Department of Molecular Genetics and Microbiology, Program in Immunology and Virology, University of Massachusetts Medical School, Worcester, MA 01655-0122, USA
1 Department of Microbiology, College of Natural Sciences, Kangwon National University, Chunchon 200-701, Korea
Correspondence to: J. Stavnezer
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Abstract |
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Keywords: antibody class switch, Elf-1, IgA
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Introduction |
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In order to understand how switching to IgA is regulated, we are studying the regulation of transcription of GL RNA. Transforming growth factor (TGF)-ß1 induces GL
transcripts in mouse splenic B cells activated by polyclonal activators, e.g. lipopolysaccharide (LPS), and, subsequently, cells in this population will undergo switch recombination to IgA (69). Similarly, in the I.29µ B lymphoma cell line, TGF-ß1 increases GL
transcripts and in the presence of LPS induces switching to IgA (10). The induction of GL
transcripts by TGF-ß1 in this cell line was shown to be due to regulation at the transcriptional level (10). Importantly, TGF-ß1-deficient mice produce very little IgA (11).
TGF-ß1 is a pleiotropic cytokine, having suppressive effects on growth, and both inhibitory and stimulatory effects on the differentiation of a variety of cell types (12,13). Receptors for the TGF-ß superfamily have serine/threonine kinase domains, and treatment with TGF-ß1 results in post-translational modification and activation of the transcription factors Smad 2 and Smad 3 (1315). TGF-ß1 also induces synthesis of other transcription factors, i.e. AP-1 (16,17) and core binding factor (CBF) 3 (18).
To attempt to understand the regulation of transcription of GL RNA, DNA segments containing the 5' flank and first initiation site of the transcripts have been analyzed by reporter gene assays. A deletional analysis indicated that the DNA segment between 130 and +46, relative to the first initiation site for mouse GL
transcripts, is sufficient for expression and TGF-ß1 inducibility of a reporter gene in two different B cell lines (19). Within this region, a novel element (TßRE), located at 128/104 relative to the first RNA initiation site in the GL
promoter, was found to be required for TGF-ß1 inducibility of a reporter gene and when multimerized was sufficient to transfer the inducibility to a minimal c-fos promoter (19). This element, which is present in both the mouse and human promoters, consists of a direct repeat unit containing binding sites for two different transcription factor families: (i) Smad and (ii) CBF
proteins (also known as Runx, AML or PEBP
) (18,2025). Additional CBF
binding sites are also present in the mouse and human GL
promoters (18,23). When activated by TGF-ß1 treatment, Smads 3 and 4 synergize with CBF
to induce the GL
promoter (22,24,25). Synthesis of one of the CBF
proteins, CBF
3, is inducible by TGF-ß1 in splenic B cells and in the B cell line I.29µ (18).
A binding site for an ATF/CREB transcription factor is also important for expression and TGF-ß1 inducibility of both the mouse and human GL promoters in reporter gene assays (19,26). Recently, CREB was shown to bind this site and to synergize with Smads 3 and 4, and CBF
to confer TGF-ß1 inducibility to the mouse promoter when over-expressed (25). Surprisingly, the B cell-specific protein BSAP was found to inhibit transcription when bound to a site in the GL
promoter (27), whereas it activates transcription from the GL
promoter (28,29).
An Ets site has been shown to be essential for expression of the human GL promoter (26), although which protein(s) functions through this site was not determined. The mouse promoter also has an Ets consensus element at a comparable position, although its function has not been previously examined. Ets sites have a core GGA sequence, with most having a GGAA core, and additional nucleotides surrounding the core affect which particular Ets protein binds at a specific site (30). However, since most cells have several different Ets proteins it is often difficult to determine which protein(s) actually binds and regulates transcription of particular genes. Furthermore, Ets proteins often require protein partners in order to bind DNA and to activate transcription (31).
In this manuscript we compare the effects of nucleotide substitutions in all the transcription factor binding sites known to be, or potentially, important for activating the mouse GL promoter. We then focus our efforts toward identifying the Ets proteins that bind the GL
promoter in B cells and their role in transcription. We demonstrate that the Ets site is important for TGF-ß1 inducibility in addition to overall activity of the promoter in I.29µ B cells. We show that although this site binds Fli-1 when present on a small DNA segment, it binds Elf-1 and PU.1 in the context of the full-length promoter, and that Elf-1 binding is inducible in splenic B cell extracts by LPS.
It has been reported that mice with targeted deletions of various NF-B genes have defects in antibody class switching. For some isotypes, e.g. IgE, the reduction in isotype production correlates with a defect in ability to synthesize GL transcripts (32,33). For IgA, it was found that NF-
B/p50-deficient mice produced 4-fold less IgA upon immunization (32) and that splenic B cells induced in culture switched ~10-fold less well to IgA than wild-type B cells (33). However, Snapper et al. (33) found that there was no decrease in the levels of GL
transcripts induced in the p50-deficient mice. In this manuscript we examine the effect of mutation of a NF-
B binding site on the activity of the GL
promoter.
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Methods |
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Cell culture
I.29µ cells were cultured as described (10). A20.3 cells were cultured at 37°C in an atmosphere of 5% CO2 in RPMI 1640 medium supplemented with 10% FBS, 50 µM 2-mercaptoethanol, 0.1 mM non-essential amino acids, 1 mM sodium pyruvate, 2 mM L-glutamine, 100 U/ml penicillin, 100 µg/ml streptomycin and 0.1 mg/ml kanamycin sulfate (the last six reagents came from Gibco, Grand Island, NY). In transfection experiments with I.29µ cells shown in Fig. 1, human or porcine TGF-ß1 (R & D Systems, Minneapolis, MN) was added twice (right after transfection and again at 7 h after transfection) at a concentration of 1 ng/ml each time. In experiments with A20.3 cells and in all other experiments with I.29µ, TGF-ß1 was added right after transfection at a final concentration of 2 ng/ml. The amount of TGF-ß1 used was determined to be in the optimal range (data not shown).
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Oligonucleotides
The sequences of the top strand of the double-stranded oligonucleotides used in electrophoretic mobility shift assays (EMSA) are given below.
Consensus transcription factor sites.
Cre (CREB-binding site): 5'-GATTCGATGACGTCAGTACGGTG-3'; B (NF-
B site): 5' CAACGGCAGGGGAATTCCCCTCTCCTT 3' (38); AP-1: 5'-AGCTTGGTGACTCATCCG-3'; CBF
: 5'-CGTATTAACCACAATACTCG-3' (20).
Primers used for PCR.
Lower case bold letters indicate mutated nucleotides; lower case plain text letters indicate nucleotides used for cloning that are not in the GL promoter. Reverse PCR primer (+14/7) 5'-cgcggatccTGGATGGGTCAGACTGAGCGC-3'; mEts, upper 5'-GGAACgatcgGTGGGTGAGGGACC-3'; lower 5'-CCCACcgatcGTTCCACCTCCACGTT-3'; m
B, upper 5'-TGGAACAGGAAGTGGGTcttctctagaACCTAGGGGCCAGAC-3'; lower 5'-GTCTGGCCCCTAGGTtctagagaagACCCACTTCCTGTTCCA-3'; mCBF3, upper 5'-CCCACCTAGGtaaactaCACACAGTCATGCAG-3'; lower 5'-agtttaCCTAGGTGGGGTCCC-3'; m(
B + CBF3) upper 5'-tctagaACCTAGGtaaactaCACACAGTCATGCA-3'; lower 5'-agtttaCCTAGGTtctagagaaGACCCACTTCCTGTTCCA-3'.
Single-stranded oligonucleotides were obtained from Operon Technologies (Alameda CA), DNA International (Woodlands TX), DNAgency (Aston, PA) or Ransom Hill Biosience (Ramona, CA).
Plasmid constructs
Mutations of the GL promoter (Fig. 1A
) were created by PCR based on an overlap extension technique (39). PCR- amplified fragments carrying the desired mutations were then cloned into SmaIBglII sites upstream of the luciferase (Luc) reporter gene in the pXP2 vector (40). Each construct was sequenced to confirm that the expected mutations had been introduced and that no extra mutations had been created. DNA sequencing was performed using the dideoxy chain termination method with Sequenase version 2.0 kits (US Biochemical, Cleveland, OH) or by automated sequencing using an Applied Biosystems sequencer and software (Foster City, CA).
Transfection
Transfection was performed by electroporation using Cell ZapII (Anderson Electronics, Brookline, MA) as described (18). The GL Luc reporter plasmids were co-transfected with one of three different internal control plasmids, depending on the experiment. pSV2CAT was used for I.29µ transfections shown in Fig. 1
(B); pPGKß-gal (41) or pCMVß-gal (Stratagene, La Jolla CA) for all other transfections. Cells were incubated for the indicated times at 37°C and then assayed for Luc and chloramphenicol acetyltransferase (CAT) or ß-galactosidase (ß-gal) activity.
Expression plasmids
cDNAs for Smads 3 and 4, cloned in pcDNA3, were obtained from M. Kawabata (The Cancer Institute, Tokyo) (42). The expression plasmid for human AML2 (CBF3) was obtained from S. Hiebert (St Jude Children's Research Hospital, Memphis, TN) (43). Mouse cDNA for Elf-1 cloned in pCDNA3 was obtained from M. Roussel (St Jude Children's Research Hospital) (44). This plasmid was used in the transient reporter assays. Human Elf-1 and PU.1 cDNAs used for in vitro transcription/translation in reticulocyte lysates were from L. Shapiro (St Jude Children's Research Hospital) (45). cDNA for mouse Fli-1, cloned in pGEM, was obtained from A. Bernstein (University of Toronto, Toronto, Canada) (46). The dominant-negative Ets expression plasmid was from E. Boulukos (Universite de Nice-Shophia Antipolis, Parc Valrose, France) (47). Rat CREB expression plasmid, pCMV2-CREB, was subcloned from the rat cDNA (48) by P. R. Dobner (University of Massachusetts Medical School).
Luc, CAT and ß-gal assays
Luc and CAT assays were performed as described (37), and ß-gal was assayed as described (49). The CAT activity was measured by a liquid scintillation counter after 4 h (for A20.3 cells) and 20 h (for I.29µ cells) incubation at 37°C, in the linear range of the assay.
Oligonucleotide probes for EMSA
To produce double-stranded oligonucleotides, complementary single-stranded oligonucleotides were annealed at 100 ng/µl each in 100 mM NaCl, 10 mM TrisHCl, pH 8.0 and 1 mM EDTA. Prior to annealing, the oligonucleotides were incubated at 95°C for 10 min to disrupt secondary structure, and then incubated at 10°C below the Tm for 1 h and slowly cooled to room temperature. The annealed oligonucleotides were ethanol precipitated and purified on a 1215% polyacrylamide gel. Purified double-stranded oligonucleotides were end-labeled with [-32P]ATP using T4 polynucleotide kinase. DNA segments corresponding to the GL
promoter from 130 to +14 were generated by PCR. They were gel-purified and end-labeled as described for the annealed oligonucleotides.
Preparation of nuclear extracts (NE)
NE for I.29µ, A20.3 and splenic B cells were prepared using the method of Schreiber et al. (50) as modified (37). In addition to several protease inhibitors, a phosphatase inhibitor Na3VO4 (Boehringer Mannheim, Indianapolis, IN) was added to buffer A and C at a final concentration of 1 mM. Protein concentration of the NE was determined using the Bradford assay (BioRad, Richmond, CA).
In vitro transcription and translation
For in vitro transcription and translation, hElf-1, hPU.1 and mFli-1 proteins were synthesized using the TNT-coupled reticulocyte lysate system (Promega, Madison, MI), according to the manufacturer's protocol. An aliquot of 1µl of the resulting lysate was used for each EMSA incubation.
EMSA
DNA binding reactions were performed in 15 µl reaction volumes containing 0.11 ng (15,00030,000 c.p.m.) end-labeled double-stranded DNA probe, 15 µg NE and 24 µg poly(dIdC) (Pharmacia, Piscataway NJ). The final concentration of NaCl in each reaction mixture was adjusted to 80 mM by adding buffer C (50). The reaction mixtures were incubated at room temperature for 30 min and then loaded onto 46% native polyacrylamide gels. All gels were electrophoresed in re-circulating 0.5xTBE buffer at 100150 V for 24 h. For supershift or competition experiments, antibody (or antiserum) (1 µl, except where indicated) or competitors were added to the entire mixture, except the probe which was added last.
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Results |
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In I.29µ cells, TGF-ß1 increases Luc activity of the plasmid driven by the wild-type GL promoter by 3.5-fold (Fig. 1B
). This level of induction is consistent with previous results (19). These results are also in accord with the finding that TGF-ß1 increases transcription of the endogenous GL
RNA in I.29µ cells by 5-fold in a nuclear run-on assay (10).
The DNA segment 130/104 contains a direct repeat dimer that has two binding sites for Smad proteins (CAGAC, indicated as SBE1 and 2, Smad-binding element, in Fig. 1A) and two binding sites for CBF
(ACCACA, indicated as CBF1 and 2). This segment has been shown to be sufficient for conferring TGF-ß1 responsiveness to a minimal c-fos promoter (19), and to be synergistically activated by Smads 3 and 4 and CBF
(18,2224). Mutation of SBE 1 and 2 in construct m1-TßRE eliminates TGF-ß1 induction but does not reduce basal activity in I.29µ cells (Fig. 1A and B
). Mutation of CBF1 and 2 (m2-TßRE) reduces TGF-ß1 induction by ~30% and basal promoter activity by ~50%. Mutation of a downstream, weak CBF
binding site (CBF3) (18) reduces basal activity by 3-fold but has no effect on TGF-ß1 induction. Consistent with published studies (18,24,25), mutation of the CREB-binding site eliminates induction by TGF-ß1 and reduces basal promoter activity by 5-fold.
Surprisingly, mutation of a sequence element that matches a consensus NF-B binding site (54/45) slightly increases promoter activity and has no effect on induction by TGF-ß1. This site does bind NF-
B/p50 (see below). Two additional sets of mutations of the
B site were also tested in the reporter assay with similar results (data not shown). Mutation of both the
B element and the third CBF
binding site has an effect similar to mutation of the third CBF
site alone (Fig. 1B
). Of all the sites in the promoter, the Ets consensus motif at 64/61 (GGAA) appears to the most important for overall activity, since mutation of this site eliminates promoter activity, both basal expression and induction by TGF-ß1.
The effects of the promoter mutations were similar but not identical when tested in A20.3 cells (Fig. 1C). TGF-ß1 increases Luc activity of the wild-type promoter by 8-fold in A20.3 cells. Mutation of both SBEs (m1-TßRE) abolishes induction and reduces basal promoter activity by 60%. Mutation of the ATF/CRE or Ets site nearly eliminates promoter activity, although TGF-ß1-induction is mostly retained. As in I.29µ cells, mutation of CBF3 reduces expression but has no effect on induction by TGF-ß1 (data not shown) and mutation of the
B site has little effect on promoter activity. As in I.29µ cells, the effect of mutation of both the
B site and CBF3 is similar to the effect of mutation of CBF3. Unlike results in I.29µ cells, however, the m2-TßRE mutation does not significantly affect promoter activity (15% reduction for TGF-ß1-induction and 10% reduction for basal activity compared with the wild-type promoter, data not shown). In both cell lines, TGF-ß1 has no effect on the promoterless pXP2 plasmid.
Thus, the effects of mutation of some of the consensus elements differ somewhat between two cell lines, only one of which (I.29µ) can express endogenous GL transcripts and switch to IgA. In both cell lines, the SBE are necessary for inducibility by TGF-ß1. However, the CBF
-binding sites 1 and 2 differ in their importance in the two cell lines, being important for both basal and induced expression in I.29µ but not in A20.3 cells. This may be because TGF-ß1 induces CBF
3 in I.29µ cells but not in A20.3 cells (18), although the constitutively expressed CBF
2 is equally abundant in these two cell lines (unpublished data). In I.29µ cells the ATF/CREB element is essential for induction by TGF-ß1 and the Ets motif is essential for all promoter activity, whereas in A20.3 these elements are do not contribute to TGF-ß1 inducibility although they are very important for overall promoter activity. Neither CREB nor Ets proteins have been reported to be inducible by TGF-ß1. The data obtained in I.29µ cells are consistent with the hypothesis that CREB and the protein(s) binding at the Ets site interact directly, or indirectly, with the TGF-ß1-inducible proteins, Smads and CBF
3, which bind at the TßRE.
To further establish the importance of the Ets site for activity of the promoter, we tested the effect of co-transfection of an expression plasmid for a dominant-negative version of Ets2 (47) along with the GL promoter Luc reporter plasmid in A20.3 cells. As shown in Fig. 2
, the dominant-negative Ets2 expression plasmid inhibits the basal expression and also TGF-ß1 induction of the promoter in a dose-responsive manner. The recombinant dominant-negative Ets protein was shown to bind to the GL
Ets site in EMSA (data not shown). These data further indicate the importance of Ets family proteins for activity of the promoter.
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A DNA probe of intermediate length, containing the 94/55 promoter segment, binds both Fli-1 and Elf-1, as both of these antibodies supershift complexes (Fig. 3C, lanes 1719). In addition, PU.1 antibody inhibits the fastest migrating complex formed with the 94/55 probe and inhibits a component of the fast-migrating major complex formed with the full-length promoter segment (Fig. 3C
, lane 20 and Fig. 3D
, lane 25). No other Ets antibody tested (Ets1/2, PEA3 and SpiB) affected complexes detected with any GL
probe (data not shown). TGF-ß1 treatment has no effect on formation of the Ets complexes (Fig. 3C
cf,. lanes 7, 8 and 15, 16; data not shown). NE from A20.3 cells gave similar results, except they have slightly lower levels of Elf-1 binding activity (data not shown).
These data indicate that the binding of Elf-1 and PU.1 to the GL promoter requires, in addition to the 71/55 segment, the flanking region. Neither of the two Ets consensus elements in the GL
promoter are optimal Elf-1 or PU.1 sites. An optimal Elf-1 site is shown in Fig. 3
(A) (44,53). It has previously been shown that Elf-1 binds to and activates transcription at non-optimal sites by interacting with other proteins, including AP-1, HMG-I(Y) and NF-
B p50 and c-Rel (5456). However, we have not found any binding sites for AP-1 or HMG-I(Y) in the GL
promoter and the NF-
B site is not required for promoter activity. Although Fli-1 binds to the small probe, it too may have a partner protein since the Fli-1 complex formed on the small probe migrates slightly more slowly than the complex formed with the 94/55 probe (Fig. 3C
cf,. lanes 14 and 15). Perhaps the partner proteins differ on these two probes.
Sequences required for Elf-1 and PU.1 binding to the GL promoter.
To attempt to identify the nucleotides required for binding of Elf-1 and PU.1 to the GL promoter, several probes of different lengths were tested in EMSA using I.29µ NE. Addition of 4 nucleotides to the 5' side of the Ets sites (75/55 segment) did not allow Elf-1 binding (data not shown). Addition of 4 nucleotides to the 3' side (75/51) only allowed weak Elf-1 binding (data not shown). By testing several additional segments, we found that the nucleotides required for nearly optimal Elf-1 binding resided within the 83/51 segment. This probe also binds PU.1. It also binds Fli-1, but to a lower extent than the 71/55 probe (Fig. 4
, lanes 17, and data not shown).
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To examine whether binding of Elf-1 to the wild-type GL promoter depends on B cell-specific proteins, we asked if individual recombinant Elf-1, Fli-1 and PU.1 obtained by in vitro translation using reticulocyte lysates would show the same binding specificity as found in B cell extracts. Reticulocyte lysates programmed by mRNA for the three different Ets proteins were used in EMSA with both the shorter and longer GL
Ets probes. Figure 5
shows that the short Ets probe, 71/55, binds recombinant PU.1 and Fli-1 but fails to binds Elf-1 (Fig. 5
, lanes 37), whereas the 83/51 probe binds all three proteins (Fig. 5
, lanes 813). Thus, recombinant Elf-1 binding retains the same specificity as it does in B cell NE. Also, although recombinant PU.1 does bind the short probe, it does not bind as well to the short probe as to the longer probe. Since the recombinant Elf-1 complex co-migrates with the complex formed with I.29µ NE (Fig. 5
, lanes 1416), it appears that if a partner protein is required, it is also present in reticulocyte extracts. Lysates programmed by the empty expression vector (pcDNA3) showed no binding to either probe. These data demonstrate that Elf-1 binding to the GL
promoter does not depend on PU.1 binding nor does PU.1 binding depend on Elf-1 binding. Furthermore, the role of the additional sequences surrounding the Ets sites is not specific to B cell lines. These data also indicate that although Elf-1 and Fli-1 bind in a reciprocal manner to the various Ets site probes, this is not simply due to Fli-1 competing with Elf-1, since when recombinant Elf-1 is tested individually, it shows the same binding specificity.
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Elf-1 binding is affected by LPS activation of splenic B cells
The above data suggest that Elf-1 or PU.1 binding is required for transcription of the GL promoter in B cell lines. Therefore, it was important to determine if NE from splenic B cells activated to switch to IgA contain Elf-1 or PU.1 binding activity. Using the 130/+14 promoter segment in EMSA, we found that NE from resting splenic B cells do not show Elf-1 binding activity, but that after activation for 14 h with LPS alone, or with LPS + TGF-ß1, binding activity is induced (Fig. 8
, cf. lanes 2, 3 and 5, 6; data not shown). PU.1 binding to the promoter was not detected. Induction of Elf-1 activity may be one of the roles of LPS in activating class switching in splenic B cells. However, LPS has additional roles since it is required for induction of CSR in I.29µ cells (10), although it is not required for Elf-1 binding activity in these cells (Fig. 8
, lanes 8 and 11).
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Because of this result it was important to demonstrate that the consensus B element actually binds NF-
B. In the EMSA shown in Fig. 3
(E), using the 130/+14 probe, we demonstrate that a consensus
B site competes a component of the fastest-migrating predominant complex (Fig. 3E
, lane 2), and also competes with this complex and an additional slower-migrating complex detected using the mEts site probe (Fig. 3E
, lane 8). Presumably, the faint upper NF-
B complex also binds the wild-type promoter segment but cannot be detected due to the background at this position in the lane. To determine whether these two complexes contain NF-
B/p50, supershift assays were performed, as shown in Fig. 3
(E, lanes 1217). Using the 130/+14 probe, anti-p50 supershifts a component of the fastest-migrating major component detected with NE from I.29µ cells (Fig. 3E
, lane 13) and also from LPS + TGF-ß1-treated splenic B cells (Fig. 3E
, lane 15). Using the mEts probe in supershift experiments with antibody to NF-
B p50, we find that both complexes are supershifted (Fig. 3E
, lane 17).
We reasoned it was possible the B mutations do not affect activity of the GL
reporter plasmid because the amount of NF-
B is too low in the B cell lines to activate the promoter. To examine this possibility, we tested the effect of over-expression of several NF-
B proteins in co-transfection reporter gene assays. None of the expression plasmids tested, those encoding p50 alone, or RelA, c-Rel or RelB in combination with p50 stimulated GL
promoter activity in I.29µ cells (data not shown). We conclude that although NF-
B can bind the GL
promoter, it does not have a significant effect in transient reporter gene assays.
Nocodazole treatment increases GL promoter activity
Smads 2, 3 and 4 have recently been shown to be associated with microtubules in the cytoplasm of epithelial, endothelial and HeLa cells lines (58). TGF-ß1 treatment of an epithelial cell line results in the release of Smads from microtubules, and subsequent phosphorylation and migration to the nucleus where they activate transcription. Dong et al. (58) were able to mimic and enhance the effect of TGF-ß1 on transcription by treatment of the epithelial cell line with nocodazole, which disrupts microtubules. We were interested to learn if Smad proteins might also be associated with microtubules in B cell lines, so we tested whether nocodazole could stimulate transcription driven by the GL promoter in a B cell line. Consistent with the results in epithelial cells, we found that nocodazole increases activity of the promoter in A20.3 B cells by 2- to 3-fold, in the presence or absence of TGF-ß1, and also when Smads 3 and 4 are over-expressed (Fig. 9
). These data suggest that in the absence of TGF-ß1, transiently transfected Smad proteins associate with microtubules in B cell lines, e.g. A20.3, and that TGF-ß1 disrupts this association.
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Discussion |
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Ets proteins have been implicated in regulation of genes during a variety of biological processes, including growth control, transformation, lymphocyte differentiation and developmental programs in many organisms. About 20 proteins have been identified that have the conserved ets domain and which bind the consensus Ets motif, found in both enhancer and core promoter regions. Ets family proteins bind to DNA as a monomer and generally bind cooperatively with other transcription factors, e.g. AP-1 (54,56,5961), BSAP (62), Pip (NF-EM5) (63,64) MafB (65), Sp1 (66), serum response factor (67) and CBF (68,69). Elf-1 and NF-
B have been shown to interact and to often bind adjacent sites on promoters (30,55,70). However, although the Ets and
B sites in the GL
promoter are also near each other, they do not appear to cooperate in that the Ets site is essential and the
B site appears to have no role in transcriptional activity. By contrast, CBF
, another protein known to interact with Ets proteins, is important for TGF-ß1-induced activity of the GL
promoter.
Consistent with a requirement for cooperative binding with another transcription factor, the Ets sites of the GL promoter bind Elf-1 and PU.1 when the probe used for EMSA contains at least 13 nucleotides 5' and 10 nucleotides 3' to the core Ets elements (GGAA). However, if a short fragment containing 7 nucleotides upstream and 6 nucleotides downstream of the 3' Ets core is used as a probe, Fli-1 binds but not Elf-1 or PU.1. These data indicate that nucleotides in addition to the 3' Ets site are required for Elf-1 and PU.1 binding. Recombinant Elf-1 and PU.1 tested individually in EMSA retain the same binding specificity, indicating that the differential binding is not due to competition among these Ets proteins for the same binding sites. From these data we conclude that Elf-1 and PU.1 are likely to be the proteins which bind in vivo. The binding differences may be due to the requirement for additional binding sites for different partner binding proteins. However, we have tested the effect of mutating several different nucleotides in the flanking regions of the 83/51 Ets probe on binding by Elf-1 and PU.1, and find that the only mutations that significantly reduce binding are those within or between the two Ets sites (Fig. 4
and data not shown). Therefore, we have been unable to obtain direct data implicating interactions between these Ets proteins and other transcription factor families in the regulation of the GL
promoter.
Although we obtained no direct evidence of proteinprotein interaction, our reporter gene assays suggest that Ets proteins which bind the GL promoter interact with other factors to regulate the TGF-ß1-inducible and constitutive activity of the promoter. Mutation of the more 3' Ets site not only eliminates basal expression but also eliminates TGF-ß1 inducibility of the GL
promoter in I.29µ cells. In addition, over-expression of a dominant-negative form of Ets 2 only inhibits basal activity of the promoter by 30%, but inhibits TGF-ß1 inducibility by 50% in A20.3 cells. Furthermore, extensive gel shift and Western blotting analyses failed to find evidence that TGF-ß1 induces Ets proteins in the two B cell lines or in splenic B cells (Fig. 3
and data not shown). Because of these data, we hypothesize that Ets proteins, presumably Elf-1 and PU.1, may interact with complexes involving TGF-ß1-induced factors and thereby contribute to TGF-ß1 induction of the promoter. Candidates for this interaction are the TGF-ß1 inducible factors that bind the GL
promoter, i.e. Smads 3 and 4 and CBF
3. This complex might also involve CREB, since this factor also contributes to TGF-ß1 induction of the promoter (Fig. 1B
) (19,25,26). Perhaps the reason we have been unable to demonstrate this interaction in co-transfection reporter gene assays is due to limiting amounts of another factor.
Elf-1 is present in two B cell lines and is inducible in splenic B cells treated with the B cell mitogen LPS
Although the binding activity of Elf-1 is constitutive in I.29µ and A20.3 cells, we found that it is inducible by LPS in splenic B cells. LPS has been shown to activate p38 MAP, ERK and JNK kinases (71,72). Elf-1 binding activity has been shown to be dependent upon serine phosphorylation and many of the phosphorylated serine residues match consensus MAP kinase sites (30). PU.1 can also be phosphorylated on serine by JNK1 and by casein kinase II in vitro, and is phosphorylated in B cells. However, no functional significance for PU.1 phosphorylation has been demonstrated (30).
In addition to being regulated by phosphorylation, Elf-1 activity is regulated by binding to the cell-cycle regulator Rb. Elf-1 binds to the hypophosphorylated form of Rb protein, but not the phosphorylated form (73). During the transition from the G1 to S phase of the cell cycle, Rb becomes phosphorylated, and the DNA binding and transcriptional activities of Elf-1 are induced in peripheral blood T cells. Since LPS induces kinase activity and B cell proliferation, it is possible that LPS induces Elf-1 activity by two mechanisms, by serine phosphorylation and by dissociation from Rb. CSR in splenic B cells requires cell proliferation (74) and during this proliferation Elf-1 may be activated. Furthermore, IgA switching in vivo occurs in the highly stimulatory environment of the intestinal lymph tissue called Peyer's patches, in which B cells are rapidly proliferating (75,76). The bacterial products endotoxin, non-methylated CpG-containing DNA and other bacterial cell wall components within this environment induce B cell activation and proliferation (77,78). Our data suggest that induction of Elf-1 binding after activation of B cells during an immune response is important for GL transcription and subsequent switching to IgA.
NF-B binds to the GL
promoter but has no effect on its activity in transient transfection reporter assays
The finding that mutation of the NF-B binding site has no effect on activity of the GL
promoter in reporter gene assays was surprising, although this finding is consistent with results showing that p50-deficient B cells synthesize normal levels of GL
transcripts (33). NF-
B sites have been found in the promoters for three other mouse IgH GL transcripts:
(29,79),
1 (37) and
3 (80). These
B sites have been shown to be essential for expression of the GL
and
1 promoters, and for their induction by IL-4 and/or by CD40L (29,37,79,81). The composition of the NF-
B complexes binding a
B site affects its function (37,38,8284). For the mouse GL
1 promoter, RelA and RelB/p50 heterodimers were found to be activating, whereas p50 homodimers and c-Rel-p50 heterodimers were found to be inhibitory (84). Although we have been unable to detect any NF-
B protein besides p50 in the two complexes that bind the GL
promoter, the migration of the complexes is consistent with that of heterodimers of RelA, RelB or c-Rel with p50 (upper complex) and p50 homodimers (lower complex). It is possible that the
B site plays a role in a process other than transcription of the GL
promoter since switching to IgA in p50-deficient B cells is greatly reduced (32,33).
In conclusion, GL transcription in the mouse B cell line I.29µ is activated by a combination of transcription factors inducible by TGF-ß1 which interact with factors constitutively expressed in this B cell line. The most important of the constitutive factors is one or more Ets protein, most likely Elf-1 and possibly PU.1. However, in resting splenic B cells, neither Elf-1 nor PU.1 binding activity is detectable, although Elf-1 binding is inducible by LPS, and the combination of LPS and TGF-ß1 induces GL
transcripts (9). Future studies will need to address the mechanism of Elf-1 induction in B cells and whether Elf-1 directly interacts with transcription factors induced by TGF-ß1.
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Acknowledgments |
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Abbreviations |
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ß-gal ß-galactosidase |
CAT chloramphenicol acetyltransferase |
CBF![]() |
EMSA electrophoretic mobility shift assay |
GL germline |
LPS lipopolysaccharide |
Luc luciferase |
NE nuclear extracts |
SBE Smad-binding element |
TßRE TGF-ß1-responsive element |
TGF transforming growth factor |
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Notes |
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Received 27 October 2000, accepted 19 February 2001.
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References |
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