6.1. Germline transcripts

Germline transcription is the production of transcripts from V, D, or J segments and adjacent regions before they undergo rearrangement (Fig. 1.2). Sense germline transcripts starting from promoters upstream of V, D, and J segments have been described in all Ig and TCR loci (Hesslein and Schatz, 2001), and their stage-specific expression patterns strongly correlate with accessibility of these transcribed elements (e.g., Yancopoulos and Alt, 1985). The precise role of sense germline transcripts is still not understood and has been debated (Krangel, 2003). Recent studies support the notion that active transcription mediates chromatin changes that render the transcribed regions accessible to the recombinase (Sen and Oltz, 2006). However, it has been debated whether germline transcripts are the cause or the effect of these chromatin changes, and neither possibility has been unequivocally proven or disproved. On one hand, the levels of germline transcripts exhibit a positive correlation with rearrangement efficiency (Sun and Storb, 2001), which could suggest that the process of transcription itself could promote RAG targeting. However, others have shown that the correlation between individual rearrangements and germline transcription is not absolute (Angelin-Duclos and Calame, 1998; Sikes et al., 2002).

The IgH locus in germline configuration is transcribed from the promoter of DQ52 (PDQ52), the 3′ most D_H segment, towards Cμ, thereby producing the so-called μ0 transcript (Fig. 1.2A). After D to J_H rearrangement, the recombined DJ_H element is transcribed (Fig. 1.2C) (Alessandrini and Desiderio, 1991; Reth and Alt, 1984); and at the same time, unrearranged V_H segments are transcribed from their promoters (Fig. 1.2B). Germline V_H transcription appears to be silenced upon a productive rearrangement (Corcoran, 2005; Yancopoulos et al., 1985).

More recently, antisense transcripts have been found to occur throughout the V_H cluster (Fig. 1.2B) (Bolland et al., 2004), in the D_H region (Fig. 1.2A and C) (Bolland et al., 2007; Chakraborty et al., 2007), and in the J_H region (Fig. 1.2A) (Bolland et al., 2007; Perlot et al., 2008). V_H antisense transcripts appear to be biallelic, and it has been argued that such transcripts are large and span several V_H segments and the adjacent intergenic regions; but formal proof of their initiation sites is still lacking. V_H antisense transcription was shown to initiate during D to J_H recombination, and to be rapidly downregulated after V_H to DJ_H recombination (Bolland et al., 2004). D_H antisense transcripts were detected in RAG-deficient pro-B-cells as well as on the D–J_H rearranged allele of B-cell lines with a functionally assembled IgH gene (Chakraborty et al., 2007). D_H antisense transcripts have been suggested to originate from the 3′ most D_H (Chakraborty et al., 2007) or from the J_H region (Bolland et al., 2007).

The functional significance of antisense transcription in the context of V(D)J recombination has not been fully elucidated. It has been postulated that antisense transcription promotes an active chromatin state rendering the locus more accessible (Bolland et al., 2004), based on the observed correlation between antisense V_H germline transcription and active V_H to DJ_H recombination (Bolland et al., 2004). Similar conclusions were reached based on the observation of reduced antisense D_H transcripts and reduced D to J_H rearrangements in mice lacking the intronic enhancer, Eμ (Afshar et al., 2006; Bolland et al., 2007). Conversely, others have raised the possibility that antisense transcripts, at least in the DSP D_H segments, could pair with low levels of postulated germline sense transcripts and elicit RNA interference-mediated transcriptional gene silencing (Chakraborty et al., 2007; Koralov et al., 2008). It should be noted that true germline sense transcripts have not been identified as yet in the germline D_H segments, but their level may be as low as those originally identified in the S. pombe centromeric repeats and may only be revealed in an RNAi-deficient background (Volpe et al., 2002).

6.2. Spatial organization and nuclear positioning of the IgH locus

The spatial organization of the Ig and TCR loci was analyzed by three-dimensional fluorescence in situ hybridization (3D FISH), in which nuclear organization remains preserved. Several groups showed that before undergoing rearrangement, the IgH locus moves from its default position at the nuclear periphery to a more central compartment (Fuxa et al., 2004; Kosak et al., 2002), going along with the observation that the nuclear periphery has a repressive effect on transcription (Andrulis et al., 1998; Baxter et al., 2002; Reddy et al., 2008) and, therefore, might keep the IgH locus in an inaccessible state. These observations are consistent with the peripheral location of the IgH locus in thymocytes which have only low level of D to J_H and no V_H to DJ_H rearrangements (Fuxa et al., 2004; Kosak et al., 2002; Kurosawa et al., 1981). The centrally located IgH locus in pro-B-cells can undergo D to J_H rearrangement; however, for rearrangements of the distant V_H elements, long-range contraction and looping of the IgH locus (Jhunjhunwala et al., 2008) seems to be crucial as lack of IgH locus contraction in Pax5-deficient pro-B-cells does not allow for rearrangements of intermediate and distal V_H families (Fuxa et al., 2004; Sayegh et al., 2005). After successful rearrangement, the IgH locus decontracts and, thereby, has been proposed to impede further V_H to DJ_H rearrangements by increasing the distance between V_H elements and the DJ_H region (Roldán et al., 2005). Therefore, it seems that one aspect of V_H to DJ_H recombination accessibility might be influenced by spatial arrangement of the IgH locus within the nucleus.

In B lineage stages subsequent to the pro-B stage, one IgH allele is positioned in close proximity to centromeric heterochromatin (Roldán et al., 2005; Skok et al., 2001). This finding was interpreted as the monoallelic silencing of the nonproductive IgH allele, because transcriptionally silent genes have been shown to associate with centromeric heterochromatin (Brown et al., 1997). However, the V_H cluster gets silenced on both alleles in the context of germline transcription, and considering the fact that rearrangements from both IgH alleles, productive and nonproductive in either DJ_H or V_HDJ_H configuration, are expressed in all B-cell stages that were examined (Daly et al., 2007; Fukita et al., 1998; Ono and Nose, 2007), monoallelic silencing might be either a short-time transient phenomenon or recruitment to centromeric heterochromatin might have other implications in the process of allelic exclusion.

Studies of interactions between IgH and Igκ alleles demonstrated coordinated patterns of action of Ig loci during B-cell development. Interactions between IgH and Igκ, predominantly in pre-B-cells, were demonstrated to reposition the interacting IgH allele to centromeric heterochromatin and induce IgH locus decompaction (Hewitt et al., 2008). Therefore, this interchromosomal interaction could play a role in IgH allelic exclusion and the transition from accessible IgH alleles to accessible Igκ alleles.

7.1. Promoter of DQ52

DQ52 is the 3′ most D segment. Its promoter becomes active before D to J_H rearrangement to generate the μ0 transcript (Fig. 1.2A) (Alessandrini and Desiderio, 1991; Kottmann et al., 1994; Schlissel et al., 1991a). This transcript runs all the way through the Cμ exons, which get spliced to the J_H1 splice donor site (Schlissel et al., 1991b). The same promoter region also gives rise to a low-level antisense transcript (Chakraborty et al., 2007). It has been suggested that the repetitive nature of the D_H region in combination with bidirectional transcription can elicit RNA interference-mediated transcriptional gene silencing that would lead to the observed inactive chromatin state of the DSP elements. However, as mentioned above, only antisense and no sense transcripts have been detected thus far in the germline D_H region (Chakraborty et al., 2007).

Every D_H element upstream of DQ52 has a bidirectional promoter which, upon D to J_H rearrangement, potentially through approximation to the Eμ enhancer, gets activated to generate an antisense transcript and a sense transcript (Fig. 1.2C) (Alessandrini and Desiderio, 1991; Chakraborty et al., 2007). The sense transcript gets spliced in a way that the rearranged DJ_H segment is joined to the Cμ exons. In one reading frame this mRNA can encode for a shorter version of the μHC (Reth and Alt, 1984), which can inhibit subsequent V_H to DJ_H rearrangements (Gu et al., 1991; Löffert et al., 1996; Malynn et al., 2002). Targeted deletion of the DQ52 promoter, which has both promoter and enhancer activity (Kottmann et al., 1994), in mice had no major impact on D to J_H rearrangement, other than a slight shift in J_H usage (Afshar et al., 2006; Nitschke et al., 2001). However, in these studies, μ0-like transcripts were still evident, suggesting that activity of the heterogeneous promoter of DQ52 was not entirely abrogated. In a different study, the intronic Eμ enhancer was replaced with a phosphoglycerate kinase promoter–neomycin resistance gene cassette (PGK-Neo^R), which resulted in complete absence of μ0 transcripts and complete inhibition of D to J_H rearrangement (Perlot et al., 2005). In this regard, targeted deletion of an analogous promoter element in the TCRβ locus, the promoter of Dβ1, led to diminished germline transcripts from this promoter and reduced Dβ1 rearrangements (Whitehurst et al., 1999), demonstrating an accessibility control function for this element in D_β–J_β recombination.

7.2. V_H promoters

Every V_H element has its own promoter that initiates V_H germline transcripts before V_H to DJ_H rearrangement (Fig. 1.2B), as well as transcripts of the assembled V_HDJ_H exon after rearrangement (Fig. 1.2D). Most V_H promoters can generate a germline transcript, in which a leader exon gets spliced to a V_H exon (Fig. 1.2B). The transcript gets polyadenylated and contains an open reading frame (Yancopoulos and Alt, 1985); however, no V_H protein or its function has thus far been demonstrated. The most conserved element across V_H promoters is the octamer ATGCAAAT (Parslow et al., 1984). This sequence element has been shown to be necessary for V_H transcription (Mason et al., 1985), and it binds the ubiquitously expressed Oct-1 and the B-cell-specific Oct-2, both POU family transcription factors. Most but not all V_H promoters contain a TATA box, an initiator (Inr) element (Buchanan et al., 1997), a heptamer, and a pyrimidine stretch (Eaton and Calame, 1987). Additionally, binding sites for a number of mostly B-lineage-specific transcription factors and chromatin remodeling complexes have been identified in V_H promoter regions (Johnston et al., 2006).

Germline transcripts from unrearranged V_H promoters are generated upon D to J_H rearrangement in pro-B-cells, and downregulated after completed V_H to DJ_H recombination and assembly and expression of a functional V_HDJ_H exon (Bolland et al., 2004; Hardy et al., 1991). The promoter of a recombined V_H element stays active throughout B-cell development, and cell line experiments showed that the first upstream unrearranged V_H segment can also be continuously expressed at reduced levels (Wang and Calame, 1985). Promoter activity of a functionally rearranged V_H element was shown to be partially dependent on the 3′ regulatory region (Pinaud et al., 2001). Thus, V_H promoters might fulfill a dual role: to help confer accessibility to germline V_H segments and to drive expression of the assembled heavy chain gene.

7.3. Intronic enhancer

The IgH intronic enhancer (Eμ) was the first cellular (as opposed to viral) eukaryotic enhancer element described (Alt et al., 1982; Banerji et al., 1983; Gillies et al., 1983). Eμ comprises a 220 bp enhancer core (cEμ) and two flanking matrix attachment regions (MARs). Targeted deletion of both MARs shows that they are dispensable for efficient V(D)J recombination within the IgH locus (Sakai et al., 1999). Deletion of Eμ in B-cells (Chen et al., 1993; Sakai et al., 1999; Serwe and Sablitzky, 1993) and in the germline of mice (Afshar et al., 2006; Perlot et al., 2005) led to reduced D to J_H rearrangement and severely impaired V_H to DJ_H rearrangement. The residual V(D)J recombination activity in the IgH locus implies that activation of IgH rearrangements may also involve one or more additional enhancer type elements. One candidate for such a compensating element is the promoter/enhancer region PDQ52, which was speculated to promote D to J_H recombination (Alessandrini and Desiderio, 1991). However, deletion of PDQ52 along with Eμ did not show increased impairment above that seen with deletion of Eμ alone (Afshar et al., 2006). However, since the deletion of PDQ52 appeared to be incomplete, this element can not yet be ruled out as having redundant functions with Eμ in conferring accessibility to the D_H–J_H region. Another candidate for cooperative function with Eμ is the 3′ IgH regulatory region, but the double knockout of Eμ and the IgH 3′RR has not been generated. By analogy, deletion of the intronic Igκ enhancer (iEκ) reduces Vk to Jκ rearrangements (Xu et al., 1996); whereas a double knockout of iEκ and the 3′Eκ enhancer completely blocks recombination of the Igκ locus (Inlay et al., 2002). The iEκ and 3′Eκ in the Igκ locus are the enhancer elements corresponding to the position of Eμ and IgH 3′RR in the IgH locus.

It has been puzzling why in Eμ knockout mice the V_H to DJ_H step is more severely impaired than the D to J_H step, even though the Eμ enhancer has no obvious effect on germline transcripts of intermediate and distal V_H families (Perlot et al., 2005). One explanation could be significant underestimation of D to J_H impairment in Eμ knockout mice. Initial very low levels of D to J_H rearrangements could limit the crucial DJ_H substrates for subsequent V_H to DJ_H rearrangements and, therefore, result in the observed strong reduction of V_H to DJ_H recombination. After a productive rearrangement, feedback regulation inhibits further V_H to DJ_H recombination, but does not block further D to J_H rearrangements (Reth et al., 1987). Therefore, D to J_H recombination might “catch up” over the course of B-cell development and mask a stronger impairment.

Notably, replacement of Eμ with a PGK-Neo^R cassette (Chen et al., 1993; Perlot et al., 2005; Sakai et al., 1999) or introduction of PGK-Neo^R cassette just 5′ of Eμ (Chen et al., 1993; Delpy et al., 2002) results in a much more severe impairment or a complete block of V(D)J recombination and concomitant complete loss of μ0 transcripts (Perlot et al., 2005). This phenomenon could be explained by a promoter competition/insulating mechanism. In such a scenario, the PGK-Neo^R gene and its promoter might compete with PDQ52 for activity from a downstream cis-element such as the IgH 3′RR, which is known to act over long distances (Pinaud et al., 2001). Similar promoter competition for the IgH 3′RR has been observed between I promoters and the PGK-Neo^R cassette introduced in the C_H region (see below). Alternatively, the PGK-Neo^R cassette could induce local chromatin changes that impede μ0 germline transcription and accessibility of D and J_H segments.

Extensive studies revealed an array of binding sites for B-lineage-specific transcription factors and also for ubiquitously expressed proteins within the Eμ enhancer and the flanking MARs (Calame and Sen, 2004). The unique combination of these factors is likely to mediate the enhancer’s predominant activity in pro-B-cells (Inlay et al., 2006). In this context, replacement of iEκ with Eμ leads to premature Igκ rearrangement in pro-B-cells and absence of Igκ rearrangements in pre-B-cells, the stage when LC rearrangement normally takes place (Inlay et al., 2006), corroborating the pro-B-cell specificity of Eμ.

Eμ was suggested to play a role in regulating antisense transcripts through the J_H and D_H region (Afshar et al., 2006; Bolland et al., 2007), and additionally a promoter region within Eμ was identified that gives rise to the Iμ transcript (Lennon and Perry, 1985; Su and Kadesch, 1990). Starting at Eμ, this transcript extends through the μ switch region and Cμ. Transcription of switch regions was shown to be necessary for CSR, probably for targeting AID, and in this regard, deletion of Eμ leads to reduced Iμ transcript levels and reduced CSR (Bottaro et al., 1998; Perlot et al., 2005). Deletion of Eμ has no obvious effect on somatic hypermutation of V_HDJ_H exons in mature B-cells (Perlot et al., 2005). An open question is how the activity of AID is specifically targeted to regions within Ig loci. It was speculated that cis-regulatory elements could determine this specificity, but neither cEμ nor the IgH 3′RR, alone (Morvan et al., 2003), seem to have a crucial role in targeting SHM to the IgH locus V_HDJ_H segments. While an absolute requirement of cEμ or the IgH 3′RR for SHM can be excluded, there is a possibility that smaller defects of SHM in these mutants are masked by selection processes during affinity maturation. Also, a combined function of cEμ and the IgH 3′RR in promoting or targeting SHM is another possibility that needs to be tested.

7.4. 3′ IgH regulatory region and I promoters

I promoters are located upstream of all switch regions (Chaudhuri et al., 2007; Lennon and Perry, 1985; Lutzker and Alt, 1988). Transcripts initiating from I promoters are processed in such a way that an I (intervening) exon, located immediately downstream of the I promoter, is spliced to the associated C_H exons. In this process, the intronic region including the S region is spliced out and the transcript gets polyadenylated. However, these transcripts appear “sterile,” as they do not contain an open reading frame and could not be shown to encode for a protein (Chaudhuri et al., 2007). Active transcription from I promoters is necessary for CSR as only transcribed S regions can become AID targets during CSR. In this context, deletion of I promoters abrogates efficient CSR to the associated C_H genes; while replacement of I promoters with a constitutively active promoter directs CSR to the associated C_H gene (Manis et al., 2002). Transcription from different I promoters prior to CSR can be induced upon stimulation with different activators or cytokines. Corresponding surface receptors for these molecules and their associated downstream signaling pathways effect different combinations of activating or repressive response elements within I promoter regions, which leads to CSR to different IgH isotypes under different stimulation conditions (Stavnezer, 2000). Most I promoters do not appear to act in isolation as efficient transcription from them also requires the IgH 3′RR (Pinaud et al., 2001) and physical interaction between the IgH 3′RR and specific I promoters has been implicated (Wuerffel et al., 2007).

The IgH 3′RR is located downstream of Cα at the very 3′ end of the IgH locus (Fig. 1.2B). This regulatory region consists of a number of DNaseI hypersensitive sites scattered over ~35 kb (Dariavach et al., 1991; Garrett et al., 2005; Lieberson et al., 1991; Matthias and Baltimore, 1993; Pettersson et al., 1990); up until now, none of them were shown to play a role in V(D)J recombination but more studies are needed (Khamlichi et al., 2000; Pinaud et al., 2001). The most striking function, control of IgH CSR, has been assigned to HS3b, HS4 within the IgH 3′RR. Targeted deletions in mice revealed severely reduced CSR to most IgH isotypes and reduced germline transcription from I promoters through the corresponding S regions (Pinaud et al., 2001), a process required for CSR (Jung et al., 1993; Zhang et al., 1993). Deletion of the more 5′ DNaseI hypersensitive sites within the IgH 3′RR HS3a and HS1,2 had no effect on CSR (Manis et al., 1998); however, replacement of HS3a or HS1,2 with a PGK-Neo^R cassette resulted in a similar defect as in the HS3b, HS4 deletion (Cogné et al., 1994, Manis et al., 1998). The latter observations suggested a potential promoter competition/insulation between I promoters and the PGK-Neo^R cassette for signals from within the IgH 3′RR. This hypothesis was strengthened by insertion of a PGK-Neo^R cassette at the Iγ2b promoter or the Cε gene, respectively. In both cases, germline transcription and class switching to C_H genes 3′ of the inserted PGK-Neo^R cassette was unaffected; while germline transcription and class switching to C_H genes 5′ of the inserted PGK-Neo^R cassette was impaired (Seidl et al., 1999). These results suggest that the inserted PGK-Neo^R cassette can interfere with the long-range control effect of IgH 3′RR on CSR in a position-dependent manner.

The IgH 3′RR is necessary for efficient expression of the rearranged HC from the promoter upstream of the assembled V_HDJ_H exon (Pinaud et al., 2001), whereas the much more proximal Eμ enhancer is not required for HC expression (Perlot et al., 2005). Because it can influence expression of rearranged V_HDJ_H segments, the IgH 3′ RR can function over a distance of at least 200 kb. Such long-range activity may be important for activating oncogenes translocated into the upstream portions of the C_H locus in lymphomas. Not all of the seven described hypersensitivity sites in the spacious 3′ regulatory region have been knocked out yet, therefore, other potential functions still remain to be discovered. Apart from the above-mentioned effects on germline transcription, CSR, and IgH expression, it has been speculated that parts of the 3′ regulatory region might have a role in long-range chromatin organization. Finally, activity of the Iγ1 promoter does not appear to be dependent on the IgH 3′RR; suggesting that it carries sufficient regulatory elements itself or that there are other long range IgH locus elements that function in CSR to be defined.

7.5. Additional potential regulatory elements

Several laboratories suggested that the IgH locus can be associated with the nuclear periphery via its 5′ region (Kosak et al., 2002; Yang et al., 2005). The 5′ end of the IgH locus does not get deleted in the course of V(D)J recombination and as such is an attractive location for a missing regulatory element that controls processes such as accessibility control of the distal V_H genes, positioning of the IgH locus, or feedback regulation. In fact, ~30 kb upstream of the most distal V_H element an array of DNaseI hypersensitive sites has been identified (Pawlitzky et al., 2006). One of these sites, HS1, was reported to be pro-B-cell specific and potentially contain binding sites for the transcription factors PU.1, Pax5, and E2A. However, preliminary knockout experiments, in which HS1 was deleted, showed no effect on the IgH locus, as targeted alleles could still undergo efficient V(D)J recombination including all V_H gene families. Furthermore, allelic exclusion was unaffected (Perlot, Pawlitzky, Brodeur, and Alt, unpublished data). Other potential functions of these sites, including acting as a boundary area as was suggested by DNA modifications confined to one side of 5′IgH hypersensitive sites (Reddy et al., 2008), are still being tested.

Another area that was speculated to harbor a regulatory element is the ~90 kb region between the V_H and the D_H clusters. This region could contain an element that ensures the ordered rearrangement of the D to J_H and V_H to DJ_H steps, such as a boundary element that influences activation of separate IgH locus domains. Moreover, the V_H to D_H intergenic region is deleted on a productively rearranged allele but remains in place on a DJ_H rearranged allele, suggesting an element might reside in this region that is responsible for shutting down the incompletely rearranged allele in the context of allelic exclusion. Potential support for such an element came from placement of a V_H segment into the D_H region, which resulted in breaking of lineage specificity, ordered rearrangement, and allelic exclusion of the introduced V_H segment (Bates et al., 2007). Preliminary studies in which this intergenic region has been deleted have provided direct support for the notion that this region contains elements important for regulation of lineage specificity of V_H to DJ_H rearrangement (Giallourakis, Franklin, and Alt, unpublished data).

We thank Cosmas Giallourakis and John Manis for critically reviewing the manuscript and for discussions. T. P. received a Boehringer Ingelheim Fonds PhD scholarship. This work was supported by National Institutes of Health Grants PO1CA092625-05 and 2PO1AI031541-15 (to F.W.A.). F.W.A. is an investigator of the Howard Hughes Medical Institute.