Jane A. Skok

Nonequivalent nuclear location of immunoglobulin alleles in B lymphocytes

Individual B lymphocytes normally express immunoglobulin (Ig) proteins derived from single Ig heavy chain (H) and light chain (L) alleles. Allelic exclusion ensures monoallelic expression of Ig genes by each B cell to maintain single receptor specificity. Here we provide evidence that at later stages of B cell development, additional mechanisms may contribute to prioritizing expression of single IgH and IgL alleles. Fluorescent in situ hybridization analysis of primary splenic B cells isolated from normal and genetically manipulated mice showed that endogenous IgH, κ and λ alleles localized to different subnuclear environments after activation and had differential expression patterns. However, this differential recruitment and expression of Ig alleles was not typically seen among transformed B cell lines. These data raise the possibility that epigenetic factors help maintain the monoallelic expression of Ig.

CTCF establishes discrete functional chromatin domains at the Hox clusters during differentiation

Keeping repressed genes repressed Hox genes confer positional identity to cells and tissues. Maintaining precise spatial patterns of Hox gene expression is vital during metazoan development. The transcriptional repressor CTCF is involved in the regulation of chromatin architecture. Narendra et al. show that a CTCF protein binding site insulates regions of active and repressed Hox gene expression from each other. This protects heterochromatin containing repressed Hox genes from the encroaching spread of active chromatin. The CTCF protein appears to organize the active and repressed chromatin regions into distinct architectural domains. Science, this issue p. 1017 A DNA binding protein insulates and protects repressed topological domains in the genome from adjacent active regions. Polycomb and Trithorax group proteins encode the epigenetic memory of cellular positional identity by establishing inheritable domains of repressive and active chromatin within the Hox clusters. Here we demonstrate that the CCCTC-binding factor (CTCF) functions to insulate these adjacent yet antagonistic chromatin domains during embryonic stem cell differentiation into cervical motor neurons. Deletion of CTCF binding sites within the Hox clusters results in the expansion of active chromatin into the repressive domain. CTCF functions as an insulator by organizing Hox clusters into spatially disjoint domains. Ablation of CTCF binding disrupts topological boundaries such that caudal Hox genes leave the repressed domain and become subject to transcriptional activation. Hence, CTCF is required to insulate facultative heterochromatin from impinging euchromatin to produce discrete positional identities.

Epigenetic ontogeny of the Igk locus during B cell development.

To become accessible for rearrangement, the immunoglobulin κ locus must undergo a series of epigenetic changes. This begins in pro–B cells with the relocation of both immunoglobulin κ alleles from the periphery to the center of the nucleus. In pre–B cells, one allele became preferentially packaged into an active chromatin structure characterized by histone acetylation and methylation of histone H3 lysine 4, while the other allele was recruited to heterochromatin, where it was associated with heterochromatin protein-γ and Ikaros. These events in cis made only one allele accessible to trans-acting factors, such as RelB, which mediated DNA demethylation, to facilitate rearrangement. These results suggest that early B lymphoid epigenetic changes generate differential structures that serve as the basis for allelic exclusion.

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.

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 (Rag2c/c mice) show dramatic disruption of Tcrα/δ locus integrity. Furthermore, all Rag2c/c p53−/− mice, unlike Rag1c/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 Rag2c/c p53−/− and Atm−/− mice.

The pre‐B‐cell receptor induces silencing of VpreB and λ5 transcription

The pre‐B‐cell receptor (pre‐BCR), composed of Ig heavy and surrogate light chain (SLC), signals pre‐BII‐cell proliferative expansion. We have investigated whether the pre‐BCR also signals downregulation of the SLC genes (VpreB and λ5), thereby limiting this expansion. We demonstrate that, as BM cells progress from the pre‐BI to large pre‐BII‐cell stage, there is a shift from bi‐ to mono‐allelic λ5 transcription, while the second allele is silenced in small pre‐BII cells. A VpreB1‐promoter‐driven transgene shows the same pattern, therefore suggesting that VpreB1 is similarly regulated and thereby defines the promoter as a target for transcriptional silencing. Analyses of pre‐BCR‐deficient mice show a temporal delay in λ5 downregulation, thereby demonstrating that the pre‐BCR is essential for monoallelic silencing at the large pre‐BII‐cell stage. Our data also suggest that SLP‐65 is one of the signaling components important for this process. Furthermore, the VpreB1/λ5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing.

C-Terminal Src Kinase Controls Acute Inflammation and Granulocyte Adhesion

To establish whether the widely expressed regulator of Src family kinases Csk contributes to the control of acute inflammation in vivo, we inactivated csk in granulocytes by conditional mutagenesis (Cre/loxP). Mutant mice (Csk-GEcre) developed acute multifocal inflammation in skin and lung. Animals were protected from the disease in a microbiologically controlled environment, but remained hypersensitive to LPS-induced shock. Csk-deficient granulocytes showed enhanced spontaneous and ligand-induced degranulation with hyperinduction of integrins. This hyperresponsiveness was associated with hyperadhesion and impaired migratory responses in vitro. Hyperphosphorylation of key signaling proteins such as Syk and Paxillin in mutant granulocytes further supported breakdown of the activation threshold set by Csk. By enforcing the need for ligand engagement Csk thus prevents premature granulocyte recruitment while supporting the motility of stimulated cells through negative regulation of cell adhesion.

Chromosome dynamics and the regulation of V(D)J recombination

Summary:  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.

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.

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.