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The Journal of Immunology, 2004, 172: 1054-1064.
Copyright © 2004 by The American Association of Immunologists

NF-{kappa}B and Oct-2 Synergize to Activate the Human 3' Igh hs4 Enhancer in B Cells1

Manuel A. Sepulveda, Alexander V. Emelyanov and Barbara K. Birshtein2

Department of Cell Biology, Albert Einstein College of Medicine, Bronx, NY 10461


   Abstract
Top
 Abstract
Introduction
 Materials and Methods
 Results
 Discussion
 References
 
In B cells, the Igh gene locus contains several DNase I-hypersensitive (hs) sites with enhancer activity. These include the 3' Igh enhancers, which are located downstream of the C{alpha} gene(s) in both mouse and human. In vivo experiments have implicated murine 3' enhancers, hs3B and/or hs4, in class switching and somatic hypermutation. We previously reported that murine hs4 was regulated by NF-{kappa}B, octamer binding proteins, and Pax5 (B cell-specific activator protein). In this study we report that human hs4 is regulated differently. EMSAs and Western analysis of normal B cells before and after stimulation with anti-IgM plus anti-CD40 showed the same complex binding pattern formed by NF-{kappa}B, Oct-1, and Oct-2 (but not by Pax5). A similar EMSA pattern was detected in mature human B cell lines (BL-2, Ramos, and HS-Sultan) and in diffuse large B cell lymphoma cell lines, although yin yang 1 protein (YY1) binding was also observed. We have confirmed the in vivo association of these transcription factors with hs4 in B cells by chromatin immunoprecipitation assays. The diffuse large B cell lymphoma cell lines had a distinctive slow-migrating complex containing YY1 associated with Rel-B. We have confirmed by endogenous coimmunoprecipitation an association of YY1 with Rel-B, but not with other NF-{kappa}B family members. Transient transfection assays showed robust hs4 enhancer activity in the mature B cell lines, which was dependent on synergistic interactions between NF-{kappa}B and octamer binding proteins. In addition, human hs4 enhancer activity required Oct-2 and correlated with expression of Oct coactivator from B cells (OCA-B).


   Introduction
Top
 Abstract
 Introduction
Materials and Methods
 Results
 Discussion
 References
 
The expression of Ig H chain (Igh)3 genes in B cells is regulated by the combined activity of cis- and trans-acting elements, including promoters, enhancers, and transcription factors. Enhancers include Eµ, located between the most downstream J gene and Cµ, and the 3' enhancers (hs3A, hs1,2, hs3B, and hs4 in mice; hs3, hs1,2 and hs4 in humans). The 3' Igh enhancers are located downstream of the C{alpha} gene in mice (reviewed in Ref.1) and downstream of both C{alpha}1 and C{alpha}2 in humans, reflecting a partial duplication of the Igh-C genes (Fig. 1A) (2, 3, 4).



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  		FIGURE 1. A, Diagram of the human Igh locus indicating the presence of a duplicated set of 3' enhancers (hs3, hs1,2, and hs4). B, Transcription factor binding sites of mouse (11 ) and human (4 ) hs4 enhancers are indicated. Note that the human hs4 enhancer is predicted to contain only one octamer binding site, and the human NF-{kappa}B site contains a mismatch that diminished its affinity with respect to the mouse sequence (25 ). Fragments of the human enhancer used as EMSA probes (311 and 148 bp), as DNA competitors (148 and 52 bp), and in reporter constructs (451 bp) are schematized.

 
The mouse enhancers have been extensively studied in vitro and in vivo (reviewed in Ref.1). Active throughout B cell development, Eµ has been shown to be required for efficient VH gene assembly and µ gene expression (5, 6). However, in the absence of Eµ, several plasma cell lines continue to support high levels of Igh expression (reviewed in Ref.1). The 3' Igh enhancers are strong candidates for regulation of Igh expression at this later stage of B cell development (7, 8). The mouse 3' enhancers show synergistic activity with each other and with Eµ when combined with a VH promoter (9, 10). Our previous studies using transient transfection assays showed that mouse hs4 enhancer activity was regulated by Pax5, NF-{kappa}B, and octamer binding sites (11).

The activity of the murine 3' enhancers is tissue specific and developmentally regulated (12, 13). Although hs3A, hs1,2, and hs3B appear to be active only in plasma cells, hs4, the 3'-most enhancer, seems to be active throughout B cell development (7). In mice, targeted deletion of either hs3A or hs1,2 had no effect on B cell development, Igh expression, or class switch recombination (CSR) (14). However, the combined deletion of hs3B and hs4 severely impaired I promoter region-driven germline transcription and class switching to most isotypes (15). Because targeted deletion of hs3A, which is virtually identical with that of hs3B (16), showed no effect on isotype expression (14), hs4 is likely to play a unique role in this phenotype. In support for a role of 3' enhancers in germline transcription, transient transfection assays have shown that both mouse (17) and human (18, 19) 3' enhancers augmented I region-driven reporter constructs. A role for hs3 and/or hs4 in somatic hypermutation has also been suggested (20); however, the targeted deletion of hs3B and hs4 revealed no effect on somatic hypermutation (21).

In addition to regulation of Igh promoters, the 3' enhancers are thought to be responsible for the up-regulation of c-myc in human Burkitt’s lymphomas and mouse plasmacytomas. In these cells a reciprocal translocation juxtaposes 3' enhancers with c-myc (reviewed in Ref.22). It has been observed that the mouse 3' enhancers, hs1,2, hs3, and hs4, could increase the expression and histone acetylation of the translocated c-myc (23, 24) and up-regulate the expression of a c-myc promoter-driven reporter construct in a human B cell line (25).

The human Igh 3' enhancers share a core of 100–200 bp homology with their mouse orthologs. However, in human hs4, only one of the two octamer binding sites found in mouse hs4 is conserved, the human NF-{kappa}B site has a 1-bp mismatch with respect to the mouse enhancer, and no Pax5 site is predicted (Fig. 1B) (4). We have therefore proposed to determine whether these differences translate into a unique mechanism of regulation for the human hs4 enhancer.

In this study we show that the human hs4 enhancer is highly active in mature Burkitt’s lymphoma B cells and that its activity is regulated by synergistic involvement of both Oct-2 and NF-{kappa}B family members (and probably Oct coactivator from B cells (OCA-B)). Chromatin immunoprecipitation assays (ChIPs) showed that the human hs4 enhancer in B cells is associated in vivo with acetylated histones H3 and H4 and with NF-{kappa}B, Oct-1 Oct-2, and yin yang 1 protein (YY1). Extensive EMSA analysis showed similar patterns in normal and malignant B cell lines, including Burkitt’s, non-Burkitt’s, and multiple myeloma cells. However, an additional novel hs4 complex, containing Rel-B and YY1, was particularly evident in cell lines derived from human diffuse large B cell lymphoma (DLBCL) patients. We have confirmed in vivo association of Rel-B and YY1 by endogenous coimmunoprecipitation.


   Materials and Methods
Top
 Abstract
 Introduction
 Materials and Methods
Results
 Discussion
 References
 
Cell and cell lines

The mouse (18-81 pre-B, M12.4.1 B, J558L and S107, both plasma cells) and human (697 pro-B, BL-2 and HS-Sultan, both Burkitt’s lymphoma cells; JJN3 myeloma, Hut-78 and Jurkat, both T cells) cell lines and normal human B cells from PBL were maintained in RPMI 1640. HS-Sultan was deposited with American Type Culture Collection (Manassas, VA) as a plasmacytoma cell line, but DNA fingerprinting has now shown that it is a derivative of the Jiyoye Burkitt’s lymphoma cell line (ATCC CCL-87). Ramos (Burkitt’s lymphoma) cells, provided by Dr. M. Scharff (Albert Einstein College of Medicine, Bronx, NY), were maintained in DMEM. Human DLBCL cells (Ly1, Ly2, Ly4, Ly7, Ly8, Ly13.2, and Ly18), originally described by Messner and collaborators (26), were a gift from Dr. H. Ye (Albert Einstein College of Medicine) and were maintained in IMDM. Expression pattern profile analysis has provided evidence for at least two distinctive subgroups of DLBCL cells: the germinal center-like and the activated B cell-like (27). NF-{kappa}B target genes are up-regulated in the activated B cell type, and the survival of cells of this subgroup, but not of germinal center-like cells, appears to depend on a constitutively active NF-{kappa}B pathway (28). EMSA showed abundant NF-{kappa}B binding in all DLBCL cells we analyzed.

Normal human B cells (four different donors) were obtained from leukocyte packs (New York Blood Center, New York, NY) and processed within 6 h of blood donation to limit differences in transcription factor expression due to extended storage time (29). Human CD19+ B cells were positively selected using magnetic beads (Miltenyi Biotec, Auburn, CA) according to the manufacturer’s instructions. Purified human B cells (>94% CD19+) were either directly analyzed or cultured in RPMI 1640 for 2 days with anti-IgM (The Jackson Laboratory, Bar Harbor, ME) and anti-CD40 (555587; BD PharMingen, San Diego, CA) (30). Stably transfected Oct-2-producing JJN3 cell lines were generated with the pCGN-CMV-Oct-2 vector (31) provided by Dr. L. Eckhardt (Hunter College of The City University of New York, New York, NY); these cells expressed a hemagglutinin- and six-histidine-tagged human Oct-2 protein. JJN3 cells (107) were electroporated with 4 µg of the XmnI-opened human Oct-2 vector together with 4 µg of the PvuI-linearized NeoR-containing plasmid (pCDNA3.1). Transfected cells were cultured in a 96-well plate. For selection, G418 was added 24 h later to a final concentration of 3.5 mg/ml. After 3 wk of selection, neomycin-resistant cells were expanded, and Oct-2 expression was evaluated. Exogenous Oct-2 was detected by virtue of its hemagglutinin tag. The subline JJN3.5 expressed high levels of Oct-2 and was used for further transfections. All cell culture media were supplemented with 10% FBS (Gemini Bio-Products, Calabasas, CA), 1% penicillin/streptomycin, and {beta}-ME. Cell lines were grown at log phase at 37°C in a humidified atmosphere containing 5% CO2.

Plasmid construction and site-directed mutagenesis

The pGL2-VH vector, in which a murine H chain variable region promoter (VH; bp 21–162 from GenBank sequence M17056) was cloned using KpnI and BglII sites, was provided by Dr. L. Eckhardt. The human BAC9378 was selected from the Incyte Genomics (St. Louis, MO) collection by screening with a probe for the human hs1,2 enhancer. A 451-bp fragment containing the human hs4 enhancer was amplified by PCR using primers SA.8A and SA.9B as described by Mills et al. (4). These primers were modified to include SmaI sites. Smaller fragments were obtained as described for EMSA. The 451-bp human hs4 enhancer was cloned into the SmaI site of the pGL2-VH construct. Transient transfection assays of enhancer activity used the 451-bp fragment, whereas EMSA was performed with smaller fragments, as indicated in each figure.

Octamer and NF-{kappa}B sites were mutated by site-directed PCR-mutagenesis in the context of the 451-bp hs4 enhancer. The octamer site mutation was designed as previously described (11). A ClaI site was created using the primer 5'-AATCGATTTGGGGGAGGTGGG-3', which covered the octamer site (mutated bases are underlined), and the 5'-CGCCATCTCGGAGGTGGGTGG-3' primer. To mutate the NF-{kappa}B site, an EcoRI site (underlined letters) was inserted using 5'-ACAGTGGTGTGGAAGAATTCACCCACCTCC-3' and its complementary primer. PCR cycles were performed in a PerkinElmer machine (Norwalk, CT) as follows: initial denaturation at 95°C for 5 min; 10 cycles of 94°C for 30 s, 55°C for 1 min, and 68°C for 30 s; and final extension at 68°C for 10 min. The PCR mix was treated with 1 µl of DpnI at 37°C for 3 h, followed by 15 min at 65°C to denature the enzyme. All products were verified by digestion with the appropriate restriction enzymes and sequencing to assure the right orientation and fidelity of the cloning. Plasmids were prepared using a Qiagen maxi-prep kit (Chatsworth, CA) as described by the manufacturer.

Transfection assays

Cells were transiently transfected by electroporation as previously described (32). At least two different plasmid preparations were used to repeat each experiment. Firefly luciferase values were normalized for transfection efficiency according to the readings of a cotransfected Renilla-expressing vector. The fold activity for the enhancer constructs was determined by dividing the normalized luciferase values by the normalized values of the enhancerless construct.

EMSA

Nuclear extract preparation and EMSA were performed as described previously (33). The competitors used were 5'-CCATTTGCATATTTGCATATTTGCATCC-3' for octamer binding proteins, 5'-GGTTCTGATCGGCCATCTTGACTCAACTCAAC-3' for YY1, and 5'-GAGAGGGGATTCCCCGATTAGCTTTCGGGGAATCCCCTCT-3' for NF-{kappa}B (34). All oligonucleotides were annealed with their respective complementary sequences, with the exception of the NF-{kappa}B site, which is self-complementary. The hs4 enhancer-containing fragments were obtained as follows: a 311-bp fragment, with the forward primer 5'-TCAGGAGGCTGGACACACTAGC-3' and the reverse primer 5'-TGGCTGGGGAGTGTGAATAG TC-3', a 148-bp fragment with the forward primer 5'-GCCCGGGACCGGGCCGCAGCTCCCACC-3' and the reverse primer 5'-GCCCGGCAATGCAAATCGCCATCTCGGAGGTG-3', and a 52-bp fragment with the forward primer 5'-CCCGGGACAGTGGTGTGGAAACC CCCA-3' and the 148-bp reverse primer (some primers contained SmaI sites, which are underlined). PCR products used as EMSA probes were pools of at least three different PCR reactions. Where needed, double-stranded oligonucleotides were labeled with [{gamma}-32P]ATP using T4 polynucleotide kinase.

Antibodies

The following rabbit Abs were used: anti-H4-Ac (06-866) and anti-H3-Ac (06-599) from Upstate Biotechnologies (Lake Placid, NY); anti-Oct-1 (sc-232X), anti-Oct-2 (sc-233), anti-p50 (sc-7178X), anti-Rel-B (sc-226X), anti-p52 (sc-298X), anti-p65 (sc-7151X), anti-c-Rel (sc-6955X), anti-YY1 (sc-1703X), and normal IgG (sc-2027) from Santa Cruz Biotechnologies (Santa Cruz, CA); anti-YY1 (1610076) and anti-c-Jun (1610011) from Geneka/Active Motif (Carlsbad, CA). In addition, goat anti-Pax5 (sc-1974) and the mouse monoclonal anti-YY1 (sc-7341) from Santa Cruz Biotechnologies were used. Anti-guanine nucleotide dissociation inhibitor (anti-GDI) was used for normalization of total proteins in each lane as previously described (35).

Western blots

Nuclear extracts were prepared as described previously (33) and separated by 12% SDS-PAGE or by 4–20% gradient gels (Amersham Pharmacia Biotech, Arlington Heights, IL). Proteins were transferred onto Hybond membranes (Amersham Pharmacia Biotech) and processed as indicated by the manufacturer. Membranes were incubated with a specific primary antiserum, followed by secondary Abs conjugated to HRP (Santa Cruz Biotechnologies). Finally, signals were revealed using the ECL system (Amersham Pharmacia Biotech) as described by the manufacturer.

Coimmunoprecipitation

Coimmunoprecipitations were performed as described previously (32). Briefly, 1 x 108 Ly8 cells were lysed in mild-RIPA buffer (50 mM HEPES (pH 7.5), 150 mM NaCl, 0.1% Tween 20, 1 mM EDTA, 1 mM EGTA, and 1 mM DTT) with protease inhibitors (1 mM PMSF, 2 µg/ml leupeptin and pepstatin, and 5 µg/ml aprotinin) for 1 h on ice. Lysates were precleared for 4 h and incubated with anti-YY1 or control antiserum overnight at 4°C in the presence of 0.1 mg/ml ethidium bromide to eliminate protein DNA interactions (36).

Chromatin immunoprecipitation (ChIP)

ChIPs were performed as previously described (37), with minor modifications. Briefly, 5 x 107 cells were brought to room temperature and exposed to a 1% formaldehyde solution for 5 min to cross-link protein and nucleic acids. Chromatin was extracted and sonicated by 30-s cycles at 30-s to 2-min intervals (18 rounds for BL-2, 12 rounds for Ly8, and 12 rounds in the presence of 0.2% SDS for Jurkat cells), at 50% amplitude (Sonic Dismembrator, model 500; Fisher Scientific, Pittsburgh, PA). Precleared chromatin was incubated for 4 h at 4°C with 2–5 µg of specific Abs for acetylated histones H3 and H4, Oct-1, Oct-2, p50, Rel-B, YY1, or control serum. Immunoprecipitates were washed and treated with RNase A and proteinase K, and cross-links were reversed overnight at 55°C. Finally, DNA was isolated using the QIAQuick kit (Qiagen) as indicated. A 311-bp PCR product containing the hs4 enhancer was obtained with primers described in the EMSA section above. As a negative control, the 3'-untranslated region (3'-UTR) region of a nonrelated gene (CRP2) was amplified from the same samples (forward primer, 5'-ATGATCCCTTCTGTGTCTGCGT-3'; reverse primer, 5'-TATTGACAGCACAAGGCTCAAC-3'). All experiments were repeated with two independent chromatin preparations.


   Results
Top
 Abstract
 Introduction
 Materials and Methods
 Results
Discussion
 References
 
The human hs4 enhancer binds NF-{kappa}B, Oct-1, Oct-2, and YY1, but not Pax5

We amplified a 451-bp fragment containing the human hs4 enhancer from BAC9378 (Incyte Genomics), which contains an ~60-kb fragment extending from S{epsilon} through the region 3' of C{alpha}2 (M. A. Sepulveda and B. K. Birshtein, unpublished observations). Our sequence of human hs4 was identical with the previously reported sequence of hs4 from downstream of C{alpha}1 (GenBank accession no. AF013725) and C{alpha}2 (AF013726) (4) and was 64% identical with mouse hs4 (7, 24). The mouse hs4 enhancer contains two octamer binding sites and single sites for NF-{kappa}B and Pax5 (Fig. 1B). In contrast, human hs4 was predicted to contain a single octamer site and the NF-{kappa}B site, but not the Pax5 binding site (Fig. 1B). The predicted octamer and NF-{kappa}B sites are 20 bp apart.

To confirm that the human hs4 enhancer contains single NF-{kappa}B and octamer binding sites, we performed site-directed mutagenesis and EMSA using nuclear extracts from Ramos (Burkitt’s lymphoma) cells and a 148-bp probe. We detected a complex pattern composed of NF-{kappa}B (Fig. 2A, compare lanes 1 and 2) and octamer binding proteins (lane 3). Mutation of the {kappa}B binding site resulted in a binding pattern (lanes 4 and 5) identical with that observed for the wild-type hs4 after competition with a high affinity NF-{kappa}B binding site (lane 2). Similarly, the EMSA pattern for the octamer mutated hs4 (lanes 7 and 9) was identical with that found for the wild-type hs4 enhancer after competition with an octamer binding site (lane 3). These experiments confirmed the presence of single sites for NF-{kappa}B and octamer binding proteins in the human hs4 enhancer. In addition, the lack of NF-{kappa}B binding had no effect on octamer binding and vice versa, indicating that the individual transcription factors bound independently to human hs4. In accord with sequence predictions (4), there was no evidence of Pax5 binding, even though Pax5 was present in these nuclear extracts and could bind to the mouse hs4 enhancer (data not shown). A slow mobility complex that was competed by both NF-{kappa}B and octamer binding sites (Fig. 2A, lane 1) was only rarely observed (compare with Fig. 2B, lane 1).



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  		FIGURE 2. A, A 148-bp hs4 fragment and nuclear extracts from Ramos cells (mature B cell line) were used to confirm the presence of single NF-{kappa}B and octamer sites in the human hs4 enhancer. Mutation of the NF-{kappa}B site abolished {kappa}B binding without affecting octamer binding and vice versa. EMSA of nuclear extracts of the mature B cell lines Ramos (B), BL-2 (C), and HS-Sultan (D) revealed binding of octamer, NF-{kappa}B family members, and YY1. Complexes were identified by competition with high affinity octamer (dsOct), NF-{kappa}B (ds{kappa}B), or YY1 (dsYY1) binding sites and by supershift/competition with anti-YY1- or anti-Oct-2-specific antisera. Oct-1, Oct-2, and YY1 bands were assigned based on supershift results and differential complex mobilities. *, Supershifted complex. Anti-c-Jun, used as a control for nonspecific interference with binding in EMSA, had no effect.

 
EMSA of other Burkitt’s lymphoma cells also showed a complex pattern (compare Fig. 2B, lane 1, with 2C, lane 1 (BL-2), and 2D, lane 1 (HS-Sultan)). The pattern for these B cell lines was essentially the same for both 148- and 311-bp probes (compare lane 1 in Fig. 2, A and B), thereby corroborating the location of the NF-{kappa}B and the octamer binding sites (Fig. 2C, compare lane 1 with lanes 2 and 3, and Fig. 2D, compare lanes 1 and 2, and lanes 3 and 4) to a 148-bp core fragment of hs4. As shown for Ramos (Fig. 2, A and B), binding was accomplished by both {kappa}B and octamer family members. Both Oct-1 and Oct-2 bound to human hs4, as evidenced by bands of different mobilities, and competition and supershift assays (Fig. 2B, compare lanes 1 and 2; and Fig. 5B, lanes 3, 4, 7, and 8).



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  		FIGURE 5. A, EMSA of nuclear extracts from normal human B cells before and after stimulation with anti-IgM plus anti-CD40 showed NF{kappa}B and octamer binding to hs4. B, Specific contributions of p50, p65, Oct-1, and Oct-2 were confirmed for normal B cells both before and after stimulation.

 
Minor complexes that were not related to NF-{kappa}B or octamer binding proteins were also detected (Fig. 2C, lane 3; Fig. 2D, lane 4). Based on our observations that YY1 binds to murine hs3 (38) as it does to the µE1 site of the mouse intronic enhancer, Eµ (39), we tested whether YY1 could bind to human hs4. In fact, competition analysis with a high affinity YY1 binding site from the murine Eµ enhancer and specific antiserum against YY1 confirmed binding of YY1 to human hs4 (Fig. 2C, lane 4; Fig. 2D, lanes 5, 7, and 8). Direct YY1 binding to human hs4 was more evident when octamer binding to human hs4 was prevented (Fig. 2C, compare lanes 1 and 2) or when a longer fragment was used as a probe. Using a transcription factor database, two putative YY1 binding sites (core sequence ATGG) were suggested to be within the 148-bp fragment of the human hs4 enhancer (40). Using 25- to 30-bp oligonucleotides containing each of the two sites as probes in EMSA, we detected binding; however, this was not confirmed as YY1, because no specific supershift was observed (not shown).

DLBCL cells showed a slow-migrating complex composed of Rel-B and YY1

We wanted to determine whether the EMSA patterns of B cells representing different B cell pathologies were the same. DLBCL is an aggressive malignancy of the mature B cell phenotype, representing roughly 40% of all non-Hodgkin lymphomas. In addition to an EMSA pattern qualitatively similar to that previously observed (Fig. 3A, compare lanes 1 and 2), we consistently observed a distinctive slow mobility complex in several DLBCL-derived cell lines (Fig. 3A).



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  		FIGURE 3. EMSA of nuclear extracts from DLBCL cell lines with human hs4 revealed multiple complexes, including a slow mobility complex composed of Rel-B and YY1. A, EMSA of multiple DLBCL cell lines compared with HS-Sultan (lane 1). B, EMSA of Ly8 using specific oligonucleotides as competitors and specific antisera for supershift/competition. Dots identify individual bands. C, Endogenous coimmunoprecipitation of YY1 and Rel-B from total cell extracts of Ly8 cells was performed in the presence of 0.1 mg/ml ethidium bromide to avoid a DNA-mediated association. Polyclonal anti-YY1 sera (sc-1703x) coimmunoprecipitated Rel-B in addition to YY1. Two Rel-B isoforms appeared to be associated with YY1 in vivo. A mAb (sc-7341) was used to identify YY1 in the Western blot.

 
Using Ly8 (Fig. 3B) and Ly2 (data not shown) as representatives of the DLBCL lines, we confirmed that all the complexes identified by EMSA were efficiently competed by both a 148-bp and a 52-bp fragment of the central region of the hs4 enhancer (Fig. 3B, compare lane 1 with lanes 2 and 3). These data located human hs4 binding sites for both slower and faster mobility complexes to a core region of 52 bp, which contained single sites for octamer and NF-{kappa}B binding proteins. Using specific anti-{kappa}B Abs, we observed that faster migrating complexes contained p50, p65, and Rel-B (potentially as p50/p65 or p50/Rel-B heterodimers), but apparently not p52 or c-Rel (Fig. 3B, lanes 10–13, and data not shown). The slow mobility complex in Ly8 was competed by a high affinity NF-{kappa}B site (Fig. 3B, compare lanes 4 and 6), but not by an octamer site (Fig. 3B, compare lanes 4 and 5). Treatment with anti-Rel-B (Fig. 3B, lane 12), but not with anti-p52 or -p65 (Fig. 3B, lanes 13 and 10) or anti-c-Rel (data not shown), eliminated the slow-migrating complex. As NF-{kappa}B family members bind DNA as dimers, and Rel-B homodimers have not been observed (41), the slow-migrating complex probably contained p50 and Rel-B. However, evaluation of the contribution of p50 to the slow-migrating complex was complicated by the fact that addition of anti-p50 resulted in a supershift of p50-containing complexes (Fig. 3B, lane 11); the additional bands created by the supershift could mask the disappearance of the slow-migrating complex.

In addition to competition with a {kappa}B site and anti-Rel-B, two different anti-YY1 antisera disrupted the slow-migrating complex, showing that YY1 was also a member (Fig. 3B, lane 7, and data not shown). However, a high affinity YY1 binding site had no effect (Fig. 3B, compare lanes 8 and 9, and data not shown). These findings suggested that YY1’s contribution to the formation of the slow-migrating complex was by interaction with Rel-B rather than through direct DNA binding. In fact, we confirmed the in vivo association of YY1 with Rel-B, but not with any other {kappa}B family member, by endogenous coimmunoprecipitation using Ly8 extracts (Fig. 3C). This association was not bridged by DNA, because it occurred in the presence of ethidium bromide (36). In additional experiments in which different Rel-B isoforms could be separated, we observed that YY1 was able to associate with two different isoforms of Rel-B (Fig. 3C, top panel). Present in several of the cell lines used in this study (Fig. 4B), these Rel-B isoforms have been shown to correspond to different levels of phosphorylation (42). Hence, the novel slow-migrating complex in DLBCL is composed of Rel-B (and probably p50) associated with YY1. We considered the possibility that DLBCL cells had unique expression patterns for Rel-B and YY1, and tested this by Western analysis. However, we found no differences in YY1 and Rel-B expression in a panel of B cell lines (Fig. 4, A and B). Thus, the formation of the slow-migrating complex in DLBCL cells was independent of the pattern of expression of Rel-B and YY1.



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  		FIGURE 4. Western blot analysis of nuclear extracts from normal B cells, B and T cell lines for YY1 (A), Oct-1 (D) Oct-2 (A, B, and D), OCA-B (C), and NF-{kappa}B family members Rel-B (B), p65 (B and D), and p50 (D). GDI expression was used as a loading control. +, Cell lines that supported hs4 enhancer activity in transient transfection experiments; -, cell lines that did not support hs4 enhancer activity in transient transfection experiments.

 
EMSA analysis of normal human B cells showed NF-{kappa}B, Oct-1, and Oct-2 binding to human hs4 enhancer

We wanted to determine the EMSA pattern of normal B cells and compare it to the EMSA patterns of Burkitt’s lymphoma and DLBCL cells. Nuclear extracts were prepared from CD19+ B cells immediately after their isolation (unstimulated) and after treatment with anti-IgM and anti-CD40 for 48 h (stimulated). This stimulatory signal has been reported to trigger the proliferation of B cells (30). Accordingly, we observed enlargement of the cells and the formation of clusters (not shown). However, no differences in EMSA patterns between unstimulated and stimulated B cells were observed (Fig. 5A, compare lanes 1 and 6). This normal B cell pattern was similar to that seen in Burkitt’s lymphoma and DLBCL cells, but lacks the slow mobility complex of DLBCL cells. Competition with high affinity octamer and {kappa}B sites abolished virtually all hs4 binding in unstimulated (Fig. 5A, compare lanes 2 and 3 with lane 1), as well as in stimulated (compare lanes 7 and 8 to lane 6) cells. We detected Oct-1 (Fig. 5B, lanes 3 and 7) and Oct-2 (Fig. 5B, lanes 4 and 8) binding to human hs4 using transcription factor-specific antisera. All the complexes associated with NF-{kappa}B could be supershifted by a p50 antiserum (Fig. 5B, lanes 1 and 5). However, the heterogeneous mobility pattern suggests that other {kappa}B family members were also present. Although no Rel-B- or c-Rel-containing complexes were detected (not shown), p65-containing complexes were observed (Fig. 5B, lane 2, and data not shown). These data showed that Oct-1, Oct-2, and p50 (and limited amounts of p65) bind to hs4 in normal human B cells. Western analysis of nuclear extracts from normal human B cells confirmed similar levels of expression of p50, p65, Oct-1, and Oct-2 in both unstimulated and stimulated B cells (Fig. 4D).

Synergistic binding to NF-{kappa}B and octamer sites controls the activity of the human hs4 enhancer in mature B cell lines

Transient transfection assays showed robust human hs4 activity in BL-2, Ramos, and HS-Sultan human mature B cell lines (Table I). Although other studies had shown that human 3' enhancers were inactive or poorly active (<4-fold), in human mature B cell lines (18, 25) these differences can be attributed to differences in promoter usage and/or efficiencies of transfection. The contribution of NF-{kappa}B and octamer binding sites to human hs4 enhancer activity in Burkitt’s lymphoma cell lines was assessed by site-specific mutagenesis (Fig. 6). Enhancer activity after mutagenesis of either the single NF-{kappa}B or octamer binding sites (in the context of a 451-bp hs4 fragment) was reduced to a level comparable to the plasmid containing the VH promoter alone (Fig. 6). These data indicated that enhancer activity was dependent on synergistic engagement of both octamer and NF-{kappa}B binding sites.


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  		Table I. Human hs4 enhancer activity in B and non-B cells

 


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  		FIGURE 6. NF{kappa}B and octamer binding sites are both essential for human hs4 enhancer activity. A, Transient transfection assays revealed synergistic contributions of the NF{kappa}B and octamer sites to hs4 enhancer activity. BL-2, Ramos, and HS-Sultan human mature B cell lines were transiently transfected with wild-type (hs4), NF{kappa}B-mutated (hs4-{kappa}B-mut), or octamer-mutated (hs4-Oct-mut) enhancer constructs. The activity of the promoter (VH) was comparable to the background of the assay. Data represent the average of three experiments, each of which was performed in triplicate. Data were normalized with CMV-Renilla or thymidine kinase-Renilla readings and compared with the activity of the wild-type hs4 enhancer (100%). Representative luciferase units (LU) for hs4 were ~18,000 for BL-2, ~50,000 for Ramos, ~22,000 for HS-Sultan, and 150 for VH.

 
Transient transfection assays showed that hs4 enhancer activity was not detected in the human T cell lines Hut-78 and Jurkat or in several other human B cell lines, i.e., the pro-B cell line 697 (data not shown), the DLBCL cell line Ly8, and JJN3 multiple myeloma cells (Table I). In parallel experiments, the mouse intronic enhancer (Eµ) was found to be active in Ly8 and JJN3 cells, suggesting that these cells did not have a generalized defect (Table I). In addition, we observed modest activity in murine 18-81 pre-B cells and M12.4.1 mature B cells (Table I). No activity was detected in the murine plasma cell lines, J558L and S107, which have known defects in NF-{kappa}B activation (43).

Oct-2 is required for hs4 enhancer activity

EMSA showed that the levels of Oct-1, NF-{kappa}B, and YY1 binding were comparable in different cell lines regardless of whether they supported hs4 enhancer activity (Fig. 7A). However, the Oct-2 band was more evident in those cell lines in which hs4 was active (Fig. 7A, lanes 1–3) and was reduced or not detected in cell lines in which hs4 was relatively inactive (Fig. 7A, lanes 4–8; JJN3, Jurkat, Hut-78, 697, and M12.4.1). Western analysis of nuclear extracts confirmed that these cell lines, in which human hs4 was inactive, had low levels of Oct-2 expression, whereas other factors, such as YY1, p65, and Rel-B, were expressed at comparable levels regardless of hs4 enhancer activity (Fig. 4, A and B). This correlation between binding of Oct-2 and enhancer activity suggested that Oct-2 was essential for human hs4 activity.



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  		FIGURE 7. Human hs4 enhancer activity requires Oct-2 binding. A, EMSA of cell lines that support hs4 enhancer activity compared with those that do not. A band previously identified as Oct-2 was not present (or was highly diminished) in cell lines in which the hs4 enhancer was inactive. B, Oct-2 stably transfected into JJN3 cells (JJN3.5) resulted in increased hs4 enhancer activity. C, Western blot analysis of total cell extracts showed that Oct-2 expression was high in BL-2 and JJN3.5 cells. The Oct-2 protein expressed in JJN3 cells was tagged with hemagglutinin and six histidines, resulting in a slightly different mobility (*). D, The increased hs4 enhancer activity in JJN3.5 cells was dependent on the NF{kappa}B and octamer sites in the enhancer. For transfection experiments in B and D, data are presented as fold activity with respect to enhancerless constructs (VH). In each transfection the data were normalized with thymidine kinase-Renilla readings. Representative normalized luciferase light units (LU) for VH were 1000.

 
To test the contribution of Oct-2 to hs4 enhancer activity, we stably transfected the low Oct-2-expressing JJN3 cells with a human Oct-2 expression construct. JJN3.5 was selected because it showed high levels of Oct-2 expression (Fig. 7C, JJN3.5 cells). By Western analysis, the levels of Oct-2 in the JJN3.5 subline were ~10-fold greater than the levels present in the original JJN3 cells and were at least 2-fold higher than the levels in BL-2 cells (Fig. 7C). Transient transfection assays of the VH promoter-driven construct containing the human hs4 enhancer in JJN3.5 cells showed a 10-fold increase in enhancer activity compared with the enhancerless construct (Fig. 7B). Interestingly, there was no significant difference in the activity of the reporter containing only the VH promoter, which has an octamer site, in the JJN3.5 subline compared with that in JJN3 cells. The hs4 enhancer activity in JJN3.5 cells (high Oct-2 expression) was completely abolished in the hs4 constructs containing single mutations for {kappa}B or octamer binding sites, as it was in Burkitt’s lymphoma cell lines (Fig. 7D), suggesting that Oct-2 and perhaps factors regulated by Oct-2 were sufficient to allow JJN3 cells to support hs4 enhancer activity. We did not detect any contribution of YY1 to enhancer activity in transient transfection assays by either analysis of site-specific mutagenesis of each of the two predicted YY1 binding sites or cotransfection with a YY1 expression vector together with the hs4 reporter construct (data not shown). Interestingly, transient transfection assays of Ly8 revealed only limited hs4 enhancer activity, although both NF-{kappa}B and Oct-2 were present. However, we observed that OCA-B was virtually absent from Ly8, supporting a role for this coactivator in hs4 enhancer activity. Most DLBCL cells appear to express OCA-B (44).

ChIP showed binding of Oct-2, Oct-1, p50, Rel-B, and YY-1 to the human hs4 enhancer

We conducted ChIP assays in BL-2 (Burkitt’s lymphoma), Ly8 (DLBCL), and Jurkat (T) cells to determine whether Oct-2 and {kappa}B family members, i.e., the transcription factors we had identified in the above experiments as essential for hs4 enhancer activity, were associated with the human hs4 enhancer in vivo. As predicted for transcriptionally active genes and regulatory regions (45, 46), ChIP assays revealed that the hs4 enhancer was associated with acetylated histones H3 and H4 in both BL-2 and Ly8 cells (Fig. 8A, lanes 3 and 10; Fig. 8B, lanes 1 and 2). However, in Jurkat human T cells, the human hs4 enhancer was not associated with acetylated histone H3, and there was reduced association with acetylated histone H4 (Fig. 8C, lanes 1 and 2; compare H4 acetylation with input signal for each of the cell lines). The transcription factors Oct-1 and Oct-2, and NF-{kappa}B family members, p50 and Rel-B, were bound to the hs4 enhancer in BL-2 (Fig. 8A, lanes 1, 2, 6, and 7, respectively) and Ly8 cells (Fig. 8B, lanes 3, 4, 5, and 7, respectively), but not in T cells (Fig. 8C). In addition, we observed YY1 associated with human hs4 in Ly8 cells (Fig. 8B, lane 8). No product was observed in the absence of Ab (Fig. 8A, lanes 5 and 12), when normal rabbit IgG was used (Fig. 8A, lanes 4 and 11; Fig. 8B, lane 9; Fig. 8C, lane 7), or when the unrelated 3'-UTR region of the CRP2 gene was analyzed for the above-mentioned transcription factors (Fig. 8, A–C, bottom panel). Based on NF-{kappa}B and Oct-2 binding, Ly8 cells fulfill our minimal requirements for hs4 enhancer activity; however, they supported no more than a 2-fold increase in the transient transfection assays. These observations are consistent with a role for OCA-B in enhancer activity. In fact, preliminary results using ChIPs have detected an association of OCA-B with the human hs4 enhancer in vivo in BL-2 cells. The ChIP data showed that the factors required for hs4 enhancer activity identified in our transient transfection assays were associated in vivo with the hs4 enhancer only in B cells.



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  		FIGURE 8. Chromatin immunoprecipitation of endogenously expressed transcription factors in BL-2 (Burkitt), Ly8 (DLBCL), and Jurkat (T) cells. The transcription factors Oct-1, Oct-2, and NF{kappa}B family members, p50 and Rel-B, were associated with the hs4 enhancer in vivo in BL-2 cells (A, lanes 1, 2, 6, and 7) and Ly8 cells (B, lanes 3–5 and 7), but not in Jurkat cells (C). YY1 was also associated with hs4 in Ly8 cells (B, lane 8). In addition, the human hs4 enhancer was associated with acetylated histones H3 and H4 in BL-2 (A, lanes 3 and 10) and Ly8 (B, lanes 1 and 2) cells, but only with acetylated histone H4 in Jurkat cells (C, lane 2). No transcription factors were found to be associated with a nonrelated gene segment CRP2/ESP1 (CRP2) in any of the cell lines. Other controls included the use of normal rabbit IgG, no antisera, and no template for the PCR reaction. PCR products were 311 bp for the human hs4 enhancer and 280 bp for CRP2–3'-UTR.

 

   Discussion
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
References
 
The combined deletion of mouse hs3B and hs4 resulted in impaired class switching to most isotypes (15). Previous observations (14, 16) indicate that hs3B is unlikely to be responsible for this phenotype and imply a critical role for hs4 in this process. Comparison of the human and mouse hs4 enhancers is therefore particularly interesting. We have previously identified contributions of Pax5, {kappa}B, and octamer binding protein sites to the regulation of murine hs4 enhancer activity in mouse B cell lines (47). In 18-81 pre-B cells, the Pax5 site was a negative regulator, whereas {kappa}B and octamer sites appeared to have an additive effect. In S194 plasma cells, the {kappa}B site accounted for most of the activity, whereas the octamer site had no contribution. The mature B cell lines analyzed showed differences in their regulation of mouse hs4 activity. In A-20 cells, Pax5, NF-{kappa}B, and octamer binding proteins contributed synergistically, whereas in M12.4.1 cells, Pax5 had no contribution, and the effects of octamer and {kappa}B binding proteins were apparently additive.

Human hs4 is regulated differently from the mouse enhancer, as determined by transient transfection assays in Burkitt’s lymphoma cell lines (BL-2, Ramos, and HS-Sultan), in which hs4 was particularly active. In these cells, concerted action of Oct-2 and NF-{kappa}B family members (but not Oct-1 or Pax5) regulated human hs4 enhancer activity. ChIP data for BL-2 cells have confirmed the endogenous binding of NF-{kappa}B family members, p50 and Rel-B, and of Oct-2 to human hs4. These data are consistent with a previously reported requirement for Oct-2 for the octamer-dependent portion of mouse 3' enhancer activity in the M12 B cell line (48) and may reflect intrinsic differences between the trans-activation domains of Oct-2 and Oct-1 (49).

Although Oct-1 was not required for enhancer activity, ChIP assays showed that it was bound to human hs4. It should be noted that of the four copies of hs4 present in normal human B cells (downstream of {alpha}1 and {alpha}2 on both alleles), only one hs4 enhancer is likely to be associated with active Igh expression. Thus, Oct-1 might play a functional role by binding to nonactive hs4 enhancers. Oct-1 has been suggested to be a negative regulator of transcription under certain circumstances (50), and in B cells appears to preferentially associate with C/EBP{beta}-3, a C/EBP isoform that has a negative regulatory function (51).

Hs4 enhancer activity is also likely to require OCA-B, a known coactivator of both Oct-1 and Oct-2 (52) and a predicted contributor to the function of the mouse 3' enhancers (53). OCA-B was present in all cell lines in which human hs4 was active, but only low levels were present in Ly8 DLBCL cells, which failed to support hs4 enhancer activity even though Oct-2 and NF-{kappa}B levels were not reduced. Based on the observation that DLBCL cells generally express Oct-2, NF-{kappa}B, and OCA-B, we predict that the hs4 enhancer will be active in this subset. In fact, the EMSA pattern formed by NF-{kappa}B and octamer binding proteins in Burkitt’s lymphoma B cell lines was similar to the faster mobility complexes observed in DLBCL cell lines, and ChIP data have confirmed the binding of NF-{kappa}B family members p50 and Rel-B and of both Oct-1 and Oct-2 to human hs4 in Ly8 DLBCL cells. Altogether, our observations suggest that NF-{kappa}B, Oct-2, and probably OCA-B might coordinate the binding of an additional factor(s) that is necessary for high levels of enhancer activity.

Interestingly, EMSA and ChIP showed that YY1 could also bind to the human hs4 enhancer in human B cell lines. Previous experiments have shown that YY1 regulates other Ig enhancers (38, 54, 55, 56). However, a specific YY1 binding site in human hs4 could not be identified, and no effect of YY1 on enhancer activity could be determined. Perhaps YY1 regulates human hs4 function in vivo in ways that were not assayed by our transient transfections, e.g., by recruiting histone acetyl transferases (57) or by facilitating tethering of hs4 to a distal promoter through YY1’s association with TATA binding protein (57).

A distinctive feature of DLBCL lines was a slow mobility complex containing YY1 and Rel-B (and perhaps p50), although the factors that promote its formation are not known. Both YY1 and Rel-B are widely expressed in DLBCL cells, which have the complex, and in non-DLBCL cells, which do not, and direct binding of Rel-B to DNA is observed in cell lines regardless of the presence of the slow mobility complex. Our experiments showed a specific association between YY1 and Rel-B, but not other {kappa}B family members. We propose, therefore, that the association of YY1 to Rel-B (which is, in turn, bound to DNA) is either promoted or prevented by specific factors in DLBCL or non-DLBCL cells, respectively. It has been proposed that BLyS treatment of primary or CD40-stimulated B cells specifically activates Rel-B/p50 complexes (such as those we propose in the slow mobility complex) that mediate BLyS signals for B cell survival (58). The presence of a stable YY1/RelB/p50 complex in DLBCL cells could reflect an antiapoptotic response, which may be related to the proliferative potential of these malignant cells in vivo. However, how YY1 modulates Rel-B’s function at the hs4 enhancer (or other {kappa}B targets) in DLBCL cells remains unknown.

Similar to our observations in human B cell lines, EMSA showed NF-{kappa}B, Oct-1, and Oct-2 binding to human hs4 in both unstimulated and stimulated normal human PBL-derived B cells. Most NF-{kappa}B complexes observed in normal B cells are likely to contain p50 homodimers, which are transcriptionally inactive (reviewed in Ref.59) (only small amounts of p65 were detected). These data suggest that the hs4 enhancer is not active in PBL-derived B cells. However, it has been shown that upon CD40 stimulation (required for class switching), active NF-{kappa}B members are up-regulated in human tonsillar (60, 61) and mouse splenic (62) B cells. In addition, most class switch recombination (CSR) is thought to occur in germinal centers where Oct-2 (63) and OCA-B (64) have also been shown to be expressed. We predict that in circumstances where NF-{kappa}B, Oct-2, and OCA-B are coexpressed, the human hs4 enhancer is likely to be active.

The 3' Igh enhancers are also predicted to be involved in the dysregulation of c-myc when it is translocated into the Igh locus. When the murine 3' enhancers were individually tested, only hs4 could drive c-myc expression in a human mature B cell line (25). Interestingly, site-specific mutagenesis in both human and mouse hs4 showed that the NF-{kappa}B site was essential for c-myc promoter-driven expression, whereas mutagenesis of the upstream octamer site had no effect on mouse hs4 activity (25). However, that particular octamer site is not conserved in humans, and based on our studies, we predict that the other, evolutionarily conserved, octamer site would also be required for human hs4 activity.

Although our data show that mouse and human hs4 enhancers are regulated differently, both enhancers require Oct-2, NF-{kappa}B, and OCA-B. Mouse models provide evidence for the role of these factors in CSR, in concert with the predicted contribution of hs4 to this process. Our earlier experiments suggested that p50’s essential role in switching to {gamma}3 and {epsilon} (65, 66) was through the regulation of murine 3' enhancers (11). In addition, it has been shown that CD40/CD40L interaction, which is essential for CSR (reviewed in Ref.67), results in OCA-B up-regulation (68). The evaluation of the specific role of Oct-2 in B cells has been complicated by the fact that Oct-2 knockout mice die shortly after birth, and there is partial compensation by Oct-1. However, when scid mice were reconstituted with Oct-2-/- B cells, there was a profound defect in circulating levels of Igs of all isotypes, except for IgA (69).

Our data support a model in which the coregulated expression of Oct-2, OCA-B, and NF-{kappa}B is necessary to activate the human hs4 enhancer. A defect in activity of any of these factors would compromise endogenous hs4 enhancer activity. In fact, analysis of patients with deficiencies in CSR, e.g., hyper-IgM syndrome, has provided direct evidence for the involvement of NF-{kappa}B through identification of mutations in NEMO (70), CD40 (71), and CD40 ligand (72). NF-{kappa}B is a downstream target of CD40 stimulation, and its release from the cytoplasm is compromised by NEMO mutations. Hence, in these hyper-IgM patients we predict that the human hs4 enhancer is inactive.


   Acknowledgments
 
We thank all the members of the Birshtein laboratory for helpful comments, and Drs. Matthew Scharff, Hilda Ye, Alberto Martin, and Diana Ronai for critical review of the manuscript. We thank Dr. Perry Bickel (Washington University, St. Louis, MO) for the anti-GDI antisera.


   Footnotes
 
1 This work was supported by National Institutes of Health Grants AI41572 and AI13509 and Albert Einstein Cancer Center Grant P30CA1330. Back

2 Address correspondence and reprint requests to Dr. Barbara K. Birshtein, Department of Cell Biology, Albert Einstein College of Medicine, 1300 Morris Park Avenue, Bronx, NY 10461. E-mail address: birshtei{at}aecom.yu.edu Back

3 Abbreviations used in this paper: Igh, Ig H chain gene; ChIP, chromatin immunoprecipitation assay; DLBCL, diffuse large B cell lymphoma; Eµ, Igh intronic enhancer; GDI, guanine nucleotide dissociation inhibitor; Pax5, B cell-specific activator protein; UTR, untranslated region; YY1, yin yang 1 protein; OCA-B, Oct coactivator from B cells; CSR, class switch recombination. Back

Received for publication March 4, 2003. Accepted for publication October 30, 2003.


   References
Top
 Abstract
 Introduction
 Materials and Methods
 Results
 Discussion
 References
 

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