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• W2023851430 abstract "The immunoglobulin J chain gene is inducibly transcribed in mature B cells upon antigen recognition and a signal from interleukin-2 (IL-2). B cell-specific activator protein (BSAP), a transcription factor that silences J chaintranscription, has been identified as a nuclear target of the IL-2 signal. The levels of BSAP progressively decrease in response to IL-2 and this change correlates with the differentiation of B cells into antibody secreting plasma cells. Here we report the binding of the upstream stimulatory factor (USF) to an E-box motif immediately upstream from the BSAP site on the J chain promoter. Mutations in the USF binding motif significantly decrease J chain promoter activity in J chain expressing B cell lines. We also show that a functional relationship exists between USF and a second J chain positive-regulating factor, B-MEF2, using co-immunoprecipitation assays and transfections. Finally, we provide evidence that the binding of BSAP prevents USF and B-MEF2 from interacting with the J chain promoter during the antigen-independent stages of B cell development. It is not until the levels of BSAP decrease during the antigen-driven stages of B cell development that both USF and B-MEF2 are able to bind to their respective promoter elements and activate J chaintranscription. The immunoglobulin J chain gene is inducibly transcribed in mature B cells upon antigen recognition and a signal from interleukin-2 (IL-2). B cell-specific activator protein (BSAP), a transcription factor that silences J chaintranscription, has been identified as a nuclear target of the IL-2 signal. The levels of BSAP progressively decrease in response to IL-2 and this change correlates with the differentiation of B cells into antibody secreting plasma cells. Here we report the binding of the upstream stimulatory factor (USF) to an E-box motif immediately upstream from the BSAP site on the J chain promoter. Mutations in the USF binding motif significantly decrease J chain promoter activity in J chain expressing B cell lines. We also show that a functional relationship exists between USF and a second J chain positive-regulating factor, B-MEF2, using co-immunoprecipitation assays and transfections. Finally, we provide evidence that the binding of BSAP prevents USF and B-MEF2 from interacting with the J chain promoter during the antigen-independent stages of B cell development. It is not until the levels of BSAP decrease during the antigen-driven stages of B cell development that both USF and B-MEF2 are able to bind to their respective promoter elements and activate J chaintranscription. During a primary immune response to foreign antigens, mature B cells become activated and differentiate into pentamer IgM-secreting plasma cells. One of the critical events in this process is the synthesis of the immunoglobulin J chain protein required for the assembly and secretion of pentamer IgM antibody (1Koshland M.E. Annu. Rev. Immunol. 1985; 3: 425-453Crossref PubMed Scopus (153) Google Scholar). Studies of both normal B cells and model B cell lines have shown that J chain synthesis is tightly regulated at the transcriptional level. For efficientJ chaintranscription to occur, activation signals from both the B cell receptor and the interleukin-2 (IL-2) 1The abbreviations used are: IL, interleukin; bp, base pair(s); nt, nucleotide(s); CAT chloramphenicol acetyltransferase, MEF-2, myocyte enhancer factor-2; BSAP, B cell-specific activator protein; USF, upstream stimulatory factor; EMSA, electrophoretic mobility shift assay1The abbreviations used are: IL, interleukin; bp, base pair(s); nt, nucleotide(s); CAT chloramphenicol acetyltransferase, MEF-2, myocyte enhancer factor-2; BSAP, B cell-specific activator protein; USF, upstream stimulatory factor; EMSA, electrophoretic mobility shift assay or IL-5 receptors are needed. Correlating with IL-2/IL-5 induced gene transcription is the appearance of a DNase I-hypersensitive site (bp −170 to +88) on theJ chain promoter of activated B cells (2Blackman M.A. Tigges M.A. Minie M.E. Koshland M.E. Cell. 1986; 47: 609-617Abstract Full Text PDF PubMed Scopus (71) Google Scholar, 3Minie M.E. Koshland M.E. Mol. Cell. Biol. 1986; 6: 4031-4038Crossref PubMed Scopus (12) Google Scholar). The full activity of the J chain promoter is contained within this hypersensitive region, as has been shown using 5′ deletion mutants in a CAT reporter system (4Lansford R.D. McFadden H.J. Siu S.T. Cox J.S. Cann G.S. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5966-5970Crossref PubMed Scopus (32) Google Scholar). Three regulatory elements have been found to be present within this region. These include a T-rich positive regulatory element denoted JA (−74 to −60), a purine-rich sequence (−58 to −47) denoted JB, and a repressive motif, JC (−127 to −110) (5Rao S. Karray S. Gackstetter E.R. Koshland M.E. J. Biol. Chem. 1998; 273: 26123-26129Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 6Shin M.K. Koshland M.E. Genes Dev. 1993; 7: 2006-2015Crossref PubMed Scopus (122) Google Scholar, 7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). The factor interacting with the JA sequence has recently been identified to be a member of the myocyte enhancer factor-2 (MEF-2) family that is expressed in the B cell lineage, denoted B-MEF2 (5Rao S. Karray S. Gackstetter E.R. Koshland M.E. J. Biol. Chem. 1998; 273: 26123-26129Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar). The JB element has previously been shown to interact with transcription factor PU.1, a member of the Ets family of transcription factors expressed in hematopoietic lineages including monocytes, macrophages, and B lymphoid cells (6Shin M.K. Koshland M.E. Genes Dev. 1993; 7: 2006-2015Crossref PubMed Scopus (122) Google Scholar). Mutational analyses in J chain positive cell lines have indicated positive regulatory roles for both PU.1 and B-MEF2; base changes that prevent either PU.1 or B-MEF2 from binding result in a 95% loss of promoter activity (5Rao S. Karray S. Gackstetter E.R. Koshland M.E. J. Biol. Chem. 1998; 273: 26123-26129Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 6Shin M.K. Koshland M.E. Genes Dev. 1993; 7: 2006-2015Crossref PubMed Scopus (122) Google Scholar). The activity mediated by the JC motif has been shown to be due to the binding of the transcription factor B cellspecific activator protein (BSAP), or Pax-5 (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). BSAP/Pax-5 is a member of the Pax family of transcription factors, which are important regulators of embryonic cell development and differentiation. Targeted gene disruption experiments have shown that BSAP expression is essential both for B cell development as well as development of the nervous system (8Strachan T. Read A.P. Curr. Opin. Genet. Dev. 1994; 4: 427-438Crossref PubMed Scopus (217) Google Scholar, 9Busslinger M. Urbanek P. Curr. Opin. Genet. Dev. 1995; 5: 595-601Crossref PubMed Scopus (75) Google Scholar). BSAP is highly expressed during the early stages of B cell development, but expression ceases during the antigen-driven stages of B cell development. BSAP is considered a “master regulator” of B cell development, and at least eight B cell-specific putative target genes have been identified so far (10Wallin J.J. Gackstetter E.R. Koshland M.E. Science. 1998; 279: 1961-1964Crossref PubMed Scopus (52) Google Scholar). Depending on the target gene, BSAP can act either as an activator, a repressor, or a docking protein (9Busslinger M. Urbanek P. Curr. Opin. Genet. Dev. 1995; 5: 595-601Crossref PubMed Scopus (75) Google Scholar, 11Neurath M. Stuber E.R. Strober W. Immunol. Today. 1995; 16: 564-569Abstract Full Text PDF PubMed Scopus (52) Google Scholar, 12Michaelson J.S. Singh M. Birshtein B.K. J. Immunol. 1996; 156: 2349-2351PubMed Google Scholar). In the case of the J chainpromoter, BSAP acts as a repressor: base changes in the BSAP-binding site result in a relief of repression in J chain negative cell lines (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). An IL-2 or IL-5 signal delivered to mature B cells has been shown to cause a progressive decrease in BSAP transcripts that extends from the presecretor immunoblast through the plasma cell stages (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). This pattern of expression inversely correlates with J chain expression. In addition to a role in J chain repression, a BSAP repression motif has also been identified in the 3′ α enhancer of the immunoglobulin heavy chain genes. Although the process of BSAP repression is not well understood, a possible mechanism has been suggested by in vivo footprinting studies by Neurath and colleagues (13Neurath M. Max E.E. Strober W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5336-5340Crossref PubMed Scopus (86) Google Scholar). This work showed that although two BSAP sites exist in the immunoglobulin 3′ α enhancer, only the most 5′ site is occupied by BSAP in mature B cells. As expected, no BSAP footprint was detected in plasma cells, due to low levels of BSAP at this cell stage. Importantly, the authors identified a second factor, NF-αP, which bound to a position 50 bp downstream of the 5′ BSAP-binding site in plasma cells, but not in mature B cells. NF-αP, a member of the Ets family of transcription factors, is expressed both in mature B and plasma cells, and is necessary for maximal activity of the 3′ α enhancer. Selective blocking of BSAP binding by triplex-forming oligonucleotides resulted in an NF-αP footprint in mature B cells and an increased level of immunoglobulin gene transcription. Thus, it appears that BSAP prevents NF-αP from activating the 3′ α enhancer until the plasma cell stage. In the work described here, a new DNA binding motif JE was identified at positions −140 to −132 of the J chain promoter. This sequence resembles the μE3 element of the immunoglobulin heavy chain enhancer and the related κE3 element of the κ light chain enhancer (14Staudt L.M. Lenardo M.J. Annu. Rev. Immunol. 1991; 9: 373-398Crossref PubMed Scopus (244) Google Scholar). We show that the helix loop helix protein upstreamstimulatory factor (USF) binds to the J chain promoter at the JE motif and positively regulates J chain transcription. Recently, it was shown that USF increases the activity of the immunoglobulin heavy chain gene intron enhancer, in combination with two other factors, PU.1 and Ets-1 (15Rao E. Dang W. Tian G. Sen R. J. Biol. Chem. 1997; 272: 6722-6732Abstract Full Text Full Text PDF PubMed Scopus (47) Google Scholar). In addition, USF factors have been shown to interact with members of the basal initiation complex (16Kokubo T. Takada R. Yamashita S. Gong D.W. Roeder R.G. Horikoshi M. Nakatani Y. J. Biol. Chem. 1993; 268: 17554-17558Abstract Full Text PDF PubMed Google Scholar, 17Chiang C.M. Roeder R.G. Science. 1995; 267: 531-536Crossref PubMed Scopus (352) Google Scholar). We show here that USF may, at least in part, be mediating its positive effect on J chaintranscription through a mechanism which necessitates interaction with B-MEF2. Although these positive-acting factors are both expressed throughout B cell development, it appears that they are only able to bind weakly to the J chain promoter in the presence of BSAP. This may provide a mechanism which ensures repression of J chain transcription until the activated B cell stages. As BSAP levels decrease during that time, USF and B-MEF2 replace BSAP on theJ chain promoter and this results in activation of transcription. PD31, 38C13, K46R, BCL1, and L cells were maintained in RPMI 1640 supplemented with 10% fetal bovine serum, 2 mml-glutamine, 50 μm2-mercaptoethanol, 100 units of penicillin/ml, and 100 μg of streptomycin/ml. MOPC315 and S194 cells were cultured in Dulbecco's modified Eagle's medium supplemented as described above. Large-scale extracts were prepared from 109 cells by the detergent lysis method of Peterson et al. (18Peterson C.L. Orth K. Calame K.L. Mol. Cell. Biol. 1986; 6: 4168-4178Crossref PubMed Scopus (37) Google Scholar) as modified by Lansfordet al. (4Lansford R.D. McFadden H.J. Siu S.T. Cox J.S. Cann G.S. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5966-5970Crossref PubMed Scopus (32) Google Scholar). All buffers contained the following mixture of protease inhibitors: 0.5 nm phenylmethylsulfonyl fluoride, 0.5 mm dithiothreitol, 0.5 mmNa2S205, aprotinin (10 units/ml), leupeptin (5 mg/ml), and pepstatin A (5 mg/ml). Mini-extracts were prepared from 107 cells as described (19McFadden H.J. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11027-11031Crossref PubMed Scopus (19) Google Scholar). Probes and DNA competitors used: J1, nt −83 to −9; J2, nt −168 to −84; JA:, −74 to −60; JB, nt −58 to −47; JC, nt −127 to −110; JE, nt −140 to −132 of the J chain promoter. For gel mobility shift assays, the specific oligonucleotides were end-labeled with [α-32P]dCTP and Klenow enzyme (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). The binding reactions with crude nuclear extracts were performed as described previously (19McFadden H.J. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1991; 88: 11027-11031Crossref PubMed Scopus (19) Google Scholar) using 8–10 μg of extract, 4–6 μg of poly(dI-dC) nonspecific competitor, and 104 cpm (0.1–1.0 ng) of probe. For the antibody gel shift assays, crude nuclear extracts were preincubated with 2 μl of a 1:10 dilution of either rabbit preimmune sera or polyclonal rabbit anti-mouse USF-2 antiserum which is specific for one of the two subunits of USF (p44) (Santa Cruz Biotechnology). In each case the protein-DNA complexes formed were resolved from free probe by electrophoresis through glycerol-containing 5% polyacrylamide gels (29:1) containing 0.25 × TBE buffer. Methylation protection footprinting was performed with crude nuclear extracts from the mature B cell line, K46R; 50 μg of nuclear protein was incubated 15 min at 0 °C with 2 μg of poly(dI-dC) and 106 cpm of DNA probe. One-half microliter of dimethylsulfate was added for 45 s and quenched by the addition of dithiothreitol to a final concentration of 23 mm. The remaining steps in the assay, isolation and sequencing of free and protein-bound DNA, were performed according to the standard protocol for methylation interference footprinting (21Ausubel F.M. Brent R. Kingston R.E. Moore D.D. Seidman J.G. Smith J.A. Struhl K. Current Protocols in Molecular Biology. Wiley-Interscience, New York1987Google Scholar). The probe J2 was an 84-bp XbaI/HindIII fragment containing the J chain sequence −168 to −84 that was end-labeled on the top or bottom strand. In the pγ42CassI vector (24Durand D.B. Bush M.R. Morgan J.G. Weiss A. Crabtree G.R. J. Exp. Med. 1987; 165: 395-407Crossref PubMed Scopus (117) Google Scholar) the CAT gene is under control of a truncated γ-fibrinogen promoter (−54 to +36) that includes a TATA box and a single Sp1-binding site. Fragments from the J chain promoter (J1, bps −83 to −9; J2, bps −168 to −84; J1-J2, bps −168 to −9) and oligonucleotides representing the JE element were synthesized withXbaI linkers and inserted either singly or in multiple copies into the polylinker upstream of the γ-fibrinogen promoter. All constructs were sequenced to determine oligomer copy number and orientation. Mutagenesis of the pγ42CassI plasmid containing the J1-J2 sequence and the XBμ plasmid was performed with the TransformerTMSite Directed Mutagenesis Kit (CLONTECHLaboratories). The sequence 5′-CGTAAGTATGAACAATCTTCGTCTTTCCAGTGTAGC-3′ (mJE) was used to introduce a 3-bp change in the JE element (the underlined region replaces the wt sequence CATGTG, see “Results”). The selected plasmid was sequenced to verify the base substitutions. Mutations introduced into the JC motif have been described previously (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Transient transfections of PD31 pre-B cells were performed by the DEAE-dextran technique (22Grosschedl R. Baltimore D. Cell. 1985; 41: 885-897Abstract Full Text PDF PubMed Scopus (288) Google Scholar) and transfections of MOPC315 myeloma cells by electroporation (23Neurath M. Strober W. Wakatsuki Y. J. Immunol. 1994; 153: 730-742PubMed Google Scholar). In each case, 107 cells in logarithmic growth phase were transfected with 9 μg of supercoiled test plasmid or a combination of plasmids. Cell extracts were prepared 44–48 h after transfection and assayed for CAT activity (4Lansford R.D. McFadden H.J. Siu S.T. Cox J.S. Cann G.S. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5966-5970Crossref PubMed Scopus (32) Google Scholar). Immunoprecipitation reactions using antibody-Sepharose, were performed according to the standard protocol (20Sambrook J. Fritsch E.F. Maniatis T. Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory, Cold Spring Harbor, NY1989Google Scholar). For Western blot analyses, nuclear extract samples were boiled for 5 min, size fractionated by SDS-polyacrylamide gel electrophoresis (10%), and transferred to a nitrocellulose filter. After pretreatment with 5% dry milk in 1 × phosphate-buffered saline, the filters were incubated for 3 h with a 1:10,000 dilution of antibody specific for BSAP, PU.1, MEF-2 (Santa Cruz Biotechnology Inc.), or USF-2 (Santa Cruz Biotechnology Inc.). The filters were then washed 3 times with 1 × phosphate-buffered saline and incubated for 1 h with 1:5,000 dilution of horseradish peroxidase-conjugated goat anti-rabbit IgG. After three 1 × phosphate-buffered saline washes, the filters were developed using an enhanced chemiluminescence kit (Amersham). J chain transcription has been shown to be regulated by at least two positive elements and one repressor element on its promoter (4Lansford R.D. McFadden H.J. Siu S.T. Cox J.S. Cann G.S. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5966-5970Crossref PubMed Scopus (32) Google Scholar, 5Rao S. Karray S. Gackstetter E.R. Koshland M.E. J. Biol. Chem. 1998; 273: 26123-26129Abstract Full Text Full Text PDF PubMed Scopus (40) Google Scholar, 6Shin M.K. Koshland M.E. Genes Dev. 1993; 7: 2006-2015Crossref PubMed Scopus (122) Google Scholar, 7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). To investigate whether additional inducible elements are present which are necessary for regulation of J chain transcription, deletion analyses of the 5′ region of the minimal J chain promoter was performed. The constructs used for the deletion analyses have been described previously and were generated through progressive 5′ deletions of upstream J chain sequences from the hypersensitive region (4Lansford R.D. McFadden H.J. Siu S.T. Cox J.S. Cann G.S. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5966-5970Crossref PubMed Scopus (32) Google Scholar, 7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These fragments were tested for their ability to drive expression of the CAT gene, as shown in Fig. 1 (4Lansford R.D. McFadden H.J. Siu S.T. Cox J.S. Cann G.S. Koshland M.E. Proc. Natl. Acad. Sci. U. S. A. 1992; 89: 5966-5970Crossref PubMed Scopus (32) Google Scholar). In addition to J chain promoter sequences and the CAT gene, each of the constructs also contained the intronic immunoglobulin μE, inserted downstream of the CAT gene in opposite transcriptional orientation (24Durand D.B. Bush M.R. Morgan J.G. Weiss A. Crabtree G.R. J. Exp. Med. 1987; 165: 395-407Crossref PubMed Scopus (117) Google Scholar). The μE sequences were included because levels of CAT expression obtained with constructs lacking a heterologous enhancer were too low to yield reliable values. The constructs were transiently transfected into the J chain-expressing and IgA-secreting myeloma MOPC315, and assayed for CAT activity (Fig. 1). Data obtained from transfection of these constructs into the J chain-silent pre-B cell line PD31 has been shown previously (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar) and is shown here for the purpose of comparison. First we compared the relative CAT activity of MOPC315 cells with theJ chain negative and BSAP positive pre-B cell line PD31. Transfection of the −192 construct containing the entire hypersensitive region resulted in approximately 50-fold higher CAT activity in MOPC315 cells compared with PD31 cells, in agreement with activity of the J chain promoter in plasma cell lines, but not in early B cell lines (data not shown). Deletion of base pairs from −192 to −136 led to an 85% decrease in J chain promoter activity in MOPC315 cells, as shown in Fig. 1. This suggested the presence of a positive regulatory element, denoted JE, located at the 5′ distal portion of the hypersensitive site, upstream from the previously identified JC motif (Fig. 1). Finally, we found that further deletion of the promoter element from −135 to −76 did not cause any additional change in CAT activity (Fig. 1). This is not surprising since the repressive factor BSAP that binds to the JC motif (base pairs −127 to −110), is undetectable in MOPC315 cells (13Neurath M. Max E.E. Strober W. Proc. Natl. Acad. Sci. U. S. A. 1995; 92: 5336-5340Crossref PubMed Scopus (86) Google Scholar). However, as had been identified previously, evidence for BSAP repression is observed in the BSAP-containing and J chain negative PD31 cells, where removal of the JC binding motif (nt −136 to −76) resulted in an increased CAT activity (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). Next we examined the presence of a putative factor NF-JE that could interact with the JE element on the J chainpromoter. Electrophoretic mobility shift assays (EMSAs) were performed using a DNA fragment covering bps −168 to −84 of the J chain promoter (J2) as a probe. This probe contains the JC and JE elements, but not the JA or JB elements (see Fig. 1). Distinct binding patterns were observed using nuclear extracts from the immature B cell line 38C.13, the mature B cell line K46R, the B presecretor BCL1, the plasmacytoma lines S194 and MOPC315, the fibroblast line L (Fig.2 A), and the pre-B cell line PD31 (not shown). Only S194 and MOPC315 express the J chain. The EMSA pattern showed three complexes on the gels. The fastest migrating band (complex 1) was detected only in extracts from 38C.13, K46R, BCL1 (Fig. 2 A, arrow 1), and PD31 (not shown). This suggested that this complex contained BSAP, based on the presence of a BSAP-binding site on this probe (JC) and its pattern of expression in the B cell lineage (see below). An intermediate migrating complex (complex 2) was detected in all cell lines of the B cell lineage (Fig.2 A, arrow 2), but not in L cells. The relative amounts of the two complexes varied between early and late B cell lines: early B cell lines had high levels of complex 1, but low levels of complex 2. In contrast, the two plasmacytoma lines had very low or undetectable levels of complex 1, but high levels of complex 2. All cell lines tested showed a slow migrating complex which could not be competed with excess unlabeled J2 DNA and thus represents nonspecific (NS) protein-probe complexes (Fig. 2 A, arrow NS). The complexes 1 and 2 (Fig. 2 A, arrows 1 and 2) were analyzed further using competition EMSAs and nuclear extracts from the pre-B cell line PD31. The unlabeled double-stranded (ds) oligonucleotides JB (nt −58 to −47) and JC (nt −127 to −110), were used as competitors and contain the PU.1 and BSAP-binding sites, respectively. As expected, the JB competitor was unable to compete for binding with either complex. The JC oligonucleotide was able to prevent binding of BSAP to the J2 probe (Fig. 2 B, arrow 1), but could not compete with the second, slower migrating complex (Fig.2 B, arrow 2). In contrast, this second complex was specifically competed by a second double-stranded oligonucleotide JE, which covers −140 to −132 of the J chain promoter. To analyze the JE region further, we performed copper phenanthroline and methylation protection footprinting using the J2 probe (Fig.3). The retained band corresponding to the JE region gave an extended footprint on the noncoding strand (Fig.3 A) from bp −138 through the BSAP-binding site to bp −124 (which also contained a footprint). The coding strand showed protection in the 5′ region of the noncoding strand footprint (Fig. 3 B) in the region between bp −138 through −134. These results are in agreement with a putative factor NF-JE binding to this region of theJ chain promoter. To search for possible candidates for NF-JE, known consensus DNA-binding motifs were compared with the sequence in the JE region. One motif that contained high homology to the sequence recognized by NF-JE (CATGTG) was one recognized by E-box family proteins, particularly the μE3 (CACATG) and κE3 (CATGTG) motifs present on the immunoglobulin μ heavy chain intron enhancer and κ light chain enhancer, respectively (14Staudt L.M. Lenardo M.J. Annu. Rev. Immunol. 1991; 9: 373-398Crossref PubMed Scopus (244) Google Scholar, 25Beckmann H. Su L.-K. Kadesch T. Genes Dev. 1990; 4: 167-179Crossref PubMed Scopus (352) Google Scholar, 26Gregor P.D. Sawadogo M. Roeder R. Genes Dev. 1990; 4: 1730-1740Crossref PubMed Scopus (434) Google Scholar). To test whether NF-JE represents an E-box factor, EMSAs were performed using a polyclonal rabbit antiserum against one of the two subunits of USF, namely USF-2. When tested with PD31 nuclear extracts this antiserum was able to block NF-JE binding to the JE oligonucleotide probe (Fig. 4, lane 4). Next, we tested whether mutation of the core binding sequence for E-box proteins would result in prevention of binding of the factor NF-JE, using competition EMSAs. A mutant JE oligonucleotide (mJE) was synthesized containing a 3-base pair mutation in the region corresponding to the consensus E motif CANNTG (changing CATGTG to AATCTT). We found that the NF·JE complex could not be inhibited by this mutant JE oligonucleotide (Fig. 4, lane 2). In Fig.2 B we already showed that a wt JE oligonucleotide could compete with this complex. DNA-protein complexes were not observed when the mJE oligonucleotide was used as a labeled probe (data not shown). Together, these findings provide evidence that NF-JE represents the helix loop helix transcription factor USF and will be referred to as such hereafter. To determine the contribution of the JE element toJ chain transcription in vivo, we performed transient transfections in the J chain-expressing plasma cell line MOPC315, using either a wild-type (wt) or mutated J chainpromoter (bp −1150 to +88) driving expression of a CAT reporter gene. The mutated J chain promoter contained a 3-bp replacement mutation of the JE sequence, mJE (see Fig. 4 and text). Comparison of relative CAT activity between wt and mutated constructs revealed a 66% reduction in CAT activity in the JE-mutated J chainpromoter, as shown in Fig. 5 A. This is in agreement with the 5′ deletion studies (Fig. 1) and indicates that JE represents a positive regulatory element on theJ chain promoter. To determine whether the JE element can act as an independent activator of transcription, the truncated rat γ-fibrinogen promoter was used to drive expression of a CAT reporter gene (pγ42CassI). This vector has basal promoter activity in J chain-expressing myeloma lines (24Durand D.B. Bush M.R. Morgan J.G. Weiss A. Crabtree G.R. J. Exp. Med. 1987; 165: 395-407Crossref PubMed Scopus (117) Google Scholar). Two or three copies of the wild-type JE sequence were cloned into the pγ42CassI construct, and the JE constructs transfected into MOPC315 cells. The presence of the JE sequences had no effect on the activity of the truncated rat γ-fibrinogen promoter, suggesting that the NF-JE factor needs to be in the context of other, J chain-specific regulatory factors in order to have transcriptional activity (Fig.5 B, bottom two constructs). We next examined the effect of the JE element in the context of other known J chain promoter regulatory motifs. For these analyses, the following sequences were inserted into the pγ42CassI vector upstream of the minimal γ-fibrinogen promoter: the J1-J2 fragment (bp −168 to −9, which contains all four regulatory motifs JA, JB, JC, and JE), the J1-J2 fragment with a mutant JE-binding site (J1-J2mE), J1-J2 with a mutant JC site (J1-J2mC), the J2 fragment alone (bp −168 to −84, which contains the JC and JE motif only), the J2 fragment with a mutant JE site (J2mE), and the J2 fragment with a mutant JC site. A 4-bp mutation (underlined sequences) changes CAGTGTAGCATGCAGT to CAGTGTAGGTCACAGT in the JC motif and this results in the absence of BSAP binding to this region (7Rinkenberger J.L. Wallin J.J. Johnson K.W. Koshland M.E. Immunity. 1996; 5: 377-386Abstract Full Text Full Text PDF PubMed Scopus (101) Google Scholar). These constructs were then transfected into MOPC315 cells. The constructs containing J1 as well as both JC and JE (J1-J2 and the J1-J2mC) induced a 5- and 5.5-fold increase in the basal" @default.
• W2023851430 created "2016-06-24" @default.
• W2023851430 creator A5007805542 @default.
• W2023851430 creator A5033108573 @default.
• W2023851430 creator A5043734626 @default.
• W2023851430 creator A5046208385 @default.
• W2023851430 creator A5051871004 @default.
• W2023851430 creator A5066242556 @default.
• W2023851430 date "1999-05-01" @default.
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• W2023851430 title "B Cell-specific Activator Protein Prevents Two Activator Factors from Binding to the Immunoglobulin J ChainPromoter until the Antigen-driven Stages of B Cell Development" @default.
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