V for HS Sultan and CL-07, and 380 V for the Jurkat cell line) (18) using either an Electro Cell Manipulator (BTX, San Diego, CA; for HS sultan and CL-07) or a gene pulser transformation apparatus (Bio-Rad, Hercules, CA). The transfected cells were left for 5 min at room temperature and then transferred into 10 ml of complete medium. The cells were harvested after 24 h of growth, washed once with PBS buffer, and resuspended in 100 µl of reporter lysis buffer (25 mM Tris-acetate (pH 7.8), 2 mM DTT, 1 mM EDTA, 10% glycerol, and 1% Triton X-100) for 15 min at room temperature. The cell debris was removed by centrifugation at 12,000 rpm for 2 min. The supernatants were transferred into another tube and used for detecting the luciferase activity and ß-gal activity. Ten microliters of the cell extracts was incubated with 100 µl of luciferase substrate and 100 µl of ATP buffer. The luciferase assay light output was detected in a microplate luminometer (Anthos Labtec Instruments, Salzburg, Austria). The human CMV-ß-gal plasmid carrying the human CMV promoter linked to the ß-gal was used to normalize the cell transfection efficiency. For most experiments, the ß-gal activity was measured by incubating 10 µl of cell lysis along with 250 µl of Z buffer [60 mM Na₂HPO₄, 40 mM NaH₂PO₄, 10 mM KCl, 1.0 mM MgSO₄, and 50 mM 2-ME pH 8.0)] and 50 µl of o-nitrophenyl-ß-D-galatopyranoside buffer (concentration: 4 mg/ml of Z buffer). The reaction was stopped by adding 250 µl of stop buffer (1 M Na₂CO₃). Five hundred microliters of the mixture was used to measure at 420 nm in a DU-20 spectrophometer (Beckman Coulter, Fullerton, CA). For some experiments, ß-galactokinase was assayed using the Galacto-Light Plus Chemiluminescent Reporter Assay kit (Tropix, Bedford, MA), according to the manufacturer’s instructions.

	Results

Top Abstract Introduction Materials and Methods Results Discussion References

 
Human 3' IgH enhancer fragments regulate the GL 1 and 2 promoters

Both the GL 1 and 2 basal promoter fragments (I1-E and I2-E) and their upstream regions (D and B elements) were inserted 5' of the modified pGL-2 basic vector and used as targets to detect the regulatory activity of the 3' enhancer fragments in the DG75 cell line. The enhancer fragments (1HS1,2, 2HS1,2, 1HS3, 2HS3, and 2HS4) were inserted about 2 kb downstream of the promoter region (Fig. 1c). The basal promoter of the construct I1-E was more active than the I1-B and I1-D constructs due to silencer activity within the B and D elements (18). Similar results were seen using the GL 2 promoter (Figs. 3 and 4). The silencers upstream of the GL 1 basal promoter, although highly homologous to the silencers in the I2 region, were stronger than those of the GL 2 basal promoter (Figs. 3 and 4), a finding which agrees with previous results (18). Fragments containing HS1,2 increased the activity of I1-E about 8-to 10-fold and the activity of the I1-B and I1-D about 5- to 6-fold in both orientations (Fig. 3). The enhancer effect on the I1-E promoter fragment was also observed in the additional B cell lines tested (CL-01, data not shown and Sultan, Fig. 5B). It is likely that the silencers within the I1-D and I1-B fragments caused the reduced enhancement by 1HS1,2 and 2HS1,2 relative to that seen in 1HS1,2/I1-E and 2HS1,2/I2-E constructs. The up-regulation of the GL 2 promoter region by 1HS1,2 and 2HS1,2 was similar to that of the GL 1 promoter (Fig. 4). Taken together, these results demonstrated that the 1HS1,2 and 2HS1,2 enhancers can activate the basal promoter fragments I1-E and I2-E. The 1HS3 fragment showed no enhancer effect on the GL 1 promoters in either orientation and neither 1HS3 nor 2HS3 fragments showed any enhancer activity with the GL 2 promoter (data not shown), whereas the 2HS4 fragment has a low effect on both GL promoters. Constructs containing I1-E-2HS1,2 and I1-E-2HS3-HS1,2-HS4 showed no enhancer activity (measured as relative fold induction as compared with promoter only constructs) in either the NALM-6 human pre-B cell line (1.06 ± 0.17 and 0.72 ± 0.16, respectively) or the Jurkat human T cell line (1.01 ± 0.26 and 0.66 ± 0.12). HS3 and HS4 showed no enhancer activity in either cell line. Thus, for constructs containing a single enhancer, only those with 1 or 2 HS1,2 are capable of activating the GL 1 and 2 promoters, and the activation is restricted to the mature B cell lineage.

View larger version (28K): [in this window] [in a new window]   FIGURE 3. The enhancer activities of 1HS1,2 and 2HS1,2 fragments on the I1 promoter in the DG75 cell line. Luciferase activity was normalized by ß-gal expression and presented as relative luciferase activity. Results represent the means ± SEM of three independent experiments. The filled bar represents the correct orientation of the inserted enhancer fragment and the shaded bar represents the reverse orientation of the inserted enhancer fragments.

View larger version (17K): [in this window] [in a new window]   FIGURE 4. The enhancer activities of 1HS1,2 and 2HS1,2 fragments on the I2 promoter in the DG75 cell line. Luciferase activity was normalized by ß-gal expression and presented as relative luciferase activity. Results represent the means ± SEM of three independent experiments.

View larger version (18K): [in this window] [in a new window]   FIGURE 5. The enhancer activities of the combination of enhancer fragments in the regulation of the GL 1 promoter. The relative fold enhancement is determined by dividing the value of the normalized luciferase which was obtained for each construct by the normalized values generated by the enhancerless I1-E construct. Results represent the means ± SD of three independent experiments. A, The DG75 cell line. B, The Sultan cell line.

The synergistic effect of the enhancer segments in regulation of the GL 1 promoter

Because the combination of mouse HS3B, HS1,2, and HS4 enhancers possesses LCR activity (44), we next investigated potential synergistic effects by linking the three human HS fragments together. The schematic map of the 1HS3-HS1,2 (1HS4 is not yet available), 2HS3-HS1,2-HS4, and 2HS3-HS1,2 complexes are shown in Fig. 1a. The recombinants were transiently transfected into the three mature B cell lines (DG75, CL-01, and HS Sultan). There was no difference between the 1HS1,2 and 1HS3-HS1,2 or 2HS1,2 and 2HS3-HS1,2 constructs with regard to activation of the GL 1 or 2 promoter in the cell line tested (DG75) (Fig. 5A and data not shown). Similarly, there was no appreciable difference between 2HS1,2 and 2HS3-HS1,2 in HS Sultan (Fig. 5B) or in CL-01 (data not shown). Although constructs containing the GL 1 promoter in combination with 2HS1,2 and 2HS3-HS1,2 and 2HS3-HS1,2-HS4 all showed similar activity in CL-01 (data not shown), in DG75 the 2HS3-HS1,2-HS4 complex induced a markedly stronger activity on both GL 1 and GL 2 promoters than the 2HS3-HS1,2 complex, 2HS1,2, 2HS3, or 2HS4 (Fig. 5A). A similar increase with the 2HS3-HS1,2-HS4 complex was observed in HS Sultan (Fig. 5B). A synergistic enhancement in the DG75 cell line was also observed in the silencer containing I1-D constructs but with lower fold induction as compared with the I1-E constructs (Fig. 5A). Since the mouse 3' enhancer fragments show a more pronounced effect on switching to IgG than IgA (43), we also investigated the effect of the 2HS3-HS1,2-HS4 enhancer combination on the activity of the GL 3 promoter. GL 3 promoter activity is not detectable when 3 µg of the enhancerless construct was used, a finding in contrast to results using the GL promoter constructs. When 5 µg of the constructs was transfected, the activity of the GL 3 promoter itself (measured as relative fold induction) (0.26 ± 0.02) is only half of that of the GL 1 promoter (0.65 ± 0.01) (Fig. 6). However, the increase in GL 3 promoter activity for the 2HS3-HS1,2-HS4-GL 3 construct relative to the enhancerless GL 3 promoter construct was approximately twice the relative increase observed for the corresponding GL 1 promoter constructs. These results imply that the GL promoter of each isotype may be differentially regulated by the 3' enhancers.

View larger version (15K): [in this window] [in a new window]   FIGURE 6. Comparison between the GL 1 and GL 3 promoters and the influence of the 2-HS3-HS1,2-HS4 fragment. Five micrograms of each construct was transiently transfected into the DG75 cell line. The relative enhancement fold induction is calculated from three separate experiments by dividing the ß-gal normalized values of the constructs by the normalized values of the basic pGL-3 vector. The error bars indicate SD.

The regulation of GL 1 and 2 promoters by the human 3' C1 and C2 enhancer is TGF-ß₁ independent

TGF-ß₁ is able to induce C GL transcripts in human B cells and to induce subsequent switch to IgA production by up-regulating the GL promoters (14, 18). Since our results showed that the human 3' enhancers could up-regulate the activities of both human GL promoters, the interaction between TGF-ß₁ and the 3' enhancers in the regulation of both human GL promoters was investigated in the DG75 cell line. TGF-ß₁ induced GL 1 promoter activity 3- to 7-fold, which agrees with previous results (18). A similar fold induction was observed after TGF-ß₁ stimulation using the enhancer-containing constructs (Fig. 7), suggesting that there are no TGF-ß₁-responsive elements in the enhancer and a sequence analysis showed no TGF-ß₁-responsive elements in the enhancer core sequences. Furthermore, stimulation with TGF-ß1 did not influence the activity of a construct containing the human GL 3 promoter (a TGF-ß₁-nonresponsive promoter) and the 2HS3-HS1,2-HS4 enhancer elements (data not shown).

View larger version (18K): [in this window] [in a new window]   FIGURE 7. Enhancer influence on the GL 1-E and 1-D promoter elements in the DG75 cell line with and without addition of TGF-ß1. The relative fold enhancement is calculated from three separate experiments by dividing the normalized values of each construct by the normalized values of the enhancerless construct (I1-E). The filled bars represent cultures without TGF-ß1; the open bars represent levels after TGF-ß1 induction. The error bars indicate SD.

	Discussion

Top Abstract Introduction Materials and Methods Results Discussion References

LCRs are defined in terms of their ability to confer position-independent activation of mouse transgenes (49) or stable integrants in cell lines (44). Similar to the enhancers comprising the mouse 3' IgH LCR (32, 43, 50), human HS3, HS1,2, and HS4 interact synergistically with each other in transient transfections and all three enhancer elements may be needed in the activation of the C genes before switching.

The two human GL promoters are 98% homologous (14), with identical TGF-ß₁-responsive elements (51), and this cytokine induces GL transcripts of both IgA subclasses in human B cells (21). These observations suggest that the GL promoter itself may not contain enough sequence information to ensure subclass-restricted expression and additional cis-elements such as the different enhancer elements, independent of the TGF-ß₁ pathway, may be needed. However, additional factors such as sequences upstream of the promoters, the chromatin configuration, or the presence of insulators may also affect the transcriptional rate.

The products of the first IGHC (3-1--1) locus are expressed to a much higher degree than those of the second block (2-4--2). The 3' 1 and 3' 2 enhancer regions are possible candidates that could differentially control the genes within the respective blocks. However, we did not find any difference in the effect of the enhancers in our transient transfection experiments. As the mouse Eµ has been shown to synergize with the 3' C enhancer (47, 50), it is possible that human Eµ and V_H promoters interact positively with the 3' 1 enhancer, stimulating genes within the upstream IGHS block, whereas an Eµ-3' 2 enhancer interaction is prevented by the larger distance involved, by an insulator, by other inhibitory element(s) downstream of the 3' C1 enhancer or by the chromatin context.

Differential effects of the enhancer on the expression of mouse Ig isotypes have previously been observed, as replacement of the HS1,2 and HS3A elements by a pgk-neo^r cassette only impaired switching to some of the isotypes (IgG2a, IgG2b, IgG3, and IgE) (32, 50). Inclusion of the human GL 3 promoter in the present study enabled us to make some interesting comparisons. The GL 3 promoter shows a weaker basal activity as compared with the GL promoters. However, introduction of the linked enhancer elements (HS3–1,2–4) resulted in a greater stimulation index for the 3 promoter. In view of the recently described "promoter competition" hypothesis (32, 43, 50) which proposes that transcription from at least a subset of GL promoters is based on the ability of the local GL promoter to "interact" with the putative 3' LCR, adding the factors that facilitate GL transcription, such as TGF-ß₁, would allow the GL 1 promoter to compete effectively with the promoters for 3' 1 enhancers, perhaps because of promoter strength, the more 3' position, or both. However, when adding up-regulating factors for GL 3 transcription, such as IL-4 and PMA, the 3 promoter would compete effectively with the 1 promoter for the enhancers, possibly due to its stronger interaction with the 3' 1 enhancer. Promoter competition may therefore be a general mechanism employed for modulation of GL transcription and differential regulation of isotype switching in the IGHC locus.

In individuals with homozygous deletions of a block of IGHC genes, including the 3' 1 enhancers (1-2-4-), the IgG1 and IgG3 levels are normal or even elevated, suggesting that the enhancer downstream of the 2 may replace the function of the 3' 1 enhancer (52). The finding of a natural deletion of either of the 3' enhancer regions may be needed to fully understand the role of these elements in the regulation of switching to IgA and other isotypes. An alternative approach involves deleting all or part of the putative 3' C LCRs in a human cell line undergoing isotype switching (46), an approach that is being actively pursued in one of our laboratories.

	Acknowledgments

 
We thank Diane Cox and Hakyung Kang (University of Alberta, Alberta, Canada) for providing clones adjacent to the enhancer region used in the early stages of this project.

	Footnotes

 
¹ This work was supported by the Swedish Medical Research Council.

² Address correspondence and reprint requests to Dr. Lennart Hammarström, Division of Clinical Immunology, Karolinska Institutet at Huddinge Hospital, SE-141 86 Huddinge, Sweden.

³ Abbreviations used in this paper: GL, germline; LCR, locus control region; ß-gal, ß-galactosidase.

Received for publication October 26, 1999. Accepted for publication April 6, 2000.

	References

Top Abstract Introduction Materials and Methods Results Discussion References

 

1. Cox, D. W., V. D. Markovic, I. E. Teshima. 1982. Genes for immunoglobulin heavy chains and for 1-antitrypsin are localized to specific regions of chromosome 14q. Nature 297:428.[Medline]
2. Brandtzaeg, P., I. N. Farstad, G. Haraldsen, F. L. Jahnsen. 1998. Cellular and molecular mechanisms for induction of mucosal immunity. Dev. Biol. Stand. 92:93.[Medline]
3. Shimizu, A., T. Honjo. 1984. Immunoglobulin class switching. Cell 36:801.[Medline]
4. Gritzmacher, C. A.. 1989. Molecular aspects of heavy-chain class switching. Crit. Rev. Immunol. 9:173.[Medline]
5. Honjo, T., T. Kataoka. 1978. Organization of immunoglobulin heavy chain genes and allelic deletion model. Proc. Natl. Acad. Sci. USA 75:2140.[Abstract/Free Full Text]
6. Kataoka, T., T. Kawakami, N. Takahashi, T. Honjo. 1980. Rearrangement of immunoglobulin 1-chain gene and mechanism for heavy-chain class switch. Proc. Natl. Acad. Sci. USA 77:919.[Abstract/Free Full Text]
7. Harding, F. A., N. Lonberg. 1995. Class switching in human immunoglobulin transgenic mice. Ann. NY Acad. Sci. 764:536.[Medline]
8. Bergstedt-Lindqvist, S., P. Sideras, H. R. MacDonald, E. Severinson. 1984. Regulation of Ig class secretion by soluble products of certain T-cell lines. Immunol. Rev. 78:25.[Medline]
9. Sideras, P., S. Bergstedt-Lindqvist, E. Severinson. 1985. Partial biochemical characterization of IgG1-inducing factor. Eur. J. Immunol. 15:593.[Medline]
10. Kameyama, K., G. Sakaguchi, H. Haga, N. Shibata, M. Uryu, K. Sugimoto. 1996. The recombination mediated by double-strand breaks in extrachromosomal DNA substrate carrying mouse immunoglobulin switch regions S µ and S 2b. Biochim. Biophys. Acta 1308:263.[Medline]
11. Oancea, A. E., F. W. Tsui, M. J. Shulman. 1995. Targeted removal of the µ switch region from mouse hybridoma cells: A test of its role in gene expression in the endogenous IgH locus. J. Immunol. 155:5678.[Abstract]
12. Stavnezer-Nordgren, J., S. Sirlin. 1986. Specificity of immunoglobulin heavy chain switch correlates with activity of germline heavy chain genes prior to switching. EMBO J. 5:95.[Medline]
13. Wakatsuki, Y., W. Strober. 1993. Effect of downregulation of germline transcripts on immunoglobulin A isotype differentiation. J. Exp. Med. 178:129.[Abstract/Free Full Text]
14. Nilsson, L., K. B. Islam, O. Olafsson, I. Zalcberg, C. Samakovlis, L. Hammarström, C. I. Smith, P. Sideras. 1991. Structure of TGF-ß1-induced human immunoglobulin C 1 and C 2 germ-line transcripts. Int. Immunol. 3:1107.[Abstract/Free Full Text]
15. Kitani, A., W. Strober. 1994. Differential regulation of C1 and C2 germ-line and mature mRNA transcripts in human peripheral blood B cells. J. Immunol. 153:1466.[Abstract]
16. Shockett, P., J. Stavnezer. 1991. Effect of cytokines on switching to IgA and germline transcripts in the B lymphoma I.29 µ: Transforming growth factor-ß activates transcription of the unrearranged C gene. J. Immunol. 147:4374.[Abstract]
17. Lebman, D. A., M. J. Park, S. Hansen-Bundy, A. Pandya. 1994. Mechanism for transforming growth factor ß regulation of mRNA in lipopolysaccharide-stimulated B cells. Int. Immunol. 6:113.[Abstract/Free Full Text]
18. Nilsson, L., S. Paschalis. 1993. The human I 1 and I 2 germline promoter elements: Proximal positive and distal negative elements may regulate the tissue specific expression of C 1 and C 2 germline transcripts. Int. Immunol. 5:271.[Abstract/Free Full Text]
19. Ichiki, T., W. Takahashi, T. Watanabe. 1993. Regulation of the expression of human C epsilon germline transcript: Identification of a novel IL-4 responsive element. J. Immunol. 150:5408.[Abstract]
20. Xu, M. Z., J. Stavnezer. 1992. Regulation of transcription of immunoglobulin germ-line gamma 1 RNA: Analysis of the promoter/enhancer. EMBO J. 11:145.[Medline]
21. Islam, K. B., L. Nilsson, P. Sideras, L. Hammarström, C. I. Smith. 1991. TGF-ß1 induces germ-line transcripts of both IgA subclasses in human B lymphocytes. Int. Immunol. 3:1099.[Abstract/Free Full Text]
22. Islam, K. B., B. Baskin, L. Nilsson, L. Hammarström, P. Sideras, C. I. Smith. 1994. Molecular analysis of IgA deficiency. Evidence for impaired switching to IgA. J. Immunol. 152:1442.[Abstract]
23. Xie, X. Q., E. Pardali, M. Holm, P. Sideras, T. Grundstrom. 1999. AML and Ets proteins regulate the I 1 germ-line promoter. Eur. J. Immunol. 29:488.[Medline]
24. Banerji, J., L. Olson, W. Schaffner. 1983. A lymphocyte-specific cellular enhancer is located downstream of the joining region in immunoglobulin heavy chain genes. Cell 33:729.[Medline]
25. Gillies, S. D., S. L. Morrison, V. T. Oi, S. Tonegawa. 1983. A tissue-specific transcription enhancer element is located in the major intron of a rearranged immunoglobulin heavy chain gene. Cell 33:717.[Medline]
26. Chen, J., F. Young, A. Bottaro, V. Stewart, R. K. Smith, F. W. Alt. 1993. Mutations of the intronic IgH enhancer and its flanking sequences differentially affect accessibility of the JH locus. EMBO J. 12:4635.[Medline]
27. Gu, H., Y. R. Zou, K. Rajewsky. 1993. Independent control of immunoglobulin switch recombination at individual switch regions evidenced through Cre-loxP-mediated gene targeting. Cell 73:1155.[Medline]
28. Su, L. K., T. Kadesch. 1990. The immunoglobulin heavy-chain enhancer functions as the promoter for I µ sterile transcription. Mol. Cell. Biol. 10:2619.[Abstract/Free Full Text]
29. Bottaro, A., F. Young, J. Chen, M. Serwe, F. Sablitzky, F. W. Alt. 1998. Deletion of the IgH intronic enhancer and associated matrix-attachment regions decreases, but does not abolish, class switching at the µ locus. Int. Immunol. 10:799.[Abstract/Free Full Text]
30. Sakai, E., A. Bottaro, F. W. Alt. 1999. The Ig heavy chain intronic enhancer core region is necessary and sufficient to promote efficient class switch recombination. Int. Immunol. 11:1709.[Abstract/Free Full Text]
31. Serwe, M., F. Sablitzky. 1993. V(D)J recombination in B cells is impaired but not blocked by targeted deletion of the immunoglobulin heavy chain intron enhancer. EMBO J. 12:2321.[Medline]
32. Cogne, M., R. Lansford, A. Bottaro, J. Zhang, J. Gorman, F. Young, H. L. Cheng, F. W. Alt. 1994. A class switch control region at the 3' end of the immunoglobulin heavy chain locus. Cell 77:737.[Medline]
33. Pettersson, S., G. P. Cook, M. Brüggemann, G. T. Williams, M. S. Neuberger. 1990. A second B cell-specific enhancer 3' of the immunoglobulin heavy-chain locus. Nature 344:165.[Medline]
34. Lieberson, R., S. L. Giannini, B. K. Birshtein, L. A. Eckhardt. 1991. An enhancer at the 3' end of the mouse immunoglobulin heavy chain locus. Nucleic Acids Res. 19:933.[Abstract/Free Full Text]
35. Dariavach, P., G. T. Williams, K. Campbell, S. Pettersson, M. S. Neuberger. 1991. The mouse IgH 3'-enhancer. Eur. J. Immunol. 21:1499.[Medline]
36. Giannini, S. L., M. Singh, C. F. Calvo, G. Ding, B. K. Birshtein. 1993. DNA regions flanking the mouse Ig 3' enhancer are differentially methylated and DNase I hypersensitive during B cell differentiation. J. Immunol. 150:1772.[Abstract]
37. Sideras, P., L. Nilsson, K. B. Islam, I. Z. Quintana, L. Freihof, A. Rosen, G. Juliusson, L. Hammarström, C. I. Smith. 1992. Transcription of unrearranged Ig H chain genes in human B cell malignancies: Biased expression of genes encoded within the first duplication unit of the Ig H chain locus. J. Immunol. 149:244.[Abstract]
38. Tumas-Brundage, K. M., K. A. Vora, T. Manser. 1997. Evaluation of the role of the 3' heavy chain enhancer [3' E(hs1,2)] in Vh gene somatic hypermutation. Mol. Immunol. 34:367.[Medline]
39. Kang, H. K., D. W. Cox. 1996. Tandem repeats 3' of the IGHA genes in the human immunoglobulin heavy chain gene cluster. Genomics 35:189.[Medline]
40. Mills, F. C., N. Harindranath, M. Mitchell, E. E. Max. 1997. Enhancer complexes located downstream of both human immunoglobulin C genes. J. Exp. Med. 186:845.[Abstract/Free Full Text]
41. Chen, C., B. K. Birshtein. 1997. Virtually identical enhancers containing a segment of homology to murine 3'IgH-E(hs1,2) lie downstream of human Ig C1 and C 2 genes. J. Immunol. 159:1310.[Abstract]
42. Pinaud, E., C. Aupetit, C. Chauveau, M. Cogne. 1997. Identification of a homolog of the C 3'/hs3 enhancer and of an allelic variant of the 3'IgH/hs1,2 enhancer downstream of the human immunoglobulin 1 gene. Eur. J. Immunol. 27:2981.[Medline]
43. Seidl, K. J., J. P. Manis, A. Bottaro, J. Zhang, L. Davidson, A. Kisselgof, H. Oettgen, F. W. Alt. 1999. Position-dependent inhibition of class-switch recombination by PGK-neor cassettes inserted into the immunoglobulin heavy chain constant region locus. Proc. Natl. Acad. Sci. USA 96:3000.[Abstract/Free Full Text]
44. Madisen, L., M. Groudine. 1994. Identification of a locus control region in the immunoglobulin heavy-chain locus that deregulates c-myc expression in plasmacytoma and Burkitt’s lymphoma cells. Genes Dev. 8:2212.[Abstract/Free Full Text]
45. Pan, Q., C. Petit-Frère, J. Stavnezer, L. Hammarström. 2000. Regulation of the promoter for human immunoglobulin 3 germ-line transcription and its interaction with the 3' enhancer. Eur. J. Immunol. 30:1019.[Medline]
46. Cerutti, A., H. Zan, A. Schaffer, L. Bergsagel, N. Harindranath, E. E. Max, P. Casali. 1998. CD40 ligand and appropriate cytokines induce switching to IgG, IgA, and IgE and coordinated germinal center and plasmacytoid phenotypic differentiation in a human monoclonal IgM⁺IgD⁺ B cell line. J. Immunol. 160:2145.[Abstract/Free Full Text]
47. Ong, J., S. Stevens, R. G. Roeder, L. A. Eckhardt. 1998. 3' IgH enhancer elements shift synergistic interactions during B cell development. J. Immunol. 160:4896.[Abstract/Free Full Text]
48. Chauveau, C., M. Cogne. 1996. Palindromic structure of the IgH 3'locus control region. Nat. Genet. 14:15.[Medline]
49. Grosveld, F., G. B. van Assendelft, D. R. Greaves, G. Kollias. 1987. Position-independent, high-level expression of the human ß-globin gene in transgenic mice. Cell 51:975.[Medline]
50. Manis, J. P., N. van der Stoep, M. Tian, R. Ferrini, L. Davidson, A. Bottaro, F. W. Alt. 1998. Class switching in B cells lacking 3' immunoglobulin heavy chain enhancers. J. Exp. Med. 188:1421.[Abstract/Free Full Text]
51. Nilsson, L., P. Grant, I. Larsson, S. Pettersson, P. Sideras. 1995. The human I 1 region contains a TGF-ß1 responsive enhancer and a putative recombination hotspot. Int. Immunol. 7:1191.[Abstract/Free Full Text]
52. Hammarström, L., A. O. Carbonara, M. DeMarchi, G. Lefranc, M. P. Lefranc, C. I. Smith. 1987. Generation of the antibody repertoire in individuals with multiple immunoglobulin heavy chain constant region gene deletions. Scand. J. Immunol. 25:189.[Medline]

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