Regulation of immunoglobulin heavy-chain gene rearrangements

Summary: Regulated assembly of antigen receptor gene segments to produce functional genes is a hallmark of B‐ and T‐lymphocyte development. The immunoglobulin heavy‐chain (IgH) and T‐cell receptor β‐chain genes rearrange first in B and T lineages, respectively. Both loci require two recombination events to assemble functional genes; D‐to‐J recombination occurs first followed by V‐to‐DJ recombination. Despite similarities in overall rearrangement patterns, each locus has unique regulatory features. Here, we review the characteristics of IgH gene rearrangements such as developmental timing, deletion versus inversion, D_H gene segment utilization, ordered recombination of V_H gene segments, and feedback inhibition of rearrangement in pre‐B cells. We summarize chromatin structural features of the locus before and during recombination and, wherever possible, incorporate these into working hypotheses for understanding regulation of IgH gene recombination. The picture emerges that the IgH locus is activated in discrete, independently regulated domains. A domain encompassing D_H and J_H gene segments is activated first, within which recombination is initiated. V_H genes are activated subsequently and, in part, by interleukin‐7. These observations lead to a model for feedback inhibition of IgH rearrangements.     

Pax5 induces V-to-DJ rearrangements and locus contraction of the immunoglobulin heavy-chain gene

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.     

Comparison of different polymerase chain reaction-based approaches for clonality assessment of immunoglobulin heavy-chain gene rearrangements in B-cell neoplasia.

Several frequently applied polymerase chain reaction strategies for analysis of immunoglobulin heavy-chain gene rearrangements were compared by analyzing 70 B-cell lymphoproliferative disorders and 24 reactive lymphoid lesions. Southern blot analysis was used as the "gold standard" for clonality assessment. For polymerase chain reaction analysis, primers directed against framework (FR) 3 (FR3-A and FR3-B), FR2, and FR1 of the variable gene segments and against joining gene segments of the immunoglobulin heavy-chain gene were used. Polymerase chain reaction products were analyzed by high-resolution fingerprinting analysis using radiolabeled nucleotides, gene scanning using fluorochrome-labeled primers, and heteroduplex analysis. All of the assays generated polyclonal patterns in the reactive tissues. The sensitivity in detecting monoclonality as defined by Southern blotting varied between 60% (heteroduplex analysis with FR3 primers) and 77% (high-resolution fingerprinting analysis with FR2 primers). Comparison of the three FR3 assays revealed that gene scanning had the highest sensitivity (69%), probably because it could detect small aberrant monoclonal amplicons. False-negative results were especially frequent in follicular center lymphoma (n = 20), but also in diffuse large B-cell lymphoma (n = 18), both renowned for having mutated variable gene segments, potentially leading to primer mismatching. For example, in follicular center lymphoma, the FR3, FR2, and FR1 region primer sets detected clonality in only 35 to 40, 65, and 50%, respectively. Combining these techniques, 17 (85%) of 20 follicular center lymphoma samples showed monoclonality. Therefore, especially in follicular center lymphoma, diffuse large B-cell lymphoma, and, to a lesser extent, in other lymphomas, multiple variable gene segment primer sets must be used for a reliable assessment of clonality. Our results also suggest that gene scanning is somewhat more sensitive than other read-out systems. Heteroduplex analysis, however, is a reliable alternative within a diagnostic setting, avoiding the use of radioactivity or expensive automated sequencing equipment and fluorochrome-labeled (oligo)nucleotides.     

Retroviral interleukin 5 gene transfer into interleukin 5-dependent growing cell lines results in autocrine growth and tumorigenicity

Two interleukin 5 (IL5)-specific retroviral expression vectors have been constructed containing the neomycin gene as selectable marker and either the mouse IL5 cDNA region or the rat genomic IL5 gene under the control of the thymidine kinase promoter. High viral titer supernatants derived from the transfected or infected packaging cell line psi 2 were used to infect the two cell lines B13 and T88M whose growth is dependent on exogenous IL 5. Infection resulted in G418 resistance and IL 5-independent growth with a high frequency. Clones were established which secrete between 2 and greater than 1000 U IL5. The proliferation of the IL5 autocrine growing cells could be inhibited by an antibody directed against the IL5 receptor indicating that they grow as a result of the endogenously produced IL5. Regardless of the amount of IL5 they produced, all of the clones were highly tumorigenic in nucle mice. The phenotype of the tumors was indistinguishable from that of the injected cells. T88M or B13 cells infected with a control virus neither produced IL5, nor became factor independent, nor produced tumors. Together, the IL5 gene transfer and expression into IL5-dependent growing cells are in accordance with the "autocrine growth" hypothesis and contrast analogous experiments with IL4.     

Diversity in DNA rearrangements and in RNA expressions of immunoglobulin gene on common variable immunodeficiency.

Six heterogeneous common variable immunodeficiency (CVID) patients were analysed for germ-line DNA, DNA rearrangements, and RNA expressions of immunoglobulin (Ig) gene by Southern or northern blotting using appropriate probes. We detected no polymorphism in neutrophil DNA hybridized to a C mu and a C gamma probe. In three patients, both serum Ig and Ig-bearing cells were scarcely detected, and by northern hybridization methods, neither mu mRNA, gamma mRNA, alpha mRNA nor kappa mRNA was detected. However, one Epstein-Barr virus-transformed B lymphoblastoid cell line (LCL) of these three patients was different from the germ line in the region of JH, C gamma, and C kappa, and expressed mu mRNA at a higher level. The B cell defects of these three patients lay on the B cell maturation stage similar to X-linked agammaglobulinaemia (XLA). In two others among the six CVID patients, serum IgM and IgM-bearing cells were detected to a certain degree, and by northern hybridization, mu mRNA was detected at a lower level, but neither mu mRNA, alpha mRNA, nor kappa mRNA was detected. One LCL of these two patients could express mu mRNA at the normal level. In the last patient, the serum IgM was normal, serum IgG and IgA were somewhat low, Ig-bearing cells were normal, mu mRNA and kappa mRNA were detected at the normal level, and gamma mRNA and alpha mRNA were detected at a lower level. The defect of this patient affected the class switch stage. These results showed that primary B cell defects in CVID occurred at several B cell differentiation stages which could be classified by expression of the Ig gene, and at the degree of clonal diversity in the B cell repertoire. Furthermore, this study provides support for the idea that the CVID defect is related to a more generalized cellular function, such as regulating the proliferation and/or clonal expansion of cells of the B lymphoid lineage.     

转录因子MAZ对c1orf109基因转录表达调控的体外研究

目的:本文旨在研究转录因子Myc相关锌指蛋白(MAZ)在体外对人类1号染色体开放阅读框109(c1orf109)基因的转录表达调控。方法:体外条件下,采用凝胶电泳迁移实验筛选c1orf109基因启动子区MAZ的结合位点,并利用本室构建的由c1orf109启动子驱动的增强型绿色荧光蛋白报告载体与MAZ和转录因子特化蛋白1(Sp1)的表达质粒共转染He La细胞,24 h后,用激光共聚焦显微镜和流式细胞术检测c1orf109基因的转录表达情况。结果:c1orf109启动子区可与MAZ结合,且有与Sp1共享的结合位点;MAZ与Sp1皆可抑制c1orf109的转录表达,且Sp1的抑制作用大于MAZ(P0.05)。结论:MAZ与Sp1两个转录因子共同调控c1orf109基因在生理及病理条件的表达,且调控方向一致。这种冗余机制调控方式的存在提示该基因的精确表达调控对于细胞行使其生物学功能可能具有重要意义     

Regulation of gene expression dynamics during developmental transitions by the Ikaros transcription factor

The DNA-binding protein Ikaros is a potent tumor suppressor and hematopoietic regulator. However, the mechanisms by which Ikaros functions remain poorly understood, due in part to its atypical DNA-binding properties and partnership with the poorly understood Mi-2/NuRD complex. In this study, we analyzed five sequential stages of thymocyte development in a mouse strain containing a targeted deletion of Ikaros zinc finger 4, which exhibits a select subset of abnormalities observed in Ikaros-null mice. By examining thymopoiesis in vivo and in vitro, diverse abnormalities were observed at each developmental stage. RNA sequencing revealed that each stage is characterized by the misregulation of a limited number of genes, with a strong preference for stage-specific rather than lineage-specific genes. Strikingly, individual genes rarely exhibited Ikaros dependence at all stages. Instead, a consistent feature of the aberrantly expressed genes was a reduced magnitude of expression level change during developmental transitions. These results, combined with analyses of the interplay between Ikaros loss of function and Notch signaling, suggest that Ikaros may not be a conventional activator or repressor of defined sets of genes. Instead, a primary function may be to sharpen the dynamic range of gene expression changes during developmental transitions via atypical molecular mechanisms that remain undefined.     

DNA polymorphism 5' to the JH region of the human immunoglobulin heavy chain gene and immunoglobulin gene rearrangements in leukemia

DNA samples of two patients with acute myeloblastic leukemia were noted to have a variant band on digestion with HindIII and hybridization to a probe for the joining region of the immunoglobulin (Ig) heavy chain gene (JH). Further analysis showed that the variant band represented a constitutional DNA polymorphism rather than an Ig gene rearrangement. The HindIII fragment detected by the JH probe includes a highly polymorphic region 5' to the Ig heavy chain locus, and hence variant germline bands may be seen on autoradiography that can be mistaken for Ig gene rearrangements. The use of several different restriction enzymes and the analysis of the constitutional DNA allows a clear distinction to be made between somatic gene rearrangements and DNA polymorphisms.     

Regulation of immunoglobulin light chain gene rearrangements during early B cell development in the human.

Southern blot analyses of immunoglobulin light chain gene rearrangements in human leukemias and myelomas indicated that lambda loci in kappa-producing cells are largely unrearranged while kappa loci in lambda producers are often rearranged and inactivated by rearrangements of the kappa-deleting element (KDE). For a systematic analysis of the regulation of light chain rearrangements during early B cell development in normal human B cells also considering functionality of the rearrangements, we used FACS-sorted single naive kappa- and lambda-expressing B cells from peripheral blood of healthy humans. V(kappa)J(kappa) and V(lambda)J(lambda) joints and rearrangements involving the KDE were amplified simultaneously from single cells and sequenced. Whereas only 2 - 3 % of kappa-expressing cells carry V(lambda)J(lambda) joints, nearly all lambda-expressing cells have rearranged kappa loci and indeed carry V(kappa)J(kappa) joints. The V(kappa)J(kappa) joints in lambda-expressing cells exhibit preferential J(kappa)4 and J(kappa)5 over J(kappa)1 and J(kappa)2 usage compared to kappa-expressing cells. Thirty percent of the V(kappa)J(kappa) joints in lambda producers are rearranged in-frame. These data indicate extensive sequential V(kappa)-J(kappa) rearrangements and inactivation of functional V(kappa)J(kappa) joints in lambda-expressing cells, presumably before V(lambda)J(lambda) joining.