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Commentary |
Address correspondence to Ranjan Sen, Lab. of Cellular and Molecular Biology, National Institute on Aging, 5600 Nathan Shock Dr., Baltimore, MD 21224. Phone: (410) 558-8630; Fax: (410) 558-8026; email: rs465z{at}nih.gov
Abstract
Enhancers regulate lineage choice and the developmental timing of antigen receptor gene rearrangements. The transcription factor NF-B has been implicated as a key component of the recombination and transcription activation potential of the immunoglobulin
chain gene intronic enhancer. Here, I discuss the implications of the new observation that an NF-
B binding sitemutated enhancer in the correct biological context does not appear to affect
gene expression.
Developmental stage-specific recombination of antigen receptor loci is regulated by cis-regulatory enhancer sequences that alter accessibility of the recombinase machinery to the gene segments. The function of enhancers is mediated by DNA binding proteins that recruit to the enhancer, via proteinprotein interactions, a multisubunit complex that is a functional enhancer.
Transcriptional Regulation of the Locus.
NF-B binds to the DNA sequence known as the
B site within the enhancer (iE
) located in the J
-C
intron of the Ig
light chain gene (Fig. 1 and reference 1). The presence of nuclear NF-
B DNA binding activity in Ig-expressing B lymphocytes, but not at earlier stages of B cell differentiation, suggested that it might play a key role in
light chain gene expression. This idea was reinforced by observations that NF-
B induction in preB cell lines coordinately activates
gene transcription and V
to J
recombination (2, 3). Conversely, both processes are adversely affected when NF-
B activation is blocked (4).
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The past two decades have witnessed enormous growth in the number of biological roles ascribed to NF-B. First described as a
geneactivating protein in lymphocytes, it is now known to function in a wide variety of physiological and pathologic processes in diverse cell types (8, 9). Perhaps as a result, the role of the
B site in the
enhancer was somewhat neglected. In this issue, Inlay et al. perform the definitive experiment to determine the role of the
B motif in its normal context (10). They used gene targeting to knock in mutated iEks at the endogenous locus and then used the mutant embryonic stem cells to complement Rag-deficient blastocysts. The status of
gene rearrangements was compared on the wild-type and mutated alleles in mature heterozygote B cells. Surprisingly, the allele bearing the
B site mutation recombined at virtually wild-type levels; in contrast, alleles bearing
E1 or
E2 mutations rearranged less efficiently. Double mutation of the
E1 and
E2 sites markedly reduced rearrangement to levels seen after deletion of iE
(11). The authors conclude that E2A proteins that bind
E1 and
E2 are essential for efficient
gene rearrangements. In contrast, the
B site and NF-
B appears to be unimportant for
gene recombination.
A Role for NF-B in Epigenetic Regulation of Rearrangement?
NF-Bdependent regulation of the
locus remains a viable proposition, in my opinion, because it provides a likely mechanism for the developmental timing of
gene rearrangements. Though low levels have been noted in proB cells, the majority of the
rearrangements occur in the preB compartment after IgH-expressing cells are selected by the preB cell receptor (pre-BCR). This bias toward late rearrangements of
genes is not easily explained by an E2A-only mechanism because these bHLH proteins are expressed in proB cells where they activate IgH rearrangements via binding sites in the JH-Cµ intron enhancer (Eµ) (12). If E2A proteins are sufficient to activate iE
and are present and functional from the earliest stages of B cell development, what prevents
genes from recombining before the preB cell stage? It is possible that E2A functions on the enhancer may be regulated differentially in proB cells and preB cells. For example, increased levels of expression or posttranslational modifications may increase the function of E2A on iE
in preB cells compared with proB cells. However, there is little evidence for such changes between proB and preB cells. Instead, there is ample evidence for NF-
B activation via the pre-BCR during the proB to preB cell transition (13, 14).
How might NF-B activation render iE
more susceptible to E2A-dependent activation at the correct developmental stage? One possibility is that Rel proteins (the subunits that make up the NF-
B dimers) bind to the
B site in proB cells (15) to prevent E2A-mediated activation of recombination (Fig. 1 B). The most likely negative regulator is the p50 homodimer. This Rel protein does not contain a classical activation domain and has been shown to recruit histone deacetylases (HDACs) to regulatory sequences to actively repress transcription by epigenetic means (16). Under some circumstances, p65 (also known as RelA) behaves similarly (17), although it is usually associated with gene activation. Pre-BCRinduced NF-
B activation in preB cells may tilt the balance toward de-repression of the
locus by replacing p50 homodimers with Rel proteins that contain transcription activation domains (such as p65 or c-Rel). These Rel proteins can recruit histone acetyl transferases (HATs) (18, 19) to counteract the effects of histone deacetylases located on the gene, resulting in gene activation. The crux of the hypothesis is to view the role of NF-
B at the
locus as mediating repression or de-repression depending on the stage of B cell differentiation, with the outcome being determined by different sets of Rel proteins. In this model, mutation of the
B site prevents binding of Rel proteins that initiate repression in proB cells, as a result of which the timing of
gene rearrangements may be altered. In the simplest scenario,
recombination may occur earlier during B cell differentiation. Such NF-
Bindependent recombination may be mechanistically analogous to that induced by ectopic E2A expression in nonlymphoid cells (20, 21).
If the B site prevents E2A-dependent recombination in proB cells, the
B mutant allele in the study by Inlay et al. (10) might be expected to recombine at higher levels than the wild-type allele because it would be accessible in both proB cells and preB cells. However, without direct analysis of the proB compartment, it may be difficult to discern whether recombination is increased, given the small numbers of proB cells relative to preB cells. Alternatively, the comparable levels of recombination on both the
B mutant and wild-type alleles in mature heterozygote B cells (10) suggests that the
E1- plus
E2-driven enhancer may not be effectively activated by the nuclear milieu of the proB cell. What could be missing in proB cells that later compensates for the loss of the
B site in preB cells? One possibility is the 3'k enhancer. Once activated in preB cells, it may increase the recombination potential of the "crippled"
B mutant iE
, in a way serving as a surrogate for the missing NF-
B that would normally be recruited to the locus. In the system developed by Inlay et al., this could be tested by analyzing the effect of the
B mutation in the absence of 3'E
(10). Clearly, however, the 3'E
cannot rescue a
E1 plus
E2 doubly mutated iE
. The proposed insufficiency of a
B-mutated enhancer in the absence of additional positive regulatory sequences is consistent with all earlier functional studies of the isolated iE
.
Further Implications.
Epigenetic repression and de-repression via NF-B proteins provides plausible explanations for several other aspects of
gene regulation. First, the
locus in a minority of proB cells may escape p50-dependent epigenetic silencing. Inefficient activation of iE
via E2A proteins in these cells may result in the low level of
recombination seen in proB cells. Second, de-repression in preB cells may be limited by the concentration of HAT-recruiting Rel family members activated by the pre-BCR. It is possible that pre-BCR signaling is not strong enough or does not last long enough to generate enough positive-acting NF-
B. Thus, de-repression might occur only in that subset of cells where the level of activating Rel proteins reaches a threshold required to counteract repression. This may be the basis for variegated
gene expression in the preB compartment that was described recently by Liang et al. (22). It is worth noting that the more uniform expression of
genes in mature B cells probably reflects transcription activation via the 3'E
. Thus, each of the two enhancers might serve different, though overlapping, functions; iE
is primarily involved in activating V
recombination in preB cells, though its deficiency can be partially, or fully, compensated depending on the severity of the mutation, whereas the 3'E
is primarily involved in activating
gene transcription. Third, the
B sitedependent epigenetic changes proposed here must be superimposed on mono-allelic DNA methylation of the
locus (23), a modification that is also associated with HDAC recruitment and gene silencing (24). Thus, multiple levels of epigenetic regulation may control the timed onset of
gene recombination.
Overall, Inlay et al.'s clean experiment and its unexpected result proves to be more thought provoking than anticipated. Most importantly, it demonstrates that the timing of gene expression during B cell development remains an open question, be it mediated by NF-
B or by a different, presently unknown mechanism.
Acknowledgments
I thank Drs. Yehudit Bergman and Mark Schlissel for taking the time to share their expertise in gene regulation during preparation of this commentary.
Submitted: 4 October 2004
Accepted: 8 October 2004
References
chain intronic enhancer in activating V
J
rearrangement. J. Exp. Med. 200:12051211.
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