Faculty Bibliography

Fluorescent in situ hybridization using DNA probes on 3-dimensionally preserved nuclei followed by 3D confocal microscopy (3D DNA FISH) represents the most direct way to visualize the location of gene loci, chromosomal sub-regions or entire territories in individual cells. This type of analysis provides insight into the global architecture of the nucleus as well as the behavior of specific genomic loci and regions within the nuclear space. Immunofluorescence, on the other hand, permits the detection of nuclear proteins (modified histones, histone variants and modifiers, transcription machinery and factors, nuclear sub-compartments, etc). The major challenge in combining immunofluorescence and 3D DNA FISH is, on the one hand to preserve the epitope detected by the antibody as well as the 3D architecture of the nucleus, and on the other hand, to allow the penetration of the DNA probe to detect gene loci or chromosome territories (1-5). Here we provide a protocol that combines visualization of chromatin modifications with genomic loci in 3D preserved nuclei.

Tight control of antigen-receptor gene rearrangement is required to preserve genome integrity and prevent the occurrence of leukaemia and lymphoma. Nonetheless, mistakes can happen, leading to the generation of aberrant rearrangements, such as Tcra/d-Igh inter-locus translocations that are a hallmark of ataxia telangiectasia-mutated (ATM) deficiency. Current evidence indicates that these translocations arise from the persistence of unrepaired breaks converging at different stages of thymocyte differentiation. Here we show that a defect in feedback control of RAG2 activity gives rise to bi-locus breaks and damage on Tcra/d and Igh in the same T cell at the same developmental stage, which provides a direct mechanism for generating these inter-locus rearrangements. Both the RAG2 C-terminus and ATM prevent bi-locus RAG-mediated cleavage through modulation of three-dimensional conformation (higher-order loops) and nuclear organization of the two loci. This limits the number of potential substrates for translocation and provides an important mechanism for protecting genome stability.

Class switch recombination (CSR) has the potential to generate genomic instability in B cells as activation-induced cytidine deaminase (AID), which mediates this process, is known to target many sites outside Igh. Nonetheless we do not fully understand what factors influence AID targeting genome-wide. Given that errors in CSR can lead to dangerous, oncogenic chromosomal translocations it is important to identify the elements that determine which genes are at risk of being "hit" and could be involved in aberrant rearrangements. Here we have investigated the influence of nuclear organization in determining "off-target" activity and the choice of fusion partners. Our studies indicate that the vast majority of known AID-mediated Igh translocation partners are found in chromosomal domains that contact this locus during class switching. Further, these interaction domains can be used to identify other genes that are hit by AID.

Ag receptor diversity involves the introduction of DNA double-stranded breaks during lymphocyte development. To ensure fidelity, cleavage is confined to the G(0)-G(1) phase of the cell cycle. One established mechanism of regulation is through periodic degradation of the RAG2 recombinase protein. However, there are additional levels of protection. In this paper, we show 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-G(2) correlate with decreased Rag expression and Rag relocalization to pericentromeric heterochromatin. 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.

V(D)J recombination in B and T cells is required for the generation of receptors with a broad spectrum of specificity to foreign antigen. A total number of three immunoglobulin (Ig) and four T cell receptor (Tcr) loci can be targeted by the recombinase enzyme (RAG1/2) in a defined series of recombination events, which drive the progression of B and T cell development. This process is regulated at multiple levels to ensure lineage specific, ordered rearrangement and allelic exclusion [1]. One key component of this is modulation of chromatin looping and locus contraction, which is important in bringing widely separated gene segments into close contact with each other to enable synapse formation for lineage and stage specific V gene rearrangement [2,3,4(*),5,6(*)]. Recent studies provide new insight into looping and its role in these processes. In this review we focus on the contribution of the 11 zinc finger nuclear protein, CTCF, in mediating loop formation and conformational changes that are important for the regulation of Ig and Tcr rearrangement.

Acute lymphoblastic leukemia (ALL) results from malignancy of lymphoid progenitor cells and affects both adults and children. It is the most common childhood cancer and despite advances in treatment that now result in above 80% cure rates for children, considerable problems remain with current therapies. These include low cure rates in children with high-risk ALL, the complexity and toxic effects of current treatments and the stubbornly poor prognosis of adults with ALL (with a less than 40% long-term survival rate). ALL can be initiated by errors in V(D)J recombination, a process which creates multiple combinations of receptor genes in B and T lymphocytes in order to target foreign pathogens. During recombination, DNA double strand breaks are introduced at the borders of two selected gene segments and repair creates a new gene combination. Chromosomal translocations can occur both by mis-targeting of the RAG recombinase proteins at cryptic recombination signal sequences, as well as illegitimate repair with a DNA break generated by alternative cellular processes. Our work has unveiled a remarkable and previously unknown control step which acts during V(D)J recombination to protect genome stability. We demonstrated that the key DNA damage response factor and serine/threonine kinase ATM (ataxia telangiectasia mutated), prevents aberrant cleavage during V(D)J recombination. In wild-type cells only one of the two homologous Ig alleles is normally cleaved at a time, whereas in ATM deficient cells both Ig alleles can be cleaved simultaneously and chromosomal aberrations are detected on two Ig alleles (Hewitt et al., Nature Immunology 2009). Our recent work has been directed at understanding how ATM and the RAG recombinase (RAG1 and RAG2 proteins) cooperate to implement allelic control of V(D)J recombination. We hypothesized that ATM may act to control RAG cleavage, either directly or indirectly. To test this, we investigated developing B cells from coreRAG1 or coreRAG2 mice; these are the shortest active forms of the proteins but lack regulatory domains. We assessed mono- versus biallelic cleavage using H2AX to indicate repair foci and as a read-out for DNA double strand breaks. In pre-B cells from coreRAG1 mice, H2AX foci were predominantly colocalized with only one Igk allele per cell, which indicates monoallelic cleavage. In contrast, biallelic colocalization was highly significant in coreRAG2 expressing pre-B cells. We have analyzed RAG2 mutants to precisely identify the protein motifs that regulate cleavage. These were introduced into Rag2-deficient pre-B cell lines by retroviral infection. Expression of a coreRAG2 construct in these cells recapitulated the biallelic cleavage seen in ex-vivo isolated pre-B cells. We found that mutation of putative serine/threonine phosphorylation motifs also resulted in significant biallelic colocalization of H2AX with Igk alleles. This suggests that RAG2 performs a similar function to ATM in restricting simultaneous RAG cleavage on the antigen receptor loci and may indeed cooperate with serine/threonine kinases. These data provide a mechanistic basis for the similarities in chromosomal abnormalities between Atm^-/- and coreRag2/p53^-/- lymphomas and will contribute to our understanding of why recurrent chromosomal translocations and lymphoid cancers arise in ATM-deficient mice and humans

Nonbiased V gene usage for V(D)J joining is essential for providing an optimal immune system, but no cis-acting sequence with this function has been uncovered. We previously identified a recombination silencer and heterochromatin targeting element in the Vkappa-Jkappa intervening sequence of germline Igkappa transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igkappa repertoire with markedly decreased distal and enhanced proximal Vkappa gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vkappa-Jkappa intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vkappa genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igkappa in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igkappa and IgH loci and still exhibited IgL isotype exclusion, we concluded that stable localization at pericentromeric heterochromatin is neither necessary nor sufficient for the establishment or maintenance of allelic exclusion. Hence, Sis is a novel multifunctional element that specifies repertoire and heterochromatin localization to Ig genes

T cell fate is associated with mutually exclusive expression of CD4 or CD8 in helper and cytotoxic T cells, respectively. How expression of one locus is temporally coordinated with repression of the other has been a long-standing enigma, though we know RUNX transcription factors activate the Cd8 locus, silence the Cd4 locus, and repress the Zbtb7b locus (encoding the transcription factor ThPOK), which is required for CD4 expression. Here we found that nuclear organization was altered by interplay among members of this transcription factor circuitry: RUNX binding mediated association of Cd4 and Cd8 whereas ThPOK binding kept the loci apart. Moreover, targeted deletions within Cd4 modulated CD8 expression and pericentromeric repositioning of Cd8. Communication between Cd4 and Cd8 thus appears to enable long-range epigenetic regulation to ensure that expression of one excludes the other in mature CD4 or CD8 single-positive (SP) cells

Misrepair of DNA double-strand breaks produced by the V(D)J recombinase (the RAG1/RAG2 proteins) at immunoglobulin (Ig) and T cell receptor (Tcr) loci has been implicated in pathogenesis of lymphoid malignancies in humans and in mice. Defects in DNA damage response factors such as ataxia telangiectasia mutated (ATM) protein and combined deficiencies in classical non-homologous end joining and p53 predispose to RAG-initiated genomic rearrangements and lymphomagenesis. Although we showed previously that RAG1/RAG2 shepherd the broken DNA ends to classical non-homologous end joining for proper repair, roles for the RAG proteins in preserving genomic stability remain poorly defined. Here we show that the RAG2 carboxy (C) terminus, although dispensable for recombination, is critical for maintaining genomic stability. Thymocytes from 'core' Rag2 homozygotes (Rag2(c/c) mice) show dramatic disruption of Tcralpha/delta locus integrity. Furthermore, all Rag2(c/c) p53(-/-) mice, unlike Rag1(c/c) p53(-/-) and p53(-/-) animals, rapidly develop thymic lymphomas bearing complex chromosomal translocations, amplifications and deletions involving the Tcralpha/delta and Igh loci. We also find these features in lymphomas from Atm(-/-) mice. We show that, like ATM-deficiency, core RAG2 severely destabilizes the RAG post-cleavage complex. These results reveal a novel genome guardian role for RAG2 and suggest that similar 'end release/end persistence' mechanisms underlie genomic instability and lymphomagenesis in Rag2(c/c) p53(-/-) and Atm(-/-) mice