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Originally published In Press as doi:10.1074/jbc.M204065200 on June 21, 2002

J. Biol. Chem., Vol. 277, Issue 36, 32640-32649, September 6, 2002
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Chromatin Structural Analyses of the Mouse Igkappa Gene Locus Reveal New Hypersensitive Sites Specifying a Transcriptional Silencer and Enhancer*

Zhi-Mei Liu, Julia B. George-Raizen, Shuyu Li, Katherine C. Meyers, Mee Young Chang, and William T. GarrardDagger

From the Department of Molecular Biology, University of Texas Southwestern Medical Center, Dallas, Texas 75390-9148

Received for publication, April 25, 2002, and in revised form, June 19, 2002

    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

To identify new regulatory elements within the mouse Igkappa locus, we have mapped DNase I hypersensitive sites (HSs) in the chromatin of B cell lines arrested at different stages of differentiation. We have focused on two regions encompassing 50 kilobases suspected to contain new regulatory elements based on our previous high level expression results with yeast artificial chromosome-based mouse Igkappa transgenes. This approach has revealed a cluster of HSs within the 18-kilobase intervening sequence, which we cloned and sequenced in its entirety, between the Vkappa gene closest to the Jkappa region. These HSs exhibit pro/pre-B cell-specific transcriptional silencing of a Vkappa gene promoter in transient transfection assays. We also identified a plasmacytoma cell-specific HS in the far downstream region of the locus, which in analogous transient transfection assays proved to be a powerful transcriptional enhancer. Deletional analyses reveal that for each element multiple DNA segments cooperate to achieve either silencing or enhancement. The enhancer sequence is conserved in the human Igkappa gene locus, including NF-kappa B and E-box sites that are important for the activity. In summary, our results pinpoint the locations of presumptive regulatory elements for future knockout studies to define their functional roles in the native locus.

    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The mouse immunoglobulin (Ig) kappa  gene locus has provided a paradigm to investigate many challenging and biologically relevant problems, including site-specific recombination (1-5), tissue-specific transcriptional regulation (1, 6, 7), somatic hypermutation (8-11), DNA methylation (12-14), the relationship between chromatin structure and function (15-23), and the evolution of DNA sequence organization (24).

The mouse kappa  locus is the largest multi-gene family locus thus far identified with respect to genomic length, spanning more than 3.5 megabases (25-30). The locus contains 96 potentially functional Vkappa genes that have been grouped into 18 families based on sequence homologies (29, 31, 32), 4 functional and 1 non-functional Jkappa regions, and a single Ckappa exon. The Vkappa families are semi-clustered but partially interspersed with other Vkappa families (25, 29). The most 5' Vkappa gene is a member of the Vkappa 24 family, some 3.5-megabases away from the Jkappa -Ckappa region (29). The most 3' Vkappa gene is Vkappa 21G (29), 18 kb1 away from Jkappa 1 gene segment (this work).

Previous studies have identified several cis-acting regulatory elements in the mouse Igkappa locus. All of these elements except for Vkappa gene promoter elements reside in a 16-kb segment near or within the Jkappa -Ckappa region toward the 3' end of the locus. These include two germ line promoter elements (33, 34), KI-KII sequences (35), two non-B cell-specific silencers (36, 37), a nuclear matrix association region (MAR) (38), an intronic enhancer (Ei) (39), and a 3' enhancer (E3') (40). In some instances targeted deletions of these elements have been performed in cell lines or mice, permitting their functional significance to be addressed in the native locus. Deletion of a germ line promoter or KI-KII sequences or both results in a suppressed recombination phenotype (35, 41, 42). Deletion of the MAR in a pre-B cell line results in hyper-recombination (13), whereas its deletion from the mouse germ line down-regulates somatic hypermutation and mildly stimulates precocious Vkappa -Jkappa joining (43). Deletion of either Ei or E3' severely reduces but does not abolish Igkappa gene rearrangement (44, 45), whereas deletion of both enhancers reveals that each has a redundant but critical role in regulating recombination in the locus (46).

Although considerable insight has been revealed on the functional significance of the above elements, the results of several investigations strongly suggest that additional crucial regulatory elements within the Igkappa locus remain to be discovered. For example, expression of rearranged Igkappa transgenes containing both enhancers is influenced by the site integration and fails to exhibit copy-number dependence (16, 47, 48). In addition, mice harboring human Igkappa germ line transgenes containing all the corresponding known regulatory elements described above exhibit only poor and erratic expression relative to the endogenous mouse Igkappa locus (49-53). However, our recent success of achieving high level expression of YAC-based mouse Igkappa transgenes indicates that regulatory elements are present in these constructs capable of conferring copy number-dependent, position-independent germ line transcription, tissue and developmental stage-specific efficient Vkappa -Jkappa rearrangement, and rearranged Igkappa gene transcription (54). These constructs contained additional upstream and downstream sequences missing from other poorly expressed transgenes, sequences residing both 5' and 3' of the Jkappa -Ckappa region.

In an effort to identify new regulatory elements we have therefore focused on the aforementioned previously unstudied regions in the mouse Igkappa locus suspected to possess transcriptional regulatory elements based on our transgenic mice studies (54). Our approach takes advantage of the observation that when cis-acting elements are functional in a particular cell lineage, they often form nuclease hypersensitive sites (HSs) in chromatin (55). We report here the identification, sequencing, and initial functional characterization of several such HSs. Interestingly, one cluster of HSs specifies transcriptional silencing in pro/pre-B cells, whereas another acts as a powerful B cell-specific transcriptional enhancer.

    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Long PCR Amplification of the Intervening Sequence (IS)---Template DNA was prepared as total DNA from yeast cells bearing either YAC FAW.A3 or YAC FAW.A3 truncated at Vkappa 21G by chromosome fragmentation (24, 25). To truncate FAW.A3, a genomic Vkappa 21 fragment was amplified by PCR (primers Vkappa 21L (5'-TGC TGC TGC TCT GGG TTC CAG GTG-3') and Vkappa cdr2r (5'-GAT TCT AGG TTG GAT GCA GGA TAG-3')). Amplification conditions were 1 min at 94 °C, 2 min at 55 °C, and 1 min at 72 °C for 30 cycles. The amplified sequence was introduced into the acentric fragmentation vector pBP81 (56), linearized, and transformed into yeast harboring FAW.A3 using standard lithium acetate transformation (57). Several transformants were analyzed using pulsed-field gel electrophoresis. Long PCR was performed using the Expand Long PCR System according to the manufacturer's recommendations (Roche Molecular Biochemicals). After equilibration in 1× PCR reaction buffer (excluding enzymes), 25 µl of master mix 1 (dNTPs plus primer) was added to gel block slices followed by incubation for 15 min at 65 °C and the subsequent addition of 25 µl of master mix 2 (including enzymes). Amplification conditions were 10 cycles of 94 °C for 30 s, 55 °C for 45 s, and 68 °C for 12 min. Twenty additional cycles were performed in which the extension cycle was increased by 10 s/cycle. Primers were Vkappa 21L (5'-TGC TGC TGC TCT GGG TTC CAG GTG-3') and Jkappa 2R (5'-TTT GAG CTT GAG TAG ACA AAT ATC C-3'). 1-2% of the total products were separated by pulsed-field gel electrophoresis on 1% agarose gels using an auto algorithm for 5-50 kb on a CHEF mapper (Bio-Rad).

Cell Culture-- Cell lines, except for S194, A20, and MPC-11, were maintained in RPMI 1640, 10% fetal bovine serum, 1% penicillin-streptomycin, and 2 mM L-glutamine. beta -Mercaptoethanol (50 µM) was added to pre-B cell cultures. S194 and A20 cells were cultured in Iscove's medium containing 5% fetal bovine serum, and MPC-11 cells were cultured in Dulbecco's modified Eagle's medium containing 20% horse serum. The engineered 103/BclII cell lines Delta N1 and Delta N7 (58) were kind gifts of Eugene Oltz (Vanderbilt University). The pre-B cell 103Bcl2 lines were maintained at 34 °C with 5% CO2; S194, A20, and MPC-11cells were maintained at 37 °C and 10% CO2; all other lines were maintained at 37 °C and 5% CO2.

Mapping DNase I Hypersensitive Sites-- Cells were permeabilized with hen egg white alpha -lysolecithin (Sigma) and treated with increasing concentrations of DNase I (Worthington Biochemicals) (0.25-8.0 units/ml) (17). After lysis, DNA was purified either using Qiagen genomic columns or by phenol:chloroform extraction (13), and samples were digested to completion with either BglII, HindIII, NcoI, or PstI as indicated below. For PstI digest mapping with probes A or B (see Fig. 1), 10-15-µg samples were electrophoresed in 0.8% agarose (SeaKem GTG, FMC Bioproducts) in 0.5× Tris-acetate-buffered EDTA running buffer overnight at 1.1 V/cm. After blotting using standard neutral transfer to Nytran Plus membranes (59), 0.2-µm pore size (Schleicher and Schuell), prehybridized filters were hybridized overnight in 6× SSC (1× SSC = 0.15 M NaCl and 0.015 M sodium citrate), 10× Denhardt's solution, 1% SDS, and 100 µg/ml herring testes DNA with a 1.8- or 0.8-kb DNA fragments, corresponding to probes A and B, respectively, labeled with [alpha -32P]dCTP using Rediprime II (AP Biotech). Wash conditions were 1× SSC, 0.5% SDS 3 times for 30 min at 65 °C or until sufficient background had been eliminated. For mapping with probes C-E, electrophoresis and blotting was as above, but pre-hybridization, hybridization, and washing were performed according to a modification of Church and Gilbert (13, 60). Membranes were exposed to Eastman Kodak Co. XAR5 film with intensifying screens at -70 °C or to PhosphorImager screens (Molecular Dynamics). Probe A was a 1.8-kb ClaI/PstI fragment gel-isolated from the IS long PCR product. Primers for probe B PCR amplification were 5'-PstL (5'-TAA AAA TCC TGG TGC CAG GGG TG-3') and 5'-PstR (5'-AGC TTA AGG ACG TCA CAT AGA CT-3'). PCR reactions were performed for 30 cycles of 1 min at 94 °C, 1 min at 55 °C, and 1 min at 72 °C. BglII or NcoI digests were used for mapping with probe C, PstI digests were used for mapping with probe D, and HindIII digests were used for mapping with probe E (see Fig. 1). Probe C consisted of a 1.3-kb BglII/HindIII fragment isolated from the pRxR-1 recombinant plasmid (61). Probe D consisted of a 1.5-kb SacI fragment isolated from the pRSB recombinant plasmid (61). pRxR-1 and pRSB were the kind gifts of Michael Reth (Max-Plank-Institut für Immunobiologie, Freiburg, Germany). Probe E was a 510-bp fragment spanning exon 6 of the mouse ribose-5-phosphate isomerase gene (62), prepared as above using the following PCR primers 5'-GCT TGC TTG GAC CTG CTG G-3' and 5'-CGG CAG AGA AGA CAA AGG ATC C-3'.

Transient Transfection Luciferase Assays-- For functional tests, various fragments were inserted into the NheI or the SpeI site of the polylinker region of KpLUC or IM.KpLUC, respectively (63), designated here as PVkappa Luc and EiPVkappa Luc, respectively. A 3.6-kb fragment encompassing hypersensitive sites HS 3-6 was amplified from the IS long PCR product to add external SpeI sites using PCR conditions similar to those described above for probe preparation. The primer pairs for fragments containing HS 3-6, HS 4-6, HS 5-6, HS 6, HS 3-4, HS 3, and HS 4-5 were, respectively: 5'-ACG CGT CGA CTA GTG TAC TCT GAA CCT TGT ATG GTG ATG-3' and 5'-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3'; 5'-ACT CGT CGA CTA GTC TCT GGG CCT GCA CAG ATT CCA C-3' and 5'-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3'; 5'-ACG CGT CGA CTA GTC TGC TAC ATA TGT GCG GGG GAG G-3' and 5'-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3'; 5'-ACG CGT CGA CTA GTC CCA CCC TCA AGA CAG GCA CAG-3' and 5'-ACG CGT CGA CTA GTG CAG GTT ATG GGC CCT CTT CC-3'; 5'-ACG CGT CGA CTA GTG TAC TCT GAA CCT TGT ATG GTG ATG-3' and 5'-ACG CGT CGA CTA GTC CTC CCC CGC ACA TAT GTA GCA G-3'; 5'-ACG CGT CGA CTA GTG TAC TCT GAA CCT TGT ATG GTG ATG-3' and 5'-ACG CGT CGA CTA GTG GAA TCT GTG CAG GCC CAG AGA C-3'; 5'-ACG CAC GCG TCG ACT AGT CTC TGG GCC TGC ACA GAT TCC AC-3' and 5'-ACG CAC GCG TCG ACT AGT GCC TGT CTT GAG GGT GGG ACT G-3'. The spacer DNA control was a 2.1-kb rat amylase cDNA fragment. PCR conditions were 1 min at 94 °C, 2 min at 55 °C, and 2 min at 72 °C for 30 cycles. For vector insertions we similarly amplified a 1-kb fragment encompassing HS 9 from recombinant plasmid pRxB5 (the kind gift of Michael Reth, Max-Plank-Institut für Immunobiologie, Freiburg, Germany) (61) using the PCR primers L10F1 (5'-CCG CCG ACT AGT CGT TAG CCC CTG TCC TTG-3') and L10R1 (5'-CCG CCG ACT AGT TGT GCA TAT GTG TGT GTA CAC ATG-3'). For testing smaller segments of the 1-kb sequences, we PCR-amplified the desired regions, again adding external SpeI sites for vector insertions as above using the primers L10F2 (5'-CCG CCG ATC AGT GAA GCC AGG GAA ATG CCA C-3'), L10R2 (5'-CCG CCG ATC AGT CTA GCT TTA CAG CTT GTC-3'), L10R3 (5'-CCG CCG ATC AGT GCT TAA GCA GCA GAC AGT G-3'), L10R4 (5'-CCG CCG ATC AGT GTG CCC TGC ACC TTC AGG-3'), and L10R5 (5'-CCG CCG ATC AGT GTG GCA TTT CCC TGG CTT C-3'). PCR conditions were 1 min at 94 °C, 30 s at 58 °C, and 75 s at 72 °C for 35 cycles. Finally, to replace the Vkappa promoter with a 420-bp BglII/HindIII fragment containing the SV40 early enhancer/promoter from the pRL-SV40 vector (Promega), the Vkappa promoter was deleted from PVkappa Luc by excision with NheI and HindIII and from PVkappa Luc containing the 3.6-kb silencer by HindIII digestion. Then the sticky ends of these two vectors were filled in with Klenow and dephosphorylated. The sticky ends of the SV40 early enhancer/promoter 420-bp fragment were also filled in with Klenow and ligated to the treated vectors to construct PSV40Luc and 3.6kbPSV40Luc. Cell lines were transiently transfected either in triplicate in the same batch or in duplicate in separate batches using either optimized DEAE dextran concentrations (100-250 µg/ml; 500 µg/ml for S194 cells) as previously described (58), LipofectAMINE-Plus, or LipofectAMINE 2000 (Invitrogen). Typically, 106-107 cells and 1-2 µg of DNA were used per transfection, adjusted for insert sizes to provide equimolar comparisons, along with 20-50 ng of pRL-CMV Renilla luciferase reporter (Promega Corp). Pre-B and more mature cell lines were harvested 24 and 48 h post-transfection, respectively. The 24-h time chosen for pre-B cells allowed for optimum reproducibility of ±10 µg/ml lipopolysaccharide (LPS) comparisons (data not shown). Cell extracts were assayed for luciferase activity using Dual-LuciferaseTM reporter assay systems (Promega) following the manufacturers' instructions. The Renilla luciferase activity was used for normalization of transfection efficiencies, except for the pre-B cell samples in Fig. 5, A and B, where extract protein levels were used. Data from a minimum of triplicate experiments are represented with error bars, whereas duplicate experiments are represented as means. Data were internally consistent between triplicates with the same batch of cells.

Mutagenesis of Ed-- Two-step PCR mutagenesis was used to create mutations of NFkappa B and E-box sites as described elsewhere (64) in a 600-bp fragment amplified with L10F1 and L10R3 primers as described above. Sense and antisense primers for NFkappa B site mutation were 5'-GAA GTC AAA TTG GTT TCC ACT GTG CCA C-3' and 5'-GAA ACC AAT TTG ACT TCA TTA CCT CAT G-3'; sense and antisense primers for first E-box mutation were 5'-CCT GCA TTT TTG CAG TGC AGA TGG AC-3' and 5'-CAC TGC AAA AAT GCA GGG CTG GAC TC-3'; sense and antisense primers for second E-box mutation were 5'-CAG TGC ATT TTG ACT TGG CAA AAG AAG-3' and 5'-CAA GTC AAA ATG CAC TGC ACA GGT G-3' (mutated bases are underlined).

    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Overall Experimental Strategy-- To locate new candidate regulatory elements within the mouse Igkappa gene locus, we assayed for the presence of DNase I-hypersensitive sites in the chromatin of B cell lines representing different stages of lymphocyte differentiation. Such an approach has been proven to be successful previously and has contributed to the discovery and functional analyses of several enhancer elements in the mouse light and heavy chain Ig gene loci (16, 17, 19, 65-68). As shown in Fig. 1, we selected for study two regions that were present in our highly expressed YAC Igkappa transgenes that have not been previously characterized (54), (i) the IS between the closest Vkappa gene segment to Jkappa 1 and (ii) the region extending downstream of E3' up to the next non-Ig related gene, which encodes ribose-5-phosphate isomerase (62). By definition, the IS represents a boundary within the locus, separating the array of upstream Vkappa gene segments from the downstream Jkappa regions. The IS either becomes deleted or translocated far upstream after Vkappa -Jkappa joining at least 260 kb, based on distance of the closest Vkappa gene with a reverse orientation (26). This region, therefore, is a likely candidate to contain an element(s) that specifies regulation by its physical location or relocation relative to other important components within the Igkappa gene locus. By contrast, the downstream region studied is maintained in the locus even after recombination, like the regions harboring Ei and E3'. Fig. 1 summarizes the physical locations of HSs, pertinent restriction endonuclease sites, the various hybridization probes used for indirect end-labeling to map HSs (55), and the newly identified novel DNA sequence motifs, LINES and SINES.


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  		Fig. 1.   Positions of HSs and other key elements in the Igkappa gene locus. Bg, H, N, and P and designate the positions of relevant BglII, HindIII, NcoI, and PstI sites. Also indicated are the hybridization probes for indirect end labeling (bold bars), a potential triplex-Z DNA forming sequence (T/Z), germ line promoters (horizontal arrows), Vkappa , Jkappa , and Ckappa exons, enhancers Ei and E3', a MAR, a potential triplex forming sequence (T), and the recombining sequence (RS) associated with deletion of the Igkappa gene locus in Iglambda producers (61). Repeat sequences were identified with a program available on the web (ftp.genome.washington.edu/cgi-bin/RepeatMasker). LINE and SINE sequences are depicted as horizontal arrows and lines, respectively.

Isolation and Sequencing of the IS of the Mouse Igkappa Gene Locus-- For the purpose of generating hybridization probes for our chromatin structure mapping experiments, it was first necessary to clone and physically map the previously uncharacterized mouse Igkappa gene IS. The Vkappa family residing closest to Jkappa 1 is Vkappa 21 (24, 26, 69, 70). Previously we performed chromosome fragmentation of Vkappa 21 gene segments within the recombinant YAC clone FAW.A3 and found that the length of the IS is about 20 kb and that the closest Vkappa 21 family member to Jkappa 1 is Vkappa 21G (24). These results suggested that it might be possible to PCR-amplify the IS, which proved to be the case. As shown in Fig. 2 (arrow), the PCR-amplified IS exhibits a mobility in pulsed-field gels of about 20 kb in length, which is a size also in agreement with the physical mapping studies of Zachau and co-workers (26). Note that before truncation of FAW.A3 at Vkappa 21G, the presence of the 5' sequence leads to additional PCR products generated primarily by priming by the Vkappa 21 primer alone (Fig. 2, compare lanes 2 and 3). For further analyses, the desired PCR product was subcloned and entirely sequenced by bi-directional primer walking in duplicate using an ABI PrismTM 377 DNA Sequencer (>= 4-fold coverage). The distance between the recombination signal sequences of Jkappa 1 and Vkappa 21G proved to be 18,023 bp in length (GenBankTM accession number AF513926).2 Inspection of the sequence for segments with potential for forming alternative DNA structures revealed an interesting region ((CT)26(GT)26) with unknown present significance (Fig. 1, T/Z), analogous to a triplex-Z DNA motif found near an origin for replication in the Chinese hamster ovary dhfr gene (71). We also found a number of LINE/L1 and SINE/B2 repeats in the IS (Fig. 1, horizontal arrows and lines). Comparison of the mouse sequence with its 23.4-kb human counterpart (GenBankTM accession number AF017732) by dot matrix analysis reveals that one upstream LINE sequence is conserved as well as the downstream 4-kb region (Fig. 3), which is known to contain the germ line promoters and KI/KII elements (33-35, 41, 42).


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  		Fig. 2.   Long PCR amplification of the Igkappa gene IS. PCR amplification products were assayed by pulsed-field agarose gel electrophoresis with ethidium bromide DNA staining. Lanes 1-3, YAC FAW.A3 as template (25); lanes 4-6, YAC FAW.A3 truncated at Vkappa 21G as template (24). The solid arrow represents the IS-amplified product.


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  		Fig. 3.   Dot matrix sequence comparisons of the sequences of the mouse and human ISs. The stringency used a window with 15 of 21 matches.

The IS Exhibits Six DNase I Hypersensitive Sites in Chromatin-- To locate candidate regulatory regions within the IS, we mapped the location of HSs in a variety of cell lines using the gentle technique of permeabilization with alpha -lysolecithin to introduce DNase I (17). A total of six HSs were found in the IS in two clusters (Fig. 1, HSs 1 and 2 and HSs 3-6). HS 1 and 2 appear to be ubiquitous among the non-B and B cell lines studied and, therefore, have not been investigated further (data not shown). Primary data illustrating the detection of HS 3-6 in several cell lines is shown in Fig. 4. HS 3 and 6 also appear to be ubiquitous, being present in P815 mastocytoma, EL-4 T cells, BASC6C2 pro-B cells, 103 Bc12 pre-B cells, and S194 plasmacytoma (Fig. 4, open arrowheads). In contrast, HSs 4 and 5, although weakly detectable in EL-4 T cells and BASC6C2 pro-B cells, are most noticeable in 103Bc12 pre-B cells (Fig. 4, closed arrows) and vary in intensity in other pre-B cell lines, including 3-1 and 1-8 and the pro-B cell line 63-12, established from RAG2 -/- animals (data not shown).


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  		Fig. 4.   Analysis of the chromatin structure of the Igkappa gene IS. Solid arrows indicate pre-B cell enhanced HSs. HSs 3-6 were mapped by Southern analysis after DNase I digestion of the chromatin of various indicated cell lines after PstI digestion and labeling with probe B (see Fig. 1).

We also analyzed the most 3' portion of the IS for HSs. This region includes the potential triplex-Z DNA-forming region described above, KI/KII sequences, and two germ line promoters (Fig. 1, T/Z and horizontal arrows) (33-35, 41, 42). Initiation of transcription from these germ line promoters is induced by LPS in cultured pre-B cell lines and has been correlated with the onset of rearrangement in the Igkappa gene locus (33, 34, 72). However, in our assays we failed to detect HSs in either of these germ line promoters either before or after LPS treatment or heat induction of 103Bc12 cells (data not shown), suggesting that these promoters may only be used transiently so that HSs are not detectable or that the entire region is preferentially sensitive to DNase I in pre-B cells. In summary, our initial analyses of chromatin structure within the IS reveal the presence of HSs 1-6 as new candidates for novel regulatory elements. A computer search of the IS sequence against known transcription factor binding sites within and surrounding HSs 1-6 was not particularly revealing.

Functional Analyses of HSs 3-6 Reveals Transcriptional Silencer Activity-- Because HSs 4 and 5 appeared to be enhanced in certain pre-B cell lines, we decided to first perform functional studies on these elements together with their nearby surrounding partners, HSs 3 and 6. To determine whether transcriptional enhancer activity might be associated with HSs 3-6, a 3.6-kb fragment spanning the entire region was inserted into a construct containing a luciferase reporter gene, upstream of a minimal Vkappa gene promoter (PVkappa Luc). In contrast to our expectations, the inserted element repressed expression of the reporter gene construct 5-10-fold in 1 pro-B and 2 pre-B cell lines (Fig. 5A, 38B9, 3-1, 103Bcl2, respectively) but was essentially innocuous in MPC-11 plasmacytoma cells (Fig. 5A). To further examine this silencing activity, the 3.6-kb fragment was inserted upstream of the intronic enhancer in a related reporter gene, EiPVkappa Luc, in either orientation (Fig. 5B). HSs 3-6 again exhibited pro/pre-B cell-specific transcriptional silencing, blocking up to 95% of activity in an orientation-independent manner (Fig. 5B). This effect was observed in all 6 pro/pre-B cell lines tested (Fig. 5B, 63-12, 103Bcl2, and 3-1; data not shown). In contrast, HSs 3-6 again had little effect on reporter gene activities in MPC-11 plasmacytoma cells (Fig. 5B) in S194 plasmacytoma cells and in A20 mature B cells (data not shown). Furthermore, pre-B cell-specific silencing could not be overcome in PVkappa Luc with or without Ei by exposure to LPS (data not shown). In addition, we have also found that the 3.6-kb silencer fragment was incapable of significantly down-regulating transcription driven by the SV40 promoter/enhancer element in P815 mastocytoma cells but could 4-fold suppress transcription in 103Bcl2 pre-B cells (data not shown). Taken together, these results indicate that silencing is clearly pro/pre-B cell-specific. We term this silencer Sis.


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  		Fig. 5.   DNA sequences encompassing HSs 3-6 in the IS exhibit pro/pre-B cell specific transcriptional silencing. A, a 3.6-kb fragment spanning the entire region was inserted into a construct containing a luciferase reporter gene upstream of a minimal Vkappa gene promoter (PVkappa Luc) and assayed in the pro-B cell line 38B9, pre-B cell lines 3-1 and 103Bcl2, and MPC-11 plasmacytoma cells. B, the 3.6-kb fragment was inserted upstream of the intronic enhancer in a related reporter gene, EiPVkappa Luc, in either orientation. The activities of the luciferase reporter genes were assayed in the pro-B cell line 63-12 derived from RAG2 -/- animals, pre-B cell lines 103Bcl2 and 3-1, and MPC-11 plasmacytoma cells. C, the luciferase activities of a series of constructs harboring 5' or 3' deletions or both in the 3.6-kb Sis and a spacer DNA length control assayed in 103Bcl2 cells. The test sequences were inserted upstream of EiPVkappa in the luciferase reporter gene. The activity of each construct was measured after transient transfection of the indicated cell lines. The level of activity of each construct is expressed as a percent of the activity of the indicated reporter gene without an insert at the upstream test site after correction for extract yields by protein assays (A and B panels) or relative transfection efficiencies by monitoring the activity of a co-transfected pRL-CMV Renilla luciferase reporter gene (panel C). Insertion in the native orientation with respect to the promoter is indicated as HS3-6 right-arrow , whereas insertion in the reverse orientation is indicated as HS3-6 left-arrow  . Spacer, cDNA length spacing control.

To pinpoint the DNA sequences responsible for silencing, we created a series of 5' or 3' deletions or both in the 3.6-kb Sis and also performed a spacer DNA control. The test sequences were inserted in the positive orientation upstream of EiPVkappa in the luciferase reporter gene. However, this analysis revealed that silencer activity appeared to require all 4 HSs; when HS 3 was deleted, silencing activity was lost, and could not be established by HS 3 by itself nor could HSs 4 and 5 silence by themselves (Fig. 5C). Furthermore, silencing was not due to a change in the DNA sequence spacing as demonstrated by insertion of a cDNA fragment (Fig. 5C). In summary, these initial functional studies reveal that Sis requires Hs 3-6 to reduce transcription from 5- to 20-fold in a pro/pre-B cell-specific manner.

The Downstream Region Exhibits a Plasmacytoma-Cell-specific HS-- To locate additional candidate regulatory regions in the Igkappa gene locus using similar techniques to those described above, we mapped the location of HSs in a several pre-B and B cell lines within a 30-kb region downstream of E3'. We took advantage of the existing restriction endonuclease physical map and recombinant plasmids bearing these downstream sequences that were kindly made available to us by Michael Reth (61).3 Probes C, D, and E failed to detect HSs in the downstream region in either 63-12 pro-B or 103Bcl2 pre-B or S194 plasmacytoma cells after indirect end labeling of chromatin DNA digests at BglII, PstI, and HindIII sites, respectively (see Fig. 1 for the detection strategy; data not shown). However, using probe C for indirect end labeling of chromatin DNA digests at an NcoI site revealed a new HS in the 30-kb region studied, designated HS 9 (Fig. 1). Primary data illustrating the detection of HS 9 is shown in Fig. 6. Interestingly, HS 9 proved to be present only in terminally differentiated plasmacytoma cells (Fig. 6, MPC-11 and S194), being absent from 3-1 pre-B cells, 103Bcl2 pre-B cells ±LPS (Fig. 6), A20 mature B cells, and EL-4 T cells (data not shown). In summary, this analysis revealed HS 9 as a plasmacytoma cell-specific HS.


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  		Fig. 6.   Analysis of the chromatin structure of the Igkappa gene downstream region. Solid arrow indicates the plasmacytoma cell-specific HS 9. Mapping by Southern analysis after DNase I digestion of the chromatin of various indicated cell lines after NcoI digestion and labeling with probe C (see Fig. 1).

The DNA Sequence Encompassing HS 9 Contains Potential NF-kappa B Binding Sites and E-boxes, Is Evolutionary Conserved, and Is Distally Flanked by a Novel Sequence Motif-- To gain insight on potential important or unusual DNA sequence motifs and potential transcription factor binding sites and assess evolutionary conservation, we sequenced a 6-kb region surrounding and encompassing HS 9 (GenBankTM accession number AF513925).2 Inspection of the sequence for segments with potential for forming alternative DNA structures revealed a highly unusual 172-bp polypurine array ((AAAG)2(A)5(G)2(A)3(GAAA)2(GA)23(A)2(GA)5(GGGA)8(GGAA)14) exhibiting domains of mirror repeat symmetry, with triplex forming potential and unwinding capabilities,4 of unknown present significance (Fig. 1, T). We also found segments of one LINE/L1 and SINE/B2 repeat in this downstream region (Fig. 1, horizontal arrow and line). Significantly, the sequence encompassing HS 9 possesses a potential NF-kappa B binding site and E-boxes (see below), characteristics shared with Ei (63, 64). Comparison of the mouse sequence with its 6-kb human counterpart (GenBankTM accession number AC096579) by dot matrix analysis reveals that several domains in the downstream region are conserved, including those encompassing HS 9 (Fig. 7A) and its potential transcription factor binding sites (Fig. 7B). However, the mouse triplex-forming motif was not found in the corresponding region of the human sequence but, interestingly, nevertheless resides in the IS of the human Igkappa gene. In summary, the observed conservation of HS 9 prompted our interest for the functional analyses described below.


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  		Fig. 7.   Comparison of the mouse Ed sequence with its human counterpart. A, dot matrix sequence comparisons of the sequences of the mouse and human Igkappa gene downstream region using a window with 13 of 21 matches. B, the sequences encompassing HS 9 possess one potential NF-kappa B binding site (GGG(A/G)NN(T/C)(T/C)CC) and two E-boxes (CANNTG), which are conserved between mouse and human.

Functional Analysis of HS 9 Reveals a B Cell-specific Transcriptional Enhancer-- To determine whether transcriptional enhancer activity might be associated with HS 9, a 1-kb fragment spanning the entire region was inserted upstream of PVkappa in the luciferase reporter gene construct. The sequence proved to enhance transcription in an orientation-independent fashion in both 103Bcl2 pre-B and S194 plasmacytoma cells (Fig. 8A). We therefore term this enhancer Ed, based on its downstream location in the locus. To determine whether such an enhancement might be related to NF-kappa B activity, we took advantage of a derivative of the 103Bcl2 cell line that was kindly made available to us by Eugene Oltz, termed Delta N1, which has been engineered to express a dominant negative form of Ikappa B (58). As shown in Fig. 8A, enhancement of transcription by the 1-kb fragment was markedly suppressed in the Delta N1 cell line. Similar results were obtained using an independently derived dominant negative expressing clone, Delta N7 (data not shown). We conclude that the enhancement of transcription in pre-B cells requires NF-kappa B.


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  		Fig. 8.   DNA sequences encompassing HS 9 specify a transcriptional enhancer. A, a 1-kb fragment spanning the entire region was inserted upstream of PVkappa in the luciferase reporter gene construct in both orientations and assayed in S194 plasmacytoma cells, pre-B cell lines 103Bcl2, and a 103Bcl2 derivative Delta N1 that possesses a dominantly inhibited NF-kappa B. B, the activity of luciferase reporter genes containing neither, either, or both Ei and Ed enhancers inserted upstream of PVkappa in 103Bcl2 pre-B cell line (in the absence or presence of LPS) and S194 and MPC-11 plasmacytoma cells. Panel B represents data normalized to PVkappa Luc. C, the 1-kb Ed fragment assayed in EL-4 T cells, 38B9 pro-B cells, and A20 mature B cells. Except for the upper diagram in panel B, all data are represented as the activity of each construct relative to the activity of the indicated reporter gene without an insert at the upstream test site after correction for relative transfection efficiencies by monitoring the activity of a co-transfected pRL-CMV Renilla luciferase reporter gene. Insertion in the native orientation with respect to the promoter is indicated as Ed right-arrow , whereas insertion in the reverse orientation is indicated as Ed left-arrow  .

To explore how the activity of Ed in the 1-kb fragment compares to or can cooperate with Ei, which is also responsive to NF-kappa B in pre-B cells (58, 63, 64), we compared on an absolute scale (normalized to PVkappa Luc) the activity of luciferase reporter genes containing neither, either, or both enhancers inserted upstream of PVkappa in the absence or presence of LPS. As shown in Fig. 8B (top panel), Ed is a more powerful enhancer than Ei, responds mildly to LPS induction as expected, and cooperates with Ei in an additive fashion. However, in S194 and MPC-11 plasmacytoma cells the two enhancers synergistically activate transcription, leading to activation severalfold higher than the multiplication products of expression levels achieved by each individual enhancer alone (Fig. 8B, lower panels).

To investigate further the cell type and developmental specificity of Ed, we assayed for its activity in several other cell lines. As shown in Fig. 8C, Ed lacks activity in EL-4 T cells but possesses enhancer activity in 38B9 pro-B cells in PVkappa Luc or EiPVkappa Luc reporter genes and in A20 mature B cells only in the PVkappa Luc reporter gene. We conclude that Ed is a B cell-specific enhancer whose activity emerges early in lymphocyte development when assayed by transient expression. This is in contrast to the plasmacytoma stage-specific appearance of HS 9 (see "Discussion").

Site-directed Mutagenesis of Ed Reveals That NF-kappa B and E-box Sites Are Important for Enhancer Activity-- To pinpoint the DNA sequences responsible for enhancement, we created a series of constructs bearing different segments of the 1-kb enhancer. Fig. 9A shows that deletion of 5' and 3' sequences together flanking the potential NF-kappa B binding site and E-boxes significantly reduced but did not fully erase enhancer activity either in the absence or presence of Ei in S194 plasmacytoma cells. The majority of Ed activity could be narrowed to a 600-bp fragment bearing a 3' deletion (Fig. 9A); similar results were obtained in 103Bcl2 pre-B cells (data not shown).


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  		Fig. 9.   The NFkappa B site and E-box are important for the Ed enhancer activity. A, the luciferase activity of a series constructs bearing different segments of the 1-kb enhancer with or without Ei in S194 plasmacytoma cells. Data represented in panels A were normalized as described in Fig. 8. B, functional analysis of the 600-bp Ed fragment with and without targeted mutations in the NF-kappa B site (Delta kappa B), the first E-box (Delta E1), or the second E-box (Delta E2) inserted upstream of PVkappa or EiPVkappa luciferase reporter gene constructs assayed in S194 plasmacytoma and 103Bcl2 pre-B cell lines. Insertion in the native orientation with respect to the promoter is indicated as  right-arrow ori, whereas insertion in the reverse orientation is indicated as black-square left-arrow  ori. GGGGGGTTTC right-arrow AATTGGTTTC is represented by the mutated Ed NF-kappa B site (Delta kappa B), CACCTG right-arrow CATTTT is represented by the mutated Ed first E-box (Delta E1), and CAGATG right-arrow CATTTT is represented by the mutated Ed second E-box (Delta E2), respectively.

To determine the role of NF-kappa B and E-box sites on Ed activity, two-step PCR mutagenesis was used to create mutations in the NF-kappa B and E-box sites in the 600-bp Ed fragment. As shown in Fig. 9B, mutations in the NF-kappa B site caused at least a 3-fold decline in enhancement in either the PVkappa Luc or EiPVkappa Luc reporter genes in both 103Bcl2 pre-B cells and S194 plasmacytoma cells. Mutations in the E-box 1 or 2 had little effect on the these reporter genes in 103Bcl2 pre-B cells, but mutated E-box 1 led to at least a 3-fold decline in expression of reporter genes in S194 plasmacytoma cells. Mutations in E-box 2 also were deleterious to expression in S194 plasmacytoma cells (Fig. 9B). In conclusion, the NF-kappa B site is important for activity regardless of the presence or absence of Ei in either pre-B or plasmacytoma cells, whereas E-box 1 and 2 are most important for maximal expression in the presence of Ei in plasmacytoma cells. These results further suggest that the synergy between Ei and Ed in plasmacytoma cells requires both the NF-kappa B site and E-boxes 1 and 2.

    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We have physically mapped, cloned, and sequenced the mouse Igkappa gene IS. Comparison of this sequence with its human counterpart reveals roughly similar lengths of 18 and 23 kb, respectively. Besides conserved LINE elements in their 5' regions, only the 3' regions share significant extended sequence homology, which correspond to segments containing the germ line promoters and KI/KII elements (33-35, 41, 42). Thus, even though we have identified several HS within the mouse IS in the chromatin of several cell lines, the sequences corresponding to these sites appear not to be heavily conserved. However, in the human IS a cluster of V-regions in closest proximity to Jkappa 1 possesses inverted orientations (73), whereas in the mouse the corresponding V-regions are in the forward orientation. The consequence of this difference is that V-J joining using these closest V-regions would simply invert the IS in the human Igkappa gene locus but lead to a deletion of the corresponding element in the mouse. The first inverted V-region in the mouse is Vkappa 19/3, residing some 260 kb upstream from Jkappa 1 (26). Hence, the mouse IS HS could have evolved a regulatory function(s) that requires its deletion or far removal from the Jkappa -Ckappa region after V-J joining and/or their ensured presence near the Jkappa -Ckappa region in germ line but not rearranged alleles.

We have demonstrated that the IS of the mouse Igkappa locus contains several previously unidentified HS in chromatin and that a subset of these sites, Hs 3-6, constituting Sis, are associated with a pro/pre-B cell-specific transcriptional silencer activity. Although an analogous region in the chicken Iglambda gene has been reported to also contain a transcriptional silencer (74-76), the mouse element that we have identified differs in two significant respects from the chicken component. The latter is not cell type-specific and does not share sequence similarity with the mouse element (74-76). Furthermore, the previously identified non-B cell-specific silencers within the mouse Igkappa gene locus share no sequence homology with Sis (36, 37). We conclude that we have identified a novel regulatory element within the Igkappa gene locus. Indeed, Igkappa germ line transgenes containing the IS in addition to all previously known regulatory elements and the Ed discovered here exhibit high level germ line transcription, tissue-specific rearrangement and subsequent transcription of rearranged genes, and apparent allelic exclusion (54), whereas other transgenes lacking the IS do not (49-53).

The mechanism of silencing is an interesting subject to consider. Possibly the IS silencer exerts its effects by disrupting the assembly of functional transcription factor complexes on promoter elements. It is also possible that the silencer works by targeting the reporter genes to a heterochromatic nuclear subcompartment, as has been associated with the process of allelic exclusion at the mouse Igkappa gene locus (77). Interestingly, the silencer contains LINE element segments, and LINES have been implicated in the process of X-chromosome inactivation (78). Although the silencer activity appears complex, requiring a combination of HSs, other DNA regulatory elements such as MARs, insulators, and polycomb response elements also appear to be quite large and often difficult to define (79).

We have identified a powerful enhancer associated with HS 9, termed Ed, in the downstream region of the Igkappa gene locus. Interestingly, enhancers also exist in related far downstream positions in the IgH locus (68). This location ensures that the element will not be deleted upon normal V(D)J joining and class-switch recombination (1). However, Ed is located 5' of the recombining sequence segment in a region that is frequently deleted in Iglambda -expressing cells. HS 9 is not present in pre- or mature B cells, yet the associated sequence exhibits enhancer activity in transient transfection experiments in these lines. We suggest that this is because the chromatin structure is more open in the transiently transfected plasmid as compared with the endogenous locus to bind NF-kappa B and E2A proteins (63, 64), which are required for Ed activity in B cells as demonstrated by expression of a dominant negative form of Ikappa B and by mutations in the corresponding cis-elements. Although both Ei and Ed possess binding sites for NF-kappa B and E2A, Ed is a stronger enhancer than Ei. This suggests that Ed may interact with additional transcription factors, other NF-kappa B subunits, other combinations of E-proteins, or different post-translational-modified forms of these proteins.

Both NF-kappa B and E2A have previously been demonstrated to be required for B cell development (for review, see Refs. 7 and 80). Knockouts of several NF-kappa B subunits in a variety of combinations leads to a complete block in B cell commitment. Surprisingly, mice lacking both the p50 and p52 subunits of NF-kappa B fail to develop mature IgM+ B cells with the earlier stages of B cell development proceeding normally (81). It is possible that NF-kappa B binding to Ed, most active in later stages of B cell development, is required for full high level expression of rearranged Igkappa alleles and final B cell development.

Our data also indicate a requirement of an intact E-box-1 for full enhancer activity. The requirement for E-box binding proteins in B cell development is well established (reviewed in Ref. 80). The alternatively spliced forms of E2A, E12, and E47 are both required for B cell development at multiple stages, including binding to the E boxes in Ei and E3' (82-84). E2A knockout mice form barely detectable levels of B220+ cells (85, 86), but E47 knock-in experiments demonstrate that E47 can allow cells to progress through to the mature B220+ IgM+ stage (87). Although roles for E2A have been identified at multiple points during the development of both B and T cells, no role has yet been found at the transition from pre-B to mature B cells. The E boxes in Ed may provide a context to identify yet another role for these transcription factors in lymphocyte development.

Previously we and others have shown that Ei and E3' act synergistically when together in expression constructs in plasmacytoma but not pre-B cells (16, 63). Here we show that Ei and Ed synergize, again in plasmacytoma but not pre-B cells, through a process requiring sites for both NF-kappa B and E2A. Although we have not explored interactions between E3' and Ed, previous studies have shown that dimerization of either Ei or E3' alone also yields to similar levels of synergy (88). Thus, interactions between unique aspects present in different enhancers are not necessary for synergy.

It is interesting to consider the relative timing of appearance and functions of Hs 7-9 during B cell development. HS 8 (E3') appears at the earliest stages and persists throughout B cell development but exhibits differentiation specific changes in its fine structure (17, 20). This enhancer is thought to play a negative role during early followed by positive roles later in B cell development (17, 20, 63, 89). HS 7 (Ei) is LPS-inducible in early B cell lines (19, 65) but becomes constitutive in plasmacytoma cells (16). Both HS 7 and HS 8 contribute to the efficiency of rearrangement at the Igkappa locus (44-46). As we have suggested above, the latest-appearing hypersensitive site, HS 9 (Ed), presumably contributes to high level transcription in terminally differentiated B cells.

After sequencing the region encompassing HS 9, we found that the segment already existed in the data base and had been termed L10 (GenBankTM accession number V 01557)2 (90). An aberrant rearrangement in MOPC 41 plasmacytoma cells resulted in the linkage of L10 to the recombination signal sequence of Jkappa 1 (90), which is now recognized to have occurred by creating an inversion in the Jkappa -Ckappa and downstream sequences in the locus (26). Besides the recombining sequence associated with deletion of the Igkappa gene locus in Iglambda producers (61), L10 is the only rearranging structure downstream of Ckappa .

Obviously, targeted deletion of Sis and Ed from the native Igkappa locus will be required to establish if they play any essential roles in Igkappa gene dynamics. To obtain a definitive answer on this point, we have instituted such an experimental plan using the YAC-based Igkappa transgenic system that we have previously developed (54). We have targeted through reverse genetics in yeast LoxP sites on either side of Hs 3-6 and have established transgenic mouse lines harboring such an engineered Igkappa transgene. Once we identify lines in which this transgene is fully functional, we will breed such animals with those that conditionally express Cre recombinase for subsequently determining the functional consequences during or after B cell development of deleting the element. We plan to carry out a similar approach for the elucidation of Ed function.

    ACKNOWLEDGEMENTS

We thank S. Hall and R. Conner for automated sequencing, A. Tizenor for aid in graphics, and Drs. Michael Reth, Brian VanNess, and Eugene Oltz for providing plasmids and cell lines.

    FOOTNOTES

* This investigation was supported by National Institutes of Health Grant GM29935 and Robert A. Welch Foundation Grant I-823 (to W. T. G.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EBI Data Bank with accession number(s) AF513925 and AF513926.

Dagger To whom correspondence should be addressed: Dept. of Molecular Biology, University of Texas Southwestern Medical Center, 5323 Harry Hines Blvd., Dallas, TX 75390-9148. Tel.: 214-648-1924; Fax: 214-648-1915; E-mail: william.garrard@utsouthwestern.edu.

Published, JBC Papers in Press, June 21, 2002, DOI 10.1074/jbc.M204065200

2 The new sequences reported here were scanned against the database and were in part matched with Celera GA x5J8B7W84WR:11000001..11340984 and L10 (GenBankTM accession number V01557).

3 M. Reth, personal communication.

4 R. Sinden, personal communication.

    ABBREVIATIONS

The abbreviations used are: kb, kilobase(s); E3', 3' enhancer; Ed, downstream enhancer; Ei, intronic enhancer; HS, hypersensitive site; IS, intervening sequence; Luc, luciferase reporter gene; MAR, matrix association region; PVkappa , minimal Vkappa gene promoter; Sis, intervening sequence silencer; YAC, yeast artificial chromosome.

    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
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