Germline Transcription Research Articles

SPI-C and STAT6 can cooperate to stimulate IgE germline transcription

SPI-C is a novel ETS protein that is expressed in B lymphocytes. No target gene for SPI-C has so far been defined. We have performed a yeast two-hybrid screen using SPI-C as bait in order to further analyze the functional role of this orphan transcription factor. We found that SPI-C interacted specifically with the C-terminus of STAT6 in yeast. By co-immunoprecipitation in transfected COS7 cells the physical interaction between SPI-C and STAT6 was confirmed. Furthermore, this protein–protein interaction is functional since we could demonstrate that SPI-C and STAT6 stimulated IL4 induced Iε transcription synergistically but only when both proteins bound to DNA. Thus, a protein interaction between SPI-C and STAT6 is the basis for a novel mechanism for regulation of IL4 induced gene expression.

Class switch recombination in selective IgA-deficient subjects.

Selective IgA deficiency is a common immunodeficiency in Caucasians, but the molecular basis of the disorder remains elusive. To address this issue we examined the molecular events leading to IgA production. Naive IgD positive B cells were purified from four donors with IgA deficiency and four control donors, all Caucasians. Stimulation of B cells from IgA-deficient donors with the cytokines transforming growth factor (TGF)-beta, interferon (IFN)-gamma or interleukin (IL)-10 in the presence of anti-CD40 antibodies showed reduced expression of both activation-induced cytidine deaminase (AID) and alpha germline transcripts (GLT) compared to controls. It was possible, however, to induce AID and alpha GLT when stimulating the cells with anti-CD40 antibody and TGF-beta in the combination with IL-10. Moreover, in anti-CD40 antibody-stimulated cultures, addition of IL-10 or IL-10 + TGF-beta in combination, induced IgA production, albeit lower than found in B cells from controls. The B cells from the IgA-deficient subjects were less effective in differentiating into CD138(+) X-box binding protein 1 (XBP-1)(+) plasma cells when stimulated with TGF-beta, IFN-gamma or IL-10. Interestingly, when adding IL-4 to TGF-beta alone or in combination with IL-10, the immunoglobulin production in B cells from IgA-deficient donors was comparable with those of normal controls. These data show that in healthy subjects in vitro IgA production can be up-regulated by addition of IL-10 to CD40-stimulated B cells, whereas a similar B cell differentiation does not occur in IgA-deficient subjects. Addition of IL-4, however, reverts this abnormality.

Bruton’s Tyrosine Kinase and SLP-65 Regulate Pre-B Cell Differentiation and the Induction of Ig Light Chain Gene Rearrangement

Bruton's tyrosine kinase (Btk) and the adapter protein SLP-65 (Src homology 2 domain-containing leukocyte-specific phosphoprotein of 65 kDa) transmit precursor BCR (pre-BCR) signals that are essential for efficient developmental progression of large cycling into small resting pre-B cells. We show that Btk- and SLP-65-deficient pre-B cells have a specific defect in Ig lambda L chain germline transcription. In Btk/SLP-65 double-deficient pre-B cells, both kappa and lambda germline transcripts are severely reduced. Although these observations point to an important role for Btk and SLP-65 in the initiation of L chain gene rearrangement, the possibility remained that these signaling molecules are only required for termination of pre-B cell proliferation or for pre-B cell survival, whereby differentiation and L chain rearrangement is subsequently initiated in a Btk/SLP-65-independent fashion. Because transgenic expression of the antiapoptotic protein Bcl-2 did not rescue the developmental arrest of Btk/SLP-65 double-deficient pre-B cells, we conclude that defective L chain opening in Btk/SLP-65-deficient small resting pre-B cells is not due to their reduced survival. Next, we analyzed transgenic mice expressing the constitutively active Btk mutant E41K. The expression of E41K-Btk in Ig H chain-negative pro-B cells induced 1) surface marker changes that signify cellular differentiation, including down-regulation of surrogate L chain and up-regulation of CD2, CD25, and MHC class II; and 2) premature rearrangement and expression of kappa and lambda light chains. These findings demonstrate that Btk and SLP-65 transmit signals that induce cellular maturation and Ig L chain rearrangement independently of their role in termination of pre-B cell expansion.

Molecular basis of common variable immunodeficiency

Common variable immunodeficiency (CVID) is the most prevalent human primary immunodeficiency requiring medical attention. Until recently, the only known genetic defect specific to CVID was the inducible costimulatory receptor (ICOS) deficiency, which accounts for less than 1% of the patients. Recently, mutations in the TNF receptor family member transmembrane activator and calcium-modulator and cyclophilin ligand interactor (TACI), which mediates isotype switching in B cells, were found to be present in 10% to 20% of patients with CVID. Mutations in TACI were also found in relatives of patients with CVID who had IgA deficiency (IgAD), as well as in a patient with isolated IgAD. In the majority of patients described to date, only one TACI allele is mutated, showing an autosomal dominant transmission of the disease. B cells from individuals with TACI mutations did not produce IgG and IgA in response to the TACI ligand a proliferation-inducing ligand (APRIL), probably reflecting impaired isotype switching. These results suggest that TACI mutations can lead to CVID and IgAD.

Germ line transcription in mice bearing neor gene downstream of I 3 exon in the Ig heavy chain locus

Class switch recombination (CSR) is preceded by germ line transcription that initiates from promoters upstream of switch (S) sequences and terminates downstream of associated constant genes. Previous work showed that germ line transcripts and their processing are required for CSR and that germ line transcription is regulated in a major part by a regulatory region located downstream of the Ig heavy chain locus. This long-range, polarized effect can be disturbed by inserting an expressed neomycine resistance (neo(r)) gene. To contribute to a better understanding of the mechanism of such a long-distance regulation, we generated knock-in mice in which a neo(r) gene was inserted downstream of Igamma3 exon leaving intact all the necessary elements for germ line transcription and splicing. We show that the expressed neo(r) gene interferes with transcription initiation from Igamma3, and that it impairs but does not block S recombination to Cgamma3. Moreover, we show for the first time that the neo(r) gene provides through chimeric neo(r)-Cgamma3 transcripts the necessary elements for splicing of germ line transcripts by activating two novel cryptic splice sites, one in the coding region of the intronless neo(r) gene and the other in the Igamma3-Cgamma3 intron.

Regulation of IgH Gene Assembly: Role of the Intronic Enhancer and 5′DQ52 Region in Targeting DHJH Recombination

The assembly of Ag receptor genes by V(D)J recombination is regulated by transcriptional promoters and enhancers which control chromatin accessibility at Ig and TCR gene segments to the RAG-1/RAG-2 recombinase complex. Paradoxically, germline deletions of the IgH enhancer (Emu) only modestly reduce D(H)-->J(H) rearrangements when assessed in peripheral B cells. However, deletion of Emu severely impairs recombination of V(H) gene segments, which are located over 100 kb away. We now test two alternative explanations for the minimal effect of Emu deletions on primary D(H)-->J(H) rearrangement: 1) Accessibility at the D(H)J(H) cluster is controlled by a redundant cis-element in the absence of Emu. One candidate for this element lies 5' to D(Q52) (PD(Q52)) and exhibits promoter/enhancer activity in pre-B cells. 2) In contrast to endpoint B cells, D(H)-->J(H) recombination may be significantly impaired in pro-B cells from enhancer-deficient mice. To elucidate the roles of PD(Q52) and Emu in the regulation of IgH locus accessibility, we generated mice with targeted deletions of these elements. We report that the defined PD(Q52) promoter is dispensable for germline transcription and recombination of the D(H)J(H) cluster. In contrast, we demonstrate that Emu directly regulates accessibility of the D(H)J(H) region. These findings reveal a significant role for Emu in the control mechanisms that activate IgH gene assembly and suggest that impaired V(H)-->D(H)J(H) rearrangement in enhancer-deficient cells may be a downstream consequence of the primary block in D(H)-->J(H) recombination.

E2A and IRF-4/Pip Promote Chromatin Modification and Transcription of the Immunoglobulin κ Locus in Pre-B Cells

The immunoglobulin kappa light chain (Igkappa) locus is regulated in a lineage- and stage-specific manner during B-cell development. The highly restricted timing of V to J gene recombination at the pre-B-cell stage is under the control of two enhancers, the intronic enhancer (kappaEi) and the 3' enhancer (kappaE3'), flanking the constant exon. E2A transcription factors have been indicated to be directly involved in the regulation of Igkappa locus activation. In this study, we utilize E2A-deficient pre-B cells to directly investigate the mechanism of E2A-mediated Igkappa activation. We demonstrate that Igkappa germ line transcription is severely impaired and recombination is blocked in the absence of E2A. Reconstitution of E2A-/- pre-B cells with inducible human E2A (E47R) is sufficient to promote chromatin modification of Igkappa and rescue Igkappa germ line transcription and Jkappa gene recombinase accessibility. Furthermore, we show that increased E2A recruitment to kappaEi and kappaE3' correlates with activation of Igkappa in pre-B cells and that recruitment of E2A to kappaE3' is in part dependent on the transcription factor IRF-4. Inhibition of IRF-4 expression in pre-B cells leads to a significant reduction of Igkappa germ line transcription and enhancer acetylation. In the absence of E2A, increased IRF-4 expression is not sufficient to promote Igkappa enhancer chromatin modification or transcription, suggesting that the sequential involvement of IRF-4 and E2A is necessary for the activation of the Igkappa locus. Finally, we provide genetic evidence in the mouse that E2A gene dosage can influence the development of pre-B cells during the phase of Igkappa gene activation.

AID-dependent histone acetylation is detected in immunoglobulin S regions

Class switch recombination (CSR) is regulated by the expression of activation-induced deaminase (AID) and germline transcripts (GLTs). AID-dependent double-strand breaks (DSBs) are introduced into switch (S) regions and stimulate CSR. Although histone acetylation (Ac) has been well documented in transcription regulation, its role in DNA damage repair remains largely unexplored. The 1B4.B6 B cell line and normal splenic B cells were activated to undergo CSR and analyzed for histone Ac by chromatin immunoprecipitation (ChIP). A detailed study of the Iγ3-Sγ3-Cγ3 locus demonstrated that acetylated histones are focused to the Iγ3 exon and the Sγ3 region but not to the intergenic areas. Histone H3 Ac is strongly correlated with GLT expression at four S regions, whereas H4 Ac was better associated with B cell activation and AID expression. To more directly examine the relationship between H4 Ac and AID, LPS-activated AID KO and WT B cells were analyzed and express comparable levels of GLTs. In AID-deficient B cells, both histones H3 and H4 are reduced where H4 is more severely affected as compared with WT cells. Our findings raise the intriguing possibility that histone H4 Ac at S regions is a marker for chromatin modifications associated with DSB repair during CSR.

The extract of syngeneic keratinocytes enhances IgE production from BALB/c mouse splenic lymphocytes in vitro

The increase of serum IgE levels is closely associated with atopic dermatitis. We have previously revealed that cellular extract of PAM212 cells (PAM-extract), BALB/c mouse keratinocyte cell line, induced a remarkable increase of serum IgE levels, in vivo, when subcutaneously injected into BALB/c mice. However, precise mechanism of IgE-increasing activity was unclear. To elucidate the mechanism of IgE-increase in sera of BALB/c mice induced by PAM-extract, we explored the direct influence of PAM-extract on immunoglobulin production and class-switching in the culture of splenic lymphocytes and purified B-cells, in vitro. Splenic lymphocytes or purified B-cells obtained from BALB/c mice were cultured with various combinations of IL-4, anti-CD40 antibody, and PAM-extract for 7 days. IgE and IgG concentrations of culture supernatants were measured by ELISA. Epsilon germ-line transcriptions were assessed by RT-PCR from the cultured cells. IgE and IgG concentrations in culture supernatant of splenic lymphocytes were increased by an addition of PAM-extract in the presence of both IL-4 and anti-CD40 antibody. Epsilon germ-line transcript was also induced in parallel to the increase of IgE production. Similar results were obtained when purified B-cells were employed in stead of whole splenic lymphocytes. The enhancement of IgE production in vitro was also observed, when splenic lymphocytes of CBAj mouse were cultured with cellular extract of KCMH-1 cells, CBAj mouse keratinocyte cell line. The cellular extract of keratinocyte promotes immunoglobulin class-switching to IgE and IgE production from mouse splenic B-cells in an IL-4- and CD40-stimuli-dependent manner. Such enhancement may account for the increase of serum IgE in patients with dermatitis in association with a Th2 microenvironment.

Immunoglobulin class-switch recombination occurs in mantle cell lymphomas

Mantle cell lymphoma (MCL) is an IgM-expressing B cell lymphoma that originates from naive B cells and responds poorly to chemotherapy. We show here that several MCLs harbour isotype-switched subclones. Similar to the situation in normal B cells, in vitro stimulation of MCL cell lines with CD40 ligand (CD40L) and interleukin-4 induced expression of activation-induced cytidine deaminase (AID) and germline transcription at the immunoglobulin heavy chain gene locus. Additionally, the occurrence of switch-circle transcripts and mature IgG transcripts after stimulation indicated ongoing class-switch recombination in mantle cell lymphoma cell lines. Furthermore, stimulation of primary MCL cells in vitro induced expression of class-switched IgG mRNA in the tumour cells. Our data indicate that mantle cell lymphomas have retained the ability to undergo class-switch recombination if appropriate stimuli, such as the CD40 ligand, are provided.

Oral administration of pulverized Konjac glucomannan prevents the increase of plasma immunoglobulin E and immunoglobulin G levels induced by the injection of syngeneic keratinocyte extracts in BALB/c mice

Immunoglobulin (Ig) E plays a key role in the pathogenesis of atopic diseases, such as asthma, atopic dermatitis and allergic rhinitis. Oral administration of pulverized Konjac glucomannan (PKGM) has recently been demonstrated to prevent both plasma IgE elevation and developing dermatitis in NC/Nga mice, a model of atopic dermatitis. To clarify the direct effect of PKGM on the increase of plasma IgE, we employed the system of BALB/c mouse that increases IgE level without developing dermatitis in response to continuous injection of the extract of syngeneic keratinocytes, PAM 212 cells (PAM extract). Three weeks after the start of feeding with either control or PKGM diet, mice were injected subcutaneously with PAM extract bi-weekly for 10 weeks. The levels of plasma Igs were measured by enzyme-linked immunosorbent assay every 2 weeks after the injection. The levels of epsilon germline transcription and the amounts of mRNA for IL-4, IFN-gamma, GATA-3 and T bet gene in the spleen were evaluated by real-time RT-PCR at the end of the experiment. On the one hand, PKGM prevented the increase of plasma IgE and IgG (IgG1, IgG2b) induced by PAM extract, and on the other hand, it enhanced the levels of plasma IgG3. However, it did not affect the level of plasma IgM. PKGM also reduced the levels of plasma ovalbumin (OVA)-specific IgE in OVA-sensitized mice. Moreover, PKGM attenuated the induction of epsilon germline transcription and expression levels of mRNA for IL-4, IFN-gamma and GATA-3 in the spleen of PAM extract-injected mice. PKGM also attenuated the induction of epsilon germline transcription and mRNA for IFN-gamma and T bet in the spleen of phosphate-buffered saline-injected control mice. These results suggested that oral administration of PKGM prevents the elevation of plasma IgE by suppressing IgE class switching in B cells and/or the commitment development of naive lymphocytes to both T-helper type 1 (Th1) and Th2.

Stat1-Dependent Synergistic Activation of T-bet for IgG2a Production during Early Stage of B Cell Activation

During the adaptive phase of an immune response, naive B cells receive multiple signals to become activated. Among them are the engagement of the B cell Ag receptor and stimulation by cytokines. Specifically for an anti-microbial response, the recognition of viral or bacterial Ags by the BCR and the stimulation of IFN-gamma result in the predominant production of IgG2a. The T-bet protein has been shown to be required for class switching to IgG2a. In this report we further investigated the regulation of T-bet gene expression during the early stage of B cell activation. We show that there is a striking synergistic activation of T-bet in primary B cells when both the BCR and IFN-gamma signaling pathways are activated. The synergistic activation of T-bet correlates with a 100% increase in the number of B cells that produce IgG2a. This transcription synergy on T-bet is transient in the first 24 h of B cell activation. Furthermore, we demonstrate that the synergistic activation of T-bet is dependent on Stat1 and that Stat1 is required for the IgG2a germline transcription and the production of IgG2a in response to the simultaneous signaling of BCR and IFN-gamma. Finally, we show that Stat1 directly regulates the expression of T-bet by binding to the T-bet promoter. These results reveal the mechanism of regulation of T-bet expression and uncover a novel physiological function of Stat1 for B cell activation.

A Very Short Mutational Hot-Spot in the IGH 5′Sμ Region Suggests That the Acquisition of a Class Switch Recombination Defect Is an Important Step Toward Lymphomagenesis.

The t(14;18), which occurs during VDJ joining, is usually seen as the primary genetic event in follicular lymphoma (FL). Pre-lymphoma cells would then migrate to the secondary lymphoid organs and acquire secondary genetic abnormalities. The mechanisms leading to these secondary lesions are unclear, but both somatic hypermutation (SHM) and class switch recombination (CSR) processes may be involved. Surprisingly, while many FL still express an IgM, aberrant switch junctions were reported on both IGH alleles, indicating dysfunctions of CSR during the onset of the neoplasm. Recently, AID mediated point mutations were shown to accumulate in the switch μ (Sμ) regions of stimulated B cells undergoing CSR. To get insights into a possible CSR deregulation in FL, we sequenced the 5′ end of the Sμ region for 73 t(14;18) positive tumours. 152 point mutations and 5 deletions were identified, for a mutation frequency of 0.31/100 bp. Notably, mutations were found for a majority of cases (49/73, 67.1%) rather than being restricted to few tumours. Significant targeting of RGYW motifs (p<10−4) and bias for transitions over transvertions (p<10−4) both suggested the involvement of AID. More importantly, our data showed a highly significant clustering effect, with 22 mutations (13,9%) found on a 4 bp cluster on the GTAA splicing donor site of the Iμ exon (p<10−6). Those 22 mutations were found in different tumors so that 30.1% of the lymphomas in our series had a mutated splicing site. Analysis of whole blood genomic DNA from 14 patients and 20 healthy donors indicated that these mutations are not polymorphisms but are instead acquired by tumor cells. Importantly, allele specific PCR experiments showed no clustering effect on the translocated alleles, while 15/79 (19%) mutations on the functional alleles were found on the Iμ splicing site (p<10−6). Such mutations were also found, but on the translocated alleles, for two additional lymphomas with complex t(3;14;18)(q27;q32;q21) and t(8;14;18)(q24;q32;q21) translocations, suggesting that they may be linked to downstream rearrangements on the IGH locus. Finally, RT-PCR experiments showed that a mutation of the Iμ splicing site leads to the expression of aberrant germline transcripts. Together, our data thus indicate that mutations leading to an incorrect splicing of the germline Sμ transcripts are probably positively selected during lymphomagenesis, and therefore that an acquired perturbation of the CSR process may have a major impact during the onset of the disease, maybe by preventing pre-lymphoma cells from switching their surface immunoglobulin and/or by favouring subsequent genetic lesions.

Allele-Specific Regulation of TCRβ Variable Gene Segment Chromatin Structure

Allelic exclusion of the murine Tcrb locus is imposed at the level of recombination and restricts each cell to produce one functional VDJbeta rearrangement. Allelic exclusion is achieved through asynchronous Vbeta to DJbeta recombination as well as feedback inhibition that terminates recombination once a functional rearrangement has occurred. Because the accessibility of Vbeta gene segment chromatin is diminished as thymocytes undergo allelic exclusion at the CD4(-)CD8(-) (double-negative) to CD4(+)CD8(+) (double-positive) transition, chromatin regulation was thought to be an important component of the feedback inhibition process. However, previous studies of chromatin regulation addressed the status of Tcrb alleles using genetic models in which both alleles remained in a germline configuration. Under physiological conditions, developing thymocytes would undergo Vbeta to DJbeta recombination on one or both alleles before the enforcement of feedback. On rearranged alleles, Vbeta gene segments that in germline configuration are regulated independently of the Tcrb enhancer are now brought into its proximity. We show in this study that in contrast to Vbeta segments on a nonrearranged allele, those situated upstream of a functionally rearranged Vbeta segment are contained in active chromatin as judged by histone H3 acetylation, histone H3 lysine 4 (K4) methylation, and germline transcription. Nevertheless, these Vbeta gene segments remain refractory to recombination in double-positive thymocytes. These results suggest that a unique feedback mechanism may operate independent of chromatin structure to inhibit Vbeta to DJbeta recombination after the double-negative stage of thymocyte development.

Germ Line Transcripts Are Processed by a Dicer-Like Protein That Is Essential for Developmentally Programmed Genome Rearrangements of Tetrahymena thermophila

Abundant approximately 28-nucleotide RNAs that are thought to direct histone H3 lysine 9 (H3K9) methylation and promote the elimination of nearly 15 Mbp of DNA from the developing somatic genome are generated during Tetrahymena thermophila conjugation. To identify the protein(s) that generates these small RNAs, we studied three Dicer-related genes encoded within the Tetrahymena genome, two that contain both RNase III and RNA helicase motifs, Dicer 1 (DCR1) and DCR2, and a third that lacks the helicase domain, Dicer-like 1 (DCL1). DCL1 is expressed upon the initiation of conjugation, and the protein localizes to meiotic micronuclei when bidirectional germ line transcription occurs and small RNAs begin to accumulate. Cells in which we disrupted the DCL1 gene (DeltaDCL1) grew normally and initiated conjugation as wild-type cells but arrested near the end of development and eventually died, unable to resume vegetative growth. These DeltaDCL1 cells failed to generate the abundant small RNAs but instead accumulated germ line-limited transcripts. Together, our findings demonstrate that these transcripts are the precursors of the small RNAs and that DCL1 performs RNA processing within the micronucleus. Postconjugation DeltaDCL1 cells die without eliminating the germ line-limited DNA sequences from their newly formed somatic macronuclei, a result that shows that this Dicer-related gene is required for programmed DNA rearrangements. Surprisingly, DeltaDCL1 cells were not deficient in overall H3K9 methylation, but this modification was not enriched on germ line-limited sequences as it is in wild-type cells, which clearly demonstrates that these small RNAs are essential for its targeting to specific loci.

Cell surface expression of a peptide encoded by the unrearranged TCR-Vβ8.2 gene

Germline transcription of T-cell receptor ( TCR) genes has been described in early lymphoid cells. The most common explanation for this phenomenon is that transcription of unrearranged Vβ genes directs gene usage during the rearrangement event. Germline transcription of the TCR-Vβ8.2 gene has been detected in a precursor T-cell line, C1-V13D, which shows no rearrangement at any of the TCR gene loci. This cell line also shows weak binding of specific anti-Vβ8.2 antibody to the cell surface, consistent with expression of a truncated TCRβ chain. RT-PCR has been used to confirm expression of spliced germline transcripts of TCR-Vβ8.2 in C1-V13D initiated from both leader ( L) 5.1 and L8.2. Transcripts initiated from L8.2 were also detectable in unspliced form. In order to test expression and subcellular localisation of any encoded peptides, amplified germline transcripts in both spliced and unspliced form were cloned into the pEGFP-N1 fusion vector for stable transfection and overexpression in C1-V13D. Cell surface expression of a fusion protein between EGFP and a Vβ peptide has been confirmed in C1-V13D but not in control COS-7 cells. Results presented here raise the possibility of a new pre-TCR structure specific to early lymphoid cells and based on TCR-Vβ8.2 gene expression.

Elucidation of IgH intronic enhancer functions via germ-line deletion

Studies of chimeric mice demonstrated that the core Ig heavy chain (IgH) intronic enhancer (iEmu) functions in V(D)J and class switch recombination at the IgH locus. To more fully evaluate the role of this element in these and other processes, we generated mice homozygous for germ-line mutations in which the core sequences of iEmu (cEmu) were either deleted (cEmu(Delta/Delta) mice) or replaced with a pgk-Neo(R) cassette (cEmu(N/N) mice). The cEmu(Delta/Delta) mice had reduced B cell numbers, in association with impaired D to J(H) and V(H) to DJ(H) rearrangement, whereas cEmu(N/N) mice had a complete block in IgH V(D)J(H) recombination, confirming that additional cis elements cooperate with iEmu to enforce D to J(H) recombination. In addition, developing cEmu(Delta/Delta) and cEmu(N/N) B lineage cells had correspondingly decreased levels of germ-line transcripts from the J(H) region of the IgH locus (mu0 and Imu transcripts); although both had normal levels of germ-line V(H) transcripts, suggesting that cEmu may influence IgH locus V(D)J recombination by influencing accessibility of J(H) proximal regions of the locus. Consistent with chimera studies, peripheral cEmu(Delta/Delta) B cells had normal surface Ig and relatively normal class switch recombination. However, cEmu(Delta/Delta) B cells also had relatively normal somatic hypermutation of their IgH variable region genes, showing unexpectedly that the cEmu is not required for this process. The availability of mice with the iEmu mutation in their germ line will facilitate future studies to elucidate the roles of iEmu in V(H)(D)J(H) recombination in the context of IgH chromatin structure and germ-line transcription.

The TCRβ Enhancer Is Dispensable for the Expression of Rearranged TCRβ Genes in Thymic DN2/DN3 Populations but Not at Later Stages

The Ebeta enhancer has been shown to be dispensable for germline transcription of nonrearranged TCRbeta segments but appears to be required for TCRbeta V to DJ rearrangement. Ebeta dependency of the subsequent expression of VDJ-rearranged TCRbeta genes in thymic subpopulations has so far not been analyzed. We generated transgenic mice, using a Vbeta8.2Dbeta1Jbeta1.3-rearranged TCRbeta bacterial artificial chromosome, which lacked Ebeta, and monitored transgene expression by flow cytometry using Vbeta-specific mAbs and an IRES-eGFP reporter. Transgene expression was found in double negative (DN)2 and DN3 but not at later stages of thymopoesis. There was no toxicity associated with the transgene given that apoptosis in DN3, DN4 was not increased, and the number of DN4 cells generated from DN3 cells in reaggregate thymic organ cultures was not diminished. The transgenic TCRbeta gave rise to a pre-TCR, as suggested by its ability to suppress endogenous TCRbeta rearrangement, to facilitate beta-selection on a TCRbeta-deficient background and to inhibit gammadelta T cell lineage development. The results suggest that the Vbeta8.2 promoter is sufficient to drive expression of rearranged TCRbeta VDJ genes Ebeta independently in DN2/DN3 but not at later stages.

Regulation of TCR δ and α repertoires by local and long-distance control of variable gene segment chromatin structure

Murine Tcrd and Tcra gene segments reside in a single genetic locus and undergo recombination in CD4−CD8− (double negative [DN]) and CD4+CD8+ (double positive [DP]) thymocytes, respectively. TcraTcrd locus variable gene segments are subject to complex regulation. Only a small subset of ∼100 variable gene segments contributes substantially to the adult TCRδ repertoire. Moreover, although most contribute to the TCRα repertoire, variable gene segments that are Jα proximal are preferentially used during primary Tcra recombination. We investigate the role of local chromatin accessibility in determining the developmental pattern of TcraTcrd locus variable gene segment recombination. We find variable gene segments to be heterogeneous with respect to acetylation of histones H3 and H4. Those that dominate the adult TCRδ repertoire are hyperacetylated in DN thymocytes, independent of their position in the locus. Moreover, proximal variable gene segments show dramatic increases in histone acetylation and germline transcription in DP thymocytes, a result of super long-distance regulation by the Tcra enhancer. Our results imply that differences in chromatin accessibility contribute to biases in TcraTcrd locus variable gene segment recombination in DN and DP thymocytes and extend the distance over which the Tcra enhancer can regulate chromatin structure to a remarkable 525 kb.

Comparative analyses of sheep and human TRG joining regions: Evolution of J genes in Bovidae is driven by sequence conservation in their promoters for germline transcription

The availability of genomic clones representative of the T cell receptor gamma ( TRG1@ and TRG2@) ovine loci enabled us to compare the germline genomic organization and nucleotide diversity of joining ( J) segments and reconstruct their evolutionary history by phylogenetic analysis of cattle, sheep and human expressed sequences. Expression profiling (RT-PCR data) in fetus and adult indicated that only the ovine J genes in which two or more of the key sequence features, such as recombination signal sequences (RSS), 3′ splice sites, and core sequences, are missing or severely altered fail to be transcribed. Comparative genomic examination of the two human with the six sheep germline transcription promoters located at 5′ of the relevant constant ( C)-distal J segments showed a strong conservation of the redundant STAT consensus motifs, indicating that TRG1@ and TRG2@ loci are under the influence of IL-7 and STAT signalling. These findings support the phylogenetic analysis of human and Bovidae (cattle and sheep) that revealed a different grouping pattern of C-distal compared to C-proximal J segments. Likewise, the phylogenetic behaviour of either C-distal and C-proximal J segments is in accordance with the Bovidae TRG clusters evolution. Comparison of sheep and human structures of recombination signal sequences (RSS) has highlighted a greater conservation in sheep 12 RSS rather than 23 RSS thus suggesting that the initial recruitment of recombination activating genes (RAG) products requires at least one relatively high-affinity RSS per recombination event.