PU.1 is a member of the ets family of transcription factors and is expressed exclusively in cells of the hematopoietic lineage. Mice homozygous for a disruption in the PU.1 DNA binding domain are born alive but die of severe septicemia within 48 h. The analysis of these neonates revealed a lack of mature macrophages, neutrophils, B cells and T cells, although erythrocytes and megakaryocytes were present. The absence of lymphoid commitment and development in null mice was not absolute, since mice maintained on antibiotics began to develop normal appearing T cells 3–5 days after birth. In contrast, mature B cells remained undetectable in these older mice. Within the myeloid lineage, despite a lack of macrophages in the older antibiotic‐treated animals, a few cells with the characteristics of neutrophils began to appear by day 3. While the PU.1 protein appears not to be essential for myeloid and lymphoid lineage commitment, it is absolutely required for the normal differentiation of B cells and macrophages.

Compaction and looping of the ∼2.5-Mb Igh locus during V(D)J rearrangement is essential to allow all V H genes to be brought in proximity with D H -J H segments to create a diverse antibody repertoire, but the proteins directly responsible for this are unknown. Because CCCTC-binding factor (CTCF) has been demonstrated to be involved in long-range chromosomal interactions, we hypothesized that CTCF may promote the contraction of the Igh locus. ChIP sequencing was performed on pro-B cells, revealing colocalization of CTCF and Rad21 binding at ∼60 sites throughout the V H region and 2 other sites within the Igh locus. These numerous CTCF/cohesin sites potentially form the bases of the multiloop rosette structures at the Igh locus that compact during Ig heavy chain rearrangement. To test whether CTCF was involved in locus compaction, we used 3D-FISH to measure compaction in pro-B cells transduced with CTCF shRNA retroviruses. Reduction of CTCF binding resulted in a decrease in Igh locus compaction. Long-range interactions within the Igh locus were measured with the chromosomal conformation capture assay, revealing direct interactions between CTCF sites 5′ of DFL16 and the 3′ regulatory region, and also the intronic enhancer (Eμ), creating a D H -J H -Eμ-C H domain. Knockdown of CTCF also resulted in the increase of antisense transcription throughout the D H region and parts of the V H locus, suggesting a widespread regulatory role for CTCF. Together, our findings demonstrate that CTCF plays an important role in the 3D structure of the Igh locus and in the regulation of antisense germline transcription and that it contributes to the compaction of the Igh locus.A ntigen receptors are created through the highly regulated lineage-specific process of V(D)J recombination, creating a diverse repertoire of Ig and T-cell receptors. The generation of the mouse Ig heavy chain in pro-B cells begins with D H -to-J H rearrangement on both alleles, followed by V H -to-D H J H rearrangement. In order for the >100 functional murine V H genes spread across ∼2.5 Mb to gain access to the single D-J rearrangement on that allele, the Igh locus undergoes contraction and looping during the pro-B-cell stage of B-cell differentiation (1-5). By measuring spatial distances between 11 small probes spread throughout the Igh locus, Jhunjhunwala et al. (2) demonstrated that distal and proximal V H genes were approximately equidistant from the D genes specifically at the pro-B-cell stage when the V H genes are rearranging. Computational as well as geometrical approaches have suggested that the locus is organized into rosette-like clusters of loops that compact during rearrangement. Several proteins have been reported to influence Igh locus compaction, including Pax5, YY1, and Ikaros (5-7). These proteins and others, such as Ezh2 (8), are also necessary for the rearrangement of distal V H genes but not proximal V H genes. This is most likely a consequence of the lack of locus compaction in the absence of these proteins. How all these proteins funct...

SummaryMuch of T and B lymphocyte receptor diversity derives from the addition of nontemplated N regions at thejunctions of receptor gene elements, although fetal T cells expressing y/b receptors lack N regions. I have sequenced immunoglobulin H chain variable regions of PCR-amplified DNA and cDNA from fetal and newborn mouse liver and spleen cells. These sequences showed an absence of N regions . Only 1/87 DNA sequences and 17/146 RNA sequences contained N regions, in striking contrast to adult Ig sequences. These data show that N region insertion is a developmentally regulated process in B cells as well as in T cells, and demonstrate that receptor diversity in neonatal B cells is limited by the absence of N regions as well as by biased usage of Vh genes.T he initial diversity in Igs is afforded by the large number of V, D, and J gene elements used to create the L and H chain variable regions (1-5). In addition, there is considerably more diversity generated at the junctions of these gene segments. Thisjunctional diversity is created by two mechanisms: (a) deletion of a variable number of nucleotides from the ends of the coding segments, presumably by exonuclease activity; and (b) subsequently, addition of a variable number of nucleotides to the V-D and D-J junctions of the H chain before ligation ofthe DNA (6) . These latter nucleotides, called N regions, are nontemplated and are thought to be added by the enzyme terminal deoxynucleotidyl transferase (TdT)t (7-10) . functional diversity increases the antibody repertoire by several orders of magnitude. The N regions and all of the D region are in CDR 3 and thus contribute significantly to the antigen-binding site.Rearrangement ofIg coding elements in B cell precursors is a highly regulated process (11). At the Ig H chain (IgH) locus, D to J rearrangements (with possible N region addition at the DJ junctions) precede V to D J rearrangement (again with possible N region addition at the VD junction) during pro-B cell development . After successful IgH rearrangement, L chain V to J rearrangement takes place at the pre-B cell stage. L chain VJjunctions do not contain N regions, which correlates with the absence of TdT in most pre-B cells (12).Other features of Ig rearrangement show developmental regulation ; e.g., B cells generated during fetal and neonatal life overexpress Jh-proximal Vh genes (13-15) . In contrast, ' Abbreviations used in thus paper. A-MuLV, Abelson-MuLV; RSS, recombination signal sequence; TdT, terminal deoxynucleotidyl transferase . adult B cells show random Vh utilization (15-19), although one recent study suggested that IgH rearrangement may be biased towards theJh-proximal Vh genes throughout life (20).Developmental regulation of N region addition at the IgH locus has not been observed in the B cell lineage, but it is intriguing that fetal T lymphocytes expressing TCR-y/b, and some of their adult progeny, lack N regions (21-23) . Extrapolating this observation to other lymphocytes is not obvious, however, since these y/b T cells express only ...

Contraction of the large Diversity in the Ig Ab repertoire is achieved through V(D)J recombination, a lineage-specific process that is highly regulated during B cell development. IgH rearrangement in pro-B cells begins with D H to J H rearrangement followed by rearrangement of a V H gene segment to D H J H . The IgH is assembled before the L chains, and Ig rearrangement precedes Ig rearrangement. Strict regulation of accessibility allows for the lineage-specific and developmentally ordered rearrangement of V(D)J gene segments. The mechanisms controlling V H to D H J H rearrangement are exceptionally complex because Ͼ100 functional murine V H genes span a 2.5 Mb region. Likewise, the 96 functional V genes cover 3.1 Mb. The question arises of how all the V genes acquire access to the small J cluster (Ͻ2 kb) in either the Igh or Ig loci. Three-dimensional fluorescence in situ hybridization studies have demonstrated that the Igh and Ig loci undergo significant contraction to position gene segments in proximity for rearrangement at the appropriate time for rearrangement (1-4). In pro-B cells, the V H genes are brought into close proximity to D H gene segments via multiple loop structures, thus facilitating rearrangement through these long-range chromosomal interactions (4). The contraction and looping of the loci raises the question of which nuclear factors could be controlling these interactions. One potential nuclear protein that participates in long-range chromosomal interactions is CCCTC-binding factor (CTCF). 3CTCF is a ubiquitously expressed 11-zinc finger nuclear protein that is associated with all known vertebrate insulators (5). Chromatin insulators or boundary elements create distinct chromosomal domains preventing outside influences on the insulated region (5). The enhancer-blocking activity of CTCF prevents the interactions between enhancers and promoters separated by the insulator. Global mapping of CTCF binding has shown that CTCF binds in regions that could separate different chromosomal domains, consistent with the idea that CTCF may insulate the spread of repressive chromatin modifications into neighboring active domains (6 -8). One of the ways that CTCF may function as an insulator and regulate gene expression is through the facilitation of long-range intrachromosomal and interchromosomal looping (9 -11).CTCF has been reported to associate with a number of factors including YY1 (12), the chromodomain helicase CHD8 (13), and cohesin subunits (14 -17). Cohesin proteins have an established role of facilitating cohesion of sister chromatids during cell division (18)

The E2A gene products, E12 and E47, are required for proper B cell development. Mice lacking the E2A gene products generate only a very small number of B220+ cells, which lack immunoglobulin DJ(H) rearrangements. We have now generated mice expressing either E12 or E47. B cell development in mice expressing E12 but lacking E47 is perturbed at the pro-B cell stage, and these mice lack IgM+B220+ B cells in both bone marrow and spleen. IgM+B220+ B cells can be detected, albeit at significantly reduced levels, in the bone marrow and spleen of mice lacking E12. Ectopic expression of both E12 and E47 in a null mutant background shows that E12 and E47 act in concert to promote B lineage development. Taken together, the data indicate that both E12 and E47 allow commitment to the B cell lineage and act synergistically to promote B lymphocyte maturation.

Immunoglobulin (Ig) and T cell receptor (TCR) genes are assembled during lymphocyte maturation through site-specific V(D)J recombination events. Here we show that E2A proteins act in concert with RAG1 and RAG2 to activate Ig VK1J but not Iglambda VlambdaIII-Jlambda1 rearrangement in an embryonic kidney cell line. In contrast, EBF, but not E2A, promotes VlambdaIII-Jlambda1 recombination. Either E2A or EBF activate IgH DH4J recombination but not V(D)J rearrangement. The Ig coding joints are diverse, contain nucleotide deletions, and lack N nucleotide additions. IgK VJ recombination requires the presence of the E2A transactivation domains. These observations indicate that in nonlymphoid cells a diverse Ig repertoire can be generated by the mere expression of the V(D)J recombinase and a transcriptional regulator.

Antigen receptor locus V(D)J recombination requires interactions between widely separated variable (V), diversity (D), and joining (J) gene segments, but the mechanisms that generate these interactions are not well understood. Here we assessed mechanisms that direct developmental stage-specific long-distance interactions at the Tcra/Tcrd locus. The Tcra/Tcrd locus recombines Tcrd gene segments in CD4 − CD8 − double-negative thymocytes and Tcra gene segments in CD4 + CD8 + double-positive thymocytes. Initial V α -to-J α recombination occurs within a chromosomal domain that displays a contracted conformation in both thymocyte subsets. We used chromosome conformation capture to demonstrate that the Tcra enhancer (E α ) interacts directly with V α and J α gene segments distributed across this domain, specifically in double-positive thymocytes. Moreover, E α promotes interactions between these V α and J α segments that should facilitate their synapsis. We found that the CCCTC-binding factor (CTCF) binds to E α and to many locus promoters, biases E α to interact with these promoters, and is required for efficient V α -J α recombination. Our data indicate that E α and CTCF cooperate to create a developmentally regulated chromatin hub that supports V α -J α synapsis and recombination.T-cell development | T-cell receptor | thymus T and B cells produce diverse antigen receptors through the recombination of variable (V), diversity (D), and joining (J) gene segments at the T-cell receptor (Tcra, Tcrb, Tcrg, and Tcrd) and Ig (Igh, Igκ, and Igλ) loci. This V(D)J recombination is initiated by the lymphoid-specific recombination-activating gene-1 (RAG-1) and RAG-2 proteins, which recognize the recombination signal sequences (RSSs) that flank all V, D, and J gene segments and then cleave the DNA between the RSSs and the adjacent coding gene segments (1). A critical feature of the reaction is the assembly of a synaptic complex composed of two RSSs before the generation of RAG-dependent DNA doublestrand breaks (DSBs). As such, lineage-and developmental stagespecific V(D)J recombination events can be regulated not only by changes in RAG protein expression and RSS accessibility to RAG proteins but also by the ability of those RSSs to undergo synapsis (2).Conformational changes of antigen receptor loci are believed to support V(D)J recombination events because they can bring distant RSSs into proximity and therefore increase the probability of RSS synapsis (2, 3). Studies using 3D-FISH have demonstrated that lineage-and development stage-specific locus contraction marks the recombination windows at antigen receptor loci (3). For example, the 3-Mb Igh locus contracts specifically in pro-B cells to support V H -to-D H J H recombination (4-7). This contracted conformation brings distal and proximal V H segments, which are separated by megabases in the linear DNA sequence, to the vicinity of the D H J H cluster, presumably allowing all V H segments a similar opportunity for recombination (8). In addition, the mapping of Igh locus DNA-DNA...

Noncoding sense and antisense germ-line transcription within the Ig heavy chain locus precedes V(D)J recombination and has been proposed to be associated with Igh locus accessibility, although its precise role remains elusive. However, no global analysis of germline transcription throughout the Igh locus has been done. Therefore, we performed directional RNA-seq, demonstrating the locations and extent of both sense and antisense transcription throughout the Igh locus. Surprisingly, the majority of antisense transcripts are localized around two Pax5-activated intergenic repeat (PAIR) elements in the distal IghV region. Importantly, long-distance loops measured by chromosome conformation capture (3C) are observed between these two active PAIR promoters and Eμ, the start site of Iμ germ-line transcription, in a lineage-and stage-specific manner, even though this antisense transcription is Eμ-independent. YY1 −/− pro-B cells are greatly impaired in distal V H gene rearrangement and Igh locus compaction, and we demonstrate that YY1 deficiency greatly reduces antisense transcription and PAIR-Eμ interactions. ChIP-seq shows high level YY1 binding only at Eμ, but low levels near some antisense promoters. PAIR-Eμ interactions are not disrupted by DRB, which blocks transcription elongation without disrupting transcription factories once they are established, but the looping is reduced after heat-shock treatment, which disrupts transcription factories. We propose that transcription-mediated interactions, most likely at transcription factories, initially compact the Igh locus, bringing distal V H genes close to the DJ H rearrangement which is adjacent to Eμ. Therefore, we hypothesize that one key role of noncoding germ-line transcription is to facilitate locus compaction, allowing distal V H genes to undergo efficient rearrangement.A ntigen receptors in lymphocytes are assembled in the highly regulated lineage-specific process of V(D)J recombination, which creates a diverse repertoire of Ig and T-cell receptors. In each precursor lymphocyte, one each of the many V, D, and J gene segments at the appropriate receptor loci are juxtaposed to create a V(D)J exon encoding the variable region of the antigen receptor. In B-lineage progenitors, rearrangement occurs first at the Ig heavy chain (Igh) locus, where D H to J H rearrangement occurs first on both alleles, followed by V H to DJ H rearrangement (1). After successful rearrangement of the Igh locus, rearrangement at the Igk light chain locus begins. These successive stages of rearrangement have been proposed to be regulated by differential accessibility of different portions of the loci at the appropriate time for rearrangement (2). Early indications of this stage-specific and lineage-specific accessibility came from the observation that unrearranged gene segments underwent noncoding transcription at the stage immediately preceding their rearrangement (3, 4).The murine Igh locus spans ∼2.8 Mb, of which ∼2.4 Mb contains the 195 V H gene segments (5). The V H genes are divided i...