Jane Skok, PhD
Associate Professor of Pathology PhD,
1985 University College London
B cell, T cell, Lymphocyte, Allelic Exclusion, Locus Contraction, Looping, Immunoglobulin and Tcr Loci, Homologous Pairing, ATM, Pax5, STAT5
B and T lymphocyte development is driven by V(D)J recombination, a process by which gene segments at the antigen receptor loci are rearranged to create a vast repertoire of antigen receptor loci. In normal circumstances lymphocytes circumvent the dangers associated with the introduction of multiple double strand breaks (DSBs) through deft co-ordination and tight control over 'where and when' breaks are introduced. Research in the Skok lab, employing a combination of sophisticated imaging techniques, molecular biology, and genetics, indicates that nuclear dynamics and pairing of homologous and heterologous chromosomal loci controls accessibility to the RAG1/2 recombinase.
Immunoglobulin(Ig) and T-cell receptor (Tcr) variable region exons are assembled from arrays of V, D, and J coding segments during the development of B and T cells, respectively. Scattered across six different chromosomal locations, the T cell receptor (Tcr)a,b,d, and g loci and immunoglobulin (Ig)heavy (Igh) and light (Igk and Igλ) chain loci each contain large arrays of V, D, and J coding segments flanked by recombination signal sequences (RSS). The lymphoid-specific recombinase, consisting of RAG1 and RAG2 (the protein products of the recombination activating genes 1 and 2), binds to a pair of conserved signal sequences that can be many kilobases apart, cleaves the DNA at the signal sequence borders, and holds the resulting DNA double-strand breaks in a post-cleavage complex. The post-cleavage complex then guides the broken DNA ends into the ubiquitous nonhomologous end joining (NHEJ) machinery for repair, ultimately forming a new antigen receptor gene.
V(D)J rearrangement is regulated by lineage and locus. Despite the fact that both B and T cell loci are similarly organized and undergo recombination through the same RAG proteins and DNA repair factors, full Ig gene recombination is confined to B cells, and Tcr gene recombination is confined to T cells. Rearrangement is also ordered within a given lineage: the Igh is rearranged at the pro-B cell stage of development prior to Igk or Igλ rearrangement in pre-B cells, and in T cells rearrangement of Tcrb occurs in CD4-CD8- double negative (DN) cells prior to Tcra rearrangement in CD4+CD8+ double positive (DP) cells. Furthermore, D-to-J recombination at the Igh and Tcrb loci must take place before V-to-DJ rearrangement can begin. Finally, recombination must also be regulated at the level of the individual allele.
Chromosome interactions play a key role in regulating V(D)J recombination. Precise spatiotemporal control of rearrangement appears to involve a number of different chromosomal interactions. As a result, V(D)J recombination has provided valuable insights into chromosomal interactions in mammalian cells.
(i) Chromosome self-association: looping and contraction:Some of the antigen receptor loci are quite long: the Ig heavy chain (Igh) and Ig kappa (Igk) loci, for example, span about 3Mb in the mouse. The Skok lab discovered that Igh, Igk, Tcrb and Tcra all undergo reversible locus contraction during recombination1,2. Contraction involves an alteration in configuration and the formation of chromatin loops, which enable synapsis and recombination between widely separated gene segments to facilitate rearrangement.
(ii) Communication between alleles: homologous pairing.Chromosome pairing is involved in X chromosome inactivation — a classic instance of monoallelic gene expression. Antigen receptor genes are also largely monoallelically expressed (‘allelically excluded') in B and T lymphocytes. The Skok lab recently discovered that homologous immunoglobulin (Ig) alleles pair up in a stage-specific manner that mirrors the stages of their recombination3. The frequency of homologous Ig pairing is substantially reduced in the absence of the RAG1/RAG2 recombinase, but is rescued in Rag1-/- developing B cells with a transgene expressing an active site mutant form of RAG1 that supports DNA binding but not cleavage. RAG-mediated cleavage on one Ig allele induces the other allele to relocate to repressive pericentromeric heterochromatin (PCH) in a manner that requires the DNA damage response factor, ATM. In the absence of ATM, repositioning at PCH is diminished and the incidence of cleavage on both alleles is significantly increased. ATM appears to be activated by the introduction of a double-strand break on one allele to act in trans on the uncleaved allele, repositioning or maintaining it at PCH to prevent bi-allelic recombination and chromosome breaks or translocations8.Morerecently we have shown that pairing and higher-order looping of Tcra homologous alleles are linked with ATM-mediated regulation of their recombination4.
(iii) Communication between different loci.We discovered that at the Igh locus, loss of intrachromosomal connections and altered locus accessibility occur as a result of an inter-chromosomal interaction with pericentromerically located Igk alleles5. Igk (located on chromosome 6) directs the unrearranged Igh allele (located on chromosome 12) to pericentromeric heterochromatin where the two loci associate, mediated by the 3' enhancer (3'Ek) of the Igk locus. Repositioning of the unrearranged Igh locus at pericentromeric heterochromatin limits access to RAG proteins while decontraction provides a physical barrier to further V gene rearrangement. Thus, the Igk locus interacts with the Igh locus to co-ordinate the transition from Igh to Igk rearrangement and thus from one stage of B cell development to the next.
(iv) Communication between different loci is linked to regulation of cleavage. Our most recent studies reveal that there is similarity between the C-terminus of RAG2 and ATM in temporally harnessing RAG activity to ensure that cleavage occurs on only one locus at a time in recombining T cells (Chaumeil et al., Nature Communications in press). Intriguingly we find that RAG mediated association of recombining loci in localized ‘recombination centers’ is linked to feedback control of RAG cleavage: the introduction of a break on one locus is coupled with repositioning of the partner locus to pericentromeric heterochromatin and inhibition of bi-locus cleavage and looping out from the territory. Although we recently showed that pairing and higher-order looping of Tcra homologous alleles is linked with regulation of their recombination6, cross-talk and regulation of heterologous loci in trans has not previously been shown to be linked to the formation of higher-order looping. Indeed higher-order loop formation was previously shown to be involved in stochastic interactions between different loci on separate chromosomes while here we have found that interactions between recombining Tcra/d and Igh are mediated by the presence of RAG.
Environmental signals and their influence on regulation of recombination
Antigen receptor diversity involves the introduction of DNA double stranded breaks during lymphocyte development. To ensure fidelity, cleavage is confined to the G0/G1 phase of cell cycle. One established mechanism of regulation is through periodic degradation of the RAG2 recombinase protein. However, there are additional levels of protection. We have found that cyclical changes in the IL-7R signaling pathway functionally segregate pro-B cells according to cell cycle status. In consequence, the level of a downstream effector of IL-7 signaling, phospho-STAT5 is inversely correlated with cell cycle expression of Rag, a key gene involved in recombination. Higher levels of phopho-STAT5 in S/G2 correlate with decreased Rag expression and Rag relocalization to pericentromeric heterochromatin (PCH). These cyclical changes in transcription and locus repositioning are ablated upon transformation with v-Abl, which renders STAT5 constitutively active across the cell cycle. We propose that this activity of the IL-7R/STAT5 pathway plays a critical protective role in development, complementing regulation of RAG2 at the protein level, to ensure that recombination does not occur during replication. Our data, suggesting that pro-B cells are not a single homogeneous population explain inconsistencies in the role of IL-7 signaling in regulating Igh recombination. We are currently extending these studies to examine the effects of differential levels of IL-7/STAT5 signaling in the fetal liver and bone marrow environments.
Maintenance of genome stability during V(D)J recombination
Regulation of V(D)J recombination occurs at multiple levels to prevent the occurrence of chromosomal translocations or deletions that can result from errors in repair and/or mis-targeting of RAG1/2 to cryptic RSSs. However, beyond degradation of RAG2 protein and our more recent findings that expression of Rag1 is regulated across cell cycle7, there been have no studies that focus specifically on auto-regulation of RAG cleavage activity in individual cells. It is clearly critical to have such mechanisms in place to ensure that further breaks are not introduced in cis,or intrans on accessible target loci that undergo recombination at overlapping stages of development (Tcrg, Tcrb, Tcrd and Igh in DN T cells and Igk and Igl in pre-B/immature B cells).
In addition to uncovering long-range chromosomal interactions between homologous and heterologous Ig alleles that are of functional importance, we have also recently discovered that the transcription factor, RUNX, mediates association of Cd4 and Cd8 in CD8 expressing T cells8. Communication between Cd4 and Cd8 appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature SP cells. We now routinely perform circularized chromosome conformation capture with deep sequencing (4C-seq) experiments to learn more about global interactions of genes of interest at different stages of development. Using this approach we have found that chromosomal domains in close proximity to Igh in the nucleusof class switching B cells are predisposed to AID mediated translocations9.
1 Skok, J. A.et al. Reversible contraction by looping of the Tcra and Tcrb loci in rearranging thymocytes. Nature Immunology 8, 378-387, doi:10.1038/ni1448 (2007). PMID: 17334367
2 Roldan, E.et al. Locus 'decontraction' and centromeric recruitment contribute to allelic exclusion of the immunoglobulin heavy-chain gene. Nat Immunol 6, 31-41 (2005). PMID: 15580273
3 Hewitt, S. L.et al. RAG-1 and ATM coordinate monoallelic recombination and nuclear positioning of immunoglobulin loci. Nat Immunol 10, 655-664, doi:ni.1735 [pii] 10.1038/ni.1735 (2009). PMID: 19448632
4 Chaumeil, J.et al. Higher-Order Looping and Nuclear Organization of Tcra Facilitate Targeted RAG Cleavage and Regulated Rearrangement in Recombination Centers. Cell reports, doi:S2211-1247(13)00031-4 [pii] 10.1016/j.celrep.2013.01.024 (2013). PMID: 23416051
5 Hewitt, S. L.et al. Association between the Igk and Igh immunoglobulin loci mediated by the 3' Igk enhancer induces 'decontraction' of the Igh locus in pre-B cells. Nat Immunol 9, 396-404, doi:ni1567 [pii] 10.1038/ni1567 (2008). PMID: 18297074
6 Chaumeil, J.et al. Higher-order looping and nuclear organization of Tcra facilitate targeted rag cleavage and regulated rearrangement in recombination centers. Cell reports 3, 359-370, doi:10.1016/j.celrep.2013.01.024 (2013). PMID: 23416051
7 Johnson, K.et al. IL-7 functionally segregates the pro-B cell stage by regulating transcription of recombination mediators across cell cycle. J Immunol 188, 6084-6092, doi:10.4049/jimmunol.1200368 (2012). PMID: 22581861
8 Collins, A.et al. RUNX Transcription Factor-Mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation. Immunity 34, 303-314, doi:S1074-7613(11)00082-3 [pii] 10.1016/j.immuni.2011.03.004 (2011). PMID: 21435585
9 Rocha, P. P.et al. Close Proximity to Igh Is a Contributing Factor to AID-Mediated Translocations. Molecular Cell, doi:10.1016/j.molcel.2012.06.036 (2012). PMID: 22864115