Pre-BCR Signals and the Control of Ig Gene Rearrangements Jamie K
Seminars in Immunology 18 (2006) 31–39
Review Pre-BCR signals and the control of Ig gene rearrangements Jamie K. Geier, Mark S. Schlissel ∗ UC-Berkeley, Department of Molecular & Cell Biology, Division of Immunology, 439 Life Sciences Addition, Berkeley, CA 94720-3200, USA
Abstract Progenitor B lymphocytes that successfully assemble a heavy chain gene encoding an immunoglobulin capable of pairing with surrogate light chain proteins trigger their own further differentiation by signaling via the pre-BCR complex. The pre-BCR signals several rounds of proliferation and, in this expanded population, directs a complex, B cell-specific set of epigenetic changes resulting in allelic exclusion of the heavy chain locus and activation of the light chain loci for V(D)J recombination. © 2005 Elsevier Ltd. All rights reserved.
Keywords: B cell development; Pre-B cell receptor (pre-BCR); Allelic exclusion; V(D)J recombination; Transcriptional regulation
1. Introduction a membrane complex with the surrogate light chain components (SLC) VpreB and 5, and the transmembrane proteins Ig-␣ and B cell differentiation is a highly dynamic process that requires -. Signaling through this receptor is associated with its internal- the coordinated action of various receptors, signaling molecules, ization and degradation [1–3]. The cytoplasmic domains of Ig-␣ transcription factors and the V(D)J recombinase. Completion of and - contain immunoreceptor tyrosine-based activation motifs immunoglobulin heavy chain (HC) gene rearrangement leads (ITAMs) which, when clustered in the membrane, provide a to surface expression of the pre-BCR complex which triggers docking site for the Syk kinase, Src family kinases (Fyn, Lyn, and a cascade of signaling events that alters the developing cell’s Blk), the Tec family kinase, Btk, and the adaptor proteins Grb2 response to growth factors and effects transcription and DNA and BLNK. BLNK (also known as BASH and SLP-65) recruits rearrangement. HC expression results in pre-B cell expansion Btk, which is then phosphorylated by Lyn, Syk, or both [4,5]. and inhibits further rearrangement at the HC locus, a process Phosphorylated Btk then phosphorylates PLC␥2, which known as allelic exclusion. Pre-B cells subsequently exit the hydrolyzes PIP2 to IP3 and diacylglycerol (DAG) causing cal- cell cycle and initiate of a second set of gene rearrangements cium mobilization and resulting in activation of calcium depen- at the immunoglobulin light chain (IgLC) loci. The timing of dent enzymes (reviewed in [6]). Proliferation is induced via acti- DNA rearrangements, which involve the generation and repair vation of MAP kinase pathways, and allelic exclusion is imposed of DNA double stranded breaks interspersed with bursts of pro- by a presently undefined mechanism [7,8]. Upon pre-BCR sig- liferative expansion, must be tightly regulated to ensure proper naling, cells initially undergo a proliferative burst accompanied development and to prevent genomic instability and leukemic by down regulation of SLC components and RAGs, followed by transformation. This review will focus on the function of the exit from the cell cycle and transition from pro-B/large pre-B pre-BCR as the critical regulator of the pre-antigenic phase of I (Hardy fraction C) to small pre-B II (Hardy fraction C) [9]. B cell development. Signaling also induces transcription factors such as NF-B, Spi- B and IRF-4 as well as re-expression of RAGs, contributing to 2. Pre-BCR signalling—an overview activation of the enhancers and germline transcription and rearrangement [10,11]. Pre-BCR signaling is initiated upon expression of a clono- typic immunoglobulin heavy chain (HC) that can assemble 3. Pre-BCR signaling does not require exogenous ligand
Once the pre-BCR assembles on the cell surface, tyrosine ∗ Corresponding author. Tel.: +1 510 643 2462; fax: +1 510 642 6845. kinase-dependent signaling begins. Ig-␣/ heterodimer surface E-mail address: [email protected] (M.S. Schlissel). expression facilitated by the HC and SLC seems to be sufficient
1044-5323/$ – see front matter © 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.smim.2005.11.001 32 J.K. Geier, M.S. Schlissel / Seminars in Immunology 18 (2006) 31–39 to initiate signaling. Truncation mutants that lack extracellular tion [25], it is widely agreed that pre-BCR signaling is required domains can function to aggregate Ig-␣/ and signal B cell dif- for the proliferative expansion of pre-B cells expressing a suc- ferentiation [12,13]. In fact, pro- to pre-B cell development can cessfully rearranged IgHC gene which can assemble with SLCs. be driven by the Ig-␣ and - ITAMS (along with other conserved This proliferation signal induces two to five rounds of cell divi- cytoplasmic motifs) targeted to the inner leaflet of the plasma sion and eliminates the dependence of developing cells on IL-7 membrane by fusion with a Lck myristoylation/palmitoylation [15]. Pre-B cells are known to be cycling in vivo based on their sequence [14]. These observations, along with the fact that a rapid incorporation of the nucleotide analog BrdU [1,26,27]. single pre-B cell in the absence of any other stromal cells can Transgenic IgHC expression induces proliferation in pro-B cells proliferate in response to pre-BCR expression [15], calls into cultured on stromal cells [28]. Targeted deletion of the Ig mem- question the relevance of various extracellular ligands proposed brane exon or disruption of the SLC genes results in the failure to interact with the pre-BCR. However, soluble recombinant of pre-B cell proliferation [25]. pre-BCR can bind stromal cells [16]. The Schiff group identi- The MAP kinase pathway is involved in pre-B cell expansion fied a human pre-BCR ligand, galectin-1, that may be involved and perhaps various aspects of differentiation as well. In mature in receptor aggregation and activation of tyrosine kinase activ- B cells, Ras signaling is induced upon BCR crosslinking [29,30]. ity localized at the pre-BCR [3]. Another study by the Jack¨ Mice expressing a constitutively activated Ras mutant transgene lab implicated stromal heparin sulfate as a ligand for murine on a RAG-null background bypass the pre-BCR checkpoint and pre-BCR [17] (see also contribution by Espeli et al. in this contain almost normal numbers of B cells in their lymph nodes issue). Recent data has revealed another potential mechanism and spleen [31]. These RAS-transgenic B cells are abnormal in for pre-BCR signaling involving the SLCs. The conserved non- that they continue to express 5 and RAG2, which are normally immunoglobulin portions of 5 and VpreB may mediate con- down-regulated upon pre-BCR signaling. Their germline tran- stitutive receptor aggregation, signaling and internalization via script levels are similar to those observed in wild-type pre-B homotypic ionic interactions [18]. Various truncated or unusu- cells, but the transgenic cells also express some surface mark- ally structured HCs may inherently allow aggregation in an ers associated with more mature stages of B cell development. SLC-independent fashion providing an explanation for the trun- When the activated Ras transgene is bred onto a RAG sufficient cated HC transgene results mentioned above. but JH background, rearrangement is induced despite a lack The absence of both Ig-␣ and - causes a developmental of HC expression [32]. The survival phenotype of Ras trans- arrest at the pro-B cell stage despite intact V(D)J recombina- genic B cell precursors is similar to that of RAG-null B cells tion and HC expression revealing the requirement for these expressing transgenic HC and Bcl-2. Indeed, levels of Bcl-2 molecules in pre-BCR signaling [19,20]. One role of the ITAMs are high in the activated Ras transgenic B cell precursors, a trait is recruitment of BLNK to Ig-␣. Fusing BLNK to a mutant Ig- characteristic of mature B cells [31] perhaps allowing survival ␣ lacking these tyrosines reconstitutes downstream signaling in the setting of both proliferation and ongoing Ig locus rear- events. Thus, simply the formation of the pre-BCR leading to rangement. the membrane localization of Ig-␣ and - is sufficient to trigger Mice expressing a transgenic human dominant negative Ras, pre-BCR signaling, allow downregulation of RAGs and SLC H-rasN17, show a developmental arrest at a stage correspond- proteins, and upregulation of transcription factors that result in ing to Hardy fraction “A”. This was not attributable to increased IgLC gene rearrangements. apoptosis. Over-expression of a membrane targeted, and thus The pre-BCR may serve yet another function, that of a molec- constitutively active, partner of Ras, Raf-1(Raf-CAAX), on ular clock which eliminates B cell precursors that fail to generate the H-rasN17 background overcame the block confirming the a functional IgLC gene within a certain window of time or that involvement of this MAP kinase pathway in B cell development express a HC that is incapable of associating with any IgLC [33]. Mimicking the pre-BCR signal with these transgenes does ([21,22]; see also contribution by C. Vettermann and H.-M. Jack¨ not, however, induce allelic exclusion since V-to-DJ rearrange- in this issue). Pre-BCR signaling is associated with internal- ment proceeds despite the premature proliferation signal. ization and degradation of the pre-BCR [1–3]. Since the genes Thus, the MAPK pathway is necessary for proper pre-BCR encoding the SLC components 5 and VpreB are inactivated signaling, and when activated can substitute for the pre-BCR upon pre-BCR signaling [23], progressively less pre-BCR com- causing proliferative expansion, survival and activation, but plex can form on the surface as cells divide, culminating in not IgHC allelic exclusion, or 5 and RAG down-regulation. reduced proliferative signals. If a pre-B cell fails to make an These experiments demonstrate the existence of two separable IgLC that associates with its HC, then HC expression on pre-BCR signaling pathways; one causing proliferation and the the surface will eventually be lost, presumably leading to the induction of some transcription factors involved in differentia- cell’s arrested development and death [24]. The existence of tion, and the other mediating allelic exclusion. this clock may help protect the genome from RAG-associated genomic instability. 5. Allelic exclusion
4. Pre-BCR signals leading to proliferation Allelic exclusion remains an enigmatic aspect of pre-BCR signaling. Preventing further HC gene rearrangement in a cell While there is debate as to whether the complete pre-BCR is that already expresses a HC is necessary to ensure that each required for IgHC allelic exclusion and pre-B cell differentia- B cell has an unique antigen specificity. It is imposed at the J.K. Geier, M.S. Schlissel / Seminars in Immunology 18 (2006) 31–39 33
VH-to-DJH step in recombination while D-to-JH rearrangement expression amongst individual HC genes could account for is unaffected [34]. The ability of RAG proteins to selectively the curious observation that some HC transgenes allelically act at different loci depending on the cell type or stage in devel- exclude very stringently while others allow continuation of opment has been attributed to regulated accessibility of each D-proximal VH gene segment rearrangement [50]. Transgene rearranging locus within chromatin structure [35]. RAG proteins expression levels may also play a role. cannot cleave a nucleosomal substrate in vitro, implicating the D, an endogenously generated truncated HC protein necessity of some sort of chromatin remodeling to render the formed by translation of a partially rearranged (DH-to-JH) IgHC DNA accessible to the recombinase [36–38]. The loci within allele [51] is also capable of signaling allelic exclusion. D sur- purified nuclei that are accessible to cleavage by RAGs in vitro face expression blocks further development of B cell precursors, depend on the source of nuclei [39]. Thus, the accessibility of a process known as D selection [42,52–54]. This selection is rearranging loci is a cell-type and stage-specific property of chro- thought to occur when D expression causes HC gene allelic matin structure. The accessibility of the IgHC locus changes exclusion, thus preventing the formation of a complete IgHC across the pro-B to pre-B cell transition, and pre-BCR signal- gene [55,56]. Proliferation is impaired in the D transgenic ing induces a permanent loss of IgH locus accessibility. The mice, again pointing to different requirements for allelic exclu- mechanism or signaling pathways employed for such chromatin sion and proliferation. D can cause increased germline and remodeling have not been delineated. diminished 5 transcription, yet it cannot promote full differ- entiation to the pre-B cell stage [56]. Considering that Ig-, 5 5.1. Requirements and signals and Syk are necessary for D to signal, D apparently forms a pre-BCR-like complex, but due to the lack of VH structure, this Surface expression of a HC induces allelic exclusion and pseudo-pre-BCR fails to mimic essential aspects of the pre-BCR mice carrying a mutation in the transmembrane region of the signal and such cells eventually die from an inability to differ- HC gene are unable to turn off IgHC gene segment rearrange- entiate perhaps due to ineffective pairing with IgLC [56,57]. ments [40]. Expression of a HC transgene in mice prevents Pairing of D to the SLC could be qualitatively different, and endogenous VH-to-DJH rearrangements [41]. It is controversial, different requirements for the maturation and surface expression however, whether SLC expression is required for IgHC allelic of the two types of pre-BCRs have been demonstrated [58]. But exclusion. One group reported that targeted disruption of the precisely how lack of the V regions in this circumstance has gene encoding 5 resulted in a failure of early pre-B cell pro- such a dramatic effect on B cell development when truncation liferation and the loss of allelic exclusion at the level of HC mutants suffice to replace full-length proteins remains a mystery gene rearrangements. Surprisingly, however, these 5-deficient [12,13]. mice still displayed allelic exclusion at the level of surface HC As mentioned above, proliferative signals transmitted expression [42]. Similar results were observed in mice lacking through the MAPK pathway are not sufficient to induce allelic VpreB1, VpreB2 and 5 [43,44]. This second set of researchers exclusion. Nor is BLNK required for allelic exclusion [59]. But detected no difference in the frequency of pre-B cells with two Syk, the tyrosine kinase recruited and activated upon pre-BCR HC V(D)J rearrangements in the presence or absence of SLCs signaling upstream of BLNK, is necessary for normal B cell suggesting allelic exclusion at the level of gene rearrangement development and allelic exclusion. Irradiated mice reconstituted does not require full pre-BCR assembly. One potential explana- with Syk−/− HC transgenic cells displayed incomplete HC tion for this observation was provided in a report showing that gene allelic exclusion, however ZAP-70, another Syk family precocious expression of IgLC could replace the need for SLCs kinase expressed in B cells, appears to play a partially redundant to promote B cell development [45]. However, more compelling role with Syk. Syk−/− ZAP-70−/− double mutants completely data shows that HC can be detected on the surface of IgLC- arrest at the pro-B cell stage according to their cell surface negative SLC-deficient cells demonstrating that some HC are marker phenotype and cannot allelically exclude in the presence capable of reaching the cell surface without SLC or LC [46–49]. of a HC transgene [60]. The few cells that are able to express HC on the surface despite BLNK is the adaptor protein downstream of Syk that is the absence of SLC would then be capable of signaling progres- involved in cessation of proliferation and the activation of sion as well as allelic exclusion. Although it is not yet clear how germline transcription and rearrangement (see contribution by a HC can reach the surface in the absence of SLC, it is possible R.W.Hendriks and R. Kersseboom in this issue). However, while that HC can pair with other chaperone-type proteins in pre-B D selection is impaired in the BLNK knock out, IgHC allelic cells, transiting to the surface and signaling allelic exclusion and exclusion in that mutant remains intact [61]. This may be due to other changes in gene regulation, but not proliferation [46–49]. another partially redundant adapter, LAT, which can substitute Differences in the ability of HC gene products to be to some extent for BLNK in pre-B cell development [62].LATis expressed on the cell surface may be responsible for the sur- able to link Syk to downstream PLC␥ signaling, just as BLNK prising observation that 4–8% of pre-B cells do express two does [63]. Coincidentally, mice heterozygous for PLC␥1 and endogenous rearranged HCs, only one of which is expressed missing PLC␥2 on an IgHC transgenic background fail to induce on the cell surface [22]. This may be due to inefficient pairing allelic exclusion as measured using a PCR assay for endoge- and thus weak surface expression of the first HC, resulting in nous VH gene-segment rearrangements [64]. The mechanism a failure of allelic exclusion followed by productive rearrange- for PLC␥s’ involvement in allelic exclusion is not yet under- ment of a second IgHC allele. In this vein, differences in surface stood, but perhaps calcium flux induces activation of relevant 34 J.K. Geier, M.S. Schlissel / Seminars in Immunology 18 (2006) 31–39 transcription factors. It would be interesting to know whether SWI-SNF, involved in heterochromatin formation, or active in over-expression of PLC␥ signaling cascade components could acetylation-dependent transcriptional regulation [72]. relieve the absolute requirement for HC surface expression in allelic exclusion. 6. Turning on kappa
5.2. Chromosomal choreography HC expression causes several rounds of cell division fol- lowed by exit from the cell cycle, silencing of the HC locus, Recently, large chromosomal movements have been corre- and up-regulation of the RAGs (among other molecules involved lated with allelic exclusion at the HC locus. Fluorescent in in differentiation), as discussed above. The final step in differ- situ hybridization (FISH) experiments revealed that the HC entiation induced by HC is Ig locus activation and V-to-J locus associates with the nuclear periphery in most T cells, recombination [34,73]. Signaling cascades emanating from the but is more centrally located in LPS-stimulated B cells [65]. pre-BCR induce transcription factors and perhaps chromatin This phenomenon was shown to be RAG independent but IL- remodeling activities that bind cis-regulatory elements within 7R␣-dependent as well as cell type specific, suggesting a role the Ig locus resulting in germline transcription and rearrange- for such chromosomal relocation in the regulation of recombi- ment. The mechanism of these events is currently the focus of nation. Remarkably, two-color FISH using probes from either intense research efforts. end of the HC locus revealed a higher level of compaction It is generally agreed that Ig rearrangement does not abso- within the locus in B cells undergoing V-to-DJ rearrangement lutely require HC or pre-BCR expression. For example, one [65,66]. This compaction was lost in B cell precursors lacking can detect low levels of Ig rearrangement in JH-deleted pro-B the B cell-restricted transcription factor Pax5 correlating with cells [74] or in IL-7-dependent HC− pro-B cell clones upon a loss of rearrangement to distal V genes [67]. In transgenic T acute withdrawal of IL-7 [75]. This has led to the suggestion that cells expressing Pax5, central nuclear relocation of IgHC genes Ig locus activation may be part of a developmental program that occurred, but the further level of compaction did not. This Pax5 is independent of pre-BCR expression. It has been proposed that induced nuclear relocation stimulated D-to-JH rearrangement in increases in Ig rearrangement that are associated with pre-BCR the T cells and allowed proximal VH gene rearrangement that expression may be a consequence of the proliferative expan- normally is not found in T cells [68,69]. Thus, nuclear relocal- sion of these cells rather than a pre-BCR signal transmitted to ization and chromatin compaction may contribute to regulation the Ig locus [25]. Strong data exists which contradicts this of the recombination machinery. Allelic exclusion requires that hypothesis, however. Pre-B cells positive for cytoplasmic HC this accessibility be reversed and indeed, FISH studies have now contain far higher levels of J locus dsDNA breaks associated demonstrated that co-localization to gamma satellite sequences with active V-to-J rearrangement than do pro-B cells on a per of one HC allele coincides with allelic exclusion and is then nanogram of DNA basis [73] and expression of a HC transgene relaxed but reestablished during LC rearrangement [68]. in RAG-deficient mice results in increases in germline transcrip- IL-7 signaling is associated with histone acetylation and tion and recombinase accessibility in sorted populations of pre- germline transcription of the distal V gene segments, and is nec- as compared to pro-B cells [34,39]. Also, there is data link- essary for the large chromosomal movements of the IgH locus ing the pre-BCR-mediated activation of the Ras-MAP kinase [65]. Loss of IL-7 responsiveness upon pre-BCR signaling is pathway to locus activation [76]. Additional experimenta- accompanied by deacetylation of the distal V gene segments, tion will be required to determine whether the pathway from although subsequent exposure to IL-7 causes the locus to become pre-BCR expression to locus activation is direct or indirect, acetylated once again [70]. Treatment of activated splenic B cells however. with IL-7 greatly diminished gamma satellite association of the The two IgLC loci, and , are expressed at a ratio of approx- IgH locus further implicating the shut-off of IL-7 receptor signal- imately 20:1 in mice [77]. The more frequently rearranged, and ing in allelic exclusion [68]. Thus, adoption of new proliferation therefore more frequently studied locus consists of approx- signals and loss of IL-7 responsiveness upon pre-BCR signaling imately 140 V gene segments, 5 J gene segments (four of may be necessary to establish allelic exclusion. which are functional), two germline transcript promoters, and Correlating with the initial activation of the IgHC locus are two enhancers (see Fig. 1). These two enhancers play partially antisense transcripts originating in intergenic regions between redundant roles in activating the LC locus as deletion of either VH gene segments [71]. These transcripts are turned off by one results in a decrease in the ratio of -to- expression (more so the time B cell precursors reach the late pro-B stage (Hardy’s in the E3 deletion), but only when both enhancers are deleted fraction C, CD19+CD43+BP-1+). Whether these antisense tran- is the phenotype dramatic. Deletion of both completely abol- scripts play a role in opening up the locus or in inducing chro- ishes recombination, leaving only positive B cells [78]. matin compaction via an RNAi-dependent pathway has yet to germline transcripts are activated in pre-B cells and correlate be determined. with rearrangements [79]. The gene expression profiles driving these developmentally regulated changes in HC locus structure remain to be dis- 6.1. Transcription factors and κ locus activation covered. A microarray study designed to elucidate changes in transcription associated with B cell differentiation identified four Many studies of Ig locus regulation have utilized Abel- genes encoding proteins each of which is either associated with son murine leukemia virus (AMuLV)-transformed pro-B cell J.K. Geier, M.S. Schlissel / Seminars in Immunology 18 (2006) 31–39 35
AMuLV transformation arrests developing B cells in what appears to be an early pre-B cell-like stage by an unknown mechanism. The viral oncogene v-Abl immortalizes these cells rendering them IL-7 independent and apoptosis resistant, but blocks their further differentiation. Upon treatment of AMuLV- transformed cells with the abl kinase inhibitor STI-571 (com- monly called Gleevec) these pre-B cells activate RAG and germline transcription and rearrange their and loci at very high rates [11]. By analyzing DNA microarray data derived from such cells prior to and after treatment with STI-571, IRF-4 and Spi-B were identified as key factors in activating the LC locus [11]. Co-transfection of cDNA expression vectors encoding the two proteins was sufficient to induce germline transcripts in an AMuLV-transformed pre-B cell line [11]. PU.1−/−Spi- B−/− primary pro-B cell cultures have lower levels of tran- scription than wild-type B cell lines, however IL-7 withdrawal still induced but not activation, suggesting that rear- rangement might be unaffected by the absence of these factors Fig. 1. Pre-BCR signaling activates pre-B cell proliferation, enforces allelic [83]. exclusion and activates Ig locus rearrangement. The upper part diagrams PU.1 and IRF4 interact with the E3 enhancer in chromatin the signaling cascades initiated by homotypic surrogate light chain (SLC)- immunoprecipitation experiments [84,85]. Increased binding of dependent interactions. Although stroma cell-associated pre-BCR ligands have these proteins was associated with the enhancer’s increased been identified, it is unclear whether this kind of interaction is critical for cells passing through the pre-BCR checkpoint. The lower part is a schematic of the accessibility to restriction endonucleases as well as increased germline locus including the V and J gene segments and the constant region association with acetylated histones in B cell lines mimicking exon (C) in light grey and the intronic (Ei) and 3 (E3) enhancers in dark grey. various stages of B cell development, leading to the suggestion Transcription factor binding sites are shown beneath their respective enhancers. that accessibility has a greater affect on enhancer occupancy Dark arrows represent germline transcript promoters. than protein levels [84]. IRF4 binds E3 but in the absence of IRF4, PU.1 binding is disrupted, suggesting coordinated bind- lines. While recapitulating certain aspects of pre-B cell biol- ing of the two molecules [82,85]. There are no binding sites ogy, these transformed cells are an imperfect model regarding in the intronic enhancer for IRF4/PU.1, thus the resulting gene regulation during the pro-to-pre-B cell transition. Treat- complete absence of rearrangements is intriguing. Interest- ment of cells with LPS or growth of temperature-sensitive ingly, IRF4,8−/− B cell precursors fail to exit the cell cycle, AMuLV-transformed pro-B cells at the non-permissive temper- cannot down-regulate SLC components, and express low lev- ature stimulates germline transcription and rearrangements els of RAG proteins, all of which could contribute to the lack [80,81]. This activation is dependent on the activation of NF-B, of activation [85]. Because of the similarity between this a transcription factor composed of the five members of the Rel- phenotype and that of the BLNK null mouse and the fact family that can form various homo- and heterodimers. Indeed, in that BLNK levels are unaffected by IRF4,8 inactivation, the vivo footprinting of AMuLV-transformed cells revealed an LPS- authors suggest that IRF4,8 might be downstream of BLNK dependent NF-B footprint in the intronic enhancer [10].A in a signaling pathway. BLNK might promote exit from the direct comparison of NF-B site occupancy in primary pro- and cell cycle and differentiation as well as E3 factor binding pre-B cells showed that the NF-B site is bound with protein in causing activation. It would be interesting to know whether both pro- and pre-B cells. Gel shift analysis of pro- and pre-B cell these IRF4,8−/− B cells are able to allelically exclude IgHC nuclei revealed different levels of the distinct NF-B complexes, rearrangement. but footprinting cannot distinguish the nature of the interacting Another well-studied transcription factor, E2A, can activate protein. Ig germline transcription and recombinase accessibility in non- Occupancy of binding sites within E3 changes across the lymphoid cell lines [86]. There are E2A binding sites (known as pro-B to pre-B cell transition [10]. A Pax5 site is occupied in E-boxes) in both Ig enhancers, and a recent study showed that pro-B cells but not in pre-B cells. Likewise, pre-B cells showed two such sites in Ei were critical for V-to-J rearrangement hypersensitivity at binding sites that were not present in pro-B [87]. While a direct role for E2A has not yet been demonstrated, cells, namely those for Ets family factors, PU.1 or Spi-B, and involvement of this factor has attracted attention because of CRE, a cyclic AMP response element. The binding sites for the abundance of E2A binding sites within Ig loci and their PU.1/IRF-4 [82], CRE, and Pax5 are in very close proximity to ability to recruit chromatin modifying factors [88]. Specific each other and the authors depict a model where Pax5 binding chromatin modifications associated with V(D)J recombination interferes with binding of other factors until pre-BCR signal- have yet to be identified, although regions of the IgHC locus ing somehow reduces Pax5 DNA binding allowing CRE and poised for recombination associate with hyper-acetylated his- PU.1/IRF-4 to bind the 3 enhancer and activate the LC locus tones H3 and H4, and hypo-methylated histone H3 Lysine 9 [10]. [89–91]. 36 J.K. Geier, M.S. Schlissel / Seminars in Immunology 18 (2006) 31–39
6.2. κ locus activation and IgLC allelic exclusion 7. Conclusion
Recent attention has focused on the mechanism of IgLC The transition of B cell precursors through the pre-BCR allelic exclusion. While productive IgLC gene rearrangement checkpoint (i.e. from the pro-B to pre-B cell stage) requires leads to the formation of a BCR and inactivation of the recom- tightly coordinated signals emanating from surface pre-BCR binase, mechanisms must exist to prevent the near simultane- that directs complex changes in the pattern of gene expres- ous rearrangement and expression of both alleles in a pre-B sion and even movement of entire chromosomes within the cell. Liang et al. [92] recently reported that high-level acti- nucleus. Alterations in nuclear transcription then redirect the vation of the locus in pre-B cells occurs in only a small V(D)J recombinase to different rearranging Ig loci creating fraction of pre-B cells at any given time. Using a promoter- molecules that then replace the originating receptor with a new less GFP cDNA reporter knocked into the coding region of molecule, the BCR. While the earlier steps in signaling cascades J1, these investigators measured the percentage of cells turn- are quickly being teased apart despite the complexity of redun- ing on a allele at each stage in development using flow dant pathways, and enhancers responsible for IgHC and locus cytometry. They found that pro-B cells from heterozygous activation are known, pathways linking signaling to retargeting mice did not express the reporter, while only 5% of small of the V(D)J recombinase remain vague at best. While over- pre-B cells did. Further experiments showed that activation expression of particular signaling molecules or transcription was mono-allelic and thus may contribute to allelic exclu- factors can mimic certain aspects of development, the nature of sion of IgLC rearrangement. The authors suggest that some signaling pathways makes over-expression and knock-out stud- threshold level of a combination of particular enhancer-binding ies difficult to interpret. This is particularly true in light of a transcription factors might allow for the rare and probabilis- stochastic model of activation, which requires the cumulative tic activation and rearrangement of a single allele at a action of multiple factors. A better understanding of chromatin time. structure’s precise role in accessibility and the coordinated activ- In contrast, Bergman and co-workers found biallelic germline ity of chromatin remodeling enzymes and transcription factors transcription in single-cell RT-PCR assays performed on pre- will help identify key components involved in transmitting sig- B cells [93]. They corroborated this finding with RNA-FISH nals from the pre-BCR to the HC and LC loci, and may also performed on AMuLV-transformed pre-B cells stimulated with determine causality of these correlated events. LPS, which revealed that 91% of cells expressed LC from both alleles. These results conflict with those from Liang et al. dis- Acknowledgements cussed above who suggest monoallelic germline transcription precedes rearrangement. Differences between these two sets of Work in the authors’ lab has been generously supported results may have to do with the amounts of germline transcripts by grants from the National Institutes of Health (HL48702, detected in two different assays (GFP expression versus high AI40221, and AI57487). We wish to thank other members of cycle-number PCR). the Schlissel lab for stimulating discussions. 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