Pax5-deficient progenitor B (pro-B) cells are thought to be severely defective for recombination of all immunoglobulin heavy chain (IgH) V gene segments, but the mechanism by which Pax5 regulates this process has not been defined. To address this issue, we have examined the assembly of the IgH locus in Pax5-deficient pro-B cells and find, unexpectedly, that 3 IgH V gene segments, which lie closest to the D-J-Cµ region, recombine efficiently, but progressively more distal V gene segments recombine progressively less efficiently. Histone acetylation and germ-line transcription correlate strongly with an open or an accessible chromatin structure thought to be permissive for V(D)J recombination, and defects in recombination are typically accompanied by deficits in these processes. We were therefore surprised to observe that distal V H gene segments in Pax5−/− pro-B cells exhibit no defect in these measures of accessibility. The finding of transcribed, histone acetylated gene segments that fail to recombine suggests that a Pax5-dependent regulatory mechanism is required in addition to standard constraints of accessibility to control V H gene recombination. During lymphocyte development, V(D)J recombination assembles the variable region of antigen receptor loci from individual V, D, and J gene segments. This reaction is initiated by binding of the RAG1 and RAG2 proteins to conserved recombination signal sequences (RSSs) that flank each of these gene segments. After binding to two RSS elements, the RAG1/RAG2 complex introduces DNA double-strand breaks between the RSSs and their flanking gene segments. Importantly, the two RSSs must first be brought into close proximity and assembled into a synaptic complex before cleavage can occur (Fugmann et al. 2000;Gellert 2002), ensuring that the recombining partners are juxtaposed before potentially dangerous DNA breaks are created. After DNA cleavage, the RAG proteins and DNA repair factors process and join the ends to complete the recombination reaction.Temporal and developmental specificity of V(D)J recombination are achieved in part by regulating the accessibility of RSSs to the recombination machinery (Yancopoulos and Alt 1985;Hesslein and Schatz 2001). Although the underlying structural basis of accessibility has not been determined, V(D)J recombination of the antigen receptor loci is tightly correlated with locus changes such as nuclease sensitivity, germ-line (sterile) transcription, DNA demethylation, and histone acetylation (Krangel 2001). Cis-acting transcriptional elements found in the antigen receptor loci have been implicated in the regulation of gene rearrangement, but the mechanisms by which these elements control V(D)J recombination are unknown.Bone marrow B-cell development has been divided into distinct stages based on cell size and surface markers by Hardy (Hardy et al. 1991) and Rolink and Melchers (Rolink et al. 1994). The earliest B lineage cells (Hardy fractions A and B or Rolink and Melcher pro/pre-B-I cells) express c-kit and B220 and actively undergo D...
Immunoglobulin heavy chain rearrangement (V H -to-DJ H ) occurs only in B cells, suggesting it is inhibited in other lineages. Here we found that in the mouse V H locus, methylation of lysine 9 on histone H3 (H3-K9), a mark of inactive chromatin, was present in non-B lineage cells but was absent in B cells. As others have shown that H3-K9 methylation can inhibit V(D)J recombination on engineered substrates, our data support the idea that H3-K9 methylation inhibits endogenous V H -to-DJ H recombination. We also show that Pax5, a transcription factor required for B cell commitment, is necessary and sufficient for the removal of H3-K9 methylation in the V H locus and provide evidence that one function of Pax5 is to remove this inhibitory modification by a mechanism of histone exchange, thus allowing B cell-specific V H -to-DJ H recombination.During hematopoiesis, transcription factors initiate and maintain lineage-specific commitment 1 . In B and T lymphocytes, the unique variable (V), diversity (D) and joining (J) (V(D)J) recombination of immunoglobulin and T cell receptor (TCR) gene segments provides critical developmental checkpoints as well as diverse, clonotypic antigen recognition capability 2 . All immunoglobulin and TCR loci use a common recombinase machinery, including proteins encoded by the recombination activating genes Rag1 and Rag2 that recognize and cut at common DNA recognition elements called recombination signal sequences (RSSs). However, V(D)J recombination proceeds with strict T lineage-B lineage specificity and developmentally determined order. The mechanisms responsible for these aspects of V(D)J regulation remain poorly understood.
Pax5 encodes BSAP, a member of the paired box domain transcription factors, whose expression is restricted to B lymphocyte lineage cells. Pax5−/− mice have a developmental arrest of the B cell lineage at the pro-B cell stage. We show here that Pax5−/− mice are severely osteopenic, missing 60% of their bone mass. The osteopenia can be accounted for by a >100% increase in the number of osteoclasts in bone measured histomorphometrically. This is not due to a lack of B cells, because other strains of B cell-deficient mice do not exhibit this phenotype. There was no difference in the number of osteoclasts produced in vitro by wild-type and Pax5−/− bone marrow cells. In contrast, spleen cells from Pax5−/− mice produce as much as five times the number of osteoclasts as control spleen cells. Culture of Pax5−/− spleen cells yields a population of adherent cells that grow spontaneously in culture without added growth factors for >4 wk. These cells have a monocyte phenotype, produce large numbers of osteoclasts when induced in vitro, and therefore are highly enriched in osteoclast precursors. These data demonstrate a previously unsuspected connection between B cell and osteoclast development and a key role for Pax5 in the control of osteoclast development.
The molecules that regulate bone cell development, particularly at the early stages of development, are only partially known. Data are accumulating that indicate a complex relationship exists between B cells and bone cell differentiation. Although the exact nature of this relationship is still evolving, it takes at least two forms. First, factors that regulate B-cell growth and development have striking effects on osteoclast and osteoblast lineage cells. Similarly, factors that regulate bone cell development influence B-cell maturation. Second, a series of transcription factors required for B-cell differentiation have been identified, and these factors function in a developmentally ordered circuit. These transcription factors have unpredicted, pronounced, and non-overlapping effects on osteoblast and/or osteoclast development. These data indicate that at least a regulatory relationship exists between B lymphopoiesis, osteoclastogenesis, and osteoblastogenesis.
The 3 regulatory region (3 RR) of the murine immunoglobulin heavy chain (IgH) locus contains multiple DNase I-hypersensitive (hs) sites. Proximal sites hs3A, hs1.2, and hs3B are located in an extensive palindromic region and together with hs4 are associated with enhancers involved in the expression and class switch recombination of IgH genes. Distal hs5, -6, and -7 sites located downstream of hs4 comprise a potential insulator for the IgH locus. In pro-B cells, hs4 to -7 are associated with marks of active chromatin, while hs3A, hs1.2, and hs3B are not. Our analysis of DNA methylation-sensitive restriction sites of the 3 RR has revealed a similar modular pattern in pro-B cells; hs4 to -7 sites are unmethylated, while the palindromic region is methylated. This modular pattern of DNA methylation and histone modifications appears to be determined by at least two factors: the B-cell-specific transcription factor Pax5 and linker histone H1. In pre-B cells, a region beginning downstream of hs4 and extending into hs5 showed evidence of allele-specific demethylation associated with the expressed heavy chain allele. Palindromic enhancers become demethylated later in B-cell differentiation, in B and plasma cells.The immunoglobulin heavy chain (IgH) gene locus undergoes a panoply of regulated DNA events during B-cell development, including VDJ joining to construct variable-region genes; class switch recombination (CSR) to achieve the production of non-IgM classes, such as IgG, IgE, and IgA; and somatic hypermutation (reviewed in reference 23). The identification of the specific cis regulatory regions that regulate these processes is of considerable interest. Candidates include promoters that are locally associated with V and D segments and also with I regions upstream of each constant-region gene, except for C␦. In addition, regulators capable of acting at considerable distances from their target sequences flank the C H genes (Fig. 1A). The first of these to be identified was the ϳ1-kb intronic enhancer E, which is situated in the intron between J H and C Targeted deletion studies with mice have revealed that E is important for VDJ joining (1,29,32,36).A 3Ј regulatory region (3Ј RR) extends ϳ35 kb downstream of the C␣ gene and includes multiple DNase I-hypersensitive (hs) sites. The murine 3Ј RR contains modules (Fig. 1A) that are developmentally regulated, as assessed by DNase I hypersensitivity, reporter assays, and engagement with active chromatin marks. The three most proximal sites, i.e., hs3A, hs1.2, and hs3B, form a 25-kb palindromic region by virtue of almost identical hs3A and hs3B sequences in inverted orientation at either end of this segment and by intervening families of locally repetitive sequences flanking the central hs1.2 enhancer (4, 34). hs3A, hs1.2, and hs3B are DNase I hypersensitive at later stages of B-cell development, i.e., in plasma cells (14,20). A second unit contains hs4, which appears to be active beginning in pro-B cells and continuing throughout B-cell development (14,20,24). A third 3Ј RR m...
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