Faculty Bibliography

The role of the transcription factor Yin Yang 1 (YY1) in development is largely unknown. Here we show that specific ablation of YY1 in mouse B cells caused a defect in somatic rearrangement in the immunoglobulin heavy-chain (IgH) locus and a block in the progenitor-B-to-precursor-B-cell transition, which was partially rescued by a prerearranged IgH transgene. Three-dimensional DNA fluorescence in situ hybridization analysis revealed an important function for YY1 in IgH locus contraction, a process indispensable for distal V(H) to D(H)J(H) recombination. We provide evidence that YY1 binds the intronic Ei mu enhancer within the IgH locus, consistent with a direct role for YY1 in V(H)D(H)J(H) recombination. These findings identified YY1 as a critical regulator of early B-cell development

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

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

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 lambda5), 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 lambda5 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 lambda5 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/lambda5 alleles undergo dynamic changes with respect to nuclear positioning and heterochromatin association, thereby providing a possible mechanism for their transcriptional silencing

To become accessible for rearrangement, the immunoglobulin kappa locus must undergo a series of epigenetic changes. This begins in pro-B cells with the relocation of both immunoglobulin kappa 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-gamma 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

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

The subnuclear location and chromatin state of the immunoglobulin heavy-chain (IgH) locus have been implicated in the control of VDJ recombination. VH-to-DJH rearrangement of distal, but not proximal V(H) genes, furthermore, depends on the B-lineage commitment factor Pax5 (BSAP). He e we demonstrate that ectopic Pax5 expression from the Ikaros promote induces proximal rather than distal VH-DJH rearrangements in Ik(Pax5/+) thymocytes, thus recapitulating the loss-of-function phenotype of Pax5-/- pro-B cells. The phenotypic similarities of both cell types include (1) chromatin accessibility of distal VH genes in the absence of VH-DJH rearrangements, (2) expression of the B-cell-specific regulator EBF, (3) central location of IgH alleles within the nucleus, and (4) physical separation of distal VH genes from proximal segments in an extended IgH locus. Reconstitution of Pax5 expression in Pax5-/- pro-B cells induced large-scale contraction and distal VH-DJH rearrangements of the IgH locus. Hence, VH-DJH recombination is regulated in two steps during early B-lymphopoiesis. The IgH locus is first repositioned from its default location at the nuclear periphery toward the center of the nucleus, which facilitates proximal VH-DJH recombination. Pax5 subsequently activates locus contraction and distal VH-DJH rearrangements in collaboration with an unknown factor that is present in pro-B cells, but absent in thymocytes

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

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

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, kappa and lambda 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