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Articles by Jane A. Skok in JoVE

 JoVE Biology

Combined Immunofluorescence and DNA FISH on 3D-preserved Interphase Nuclei to Study Changes in 3D Nuclear Organization

1Department of Pathology, New York University School of Medicine, 2New York University Center for Health Informatics and Bioinformatics, 3NYU Cancer Institute, 4Department of Pathology and Yale Cancer Center, Yale University School of Medicine


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Here we describe a protocol for simultaneous detection of histone modifications by immunofluorescence and DNA sequences by DNA FISH followed by 3D microscopy and analyses (3D immuno-DNA FISH).

Other articles by Jane A. Skok on PubMed

Subnuclear Compartmentalization of Immunoglobulin Loci During Lymphocyte Development

Immunoglobulin (Ig) loci are selectively activated for transcription and rearrangement during B lymphocyte development. Using fluorescence in situ hybridization, we show that Ig heavy (H) and Igkappa loci are preferentially positioned at the nuclear periphery in hematopoietic progenitors and pro-T cells but are centrally configured in pro-B nuclei. The inactive loci at the periphery do not associate with centromeric heterochromatin. Upon localization away from the nuclear periphery in pro-B cells, the IgH locus appears to undergo large-scale compaction. We suggest that subnuclear positioning represents a novel means of regulating transcription and recombination of IgH and Igkappa loci during lymphocyte development.

Locus 'decontraction' and Centromeric Recruitment Contribute to Allelic Exclusion of the Immunoglobulin Heavy-chain Gene

Allelic exclusion of immunoglobulin genes ensures the expression of a single antibody molecule in B cells through mostly unknown mechanisms. Large-scale contraction of the immunoglobulin heavy-chain (Igh) locus facilitates rearrangements between Igh variable (V(H)) and diversity gene segments in pro-B cells. Here we show that these long-range interactions are mediated by 'looping' of individual Igh subdomains. The Igk locus also underwent contraction by looping in small pre-B and immature B cells, demonstrating that immunoglobulin loci are in a contracted state in rearranging cells. Successful Igh recombination induced the rapid reversal of locus contraction in response to pre-B cell receptor signaling, which physically separated the distal V(H) genes from the proximal Igh domain, thus preventing further rearrangements. In the absence of locus contraction, only the four most proximal V(H) genes escaped allelic exclusion in immature mu-transgenic B lymphocytes. Pre-B cell receptor signaling also led to rapid repositioning of one Igh allele to repressive centromeric domains in response to downregulation of interleukin 7 signaling. These data link both locus 'decontraction' and centromeric recruitment to the establishment of allelic exclusion at the Igh locus.

Transcriptional Regulation in Early B Cell Development

Transcription factors and signalling molecules are important for both lineage commitment and lineage-specific regulation. The B cell specification factor Pax5 plays a dual role in B lineage commitment. Simultaneously, it potentiates and limits lineage choice by activating genes that are required for the B cell program while repressing lineage-inappropriate genes; more than 100 of the latter have now been identified. In this context, repression of the tyrosine kinase Flt3 has been shown to be essential for B lineage commitment. Regulation of antigen receptor recombination constitutes another level at which lineage specificity is determined, and the identification of two factors, E47 and FOXP1, which regulate the activity of the recombinase enzymes in B lineage cells, provides insight into the mechanisms that determine this. New information regarding the control of ordered recombination and allelic exclusion comes from studies of cis-acting elements within the Ig loci.

Reversible Contraction by Looping of the Tcra and Tcrb Loci in Rearranging Thymocytes

Reversible contraction of immunoglobulin loci juxtaposes the variable (V) genes next to the (diversity)-joining-constant ((D)JC) gene domain, thus facilitating V-(D)J recombination. Here we show that the T cell receptor beta (Tcrb) and T cell receptor alphadelta (Tcra-Tcrd) loci also underwent long-range interactions by looping in double-negative and double-positive thymocytes, respectively. Contraction of the Tcrb and Tcra loci occurred in rearranging thymocytes and was reversed at the next developmental stage. Decontraction of the Tcrb locus probably prevented further V(beta)-DJ(beta) rearrangements in double-positive thymocytes by separating the V(beta) genes from the DJC(beta) domain. In most double-negative cells, one Tcrb allele was recruited to pericentromeric heterochromatin. Such allelic positioning may facilitate asynchronous V(beta)-DJ(beta) recombination. Hence, pericentromeric recruitment and locus 'decontraction' seem to contribute to the initiation and maintenance of allelic exclusion at the Tcrb locus.

Dynamic Changes in Accessibility, Nuclear Positioning, Recombination, and Transcription at the Ig Kappa Locus

The 3-megabase Igkappa locus undergoes differentially controlled nuclear positioning events and chromatin structural changes during the course of B cell development. The temporal association of chromatin structural changes, transcription, and recombination at the Igkappa locus was determined in a murine pre-B cell line that can be induced to recombine at the Igkappa locus and in ex vivo-cultured murine pre-B cells. Additionally, the timing of nuclear positioning relative to the temporal order of chromatin structural changes and recombination and transcription was determined. We demonstrate that before induction, the Igkappa locus was poised for recombination; both alleles were in a contracted state, and the enrichment of histone modifications and germline transcripts of specific Vkappa genes were observed. Histone modifications of the Vkappa genes did not vary upon induction but the levels of modifications correlated with the levels of germline Vkappa gene transcripts and recombination. Upon induction, but before VkappaJkappa recombination, centromeric recruitment of single Igkappa alleles occurred. DNase I sensitivity of the entire locus increased gradually over the course of differentiation while the enrichment of histone modifications downstream of the Vkappa genes was increased in the silencer regions upstream of Jkappa1, within the Igkappa sterile transcript, the kappa constant region, the Ekappai and Ekappa3' enhancers, and the recombining sequence. The ex vivo pre-B cells showed similar patterns of histone modifications across the locus except at the Vkappa genes. In this study, H3 acetylation correlated with levels of germline transcripts while H3 methylation correlated with levels of recombination.

Regulation of Immunoglobulin Light-chain Recombination by the Transcription Factor IRF-4 and the Attenuation of Interleukin-7 Signaling

Productive rearrangement of the immunoglobulin heavy-chain locus triggers a major developmental checkpoint that promotes limited clonal expansion of pre-B cells, thereby culminating in cell-cycle arrest and rearrangement of light-chain loci. By using Irf4-/-Irf8-/- pre-B cells, we demonstrated that two pathways converge to synergistically drive light-chain rearrangement, but not simply as a consequence of cell-cycle exit. One pathway was directly dependent on transcription factor IRF-4, whose expression was elevated by pre-B cell receptor signaling. IRF-4 targeted the immunoglobulin 3'Ekappa and Elambda enhancers and positioned a kappa allele away from pericentromeric heterochromatin. The other pathway was triggered by attenuation of IL-7 signaling and activated the iEkappa enhancer via binding of the transcription factor E2A. IRF-4 also regulated expression of chemokine receptor Cxcr4 and promoted migration of pre-B cells in response to the chemokine ligand CXCL12. We propose that IRF-4 coordinates the two pathways regulating light-chain recombination by positioning pre-B cells away from IL-7-expressing stromal cells.

Association Between the Igk and Igh Immunoglobulin Loci Mediated by the 3' Igk Enhancer Induces 'decontraction' of the Igh Locus in Pre-B Cells

Variable-(diversity)-joining (V(D)J) recombination at loci encoding the immunoglobulin heavy chain (Igh) and immunoglobulin light chain (Igk) takes place sequentially during successive stages in B cell development. Using three-dimensional DNA fluorescence in situ hybridization, here we identify a lineage-specific and stage-specific interchromosomal association between these two loci that marks the transition between Igh and Igk recombination. Colocalization occurred between pericentromerically located alleles in pre-B cells and was mediated by the 3' Igk enhancer. Deletion of this regulatory element prevented association of the Igh and Igk loci, inhibited pericentromeric recruitment and locus 'decontraction' of an Igh allele, and resulted in greater distal rearrangement of the gene encoding the variable heavy-chain region. Our data indicate involvement of the Igk locus and its 3' enhancer in directing the Igh locus to a repressive nuclear subcompartment and inducing the Igh locus to decontract.

Silencing and Nuclear Repositioning of the Lambda5 Gene Locus at the Pre-B Cell Stage Requires Aiolos and OBF-1

The chromatin regulator Aiolos and the transcriptional coactivator OBF-1 have been implicated in regulating aspects of B cell maturation and activation. Mice lacking either of these factors have a largely normal early B cell development. However, when both factors are eliminated simultaneously a block is uncovered at the transition between pre-B and immature B cells, indicating that these proteins exert a critical function in developing B lymphocytes. In mice deficient for Aiolos and OBF-1, the numbers of immature B cells are reduced, small pre-BII cells are increased and a significant impairment in immunoglobulin light chain DNA rearrangement is observed. We identified genes whose expression is deregulated in the pre-B cell compartment of these mice. In particular, we found that components of the pre-BCR, such as the surrogate light chain genes lambda5 and VpreB, fail to be efficiently silenced in double-mutant mice. Strikingly, developmentally regulated nuclear repositioning of the lambda5 gene is impaired in pre-B cells lacking OBF-1 and Aiolos. These studies uncover a novel role for OBF-1 and Aiolos in controlling the transcription and nuclear organization of genes involved in pre-BCR function.

RAG-1 and ATM Coordinate Monoallelic Recombination and Nuclear Positioning of Immunoglobulin Loci

Coordinated recombination of homologous antigen receptor loci is thought to be important for allelic exclusion. Here we show that homologous immunoglobulin alleles pair in a stage-specific way that mirrors the recombination patterns of these loci. The frequency of homologous immunoglobulin pairing was much lower in the absence of the RAG-1-RAG-2 recombinase and was restored in Rag1-/- developing B cells with a transgene expressing a RAG-1 active-site mutant that supported DNA binding but not cleavage. The introduction of DNA breaks on one immunoglobulin allele induced ATM-dependent repositioning of the other allele to pericentromeric heterochromatin. ATM activated by the cleaved allele acts in trans on the uncleaved allele to prevent biallelic recombination and chromosome breaks or translocations.

Chromosome Dynamics and the Regulation of V(D)J Recombination

Perhaps no process has provided more insight into the fine manipulation of locus accessibility than antigen receptor rearrangement. V(D)J recombination is carried out by the lymphoid-specific recombination-activating (RAG 1 and 2) proteins and the non-homologous end joining machinery; yet, it occurs only at specific loci (or portions of loci) during specific developmental stages. This spatiotemporal restriction of recombination is achieved through precise alterations in locus accessibility. In this article, we discuss the work of our laboratory in elucidating how nuclear sublocalization, chromosome conformation, and locus interactions contribute to regulating this complex process. We also discuss what is known about how key factors in B-cell development (such as the ubiquitously expressed helix loop helix protein E2A, the B-cell specific transcription factors EBF1 and Pax5, and the interleukin-7 cytokine signaling pathway) exert their effects through changes in nuclear dynamics.

Epigenetic Regulation of V(D)J Recombination

Chromosome breaks are dangerous business, carrying the risk of loss of genetic information or, even worse, misrepair of the break, leading to outcomes such as dicentric chromosomes or oncogenic translocations. Yet V(D)J recombination, a process that breaks, rearranges and repairs chromosomes, is crucial to the development of the adaptive immune system, for it gives B- and T-cells the capacity to generate a virtually unlimited repertoire of antigen receptor proteins to combat an equally vast array of antigens. To minimize the risks inherent in chromosomal breakage, V(D)J recombination is carefully orchestrated at multiple levels, ranging from DNA sequence requirements all the way up to chromatin conformation and nuclear architecture. In the present chapter we introduce various regulatory controls, with an emphasis on epigenetic mechanisms and recent work that has begun to elucidate their interdependence.

It Takes Two: Communication Between Homologous Alleles Preserves Genomic Stability During V(D)J Recombination

Chromosome pairing is involved in X chromosome inactivation, a classic instance of monoallelic gene expression. Antigen receptor genes are also monoallelically expressed ("allelically excluded") by B and T lymphocytes, and we asked whether pairing contributed to the regulation of V(D)J recombination at these loci. We found that homologous immunoglobulin (Ig) alleles pair up during recombination. Homologous Ig pairing is substantially reduced in the absence of the RAG1/RAG2 recombinase, but a transgene expressing an active site RAG1 mutant (which binds but does not cleave DNA) rescues pairing in Rag1(-/-) developing B cells. RAG-mediated cleavage on one allele induces the other allele to relocate to pericentromeric heterochromatin (PCH), likely to ensure that only one allele is cut at a time. This relocation to PCH requires the DNA damage sensor ATM (ataxia telengiectasia mutated). 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 chromosomal translocations.

V(D)J Recombination: a Paradigm for Studying Chromosome Interactions in Mammalian Cells

The RAG2 C Terminus Suppresses Genomic Instability and Lymphomagenesis

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 Tcrα/δ 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 Tcrα/δ 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.

RUNX Transcription Factor-mediated Association of Cd4 and Cd8 Enables Coordinate Gene Regulation

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.

A Multifunctional Element in the Mouse Igκ Locus That Specifies Repertoire and Ig Loci Subnuclear Location

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 Vκ-Jκ intervening sequence of germline Igκ transgenes, which we termed Sis. We now have generated Sis knockout mice in the endogenous locus. Intriguingly, Sis(-/-) mice exhibit a skewed Igκ repertoire with markedly decreased distal and enhanced proximal Vκ gene usage for primary rearrangement, which is associated with reduced occupancy of Ikaros and CCCTC-binding factor in the Vκ-Jκ intervening sequence in pre-B cells, proteins believed to be responsible for dampening the recombination of nearby Vκ genes and altering higher-order chromatin looping. Furthermore, monoallelic heterochromatin localization is significantly reduced in Sis(-/-) mice for Igκ in cis and IgH loci in trans in pre-B cells. Because Sis(-/-) mice still allelically excluded Igκ 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.

Equal Opportunity for All

The Role of CTCF in Regulating V(D)J 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. 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.

IL-7 Functionally Segregates the Pro-B Cell Stage by Regulating Transcription of Recombination Mediators Across Cell Cycle

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.

Close Proximity to Igh is a Contributing Factor to AID-mediated Translocations

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.

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