Ying Yang 1 (YY1) is a ubiquitously expressed transcription factor shown to be essential for pro–B-cell development. However, the role of YY1 in other B-cell populations has never been investigated. Recent bioinformatics analysis data have implicated YY1 in the germinal center (GC) B-cell transcriptional program. In accord with this prediction, we demonstrated that deletion of YY1 by Cγ1-Cre completely prevented differentiation of GC B cells and plasma cells. To determine if YY1 was also required for the differentiation of other B-cell populations, we deleted YY1 with CD19-Cre and found that all peripheral B-cell subsets, including B1 B cells, require YY1 for their differentiation. Transitional 1 (T1) B cells were the most dependent upon YY1, being sensitive to even a half-dosage of YY1 and also to short-term YY1 deletion by tamoxifen-induced Cre. We show that YY1 exerts its effects, in part, by promoting B-cell survival and proliferation. ChIP-sequencing shows that YY1 predominantly binds to promoters, and pathway analysis of the genes that bind YY1 show enrichment in ribosomal functions, mitochondrial functions such as bioenergetics, and functions related to transcription such as mRNA splicing. By RNA-sequencing analysis of differentially expressed genes, we demonstrated that YY1 normally activates genes involved in mitochondrial bioenergetics, whereas it normally down-regulates genes involved in transcription, mRNA splicing, NF-κB signaling pathways, the AP-1 transcription factor network, chromatin remodeling, cytokine signaling pathways, cell adhesion, and cell proliferation. Our results show the crucial role that YY1 plays in regulating broad general processes throughout all stages of B-cell differentiation.

The diverse antibody and T cell receptor (TCR) repertoires are created through the highly regulated process of V(D)J recombination. These lymphocyte receptor loci are comprised of many V (variable), D (diversity) and J (joining) gene segments. For example, in the immunoglobulin heavy chain locus (Igh), there are ~100 functional VH genes spread over 2.4 Mb, 13 DH genes and 4 JH genes. In pro-B cells, the first rearrangement is a DH to JH rearrangement on both alleles, followed by VH to DJH rearrangement. After heavy chain rearrangement is complete, several rounds of proliferation occur, followed by rearrangement in the light chain locus during the pre-B cell stage. A similar precise order of events, linked with sequential T cell differentiation stages, occurs for the rearrangement of TCR genes.In addition to the specific order of rearrangement, V(D)J rearrangement is also regulated in lineage-specific manner. Although the same RAG recombinase recombines both TCR and Ig genes, the rearrangements only occur in the correct lineage of cells, other than some DH-JH rearrangement in thymocytes. This lineage and developmental stage specificity of V(D)J rearrangement suggests precise control over the accessibility of various portions of the Ig and TCR loci such that only the gene segments that should recombine at that developmental stage will be accessible to the RAG recombinase. As demonstrated many years ago, this accessibility is manifest by the presence of germline transcription from unrearranged genes at the stage when that particular group of gene segments is poised to undergo rearrangement 1.Given the enormous size of the receptor loci, the question arises as to how a single DJ rearrangement can have approximately equal access to V genes spread over a 2-3 Mb region, since we know that V genes throughout the loci are utilized. Early clues to this issue came from three dimensional (3D) FISH studies that demonstrated that the large receptor loci compact at the appropriate stage for rearrangement, and that the loci extend again after rearrangement is completed 2, 3. The identification of the proteins responsible for these large-scale 3D structural changes in lymphocyte receptor loci has been area of great interest. For the Igh locus, the transcription factors YY1 and Pax5 have been shown to be required to for locus compaction 3. Deficiency of these proteins also leads to reduced rearrangement of distal VHJ558 gene family, suggesting that locus compaction is necessary to bring distal VH genes in proximity to the DJH rearrangement. However, the manner in which these transcription factors effect the change in the 3D structure of the locus is still unclear.In addition to transcription factors, the involvement of proteins that regulate higher order chromatin structure and nuclear architecture has been speculated 4. One such protein is CTCF, an 11-zinc finger protein associated with all vertebrate insulators, and it has been demonstrated to produce large-scale looping within β-globin and other loci, often with the aid of cohesin 5. Cohesin is a complex of 4 subunits, which forms a ring around sister chromatids, holding them together during mitosis. ChIP-chip studies have demonstrated that cohesin is bound to a subset of CTCF sites genome-wide 6. Indeed, the binding of CTCF and cohesin at many sites in the Igh locus has also been demonstrated, and knockdown of CTCF led to reduced Igh locus compaction 4, 7. In addition to the many CTCF sites throughout the VH part of the locus, only 2 other regions of CTCF binding are present in the Igh locus: one set of 2 sites just upstream of the most 5′-functional DH gene (DFL16.1), and a set of 9 sites downstream of the most distal 3′-enhancer 4, 7. These sites flank the region containing all the functional DH and JH genes and the intronic enhancer Eμ. Thus, it was proposed that these sites create a loop or domain in which the first step of V(D)J rearrangement, that of DH to JH, would take place 4, 8, 9, and this was recently demonstrated by chromosome conformation capture (3C) 7. This domain would not include the VH genes, and thus would help enforce ordered rearrangement.In a recent issue of Nature, Alt and colleagues tested the role of the two closely spaced CTCF sites upstream of DFL16.1 by making a 4.1 kb germline deletion of them, or mutating them in the germline 10. Both sets of mice showed similar phenotypes. The level of rearrangement of the proximal VH7183 and VHQ52 families was greatly increased, whereas the rearrangement frequency of the distal VHJ558 family was reduced. Sequencing revealed that > 90% of the VH7183 rearrangements were to the most 3′-functional VH gene, 81X. Likewise, > 85% of the VHQ52 rearrangements were to the most 3′-functional VHQ52 gene, Q52.2.4. Importantly, ordered rearrangement was also disrupted, in that VH81X now underwent rearrangement to a DH gene that had not previously rearranged to a JH gene. In agreement with the greatly heightened rearrangement of these two VH genes, Guo et al. demonstrated that the level of germline transcription for these proximal VH families was significantly increased. In addition, deletion of these CTCF sites resulted in loss of lineage specificity, with rearrangement of proximal VH genes in deficient thymocytes. These findings support the hypothesis that interaction of CTCF sites upstream of DFL16.1 with those flanking the 3′ RR region by loop formation creates a separate functional domain containing DH and JH regions along with Eμ, and that this loop excludes the VH region. Thus, it appears that this 4 kb region with two CTCF sites regulates and restrains the activity of the DH-proximal VH genes that are located ~100 kb upstream. In its absence, upregulated proximal VH gene germline transcription, upregulated proximal VH gene rearrangement, and inappropriate rearrangement to unrearranged D genes, or in the wrong lineage (thymocytes), are observed.Another recent paper in Nature also demonstrated the role of the CTCF/cohesin complex in VDJ rearrangement. Merkenschlager and colleagues utilized mice in which the cohesin component Rad21 was conditionally deleted in the CD4+CD8+ double positive (DP) stage of thymic differentiation, the stage at which TCRα rearrangement takes place 11. Since cells cannot survive cell division without cohesin, the authors cleverly chose to analyze DP thymocytes, which do not undergo cell division during TCRα rearrangement, thereby studying a cell division-independent role for cohesin. Firstly, they mapped cohesin (Rad21) binding in TCRα locus by ChIP-sequencing in DP thymocytes, and their analysis showed that cohesin was abundant at many key positions in the TCRα locus. The TCRα locus is different in structure from all other receptor loci, in that there are 61 Jα gene segments, with a similar number of Vα gene segments. The reason for this is that T cells often undergo multiple rounds of rearrangements before they successfully pass the positive selection step of differentiation. This successive series of rearrangements is regulated by having the initial rearrangements occurring between one of a cluster of Vα-proximal Jα genes and one of a cluster of Jα-proximal Vα genes. Subsequent rearrangements utilize more upstream Vα and downstream Jα genes. The various clusters of Jα genes each have their own germline promoter that regulates transcription of ~10 downstream Jα genes. The first round of rearrangement occurred normally in these conditional Rad21 deletion mice, since Rad21 was not fully deleted initially. However, Rad21 was deleted for all subsequent rounds of rearrangement, and these rearrangements were substantially impaired. Seitan et al. showed that normally the TEA promoter upstream of the Jα genes forms a long-range loop with the downstream enhancer Eα, presumably via the cohesin bound to both. In agreement with this hypothesis, the Rad21-deleted thymocytes had greatly reduced TEA-Eα interactions. The authors demonstrated significantly decreased germline transcription through the middle and distal Jα genes and decreased levels of H3K4me3 on those genes. This histone modification is of significance for V(D)J rearrangement since RAG2 is recruited to H3K4me3. Hence, rearrangement to the middle and distal Vα genes was also reduced. RNA-seq demonstrated that transcription of the downstream gene Dad1 was increased while germline transcription of Cα was decreased, presumably due to the loss of insulator function of the CTCF/cohesin sites demarcating those domains, and loss of TEA-Eα promoter-enhancer interactions. Therefore, cohesin controls TCRα locus rearrangement at multiple levels.Together these two recent papers in Nature further strengthen the hypothesis that the CTCF/cohesin complex plays an important role in the 3D chromatin structure within the Igh and TCRα loci by creating domains and insulation boundaries. This structural and functional compartmentalization within the large antigen receptor loci is critical to maintain the appropriate accessibility of gene segments in the complex process of V(D)J recombination. The evidence that the CTCF/cohesin complex regulates germline transcription and histone modifications further provides an example of locus-specific roles of this ubiquitous complex. Thus, the highly ordered, lineage-specific process of V(D)J recombination has yet another layer of regulation imposed by long-range 3D structures.

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germ-line transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage- and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1−/− pro-B cells are greatly impaired in distal VH gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR–Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal VH genes close to the DJH rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal VH genes to undergo efficient rearrangement.

Compaction and looping of the ~2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all VH genes to be brought in proximity with DH-JH segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ~60 sites throughout the VH region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a DH-JH-Eμ-CH domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the DH region and parts of the VH locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.

The usage of >100 functional murine Ig heavy chain VH genes, when rearranged to DHJH genes, generates a diverse antibody repertoire. The VH locus encompasses 2.5 Mb, and rearrangement of VH genes in the DH-distal half of the locus are controlled very differently from the VH genes in the proximal end of the locus. The rearrangement of distal but not proximal VH genes is impaired in mice deficient in the cytokine IL-7 or its receptor, in the transcription factor Pax5, or in Ezh2, a histone methyltransferase for Lys-27 of histone H3 (H3K27). The relative role of IL-7, Pax5, and Ezh2 in regulating distal vs. proximal VH rearrangement is not clear. Here, we show by ChIP and ChIP-on-chip that the active histone modification H3K36me2 is most highly associated with distal VH segments and the repressive histone modification H3K27me3 is exclusively present on proximal VH segments. We observed an absence of H3K27me3 in fetal pro-B cells, which predominantly rearrange proximal VH genes. Absence of IL-7 signaling reduces H3K36me2, and overexpression of IL-7 increases H3K36me2. In contrast, the major effect of the absence of Pax5 is the reduction in H3K27me3. Our data indicate that the cytokine IL-7 and the transcription factor Pax5 influence the rearrangement of the two regions of the VH locus by differentially modulating two reciprocal histone modifications during B lymphocyte development.

V(D)J recombination is a crucial component of the adaptive immune response, allowing for the production of a diverse antigen receptor repertoire (Ig and TCR). This review will focus on how epigenetic regulation and 3-dimensional (3D) interactions may control V(D)J recombination at Ig loci. The interplay between transcription factors and post-translational modifications at the Igh, Igκ, and Igλ loci will be highlighted. Furthermore, we propose that the spatial organization and epigenetic boundaries of each Ig loci before and during V(D)J recombination may be influenced in part by the CTCF/cohesin complex. Taken together, the many epigenetic and 3D layers of control ensure that Ig loci are only rearranged at appropriate stages of B cell development.

Recent studies of the regulation of antigen receptor rearrangement have revealed several completely new levels of control. Not only do antigen receptor loci undergo changes in histone modifications as they become accessible for recombination, but also the number of different histone modifications and the variation at different parts of each receptor locus reveal great complexity. RAG2 is now known to bind to one of these histone modifications, H3K4me3, and this targets the initial RAG binding events to the J genes. The large megabase receptor loci undergo 3D changes in their structure during rearrangement, and receptor loci move throughout the nucleus, transiently binding to heterochromatin, and transiently pairing with each other. RAG-mediated DNA breaks promote some of these movements, and also result in widespread changes in the transcriptional profile promoting differentiation.

The molecular mechanisms that control the temporal and lineage-specific accessibility, as well as the rearrangement frequency of VH genes for VH-to-DJH recombination, are not fully understood. We previously found a positive correlation between the extent of histone acetylation and the differential rearrangement frequency of individual VH genes. Here, we demonstrated that poorly rearranging VH genes are more highly associated with histone H3 dimethylated at lysine 9, a marker of repressive chromatin, than frequently rearranging VH genes. We also observed a positive relationship between the differential binding of Pax5 to individual VHS107 genes and rearrangement frequency. Furthermore, we showed that accessibility of the regions flanking the Pax5 binding site and the recombination signal sequence (RSS) to restriction enzyme cleavage correspond with the differential rearrangement frequency of the VHS107 family members. In addition, we found that the CpG sites located in the coding regions of VH genes are methylated in general, while the extent of DNA methylation drops dramatically near the RSS. For the VHS107 family, one CpG site located 101 bp upstream of the RSS showed variable methylation that correlates with rearrangement frequency, and the methylation status of a CpG site located 34 bp downstream of the RSS could also favor the rearrangement of V1 over V11. These findings suggest that the extent of histone modifications, chromatin accessibility, DNA methylation, as well as the differential binding of Pax5 to VH coding regions, could all influence the rearrangement frequency of individual VH genes, although some of these mechanisms are not strictly B cell specific.