A negative regulatory element in the rabbit 3'IgH chromosomal region

Veronica Volgina1, Pi-Chen Yam and Katherine L. Knight

Department of Microbiology and Immunology, Stritch School of Medicine, Loyola University Chicago, Maywood, IL 60153, USA
1 Present address: Department of Molecular Genetics and Cell Biology, University of Chicago, Chicago, IL 60637, USA

Correspondence to: K. L. Knight; E-mail: kknight{at}lumc.edu


    Abstract
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Mouse and human IgH loci contain several 3'IgH enhancers. In rabbit, a single hs1,2 enhancer is located 3' of the distal germ line C{alpha} gene, C{alpha}13. We searched for additional regulatory elements in this region by using a luciferase reporter assay and nucleotide sequence analysis. Within 8 kb 3' of C{alpha}13, we identified a 1-kb fragment that negatively regulated the hs1,2 enhancement of the I{alpha} promoter. This negative regulatory element, C{alpha}-NRE, contains a conserved 300-bp region that is associated with 8 of the 13 germ line C{alpha} genes. This conserved region contains an E box that, by electrophoretic mobility shift assay, binds an E47-like protein. At the 5' end, C{alpha}-NRE also includes a 270-bp region with 20-bp repeats nearly identical to those 3' of mouse and human C{alpha} genes, and these repeats bind unidentified nuclear protein(s). C{alpha}-NRE appears to be a novel regulatory element that may contribute to the regulation of IgH gene expression.

Keywords: B cells, gene regulation, molecular biology, rabbit, transcription factors


    Introduction
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
The interaction between positive and negative cis-acting elements, such as promoters, enhancers and silencers, regulates Ig genes. The upstream elements, including VH promoters, the DQ52 promoter–enhancer and the Eµ enhancer, mainly regulate Cµ germ line transcription, DJ and VDJ gene rearrangements and expression of rearranged µ genes. In addition to the upstream elements, the 3' regulatory region includes transcriptional enhancers hs1,2, hs3 and hs4, which are located downstream of the C{alpha} gene in mice and rats (13) and downstream of both C{alpha} genes, C{alpha}1 and C{alpha}2, in humans (46). The hs1,2 affects germ line I{alpha}, {gamma}2b and {varepsilon} promoters (7, 8) and thereby may regulate germ line transcription. When both mouse hs3B and hs4 enhancers were deleted, germ line transcription, isotype switch and somatic hypermutation were impaired (9, 10). Spontaneous deletion of the entire 3' enhancer region caused a reduction in IgH gene expression in a mouse plasmacytoma cell line (11), although a naturally occurring deletion of hs3A and hs1,2 did not affect IgH gene expression or class switch recombination (12).

The cis-acting elements that negatively regulate Ig genes include a silencer associated with chicken {lambda} light chain genes (1316), silencers 5' of human I{alpha} promoters (8, 17) and at least two negative regulatory elements (NREs) in the mouse Ig{kappa} locus (1821). Inhibition of transcription appears to be associated with some repeat sequences, such as the B1 repeats in the NRE associated with {kappa} light chain genes and the short repeats in the DH-JH region that suppress Eµ-mediated promoter activity (21, 22). The IgH locus in mice and humans, especially the 3'{alpha} region, contains abundant repetitive sequences (4, 2325) that may contribute to negative regulatory activity.

The rabbit genome contains 13 non-allelic C{alpha} genes located at the 3' end of the IgH locus (26). Each of the C{alpha} genes is associated with an I{alpha} region that includes an I{alpha} promoter. The C{alpha} genes are differentially expressed in various tissues including lung and tonsil in which C{alpha}4, the 5'-most C{alpha} gene, is the predominantly expressed one (27). While the upstream regulatory elements in the rabbit IgH locus, including germ line promoters (28) and the Eµ intronic enhancer (29), are similar to those of other species, the organization of the 3' regulatory region is different. Unlike the human genome, where two 3'{alpha} regulatory regions are associated with C{alpha} genes, C{alpha}1 and C{alpha}2, the rabbit genome contains a single 3'{alpha} regulatory region, even though it contains 13 differentially expressed C{alpha} genes (26, 27, 30). Spieker-Polet et al. (28) reported that the differential expression of the C{alpha} genes cannot be explained simply by differences in the I{alpha} promoter. Also, it cannot be explained by differential regulation by a single hs1,2 enhancer. The rabbit CH locus spans ~200 kb, and it is likely that regulatory elements in addition to hs1,2 are required to regulate expression of this complex locus. Accordingly, we hypothesized that the 3'{alpha} regulatory region and/or introns within the C{alpha} locus contain additional cis-regulatory elements. In this study we identified a novel C{alpha} negative regulatory element (C{alpha}-NRE) located in the 3'{alpha} region. C{alpha}-NRE regulates the I{alpha} promoter as well as hs1,2 enhancement of the I{alpha} promoters in vitro.


    Methods
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Cloning fragments from the 3'{alpha} region and preparation of reporter constructs
The four SacI fragments (2.4 kb, 1.4 kb, 1.3 kb and 1 kb) from the 8-kb DNA fragment of Fos15B (31) located between C{alpha}13 and hs1,2 were subcloned, and their nucleotide sequences were determined by using the ABI PrismTM 310 Genetic Analyzer. Constructs containing the SacI fragments and the SV40 promoter were generated by using the SV40 pGL3 vector containing the luciferase reporter gene (Promega, Madison, WI, USA). Rabbit I{alpha} and VH promoters and hs1,2 (28, 30) were inserted in the XhoI/SacI, HindIII and BamHI sites of pGL3, respectively. Truncated fragments of the 1-kb SacI fragment were PCR amplified and cloned 3' of the luciferase gene and 5' of hs1,2 (30) in pGL3. The following primers were used for PCR: 1-kb SacI—5' primer: 5'-CTTAACTGAGCAGATTCGT-3' and 3' primer: 5'-GGTGGGTGCCGTCCGTGAAACC-3'; 270 bp—5' primer: 5'-CTTAACTGAGCAGATTCGT-3' and 3' primer: 5'-CATCAAGACCAGGGATCA-3'; 156 bp—5' primer: 5'-CACTCCGTCCTCTCAGGCCCT-3' and 3' primer: 5'-TGAAGAAGGAGCAACACAGGG-3'.

A 710-bp fragment from the human 3'IgH regulatory region of C{alpha}2 that contained the region immediately 5' of hs3, including the 20-bp repeats and a TATA box, was PCR amplified from a 1.2-kb KpnI/SmaI fragment subcloned from BAC clone #9378 (a kind gift from B. Birshtein), with the following primers: 5'-GATCCCTATTCCTCATAAA-3', 3'-CTAAGTCGAGACCTGGCGA-5'. The nucleotide sequence of the cloned PCR product was identical to that provided by B. Birshtein and verified the presence of 20-bp repeats.

Transient transfection assays
Transient transfection assays were performed with a rabbit B cell line, 55D1, derived from a B cell tumor that developed in a c-myc transgenic rabbit (32), and a hypermutating human B cell line, Ramos (33); both cell lines were grown in RPMI 1640 supplemented with 10% FCS. The 55D1 cell line has been shown to express a low level of I{alpha} germ line (sterile) transcripts and undergo spontaneous isotype switch to IgA (28). The reporter constructs (10 µg) were transfected by electroporation into unactivated or activated 55D1 cells (2 x 107). All transfections were performed three times with each construct except as indicated. Luciferase activity was measured in duplicate (according to the manufacturer's protocol; Promega), and values were normalized to Renilla luciferase activity. To estimate the activity of reporter plasmids, the luciferase activity of constructs containing promoter, enhancer and tested fragments was compared with reporter gene activity of the promoter-only control plasmid. The raw data from the transient transfection assays were statistically analyzed by the Student's t-test and reported as percentage relative to promoter-only control plasmid.

Gel-shift analysis
Gel-shift analyses were performed as described by Spieker-Polet et al. (28). Briefly, nuclear extracts (1–5 µg of protein) were prepared as described in Dignam et al. (34) and were incubated in binding buffer with poly dI-dC (2.5 µg per reaction) for 20 min at 20°C with 32P-labeled probes (10–30 x 103 counts per minute) prepared with Klenow or T4 Polymerase (Promega). The following probes were used: (i) the 156-bp PCR product from the 1-kb SacI fragment, (ii) a 170-bp region prepared by ScaI digestion of the 270-bp PCR product from the 1-kb SacI fragment and (iii) the human 240-bp probe prepared by BamHI/SacI digestion of the 710-bp fragment of human 3'{alpha}E. The 80-bp fragments of the 156-bp region were PCR amplified with primers: 80 up -5'-CACTCCGTCCTCTCAGGCCCT-3', 5'-ACTGAGTCACCTCAGGCAG-3' and 80 down - 5'-CTGCCTGAGGTGACTCAGT-3', 5'-TGAAGAAGGAGCAACACAGGG-3'. Rabbit genomic DNA was sonicated five times for 10 s at 4°C, and the fragments with sizes 500 bp to 1 kb were used as competitors. Synthesized double-stranded probes used for competition assays contained E boxes, CREB sites (28) or two GATA-binding sites TAATTC/ATAGCCTTGATC/AACTGA (35). Since anti-rabbit E47 antibodies are not available, we used nuclear extracts from human cells (Ramos) and anti-human E47 antibody (rabbit polyclonal, N-649; Santa Cruz Biotecnology, Santa Cruz, CA, USA) for inhibition assays. Mouse anti-rabbit Ig prepared in our laboratory was used as the negative control.

Cloning C{alpha}-NREs associated with C{alpha} genes
Restriction digests of C{alpha} cosmid DNA (26) representing the entire C{alpha} cluster (~150 kb) were separated on 1% agarose gels, and Southern blots were hybridized with 300-bp (PstI/HindIII) and 700-bp (SacI/PstI) probes prepared from the 1-kb SacI fragment from the 3'{alpha} region. Fragments that hybridized with these probes were subcloned in pUC19 vector, and the nucleotide sequences were determined.


    Results
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Search for regulatory elements in the 3'C{alpha} region: identification of a NRE
To identify new regulatory elements downstream of the rabbit C{alpha} locus, we analyzed an 8-kb segment between the 3'-most C{alpha} gene, C{alpha}13, and hs1,2 (Fig. 1). Four SacI fragments were subcloned and examined for the presence of sequences homologous to known murine and human 3'{alpha} enhancers, hs3 and hs4. Although we found no evidence of enhancer-like elements, we found that the 1-kb SacI fragment contained several 20-bp repeats nearly identical to those 3' of human C{alpha}2 and mouse C{alpha} genes (Fig. 1). Because small repeats are thought to possess transcriptional regulatory activity (20, 36, 37), we used a luciferase reporter assay to determine whether this region had such activity. Luciferase expression from all constructs was compared with that of the luciferase gene driven by the I{alpha} promoter alone (Fig. 2A, construct 1, 100%). We cloned the 1-kb SacI fragment that contained the 20-bp repeats 3' of the luciferase gene driven by the I{alpha} promoter (Fig. 2A, construct 2) and used it to transfect 55D1 rabbit B cells. The 1-kb SacI fragment decreased luciferase activity by 3- to 6-fold, indicating that the 1-kb SacI fragment has a negative effect on the I{alpha} promoter. In the presence of hs1,2 which by itself increased luciferase activity ~4-fold (construct 3), the 1-kb SacI fragment essentially eliminated the expression of luciferase (construct 4). The complete inhibition is not due solely to the negative activity of the SacI fragment (construct 2) on the I{alpha} promoter; instead, we suggest that the 1-kb SacI fragment combined with hs1,2 inhibited expression from the I{alpha} promoter, while the SacI fragment alone partially suppressed I{alpha} promoter activity. These data indicate that the 1-kb SacI fragment has a negative regulatory effect not only on the I{alpha} promoter but also on hs1,2 enhancement of this promoter.



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Fig. 1. Genomic map of the region 3' of C{alpha}13 and of the consensus nucleotide sequence of the seven 20-bp repeats within the 1-kb SacI fragment. Open boxes represent C{alpha} genes. Oval = hs1,2; nucleotide sequences of the 3'{alpha} 20-bp repeats of human (GenBank L43337) and mouse (GenBank U26400) are compared with rabbit (C{alpha}13); identical nucleotides are designated by dots. The shaded box represents the 300-bp fragment.

 


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Fig. 2. Luciferase reporter assay for activity of the 1-kb SacI fragment in 55D1 B cells. Lane numbers correspond to construct numbers shown on the right. The percentage of luciferase activity was determined relative to a construct with luciferase driven by the I{alpha} promoter, 100% which was ~9000 light units (A and B), or the SV40 promoter, 100% which was ~20 000 light units (C). (A) The 1-kb SacI fragment, hs1,2 and the I{alpha} promoter are in germ line orientation relative to each other; arrow indicates orientation. (B) The 1-kb SacI fragment was replaced with a 710-bp fragment cloned from human 3'{alpha}E (see Methods). (C) The I{alpha} promoter was replaced by the SV40 promoter. The standard deviation was determined from the average of duplicate samples from three independent transfections. (D) Rabbit 55D1 B cells were transfected with luciferase constructs containing the I{alpha} promoter as well as the 1-kb SacI fragment and/or hs1,2 and after 2 h cells were activated with anti-IgM (bottom), and luciferase activity was measured 24 h later. Lane numbers refer to construct numbers. Data for unactivated B cells (top) are the same as in lanes 2–4 in (A). Percentage of luciferase activity was compared with a construct with luciferase driven by the I{alpha} promoter (100% = ~14 000 light units). The standard error was determined from the average of two samples from two independent transfections.

 
To determine whether the negative regulatory effects were position dependent, we cloned the 1-kb SacI fragment 5' of the I{alpha} promoter in the luciferase reporter construct. In this position it demonstrated promoter-like activity with increased expression of luciferase (Fig. 2A, construct 5), in contrast to the decreased expression obtained with it in the 3' position (construct 2). However, in the presence of hs1,2, the luciferase activity was eliminated with the 1-kb SacI fragment in the 5' position (Fig. 2A, compare constructs 5 and 6). Thus, in both the 5' and 3' positions, the 1-kb Sac1 fragment completely inhibited the expression of luciferase driven by hs1,2-enhanced I{alpha} promoter. These data indicate that although the promoter-like activity of the 1-kb SacI fragment is position dependent, the negative regulatory effect of this fragment on hs1,2 enhancement of I{alpha} promoter activity is position independent. We also tested the 1-kb SacI fragment in the opposite orientation and found that it has virtually identical inhibitory activity (Supplementary Figure 7, available at International Immunology Online). Thus, the negative regulatory effect of the 1-kb SacI fragment on hs1,2 enhancement of the I{alpha} promoter was both orientation and position independent. On the other hand, the promoter-like activity of the 1-kb SacI fragment shown in Fig. 2A (construct 5) was orientation independent (Supplementary Figure 7, available at International Immunology Online, bar 5). In this study we did not pursue further investigation of the promoter activity of this fragment; instead, we investigated the negative effect of the 1-kb SacI fragment.

To test whether the negative regulatory activity was specifically associated with the 1-kb SacI fragment, we replaced this fragment with a similarly sized fragment (710 bp) of human DNA containing the 20-bp repeats from the region 3' of C{alpha}2 (5, 24). We found that this segment of DNA had no negative regulatory effect on the rabbit I{alpha} promoter (Fig. 2B, construct 2) or on hs1,2-mediated enhancement of the I{alpha} promoter (compare constructs 3 and 4). The difference in hs1,2 enhancement (Fig. 2A and B, construct 3) likely represents variation between two separate experiments (the differences are not significant, P > 0.05). We conclude that the negative regulatory activity associated with the rabbit 1-kb SacI fragment is specific. We designate this NRE as C{alpha}-NRE (GenBank accession number AY655722).

To determine whether C{alpha}-NRE is specific for the I{alpha} promoter, we replaced the I{alpha} promoter with the SV40 promoter. We found a slight and statistically insignificant change in luciferase activity in the presence of C{alpha}-NRE in either the 5' or 3' position (Fig. 2C, constructs 2 and 3, P = 0.05), indicating that the C{alpha}-NRE had minimal effect on the SV40 promoter. In contrast, C{alpha}-NRE inhibited hs1,2 enhancement of the SV40 promoter (Fig. 2C, compare constructs 4 and 5, P = 0.005), indicating that C{alpha}-NRE can affect hs1,2 enhancement of a non-Ig promoter as well as the I{alpha} promoter.

The previous data demonstrate that C{alpha}-NRE has negative regulatory activity in non-activated B cells. To determine if it functions similarly in activated B cells, we treated 55D1 B cells with anti-IgM and found that C{alpha}-NRE inhibition of hs1, 2-enhanced I{alpha} promoter activity was also diminished (Fig. 2D, lower, compare bars 2 and 4); however, the inhibition was less than that observed in unactivated B cells (Fig. 2D, top). These data suggest that upon activation B cells may express a factor(s) that partially counteracts the inhibitory effect of C{alpha}-NRE.

C{alpha}-NRE associated with other germ line C{alpha} genes
We began this study to identify new regulatory elements within the C{alpha} gene cluster that could explain the differential expression of the 13 germ line C{alpha} genes. Because C{alpha}-NRE regulates both I{alpha} promoters and hs1,2, we hypothesized that a C{alpha}-NRE might be associated with each C{alpha} gene. We hybridized Southern blots of restriction enzyme-digested germ line C{alpha}-containing cosmid and phage DNA (26) with two probes, a (3' end) and b (5' end) of the 1-kb SacI fragment (Fig. 3A) as well as with probes representing the three other SacI fragments from the 3' C{alpha}13 region. Only the a and b probes hybridized with DNA from the germ line C{alpha} regions other than the C{alpha}13 region containing the four SacI fragments. Representative data for probe a are shown in Fig. 3(B). We found that probe a hybridized with DNA adjacent to 8 of the 11 C{alpha} genes tested (C{alpha}1, C{alpha}2, C{alpha}4, C{alpha}5, C{alpha}7, C{alpha}8, C{alpha}10 and C{alpha}13) but not with DNA adjacent to C{gamma} or C{varepsilon} genes. Except for C{alpha}13, the regions that hybridized with probe a are between the C{alpha} transmembrane exon of one C{alpha} gene and the I{alpha} exon of the next C{alpha} gene (Fig. 3A). We determined the nucleotide sequence of the fragments that hybridized with probe a and found a region of 300 bp that was nearly identical among the eight C{alpha} genes (Figs 1 and 4). These data show that this region has been maintained during evolution and suggest that C{alpha}-NRE has important biologic function.



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Fig. 3. Localization of C{alpha}-NRE-hybridizing regions within the C{alpha} chromosomal region. (A) Diagram of the rabbit C{alpha} locus [revised from (32)]. Open boxes = C{alpha} genes; small vertical ovals = switch region; solid boxes = C{alpha}-NRE; arrows = I{alpha} promoter; large horizontal oval = hs1,2; dots = 20-bp repeats; striped box = 300-bp region that is highly conserved among 8 of the 13 C{alpha} genes (see Fig. 4). C{alpha}-NREs are located, on an average, 4 kb downstream of C{alpha} genes. (B) Image of Southern blot of HindIII-digested rabbit C{alpha}-containing germ line phage and cosmid DNA probed with probe a. Lane 1 = C{alpha}1, C{alpha}4, C{alpha}5; lane 2 = C{alpha}1, C{alpha}2; lane 3 = C{alpha}13; lane 4 = C{alpha}14; lane 5 = C{gamma} and lane 6 = C{varepsilon}. Positions of {lambda}/HindIII markers are marked at the left (kilobase).

 


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Fig. 4. Nucleotide sequence comparison of C{alpha}-NREs from eight C{alpha} genes. Dots represent nucleotide identity; slashes represent gaps introduced to increase sequence similarity. The 300-bp fragment is underlined; arrows mark the position of primers used for PCR amplification of the 156-bp fragment. The two boxed regions are E boxes, the asterisk indicates the functional E box. The sequence of the 20-bp repeat region is shortened; complete sequences of repetitive regions associated with eight C{alpha} genes are available Online (GenBank Accession Numbers AY655722, AY685001, AY685002, AY685003, AY685004, AY685005, AY685006 and AY685007).

 
Probe b, which contains the 20-bp repeats of the 1-kb SacI fragment (Fig. 3A), hybridized with DNA adjacent to C{alpha}4 and C{alpha}13 but not with DNA associated with any other C{alpha} gene or with C{gamma} and C{varepsilon} genes (data not shown). By nucleotide sequence analysis we found that the region 3' of C{alpha}4 contained 20-bp repeats with a consensus sequence identical to that found in the 20-bp repeats 3' of C{alpha}13 as well as those in human and mouse 3'{alpha} regions (Fig. 1).

Localization of C{alpha}-NRE within the 1-kb SacI fragment
To further localize C{alpha}-NRE activity, we PCR amplified and cloned several fragments from the 1-kb SacI fragment and tested them in the luciferase reporter assay. Although we found several fragments that partially inhibited hs1,2-enhanced luciferase expression, no single fragment had activity comparable to that of the 1-kb SacI fragment (data not shown). The highest NRE activity was found in the 270-bp repeat fragment and in a 156-bp fragment derived from the 300-bp fragment described in Fig. 4. The 270-bp repeat fragment and the 156-bp fragment inhibited ~50 and 75%, respectively, of hs1,2 enhancement of the I{alpha} promoter (Fig. 5, compare construct 3 to constructs 4 and 5). When the mutations GT to CA in one of the E boxes within this fragment (see below) were introduced, the 156-bp fragment lost inhibitory activity (Fig. 5, construct 6). We suggest that the C{alpha}-NRE within the 1-kb SacI fragment includes at least two elements, one in the 20-bp repeat region and one in the 156-bp region.



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Fig. 5. Luciferase reporter assay to localize negative regulatory activity within C{alpha}-NRE. (A) Location of 270-bp (containing all seven 20-bp repeats) and 156-bp fragment (from the 300-bp region identified in Fig. 4) within the 1-kb SacI region used in the reporter assay. (B) Luciferase activity of the fragments with the I{alpha} promoter and hs1,2. In the 156-bp mutation lane 6, CA was mutated to TG in the second E box (see Fig. 4). Percentage of luciferase activity was compared with a construct with luciferase driven by the I{alpha} promoter (100% was ~9000 light units). The standard deviation was determined from 10 independent transfections, except for the 156mut construct, for which the standard error was determined from the average of two samples from two independent transfections.

 
Gel-shift analysis of the 20-bp repeats and the 156-bp fragment
If C{alpha}-NRE contains cis-element(s), we expected it to bind transcription factors. Because negative regulatory activity was associated mostly with the 156-bp and the 270-bp fragments, we performed gel-shift analysis with those DNA fragments and found shifted bands with the nuclear extract prepared from rabbit 55D1 B cells (Fig. 6A, lane 1 and Fig. 6B).We also found that the highly homologous human 20-bp repeat probe bound transcription factors (Fig. 6B). The DNA–protein complexes were not disrupted by the addition of increased concentrations of poly dI-dC (Fig. 6C) or by comparably sized genomic DNA fragments (Fig. 6D), indicating that the DNA–protein complexes formed with both 156-bp and 20-bp repeat probes were specific.



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Fig. 6. Gel-shift assay of C{alpha}-NRE and human 20-bp repeats 3' of C{alpha}2. Nuclear extracts from 55D1 cells (5 µg) was incubated with the 156-bp probe (A and E) and rabbit and human 20-bp repeat probes (B–D). (A) Competition assay using probe : competitor molar ratios of: 1 : 180 for the 156-bp probe (lane 2) and for the 80-bp up and down fragments (lanes 3 and 4); 1 : 300 for the 156-bp mutant competitor probe in which CA of the 3' E box (see boxes in Fig. 4) was mutated to TG (mutant TG) and GT of the 3' E box was mutated to CA (mutant CA) (lanes 5 and 6); rabbit anti-human E47 antibodies (3 µg) (lane 7); mouse anti-rabbit Ig antibody (negative control) (lane 8). (B) Left lane = probe only; right lane = extract with the rabbit and human 20-bp repeat probes. (C) The rabbit 20-bp repeat probe with non-specific competitor poly dI-dC (1.2, 2.5 and 5 µg per reaction). Similar results were obtained for the 156-bp probe (data not shown). (D) Competition of the rabbit 20-bp repeat probe with increasing amounts of genomic DNA as competitor (0-, 10-, 100-, 500- and 1000-fold molar excess). Similar results were obtained for the 156-bp probe (data not shown). (E) Competition of the 156-bp probe with increasing amount of double-stranded (ds) CREB-containing responsive element (10-, 100-, 200-fold molar excess) and ds GATA-responsive element (10-, 100-, 200-fold molar excess). Similar results were obtained for the 20-bp repeat probe (data not shown).

 
We began identifying the transcription factor(s) responsible for the observed binding by mapping potential DNA-binding sites using the Transfac 4.0 program (37). We found two putative E boxes and several CREB-binding sites within the 156-bp region. In addition, we found several putative GATA-binding sites in the 20-bp repeat region at the 5' end of C{alpha}-NRE. To determine whether the CREB or GATA sites were responsible for the binding of transcription factors in the electrophoretic mobility shift assay, we tested CREB- and GATA-containing double-stranded oligomers for their ability to compete for binding of extracts from B cells by using the 156-bp and 20-bp repeat probes, respectively. We found that neither the CREB nor GATA oligomers disrupted the DNA–protein complexes (Fig. 6E). Although, we did not have a positive control for inhibition by these oligomers, the data suggest to us that these transcription factors are not responsible for the gel shifts of C{alpha}-NRE.

To test whether the E box sites were responsible for binding of transcription factors to the 156-bp region, we performed gel-shift assays with anti-human E47 antibodies. We found that this antibody inhibited the binding of the 156-bp probe to nuclear proteins from human B cells (Ramos); the binding was not inhibited by control mouse anti-rabbit Ig antibodies (Fig. 6A, lanes 7 and 8). These data indicate that at least one of the E boxes bound transcription factors. To determine which of the two E boxes was responsible for the gel-shift, we tested two 80-bp probes in a competition gel-shift assay, one (80 bp up) spanning the 5' E box and the other (80 bp down) containing the 3' E box. We found that the DNA–protein complex with the 156-bp probe was disrupted by the 80-bp down probe but not by the 80-bp up probe, indicating that the 3' E box was responsible for the gel-shift (Fig. 6A, lanes 3 and 4). Further evidence that the 3' E box is responsible for the binding was obtained by using, as probes, the 156-bp fragment in which CA was mutated to TG and GT was mutated to CA in the 3' E box (CAGGTG). We found that both mutations abrogated in part, or completely, the inhibitory activity (Fig. 6A, lanes 5 and 6), indicating that these nucleotides within the E box were required for transcription factor binding.

These data, taken together with the finding that the inhibitory effect of C{alpha}-NRE in the luciferase assay was decreased by nearly 75% when the E box was mutated (Fig. 5B, lane 6), indicate that the E47 protein (or E47-like proteins) binds the 3' E box within the 156-bp region and likely regulates rabbit C{alpha}-NRE. The presence of a functional E box, which is a common attribute of cis-elements, supports the finding that C{alpha}-NRE contains regulatory element(s).


    Discussion
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
IgH genes are controlled by multiple enhancers located within and 3' of the IgH transcription unit. In mouse, the 3' regulatory region includes hs3A, hs1,2, hs3B and hs4 and resides 3' of the C{alpha} gene. In humans, the 3' regulatory regions are located 3' of both germ line C{alpha} genes and contain hs3, hs1,2 and hs4. Previously, we showed that rabbits have a single hs1,2 enhancer; hs3- and hs4-like enhancers were not found (30), although, Ros et al. (38) reportedly found a sequence similar to hs4. We thought that the C{alpha} chromosomal region of rabbits would have additional regulatory elements because it contains 13 differentially regulated non-allelic C{alpha} genes and is the most complex C{alpha} locus described in any species. We conducted this study to search for such elements within the 8-kb region 3' of the most distal C{alpha} gene, C{alpha}13. Neither the luciferase reporter gene assay nor nucleotide sequence analyses reported by us and by Ros et al. (38) revealed putative enhancer(s) comparable to mouse and human hs3. Instead, we found a regulatory element, C{alpha}-NRE, that suppresses hs1,2 enhancement of the I{alpha} promoter and down-regulates the I{alpha} promoter itself. Several silencers and NREs have been identified in the IgH locus (21, 39, 40); C{alpha}-NRE provides another example of a cis-element that contributes to the control of Ig genes.

Classically, DNA regulatory elements have been identified by DNase hypersensitivity (hs). However, C{alpha}-NRE was not identified by the presence of a DNase hs site. In fact, we did not find DNase hs sites in the 8-kb region downstream of the distal C{alpha} gene (data not shown). Instead, evidence for regulatory activity of C{alpha}-NRE was obtained from a combination of gel-shift and luciferase reporter gene assays. In gel-shift assays we found two fragments that bound transcription factors, a 270-bp fragment containing the 20-bp repeats and a 156-bp fragment containing a functional E box, CAGGTG, which is found in a variety of cis-elements, including all Ig enhancers (41). Binding of transcription factors to the E box was inhibited by anti-E47 antibodies. E47 is likely involved in the opening of chromatin around Ig genes (42, 43) and is known to be essential for the early stages of B cell development (44). The presence of an E47-binding E box in combination with the inhibitory activity of hs1,2 enhancement of I{alpha} promoter activity in luciferase assays indicates that C{alpha}-NRE is, indeed, a regulatory element.

The negative regulatory activity of C{alpha}-NRE was localized to a 1-kb SacI fragment; however, the activity was not confined to a single small fragment within this region. Instead, we found that several small fragments down-regulated the hs1,2 enhancer. In particular, two different fragments of 270 bp (containing 20-bp repeat fragment) and 156 bp had ~50 and 80%, respectively, of the activity found in the entire 1-kb SacI fragment. It appears that both of these fragments contribute to the activity of C{alpha}-NRE, while only the full-size 1-kb fragment has maximum negative regulatory activity. Similarly, a regulatory element with combined activity, MINE, was found to possess insulator and silencer activity (45). We do not know whether the 270-bp and the 156-bp fragments are part of the same regulatory element or if they represent a complex set of cis-elements.

One region of the rabbit C{alpha}-NRE, the 20-bp repeats, is also found in the 3'C{alpha} regions of human and mouse and by gel-shift analysis we showed that this region bound transcription factors. The identities of the transcription factor(s) that bind this region remain to be determined. The conservation of 20-bp repeats in mouse, human and rabbit indicates that this repetitive region may be associated with regulatory activity. However, the precise nature of this activity remains to be identified.

In reporter assays, C{alpha}-NRE suppressed the activity of the hs1,2 enhancer when driving the I{alpha} promoters. We speculate that one role of C{alpha}-NRE is to silence or suppress the expression of C{alpha} genes before activation of B cells and isotype switch. If so, one might expect that this suppression would be eliminated upon activation of B cells. Indeed, we found that the suppressive effect of C{alpha}-NRE, as determined by I{alpha} promoter activity, was decreased after activation of B cells by anti-IgM. We do not know, however, whether C{alpha}-NRE is a B cell-specific regulator. We attempted to test C{alpha}-NRE activity in one T cell line, 484 (a gift from P. Medveczky, University of South Florida, Tampa, FL, USA), but because hs1,2 is a B cell-specific enhancer we were unable to test C{alpha}-NRE's effect on hs1,2 enhancement of I{alpha} promoters, although C{alpha}-NRE itself inhibited I{alpha} promoter activity in this background.

C{alpha}-NRE is active in a B cell line that produces low level of germ line transcript and is poised to undergo isotype switch (28). Although it would be interesting to test whether C{alpha}-NRE acts in rabbit progenitor B cells, no such cell lines are available. Even so, we predict that C{alpha}-NRE will not be active in these cells because C{alpha}-NRE requires active hs1,2 which is known to be active only at later stages of B cell development (46).

C{alpha}-NRE as a novel NRE
C{alpha}-NRE is distinct from other NREs identified in the Ig locus. We found no nucleotide sequence similarity between C{alpha}-NRE and the B1 repetitive sequences associated with negative regulatory activity in the murine {kappa} light chain locus (21) or the transcriptional silencer of the chicken Ig{lambda} locus (14, 15). In addition, C{alpha}-NRE is not dependent on an octamer sequence as is the chicken Ig{lambda} silencer. A silencing element has been reported within 1 kb 5' of human I{alpha} exons (17); however, by nucleotide sequence analysis, C{alpha}-NRE is not likely a homologue of this silencer. In addition, luciferase constructs containing up to 900 bp 5' of the rabbit I{alpha}4 exon did not have negative regulatory activity (31) (our unpublished data), and furthermore, C{alpha}-NRE resides as much as 3 kb 5' of I{alpha} promoters.

The hs1,2-mediated effect of C{alpha}-NRE is quite different from that of known silencers because silencers act directly on promoters rather than on enhancers, and C{alpha}-NRE acts on both promoters and enhancers. Silencers are described as elements that act on promoters to inhibit transcription in a position- and orientation-independent manner (47, 48). Since the inhibitory activity of C{alpha}-NRE on the I{alpha} promoter is position dependent, we suggest that C{alpha}-NRE is not a silencer. In addition, C{alpha}-NRE affects enhancer activity of hs1,2, a function not attributed to silencers (47).

Because C{alpha}-NRE acts on the hs1,2 enhancer, we considered whether C{alpha}-NRE might be an insulator, which, as described, acts on enhancers (6, 4952). Direct comparison of these elements is complicated by the use of different assays to test the functional activity of these regulators. However, unlike C{alpha}-NRE, which directly inhibits I{alpha} promoters, insulators have not been shown to directly inhibit promoters. By a computer search, we did not find consensus CTCF-binding sites like those found in mammalian insulators (50); however, the CTCF-binding sites may be missed in computer search. Because two copies of insulator rescue promoter–enhancer activity, we tested the construct containing two copies of 1-kb SacI fragments placed 5' of I{alpha} promoter and 3' of hs1,2 in the luciferase assay. We found that luciferase activity was still inhibited in this construct (data not shown) and that no restoration of luciferase activity was observed. Thus, we found no evidence for insulator activity in the 1-kb-SacI fragment using the luciferase assay.

Taking together the analyses of silencers and insulators, it appears that C{alpha}-NRE has activity different from that of previously described NREs, and we suggest that C{alpha}-NRE represents a novel NRE. However, we cannot rule out the possibility that because C{alpha}-NRE activity is found in several regions of the 1-kb SacI fragment, the novelty of the element is in part due to a complex of regulatory elements comprised of more than one kind of regulatory element.

Association of C{alpha}-NRE with multiple C{alpha} genes
Nucleotide sequence analysis showed that the 156-bp fragment containing C{alpha}-NRE activity is conserved among 8 of the 13 C{alpha} genes. Although we did not assay the 156-bp fragment from each C{alpha} gene for negative regulatory activity, it is reasonable to conclude that each of these has negative regulatory activity. Considering that the multiple C{alpha} genes arose by duplication, we think that the C{alpha}-NREs duplicated together with C{alpha} genes during evolution. The presence of multiple NREs within the C{alpha} locus leads to the idea that these multiple regulatory elements are involved in regulating expression of the C{alpha} genes that span over 150 kb. At present, however, we do not know how these C{alpha}-NREs could interact with hs1,2 and C{alpha} genes to control C{alpha} expression. We think that C{alpha}-NRE may be part of the complex regulation of hs1,2 in the context of the locus control region or that the suppression of gene activity is due to concerted repression by hs1,2 and C{alpha}-NRE (46).

We considered the possibility that identification of C{alpha}-NRE using the luciferase assay might not reflect the interaction of cis-elements in vivo. We identified transcripts of C{alpha}-NRE in a B cell line by northern blot analysis (data not shown) suggesting that C{alpha}-NRE may function though an RNA transcript. Such a transcript could, for example, alter the PolII complex and block the transcription of C{alpha} genes.

In this study, we identified a complex regulatory element associated with rabbit germ line C{alpha} genes that negatively regulates the I{alpha} promoter and hs1,2 enhancement of this promoter. The C{alpha}-NRE suppresses hs1,2, and to our knowledge this is the first report of a regulator for hs1,2. The highest level of negative regulatory activity is found within two segments—a 156-bp region that is conserved among 8 of the 13 C{alpha} genes and a segment containing 20-bp repeats that are nearly identical to those associated with human C{alpha}2 genes. The conservation of the 156-bp region during duplication of rabbit C{alpha} genes and the conservation of the 20-bp repeats during human and rabbit evolution suggest that these elements are functionally important. We suggest that C{alpha}-NRE provides negative control of C{alpha} gene expression in B cells by inhibiting I{alpha} promoters and/or hs1,2-mediated enhancement of the I{alpha} promoters. The activity of C{alpha}-NRE may contribute to the differential expression of the multiple C{alpha} genes in rabbit and in human.


    Supplementary data
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 
Supplementary data are available at International Immunology Online.

Supplementary Figure 7. Luciferase reporter assay for activity of the 1-kb SacI fragment in 55D1 B cells. Lane numbers correspond to construct numbers shown on the right. The percentage of luciferase activity was determined relative to a construct with luciferase driven by the I{alpha} promoter, 100% which was ~9000 light units. The hs1,2, and the I{alpha} promoter are in germline orientation relative to each other; the arrow indicates reverse orientation of 1-kb SacI fragment relative to hs1,2 and the I{alpha} promoter.


    Acknowledgements
 
We are grateful to Barbara Birshtein (Albert Einstein College of Medicine, NY, USA) for insightful comments during the preparation of this manuscript. This work was supported by grants from the National Institutes of Health: AI11234 and AI50260.


    Abbreviations
 
C{alpha}-NRE   C{alpha} negative regulatory element
hs   hypersensitivity
NRE   negative regulatory element

    Notes
 
Transmitting editor: W. Strober

Received 15 December 2004, accepted 6 May 2005.


    References
 Top
 Abstract
 Introduction
 Methods
 Results
 Discussion
 Supplementary data
 References
 

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