INTRODUCTION

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The immunoglobulin (Ig) µ heavy-chain gene enhancer activates transcription and V(D)J recombination at the Ig heavy-chain (IgH) locus in precursor B cells. Enhancer function is mediated by multiple DNA binding proteins that interact with the µ enhancer. Most of these factors are ubiquitously expressed in both B cells (where the enhancer is active) and non-B cells (where the enhancer is inactive), while a smaller subset of factors have more restricted tissue distribution (9, 19, 20). The basic helix-loop-helix (bHLH) family of proteins constitute a major portion of the ubiquitously expressed factors, whereas ETS and POU domain genes are examples of the second subset. However, no single factor that can account for the cell type specificity of IgH gene expression has been identified. It is likely, therefore, that activation of the enhancer at a precise developmental stage during B-cell ontogeny is governed by the combinatorial properties of several proteins.

In principle, identification of proteins that define a B-cell-specific transcriptional unit would allow further reconstruction of the functional multiprotein-DNA complex. However, mechanistic analysis of µ enhancer function is severely complicated by the multiplicity of protein binding sites within the enhancer. Several deletion and mutational studies have defined regions of the enhancer that activate transcription and the importance of specific protein binding sites within each enhancer fragment (13, 14). A feature of the enhancer revealed by these studies was that several binding site mutations affected enhancer activity minimally. For example, mutation of the µE3 element in the context of a 250- or 470-bp enhancer fragment decreased enhancer activity by 25 to 30%. Similarly, mutation of the µE1 or µE2 element individually did not affect µ enhancer activity significantly. These observations have been interpreted to mean that there is functional redundancy among various elements, so that absence of a particular element is functionally compensated for by other elements. The presence of functional redundancy further complicates enhancer analysis by making it difficult to discern which combination of factors is responsible for enhancer activity.

To systematically circumvent these problems, we first identified the smallest domain of the enhancer that conferred B-cell-specific transcriptional activity; such an enhancer should contain no redundant elements. Characterization of the tripartite 70-bp minimal enhancer (µ70) resulted from these studies (7, 8, 20-22, 24). The µA and µB sites within µ70 bind ETS domain proteins, and the intervening µE3 element binds various leucine zipper-containing bHLH proteins, as well as the core binding factor (CBF). The µ70 enhancer has several features that suggest it is an appropriate starting point for the analysis of the full enhancer. First, like the full enhancer, it is composed of elements that bind tissue-restricted proteins and one that binds a ubiquitous protein. Second, none of the three elements can individually activate transcription in B cells either as a monomer or as multimers, suggesting that transcription activation in B cells is a combinatorial property. Third, activity in B cells requires all three elements, which indicates that there is no redundancy in this enhancer. Presumably, the necessity of µE3 in this context is due to the absence of any other µE motif in the minimal enhancer which can functionally substitute for it.

We then incorporated a second µE element, µE2, into our analysis (4). Addition of a second µE element was expected to increase the activity of the tripartite µ70 enhancer. Furthermore, because the sequences of the µE2 and µE3 elements are very similar, it was possible that the µE3 element would be dispensable in the context of the four-part enhancer; that is, µE2 could functionally substitute for µE3. As expected, inclusion of µE2 substantially enhanced transcriptional activity. However, we found that the µE3 element was still absolutely essential for function. These observations demonstrated that the µE2 and µE3 elements were functionally not equivalent and showed that µE2 activity was dependent on the µE3 site, providing evidence for communication between µE elements. We proposed a plausible mechanism for µE2-µE3 synergy based on in vitro DNA binding analyses.

Despite the insights gained from the analysis of the three- and four-part enhancers, these studies did not address two important properties of the µ enhancer. First, both enhancers described above were only weakly active as monomers, and dimerization was necessary for robust transcriptional enhancement. Second, these studies did not provide insights into the basis for functional redundancy among the µE elements. Our goal in the present study was to characterize the smallest enhancer fragment that is active as a monomer and identify the motifs that compensate for the loss of elements such as µE2 and µE3.

We found that the smallest monomeric enhancer contains five elements, µA, µB, µE2, µE3, and µE5. All five elements were essential for enhancer activity, suggesting that this was a minimal monomeric enhancer with no functionally redundant motifs. The B-cell-specific transcriptional activity of this fragment could be reconstituted in COS cells by coexpression of the ETS proteins PU.1 and Ets-1 and the bHLH protein E47. The question of redundancy was explored by extending the enhancer fragment both 5' and 3'. Surprisingly, a fragment containing a fourth µE element, µE1, still required µE3 for activity; that is, even in the presence of µE1 to µE3 and µE5, no other µE element could functionally substitute for µE3. However, an enhancer extending further 3', but incorporating no additional µE elements, showed a reduced requirement for µE3. Deletions and point mutations were used to localize the putative element, which was shown to be a binding site for the family of interferon regulatory factors (IRFs) (15, 16, 26). These observations identify the basis for redundancy of µE elements and dispel the assumption that µE elements functionally substitute for each other. In addition, we provide the first evidence for a role of IRF family proteins in Ig gene expression.

	MATERIALS AND METHODS

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Plasmids. The wild-type (WT), µA, µE3, and µB µ170 plasmids have been described previously (18, 21). The µE2, µE2 µE3, C2, C2 µE3, µ3', µ3' µE3, and µ3' µE2 µ170 plasmids were generated by first subcloning the corresponding mutant HinfI-DdeI fragments (nucleotides 346 to 518 according to the numbering system of Ephrussi et al. [6]) into the EcoRV site of pSP72. The BglII-ClaI fragments isolated from these subclones were then blunted with Klenow fragment and cloned into the SalI site of reporter plasmid 56CAT.

Transfections. Murine S194 and human DHL-9 cells were grown in RPMI medium supplemented with 5% fetal bovine serum, 5% calf serum, and 50 µg each of penicillin and streptomycin per ml. COS cells were grown in Dulbecco modified Eagle medium containing 10% newborn bovine serum and antibiotics in the amounts specified above. Transfections, cell extract preparation, and chloramphenicol acetyltransferase (CAT) activity measurements were carried out as previously described (4).

In vitro translation. In vitro translation reactions were performed with 1 µg of plasmid HAPip1-380 (2) or pSP64-IRF1 and a TNT T7 quick coupled transcription-translation kit or TNT SP6 coupled transcription-translation kit (Promega), respectively.

Electrophoretic mobility shift assay (EMSA). The µE3 Pst-C2 fragment used in Ets-1 and CBF binding was generated by digesting with PstI the PCR product used for cloning plasmid µ128. The sequence for the B probe used in the binding of HAPip1-380 is 5'-GAGAAATAAAAGGAAGTGAAACCAAG-3'. Binding conditions were as previously described (4).

	RESULTS

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Minimal monomeric µ enhancer. A schematic of the µ enhancer region is shown in Fig. 1A. A central region of approximately 170 nucleotides is densely packed with protein binding sites that are indicated by assorted geometric shapes. Flanking the core enhancer are matrix attachment regions which have been proposed to increase the distance over which the central region can exert its influence (11). Our earlier studies have assayed a 70-bp enhancer (µ70) containing three elements and a 59-bp enhancer (µ59) that contains four elements. Both fragments needed to be dimerized to enhance transcription significantly. To identify a minimal monomeric enhancer, we extended µ59 at the 5' end to incorporate the adjacent µE5 element (µ74) and tested its activity by transient transfection into S194 plasma cells. Whereas µ59 monomer was a very weak activator (approximately four- to fivefold above the background of the enhancerless reporter plasmid [data not shown]), addition of one more element made µ74 a strong enhancer (Fig. 1B). Mutations in any of the five elements significantly decreased enhancer activity, showing that there were no redundant elements in this fragment (Fig. 1B). Thus, µ74 is the smallest µ enhancer fragment that is active as a monomer.

View larger version (18K): [in this window] [in a new window]   FIG. 1.   (A) Schematic representation of µ enhancer derivatives used in this study. Squares represent CAXXTGG motifs that are binding sites for ubiquitous transcription factors. The bHLH protein, E47, binds µE5 and µE2 elements; several leucine zipper bHLH proteins, such as TFE3 and USF, bind to µE3; the hematopoietic cell transcription factor, CBF, also binds to µE3; the µE1 site binds the factor YY-1 (23). The µA and µB sites bind proteins belonging to the ETS family: PU.1, a macrophage- and B-cell-specific ETS protein, binds to µB; several proteins, such as Ets-1, Erg-3, and Fli-1, bind to µA. C1 to C3 designate three other CBF binding sites; in all enhancer derivatives used here, the C1 site has been mutated. The oval marked µ3' represents a new element identified in this study. This motif functionally compensates for the loss of a µE3 or µE2 element and binds the proteins IRF-1 and IRF-2. Hf, HinfI. (B) The smallest µ enhancer fragment active as a monomer contains five essential motifs, as determined by transient transfection analysis of the WT and mutated µ74 enhancer derivatives in S194 plasma cells. 56 is the enhancerless reporter containing a c-fos gene promoter extending 56 nucleotides 5' of the transcription initiation site. All enhancer derivatives were cloned upstream of the minimal promoter, and 5 µg of plasmid DNA was used for transfection. Results shown are averages from three experiments done in duplicate and normalized to the activity of the WT enhancer, which is assigned a value of 100.

Reconstruction of µ74 activity in nonlymphoid cells. We have previously shown that the µ70 dimer is activated by coexpression of PU.1 and Ets-1 in nonlymphoid cells. Transactivation required all three elements of this enhancer and utilized an endogenous µE3 binding protein. We sought to reconstitute the activity of the µ74 enhancer in similar assays. Cotransfection of only the ETS genes activated this enhancer weakly (Fig. 2A, bar 4). Because endogenous µE3 binding proteins can work with the transfected ETS genes, the missing components were likely to be µE2 and µE5 binding proteins. Several earlier observations indicated the choice of E47 as a µE2/µE5 binding protein. E47 was originally cloned as a µE5 binding protein (3, 17), and the µE2 element falls within its consensus recognition site (25). E47 is known to be of importance in B-cell development (1, 27), and it can synergize with µE3 binding proteins to activate transcription (3, 4). Coexpression of E47 with either Ets-1 or PU.1 also activated the enhancer weakly (Fig. 2A, bars 2 and 3). However, inclusion of both ETS genes and E47 resulted in significant transcriptional activity (Fig. 2A, bar 5), which was dependent on all five sites of the µ74 enhancer (Fig. 2A, bars 6 to 10). The mutational analysis in COS cells was very similar to the pattern seen in B cells, suggesting that this "heterologous" assay reproduced several aspects of the transcriptional activity seen in B cells. Furthermore, the requirement for the µE3 site showed that a COS cell protein was recruited to this site, because none of the three exogenously expressed proteins bound significantly to the µE3 site.

View larger version (15K): [in this window] [in a new window]   FIG. 2.   Reconstitution of monomeric µ enhancer activity in nonlymphoid cells. (A) Reporter (2 µg) containing the µ74 enhancer (WT) was cotransfected into COS cells together with expression vectors for E47 (µE2/µE5 binding protein), Ets-1 (µA binding protein), and PU.1 (µB binding protein) in various combinations as noted below the graph. Total transfected DNA was kept constant at 5 µg by using pEVRF. Reporters (2 µg) containing mutated µ74 derivatives (µE5, µE2, µA, µE3, and µB) were similarly assayed in the presence of all three transactivators (last five bars). Results shown were obtained by averaging three experiments done in duplicate and were normalized to the activity of the WT µ74 reporter in the presence of all three transactivators. (B) Domains of Ets-1 (440 amino acids) and PU.1 (272 amino acids) are shown at the top. The N-terminal Ets-1 deletion mutants used for the analysis are shown by the arrows marked 167, 231, and 286. The shaded box marked TD is a previously identified transcription activation domain, the box marked INH is an autoinhibitory domain for DNA binding, and the hatched box at the C terminus is the DNA binding ETS domain. A TD in PU.1 is shown as the cross-hatched box, and the DNA binding domain is shown as a hatched box. The deletion mutant of PU.1 contains the ETS domain plus C-terminal 14 amino acids. For cotransfection analysis of COS cells, the WT µ74 reporter was transfected along with E47, Ets-1, PU.1, or deletion mutants of the latter two, as indicated below the graph. Results shown are averages of three experiments carried out in duplicate, normalized to µ74 activity in the presence of all three full-length proteins.

µE3 redundancy. Mutational analysis of the µ74 enhancer in B cells showed that µE3 was an essential component of this five-part enhancer. Yet, it is well established that mutation of µE3 in the context of longer enhancer fragments does not significantly reduce enhancer activity; that is, its function can be largely compensated for by other, presently undefined elements. To identify elements that make µE3 redundant, we assayed two other µ enhancer fragments and mutants thereof by transient transfection into S194 cells. µ87 contains one more µE element than µ74 (Fig. 1A) and was a strong enhancer (Fig. 3A, bar 2). Despite the presence of four µE elements in this fragment, mutation of µE3, µE2, or µE5 abolished enhancer function (Fig. 3A), showing that µE3 was still essential.

View larger version (14K): [in this window] [in a new window]   FIG. 3.   Elements that contribute to µE3 redundancy. (A) µE3 redundancy does not depend on other related µE elements. Activity of the µ87 enhancer (WT) that contains four µE elements and mutations thereof was analyzed by transient transfection into S194 plasma cells. Averaged CAT activity from three independent transfection experiments is shown normalized to the activity of the WT µ87 enhancer. (B) 3' enhancer sequences that contain no µE-related motif compensate functionally for µE3, as determined by transient transfection analysis of µ170 enhancer and its mutated derivatives in S194 plasma cells. Results from three independent experiments are shown. (C) µ enhancer sequences that compensate for µE3. Deletion mutants of the µ170 enhancer shown schematically above the graph, or the µE3 mutated derivative of each, were transfected into S194 followed by CAT enzyme analysis. C2µ170 refers to a C2-mutated µ170 enhancer. Data shown are averages of three independent transfections carried out in duplicate and are normalized to the activity of the WT µ170 enhancer.

Factors that regulate µE3 redundancy. The mutational analyses described above showed that two regions of the µ enhancer could functionally substitute for µE3. The stronger element is located at the 3' end of the µ170 fragment and is removed by the first 3' deletion mutant, µ152. The residual µE3 redundancy appears to be conferred by the second core homology. A role for C2 was intriguing in light of our recent observation that the human Ig µ enhancer lacks a recognizable µE3 element but contains instead a functional core-like element between the µA and µB motifs (7). In that enhancer, therefore, a core binding protein can substitute for the lack of a µE3-like element. We surmised that C2 may behave similarly, although with reduced efficiency because of its distance from the essential µA-µB combination.

View larger version (58K): [in this window] [in a new window]   FIG. 4.   Cobinding of Ets-1 and CBF to the µ enhancer. Ets231 and the DNA binding domain of CBF (CBF.DBD) were purified from bacteria. In vitro binding assays were carried out with either protein alone or both together, as indicated above the lanes, using a 96-bp DNA probe derived from the enhancer (see Materials and Methods). The probe is mutated at µE3 and C1; thus, the only remaining CBF binding site is C2. Single protein-DNA complexes are indicated with arrows labeled Ets and CBF, and a two-protein-DNA complex is labeled Ets/CBF. Lane 1, no proteins added; lanes 2 to 4, binding reactions with increasing amounts of Ets231; lane 5, CBF alone; lanes 6 to 8, constant amount of CBF as in lane 5 with increasing amounts of Ets231 as in lanes 2 to 4.

An IRF binding site in the µ enhancer. We noticed that the major µE3-substituting element at the 3' end of the µ170 fragment contained a strong match to the consensus recognition site of IRFs (Fig. 5A). To test whether this region bound IRF proteins, IRF-1 was produced by in vitro translation and used in EMSA. An oligonucleotide probe spanning the 3' end of the µ170 fragment generated a unique nucleoprotein complex (Fig. 5B, lane 2) which was efficiently competed away by inclusion of nonradioactive self-competitor DNA (lanes 3 and 4). Three other oligonucleotides containing clustered mutations in the µ enhancer IRF consensus sequences as well as the IRF binding site from the beta interferon (IFN-) gene were also used in competition assays. Mutations (M1 and M3) that changed residues within the conserved region did not compete for IRF-1 binding (lanes 5, 6, 9, and 10), whereas the flanking mutation M2 and the IFN- site competed efficiently (lanes 7, 8, 11, and 12). We also evaluated IRF-2 binding to the µ3' site and found that the pattern was indistinguishable from that of IRF-1 (data not shown). In contrast, a third IRF family member, Pip-1, did not bind to the µ enhancer site (Fig. 5C) but bound well to the B sequence from the Ig  light-chain gene enhancer. We conclude that the µ3' sequence binds a subset of IRF family proteins, and the location of this site coincides closely with the 3' µE3 substituting element.

View larger version (27K): [in this window] [in a new window]   FIG. 5.   A binding site for IRF in the µ enhancer. (A) The 18 nucleotides deleted between µ170 and µ152 (line 2, µ3') contain a sequence homologous to the consensus IRF binding site (top line). µ3'M1, µ3'M2, and µ3'M3 are mutated µ enhancer sequences within and outside the IRF consensus. The altered nucleotides are shown as outlined letters. The IRF binding site from the IFN- gene is shown on the last line. (B) IRF-1 binding to the µ enhancer. IRF-1 protein was generated by in vitro transcription and translation for use in EMSA. The radioactive probe was the µ3' oligonucleotide (A). Binding reactions were carried out in the absence of competitor DNA (lane 2) or in the presence of the µ3' oligonucleotide (lanes 3 and 4), µ3'M1 (lanes 5 and 6), µ3'M2 (lanes 7 and 8), µ3'M3 (lanes 9 and 10), and IFN- (lanes 11 and 12); 10 and 50 ng of competitor oligonucleotides were used. (C) The DNA binding domain of Pip produced by in vitro translation was used in EMSA with a µ3' probe (lanes 1 to 5) or a probe containing the B site (lanes 6 to 10) of the Ig  light-chain gene enhancer. Lanes 1 and 6, probe alone; lanes 2 and 7, reticulocyte extract alone; lanes 3 to 5 and 8 to 10, increasing amounts of Pip containing in vitro translation reactions. The specific DNA-protein complex generated by Pip is marked with an arrow at the right.

View larger version (13K): [in this window] [in a new window]   FIG. 6.   Effects of µ3' IRF site mutations on µ enhancer activity. (A and B) µ170 enhancer derivatives, as noted below the bars, were tested after cloning into the 56CAT reporter plasmid by transient transfection of S194 plasma cells followed by CAT enzyme analysis. 56 represents the enhancerless reporter plasmid, and WT is a reporter carrying an unmutated µ170 enhancer. The mutations tested (µE2, µE3, C2, and µ3') were in the motifs shown in the schematic at the top; double mutations are indicated with two of these notations. Results shown are averages of three transfections carried out in duplicate. (C) Analysis of IRF site mutations in DHL-9 B-lymphoma cells. Reporter plasmids as indicated below the graph were transiently transfected into DHL-9 cells, which were then subjected to CAT enzyme analysis. Results shown are averages of two transfections carried out in duplicate.

View larger version (87K): [in this window] [in a new window]   FIG. 7.   In vitro binding of Ets-1 and IRF-1 to the µ enhancer. Ets-1 was expressed as a GST fusion protein and purified from bacterial extracts. IRF-1 was obtained by in vitro translation in rabbit reticulocyte extracts. The DNA probe was obtained by PCR amplification and encompasses residues 376 to 519 (for WT) or 367 to 519 (for µ3' and µA) of the µ enhancer (numbering as specified by Ephrussi et al. [6]); the µA element is located between nucleotides 386 and 396, and the µ3' element is located between nucleotides 501 and 512. In vitro binding reaction mixtures contained the following: lane 1, no proteins; lane 2, 0.5 µl of reticulocyte extract; lanes 3 to 5, 0.5 µl of reticulocyte extracts plus 50, 100, and 200 ng of GST-Ets-1; lane 6, 0.5 µl of reticulocyte extracts containing in vitro-translated IRF-1; lanes 7 to 9, IRF-1 as in lane 6 plus GST-Ets-1 as in lanes 3 to 5; lanes 10 and 11, GST-Ets-1 plus IRF-1 as in lane 9 with mutated probes µ3' (lane 10) and µA (lane 11). Binary nucleoprotein complexes are labeled 1 and 2, and the ternary complex is labeled 3.

View larger version (16K): [in this window] [in a new window]   FIG. 8.   Transactivation of the µ enhancer by IRF family members. COS cells were transfected with a µ170 enhancer-containing reporter plasmid together with expression vectors for IRF family members as well as the µ enhancer binding proteins as shown below the graph. Where indicated (+), 1 µg of PU.1 and 0.25 µg of E47 expression vectors were used. IRF expression plasmids were used at two different amounts (shown in micrograms). Results shown are averages of three transfections carried out in duplicate.

	DISCUSSION

Top Abstract Introduction Materials & Methods Results Discussion References

Our earlier studies did not address two key aspects of transcriptional regulation by the Ig µ heavy-chain gene enhancer. First, µ enhancer fragments that contained up to four protein binding sites needed to be dimerized to significantly activate transcription in B cells. Therefore, it was important to identify (i) the smallest enhancer fragment that activated transcription as a monomer and (ii) the factors that mediated this activity. Second, because our effort so far had focused on identifying enhancers in which every site was necessary for function, the basis for redundancy among enhancer elements had not been systematically addressed. In this report, we examined these questions.

We found that addition of µE5 to the previously studied four-part enhancer generated a B-cell-specific transcriptional enhancer that was active as a monomer. All five elements were necessary for activity, indicating that this five-part enhancer had no redundant elements. The reason for the jump in monomeric enhancer activity between the four- and five-part enhancers is unclear at present; however, it is unlikely to be a consequence simply of increasing the numbers of elements in the enhancer. For example, a dimer of a µE3 four-part enhancer is inactive, despite retaining six functional factor binding sites. Similarly, when the µA and µB sites are moved apart in the context of µ170, enhancer activity is abolished even though all nine elements remain intact and capable of binding proteins. These observations underscore the importance of the organization of sites within the enhancer. In this regard it is interesting that the three µE elements in this enhancer fragment are aligned roughly on the same side of the DNA helix, whereas the alignment of the µA and µB sites with respect to the µE elements is shifted by approximately half a helical turn. Perhaps three appropriately positioned bHLH protein TDs can recruit a requisite coactivator significantly better than two such domains on a four-part enhancer.

The monomeric µ enhancer could be activated in nonlymphoid cells by the coexpression of PU.1, Ets-1, and E47 proteins, and as seen in B cells, all sites were necessary for enhancer function. Furthermore, we defined the domains of PU.1 and Ets-1 that were required to activate this enhancer and found them to be similar to those we had previously shown to be required to activate a dimerized tripartite enhancer. We envisage that the ETS domain of PU.1 participates in the nucleoprotein complex by bending the enhancer DNA and making direct contacts with Ets-1 bound at µA. Ets-1 at the µA site has a very different role. Unlike PU.1, N-terminal domains of Ets-1 (including a previously identified TD) serve at least two functions. First, the Ets-1 TD works together with the TFE3 TD bound at µE3 to accentuate the transcriptional potential of this pair of sites, perhaps by presenting a composite TD to the basal machinery. Second, the non-DNA binding N-terminal region of Ets-1 couples the activation potential of E47 and TFE3 bound to the µE2 and µE3 elements, respectively. In addition to providing a TD, TFE3 protein directly interacts with Ets-1 to stabilize its DNA binding as well as to alter Ets-1 conformation in a way that enhances E47 binding to the µE2 site (4). Thus, each protein has multiple jobs in the nucleoprotein complex that comprises the functional enhancer.

Redundancy among µ enhancer elements was functionally defined by the observation that mutation of certain elements such as µE2 and µE3 did not significantly diminish enhancer activity. The simplest interpretation of these results had been that another µE element functionally substituted for the loss of the mutated element. Unexpectedly, we found that the element that contributed most to making either the µE2 or µE3 element redundant was a novel IRF binding site, µ3', that is located approximately 100 bp away from µE3. In the presence of the IRF site, a µE2 or µE3 mutation had proportionately less effect on enhancer activity, whereas in the absence of the IRF site, loss of either element crippled the enhancer significantly; that is, µE2 and µE3 are essential when the IRF site is missing. In addition, the second core homology (C2) also contributed to µE3 redundancy, though to a lesser extent. The µ3' element was also active in DHL-9 B-lymphoma cells, indicating that redundancy was not a feature only of plasma cells such as S194. Finally, we showed that IRF-1, but not IRF-2 or Pip, activated the µ enhancer together with ETS and bHLH proteins in cotransfection assays. These observations suggest that IRF-1 is a likely candidate for being a functional µ enhancer binding protein but do not rule out the possibility that other IRF family members also activate this enhancer.

How do IRF proteins participate in µ enhancer activation? Two hypotheses are proposed below. First, proteins bound to µ3' may directly interact with proteins bound at the µE2-A-E3-B region. From such a complex, if either a µE2 or µE3 binding protein is missing, its loss would be compensated by the IRF protein. It is interesting that the µE2 µE3 enhancer is approximately as active as the µ3' enhancer, which suggests that either IRF or the µE2-µE3 combination can activate the µA/µB core to similar levels. Alternatively, it is possible that IRFs interact with protein bound to C2, C3, and the intervening PU.1 binding site that are located within 30 bp of µ3'. Maybe this complex of proteins can activate the µA/µB core just as the µE2-µE3 complex does. In both models, the nonessential 3' components are visualized as interacting with the essential µA/µB core. The main difference between the two models is that in the first model IRF is envisaged as working by itself, whereas in the second model it works along with other factors.

The importance of the newly identified IRF site was underscored by the observation that this site is conserved between the rodent and human µ enhancers. Furthermore, in their earliest in vivo methylation protection experiments, Ephrussi et al. (6) observed a protection over a guanosine residue that corresponds to the newly identified IRF binding site. These results suggest that IRF proteins interact with the µ enhancer in vivo. These characteristic hallmarks of functional significance of the IRF site raised the question as to why it is so important to ensure redundancy in the enhancer. We suggest that the property of the enhancer measured as a redundancy in transcription factor requirements reflects a more fundamental biologic characteristic, such as the need to modulate Ig expression during B-cell differentiation or activation. For example, there are several stages in the functional life of a B cell where Ig expression is known to be regulated. First, the transition from immature to mature B cells is accompanied by increased surface IgM expression; second, surface Ig expression is decreased in activated B cells present in germinal centers, presumably to select for high-affinity somatic mutants; lastly, IgH transcription is increased a few fold in terminally differentiated plasma cells. One way to achieve such quantitative differences is for the enhancer to contain more than the minimal number of protein binding sites; the activity of such an enhancer can then be up- or down-regulated by changing the nuclear concentration of one or more limiting transcription factors.