Germline Transcription Research Articles

The Igκ gene enhancers, E3' and Ed, are essential for triggering transcription.

The mouse Igκ gene locus has three known transcriptional enhancers: an intronic enhancer (Ei), a 3' enhancer (E3'), and a further downstream enhancer (Ed). Previous studies on B lymphocytes derived from mutant embryonic stem cells have shown that deletion of either Ei or E3' significantly reduces Igκ gene rearrangement, whereas the combined deletion of both Ei and E3' eliminates such recombination. Furthermore, deletion of either E3' or Ed significantly reduces rearranged Igκ gene transcription. To determine whether the combined presence of both E3' and Ed are essential for Igκ gene expression, we generated homozygous double knockout (DKO) mice with targeted deletions in both elements. Significantly, homozygous DKO mice were unable to generate κ(+) B cells both in bone marrow and the periphery and exhibited surface expression almost exclusively of Igλ-chains, despite the fact that they possessed potentially functional rearranged Igκ genes. Compared with their single-enhancer-deleted counterparts, Igκ loci in homozygous DKO mice exhibited dramatically reduced germline and rearranged gene transcription, lower levels of gene rearrangement and histone H3 acetylation, and markedly increased DNA methylation. This contributed to a partial developmental block at the pre-B cell stage of development. We conclude that the two downstream enhancers are essential in Igκ gene expression and that Ei in homozygous DKO mice is incapable of triggering Igκ gene transcription. Furthermore, these results reveal unexpected compensatory roles for Ed in E3' knockout mice in triggering germline transcription and Vκ gene rearrangements to both Jκ and RS elements.

The effect of CD14 C159T polymorphism on in vitro IgE synthesis and cytokine production by PBMC from children with asthma

Even though the genotype at the promoter region of the CD14 molecule is known to affect the atopic phenotypes, the cellular and molecular basis of this association is largely unknown. To investigate the effect of lipopolysaccharide (LPS) on IgE production and cytokine profile by peripheral blood mononuclear cells (PBMC) obtained from asthmatic children with the TT and the CC genotypes at position -159 of the CD14 gene. Peripheral blood mononuclear cells from asthmatic children with alternative genotypes at CD14 C159T locus were stimulated with 2 and 200 ng/ml LPS in vitro. The IgE, IgG and, IgM response was determined by ELISA and Ig έ-germline, IgG, and IgM transcription by real-time PCR. A cluster of cytokines was measured by cytometric bead array. Asthmatic children with the TT genotype but not those with the CC genotype responded with increased IgE synthesis and germline transcription to LPS stimulation. There were no genotype-related differences in IgG and IgM. TT but not the CC genotype was associated with significantly increased interleukin (IL)-4/IL-12 and IL-4/interferon-gamma (IFN-γ) ratios in the culture supernatant. There were no genotype-related differences in IL-1β, IL-7, IL-10, IL-13, IL-17A, granulocyte colony stimulating factor, granulocyte macrophage colony stimulating factor, monocyte chemotactic protein, and tumor necrosis factor alpha. Peripheral blood mononuclear cells from asthmatic children with the TT genotype at position -159 of the CD14 gene make more IgE than those with the CC genotype following LPS stimulation because of increased germline transcription and have an augmented Th2 cytokine profile.

Insulin/FOXO Signaling Regulates Ovarian Prostaglandins Critical for Reproduction

Abnormalities in insulin/IGF-1 signaling are associated with infertility, but the molecular mechanisms are not well understood. Here we use liquid chromatography with electrospray ionization tandem mass spectrometry to show that the C. elegans insulin/FOXO pathway regulates the metabolism of locally acting lipid hormones called prostaglandins. C. elegans prostaglandins are synthesized without prostaglandin G/H synthase homologs, the targets of nonsteroidal anti-inflammatory drugs. Our results support the model that insulin signaling promotes the conversion of oocyte polyunsaturated fatty acids (PUFAs) into F-series prostaglandins that guide sperm to the fertilization site. Reduction in insulin signaling activates DAF-16/FOXO, which represses the transcription of germline and intestinal genes required to deliver PUFAs to oocytes in lipoprotein complexes. Nutritional and neuroendocrine cues target this mechanism to control prostaglandin metabolism and reproductive output. Prostaglandins may be conserved sperm guidance factors and widespread downstream effectors of insulin actions that influence both reproductive and nonreproductive processes.

Elements between the IgH variable (V) and diversity (D) clusters influence antisense transcription and lineage-specific V(D)J recombination.

Ig and T-cell receptor (TCR) variable-region gene exons are assembled from component variable (V), diversity (D) and joining (J) gene segments during early B and T cell development. The RAG1/2 endonuclease initiates V(D)J recombination by introducing DNA double-strand breaks at borders of the germ-line segments. In mice, the Ig heavy-chain (IgH) locus contains, from 5' to 3', several hundred V(H) gene segments, 13 D segments, and 4 J(H) segments within a several megabase region. In developing B cells, IgH variable-region exon assembly is ordered with D to J(H) rearrangement occurring on both alleles before appendage of a V(H) segment. Also, IgH V(H) to DJ(H) rearrangement does not occur in T cells, even though DJ(H) rearrangements occur at low levels. In these contexts, V(D)J recombination is controlled by modulating substrate gene segment accessibility to RAG1/2 activity. To elucidate control elements, we deleted the 100-kb intergenic region that separates the V(H) and D clusters (generating ΔV(H)-D alleles). In both B and T cells, ΔV(H)-D alleles initiated high-level antisense and, at lower levels, sense transcription from within the downstream D cluster, with antisense transcripts extending into proximal V(H) segments. In developing T lymphocytes, activated germ-line antisense transcription was accompanied by markedly increased IgH D-to-J(H) rearrangement and substantial V(H) to DJ(H) rearrangement of proximal IgH V(H) segments. Thus, the V(H)-D intergenic region, and likely elements within it, can influence silencing of sense and antisense germ-line transcription from the IgH D cluster and thereby influence targeting of V(D)J recombination.

Imatinib Mesylate Directly Impairs Class Switch Recombination through Downregulation of AID: Its Potential Efficacy as An AID Suppressor.

AbstractAbstract 3424Activation-induced cytidine deaminase (AID), is essential for class switch recombination(CSR) and somatic hypermutation(SHM). Deregulated expression of AID acts as a genomic mutator that contributes to various tumorigenesis through chromosomal translocation and aberrant SHM. Previously, we showed that the titer of serum immunoglobulin(Ig)G and IgA in the CML patients treated with imatinib mesylate was lower than in those with interferon-ƒ¿, whereas that of IgM was higher, implying that imatinb mesylate, the abl kinase inhibitor, impairs CSR.Here we explored the effect of imatinib mesylate on CSR both in vitro and in vivo and revealed that AID was responsible for the impairement of CSR by imatinib mesylate.CSR is induced in the mouse splenic B cells by stimulation of IL-4 and LPS. In this system, IgG1 expression of spleen cells without imatinib mesylate was □‘15%, whereas that with 10ƒÊM imatinib mesylate significantly reduced to □‘3%. The reduction was observed in dose dependent manner (Figure.1). Imatinib mesylate has been reported to affect various immunomoduratory cells including dendritic cells and T cells. Our observation elucidated that imatinib mesylate has a direct effect on B cells. [Display omitted] Next, the expression of AID and the germline transcript of IgG1, which are required for CSR was examined by use of PCR. The expression of AID significantly decreased with imatinib mesylate, whereas that of the germline transcript of IgG1 did not change with and without 10 ƒÊM imatinib mesylate (Figure.2). Since the germline transcript of IgG1 remained unchanged, it was elucidated that downregulation of AID causes the inhibition of CSR by imatinib mesylate. The similar results were obtained in the experiments where the mice injected SRBCs (a immunogen) with imatinib mesylate showed significant reduced CSR and expression of AID in the spleen. [Display omitted] [Display omitted] Furthermore, we investigated whether exogenous expression of AID could rescue the inhibition of CSR by imatinib mesylate. IgG1 expression in the spleen cells without imatinib mesylate was about 36%, whereas that with 10 ƒÊM imatinib mesylate reduced to about 10.2%. When AID was exogenously expressed, IgG1 expression with 10 ƒÊM imatinib mesylate reincreased to 46.2%. It was clearly documented that the reexpression of AID could almost completely cancel the effect of imatinib mesylate on CSR.Finally, trying to understand the mechanism of downregulation of AID, the expressions of the key transcription factors, such as PAX5, E2A, E2f7 and E2f8, which bind Aicda promoter region, were examined by use of the quantitative PCR. Surprisingly all of these transcriptional factors were downregulated by imatinib mesylate. Especially the expression of E2A was dramatically reduced by imatinib mesylate. E2A is the crucial transcriptional activator for AID expression, suggesting that the remarkable downregulation of E2A by imatinib mesylate may lead to the downregulation of AID expression.Taken together, these observation lead us to the conclusion that suppression of AID via E2A is responsible for inhibition of CSR by imatinib mesylate.Our findings shed light on the etiology of hypogammaglobulinemia, an adverse effect of imatinib mesylate frequently observed in the clinical settings. The assessment and close examination of the adverse effects of the kinase inhibitors is important for the future treatment for BCR-ABL leukemias, because the treatment by multiple tyrosine kinase inhibitors like tuberculosis and HIV infection is assumed to be introduced in order to overcome the problem of drug resistance induced by the mutation such as T315I or other.It is also suggest that imatinib mesylate is the potential drug for clinical usage as AID suppressor, which deregulation of CSR and SHM with the genomic instability is observed in many B cell malignancy. Disclosures:No relevant conflicts of interest to declare.

Interleukin-17A Promotes IgE Production in Human B Cells

Recently the Th1/Th2 concept has been revised and Th17 cells have been implicated in allergy. Despite clear correlative evidence, the cellular and molecular basis for the connection between increased IL-17A and IgE in allergy has not been elucidated. Here we show using flow cytometry that allergic patients have higher numbers of IL-17A+ cells compared to nonallergic donors. The selective removal of IL-17A+ cells from peripheral blood mononuclear cells of allergic donors after an IL-17A secretion assay reduces IgE levels, whereas re-addition of recombinant IL-17A restores it, as measured by ELISA, showing their important functional implication for IgE production. In addition, IL-17A directly promotes the differentiation of IgE-secreting cells and IgE production upon anti-CD40/IL-4 costimulation, as shown by enzyme-linked immunospot technique and ELISA. IL-17A triggers rapid degradation of IκBα and subsequent translocation of NF-κB into the B-cell nucleus, followed by transcription of epsilon germ-line, activation-induced cytidine deaminase, and IFN regulatory factor 4, as analyzed by flow cytometry, western blot, and quantitative real-time RT-PCR, respectively. Our study shows that IL-17A+ cells promote IgE production and that IL-17A exerts its pro-allergic effect directly at the level of B cells. Therefore, IL-17A might be a target for the treatment of IgE-dependent diseases, including atopic dermatitis.

Intronic AT Skew is a Defendable Proxy for Germline Transcription but does not Predict Crossing-Over or Protein Evolution Rates in Drosophila melanogaster

Recent evidence suggests that germline transcription may affect both protein evolutionary rates, possibly mediated by repair processes, and recombination rates, possibly mediated by chromatin and epigenetic modification. Here, we test these propositions in Drosophila melanogaster. The challenge for such analyses is to provide defendable measures of germline gene expression. Intronic AT skew is a good candidate measure as it is thought to be a consequence, at least in part, of transcription-coupled repair. Prior evidence suggests that intronic AT skew in D. melanogaster is not affected by proximity to intron extremities and differs between transcribed DNA and flanking sequence. We now also establish that intronic AT skew is a defendable proxy for germline expression as (a) it is more similar than expected by chance between introns of the same gene (which is not accounted for by physical proximity), (b) is correlated with male germline expression, and (c) is more pronounced in broadly expressed genes. Furthermore, (d) a trend for intronic skew to differ between 3' and 5' ends of genes is particular to broadly expressed genes. Finally, (e) controlling for physical distance, introns of proximate genes are most different in skew if they have different tissue specificity. We find that intronic AT skew, employed as a proxy for germline transcription, correlates neither with recombination rates nor with the rate of protein evolution. We conclude that there is no prima facie evidence that germline expression modulates recombination rates or monotonically affects protein evolution rates in D. melanogaster.

Vitamin D receptor binds to the ε germline gene promoter and exhibits transrepressive activity

Recently, an increased incidence of allergic diseases has been associated with vitamin D deficiency. We demonstrated previously that calcitriol, the active form of vitamin D, inhibits ε germline transcription, a prerequisite for IgE production. However, the underlying mechanisms remain unexplored. We sought to investigate whether the ε germline gene promoter (Iε) represents a primary vitamin D receptor (VDR) target. Therefore we investigated VDR binding to Iε, analyzed VDR-complex composition in more detail, and delineated its functional consequences. The VDR binding to Iε in human B cells, the composition of the VDR-recruited complex, and the acetylation pattern were investigated by means of chromatin immunoprecipitation. The calcitriol-mediated action on Iε was analyzed by using a reporter gene assay. We demonstrate that Iε is a possible VDR target. Calcitriol-activated VDR binds together with retinoid X receptor α to the Iε region. The heterodimer interacts with silencing mediator for retinoid and thyroid hormone receptors, which recruits histone deacetylase (HDAC) 1 and HDAC3. The inhibition of silencing mediator for retinoid and thyroid hormone receptors or HDACs reversed the site-specific deacetylation of histones 3 and 4 and the calcitriol-driven inhibition of the ε germline transcription. The VDR-complex transrepressive actions on Iε were confirmed in a reporter assay. We show here that inhibition of IgE production by calcitriol is mediated by its transrepressive activity through the VDR-corepressor complex affecting chromatin compacting around the Iε region. Our findings shed new light on mechanisms of VDR transrepression and understanding of IgE regulation.

The Center of Accessibility: Dβ Control of V(D)J Recombination

Developmental patterning of antigen receptor gene assembly in lymphocyte precursors correlates with decondensation of the chromatin surrounding individual gene segments. Ongoing V(D)J recombination is associated with hyperacetylation of histones H3 and H4 and the expression of sterile germline transcripts across the region of recombinational accessibility. Likewise, histone acetyltransferase and SWI/SNF chromatin remodeling complexes each appear to be required for recombination, and the PHD-finger of RAG-2 preferentially associates with recombination signal sequence (RSS) chromatin that contains H3 trimethylated on lysine 4. However, the regulatory mechanisms that direct chromatin alteration and rearrangement have proven elusive, due in large part to the interdependency of individual stages in gene activation, our limited understanding of functional significance of changes to the histone code, and the difficulty of modeling recombinational accessibility in existing experimental systems. Examining Tcrb assembly in developing thymocytes, we review the central roles of RSS elements and germline promoters as foci for epigenetic reorganization of recombinationally accessible gene segments in light of recent findings and persistent questions.

Genomic signatures of germline gene expression

Transcribed regions in the human genome differ from adjacent intergenic regions in transposable element density, crossover rates, and asymmetric substitution and sequence composition patterns. We tested whether these differences reflect selection or are instead a byproduct of germline transcription, using publicly available gene expression data from a variety of germline and somatic tissues. Crossover rate shows a strong negative correlation with gene expression in meiotic tissues, suggesting that crossover is inhibited by transcription. Strand-biased composition (G+T content) and A → G versus T → C substitution asymmetry are both positively correlated with germline gene expression. We find no evidence for a strand bias in allele frequency data, implying that the substitution asymmetry reflects a mutation rather than a fixation bias. The density of transposable elements is positively correlated with germline expression, suggesting that such elements preferentially insert into regions that are actively transcribed. For each of the features examined, our analyses favor a nonselective explanation for the observed trends and point to the role of germline gene expression in shaping the mammalian genome.

Chlorogenic acid suppresses pulmonary eosinophilia, IgE production, and Th2-type cytokine production in an ovalbumin-induced allergic asthma: Activation of STAT-6 and JNK is inhibited by chlorogenic acid

Asthma is a chronic inflammatory disease of the airways characterized by reversible airway obstruction, airway hyperreactivity, and remodeling of the airways. Chlorogenic acid (CGA), an ester of caffeic acid with quinic acid, is one of the most abundant polyphenol compounds in various agricultural products. CGA shows various biological properties, such as anti-oxidant, anti-viral, anti-carcinogenic and anti-inflammatory activities. We investigated suppressive effects of CGA on ovalbumin (OVA)-induced allergic asthma in mice and underlying mechanisms of them. CGA significantly reduced pulmonary eosinophilia and expression of IL-4, IL-5 and TNF-α in the lung as well as the serum levels of total and OVA-specific IgE, while CGA enhanced those of total and OVA-specific IgG3, of which isotype switching is down-regulated by IL-4. In vitro IgE production from LPS/IL-4-stimulated splenocytes was remarkably reduced by CGA, while that of IgG3 was enhanced. The Cε germ line transcription, which is necessary for IL-4 mediated IgE isotype switching, was reduced by CGA in LPS/IL-4-stimulated splenocytes. IgE isotype switching is mediated via several transduction pathways, activating several molecules including STAT-6, NF-κB, ERK1/2, and JNK. Among the molecules, which were activated by IL-4/LPS, activation of STAT-6 and JNK was inhibited by CGA.

Transcription-Dependent Mobilization of Nucleosomes at Accessible TCR Gene Segments In Vivo

Accessibility of chromosomal recombination signal sequences to the RAG protein complex is known to be essential for V(D)J recombination at Ag receptor loci in vivo. Previous studies have addressed the roles of cis-acting regulatory elements and germline transcription in the covalent modification of nucleosomes at Ag receptor loci. However, a detailed picture of nucleosome organization at accessible and inaccessible recombination signal sequences has been lacking. In this study, we have analyzed the nucleosome organization of accessible and inaccessible Tcrb and Tcra alleles in primary murine thymocytes in vivo. We identified highly positioned arrays of nucleosomes at Dbeta, Jbeta, and Jalpha segments and obtained evidence indicating that positioning is established at least in part by the regional DNA sequence. However, we found no consistent positioning of nucleosomes with respect to recombination signal sequences, which could be nucleosomal or internucleosomal even in their inaccessible configurations. Enhancer- and promoter-dependent accessibility was characterized by diminished abundance of certain nucleosomes and repositioning of others. Moreover, some changes in nucleosome positioning and abundance at Jalpha61 were shown to be a direct consequence of germline transcription. We suggest that enhancer- and promoter-dependent transcription generates optimal recombinase substrates in which some nucleosomes are missing and others are covalently modified.

Accessibility control of TCR V region by STAT5

The signal of the IL-7R and signal transducers and activators of transcription (STAT) 5 plays an essential role in gammadelta T-cell development by inducing V-J recombination in the TCRgamma locus. Previously, we have shown that STAT5 binds to the Jgamma promoters and controls chromatin accessibility by histone acetylation. However, little is known on control mechanism of Vgamma region by the IL-7R. To elucidate the regulation by STAT5, we first analyzed the chromatin status of Vgamma region in primary thymocytes. The levels of histone H3 acetylation are high at Vgamma5, HsA element and Vgamma2 in Rag2(-/-) thymocytes but low in IL-7R alpha-chain (IL-7Ralpha)-deficient early thymocytes, suggesting that IL-7R signaling controls the accessibility of the Vgamma region. In addition, high levels of histone H3 acetylation and germ line transcription were induced at Vgamma5 and HsA by cytokine and STAT5 in cytokine-dependent Ba/F3 and other hematopoietic cell lines. Importantly, the chromatin accessibility of Vgamma5 gene is increased by cytokine signal. Furthermore, STAT5 was not recruited to a non-canonical STAT-binding motif in the endogenous chromatin of the Vgamma5 promoter by cytokine stimulation, while STAT5 binds to a consensus motif in the HsA element. In accordance with this result, STAT5 does not directly activate the Vgamma5 promoter by reporter assay. These results suggested that while STAT5 directly binds to HsA element and induces its histone acetylation, STAT5 indirectly activates the Vgamma5 promoter. Thus, this study implies a potential role of STAT5 in accessibility control of Vgamma region, especially at Vgamma5 and HsA.

Switch Region Identity Plays an Important Role in Ig Class Switch Recombination

Ig class switch recombination (CSR) is regulated through long-range intrachromosomal interactions between germline transcript promoters and enhancers to initiate transcription and create chromatin accessible to activation-induced deaminase attack. CSR occurs between switch (S) regions that flank Cmu and downstream C(H) regions and functions via an intrachromosomal deletional event between the donor Smicro region and a downstream S region. It is unclear to what extent S region primary sequence influences differential targeting of CSR to specific isotypes. We address this issue in this study by generating mutant mice in which the endogenous Sgamma3 region was replaced with size-matched Sgamma1 sequence. B cell activation conditions are established that support robust gamma3 and gamma1 germline transcript expression and stimulate IgG1 switching but suppress IgG3 CSR. We found that the Sgamma1 replacement allele engages in micro-->gamma3 CSR, whereas the intact allele is repressed. We conclude that S region identity makes a significant contribution to CSR. We propose that the Sgamma1 region is selectively targeted for CSR following the induction of an isotype-specific factor that targets the S region and recruits CSR machinery.

Role of the 3’ heavy chain enhancers in germline transcription and switch recombination (88.1)

Abstract Class switch recombination (CSR) is preceded by germline transcription and then mediated by a DNA recombination event. To study germline transcription and CSR we utilize transgenic mice with a 230 kb bacterial artificial chromosome that includes a rearranged VDJ gene, the entire heavy chain C region locus, and all known 3’ enhancers. In the intact transgene, germline transcription of and CSR to the γ, ϵ, and α heavy chain genes are regulated in parallel to the endogenous genes. We have reported that deletion of a 28 kb segment, including the 3’ enhancers HS3A, HS1,2, HS3B, and HS4, results in a reduction of CSR to all heavy chain genes to about 1% of the level of the intact transgene. We have derived four new versions of the heavy chain transgene: with 1 kb deletions of (i) HS1,2 and HS3B, (ii) HS1,2, HS3B, and HS4, (iii) HS3A, HS1,2, and HS3B, and (iv) HS3A, HS1,2, HS3B, and HS4. Comparing levels of CSR in these deletion variants to intact transgenes, we find that deletion of more enhancers results in a reduction in CSR for more heavy chain genes, and the reduction is more severe for a given heavy chain gene. Nevertheless, different enhancers have different effects on different heavy chain genes. Finally, there are enhancer deletions for which the reduction in CSR is much greater than the reduction in germline transcription. This suggests that the 3’ enhancers have a role in CSR that is distinct from the enhancement of germline transcription.

In Vivo Redundant Function of the 3′ IgH Regulatory Element HS3b in the Mouse

In the mouse, the regulatory region located at the 3' end of the IgH locus includes four transcriptional enhancers: HS3a, HS1-2, HS3b, and HS4; the first three lie in a quasi-palindromic structure. Although the upstream elements HS3a and HS1-2 proved dispensable for Ig expression and class switch recombination (CSR), the joint deletion of HS3b and HS4 led to a consistent decrease in IgH expression in resting B cells and to a major CSR defect. Within this pair of distal enhancers, it was questionable whether HS3b and HS4 could be considered individually as elements critical for IgH expression and/or CSR. Studies in HS4-deficient mice recently revealed the role of HS4 as restricted to Igmicro-chain expression from the pre-B to the mature B cell stage and left HS3b as the last candidate for CSR regulation. Our present study finally invalidates the hypothesis that CSR could mostly rely on HS3b itself. B cells from HS3b-deficient animals undergo normal proliferation, germline transcription, and CSR upon in vitro stimulation with LPS; in vivo Ag-specific responses are not affected. In conclusion, our study highlights a major effect of the global ambiance of the IgH locus; enhancers demonstrated as being strongly synergistic in transgenes turn out to be redundant in their endogenous context.

Requirement for Runx Proteins in IgA Class Switching Acting Downstream of TGF-β1 and Retinoic Acid Signaling

IgA is a specific isotype required for mucosal immunity and is the most abundant Ab produced in vivo. Recently, several inductive signals for IgA class switch recombination have been identified; however, the molecular details of the action of these signals and the specific factors acting in B cells remain elusive. In this study, we show that combination of retinoic acid (RA) and TGF-beta1 with other factors induced a much higher frequency of IgA-switched cells than reported previously. In addition, IgA production is severely impaired in Runx2-Runx3 double-deficient mice. In Runx2-Runx3-deficient B cells, both RA- and TGF-beta1-dependent inductions of alpha germline transcription are completely blocked. These data suggest that Runx proteins play an essential role in IgA class switching acting downstream of RA and TGF-beta1 signaling.

The Mouse Immunoglobulin Heavy Chain V-D Intergenic Sequence Contains Insulators That May Regulate Ordered V(D)J Recombination

During immunoglobulin heavy chain (Igh) V(D)J recombination, D to J precedes V to DJ recombination in an ordered manner, controlled by differential chromatin accessibility of the V and DJ regions and essential for correct antibody assembly. However, with the exception of the intronic enhancer Emu, which regulates D to J recombination, cis-acting regulatory elements have not been identified. We have assembled the sequence of a strategically located 96-kb V-D intergenic region in the mouse Igh and analyzed its activity during lymphocyte development. We show that Emu-dependent D antisense transcription, proposed to open chromatin before D to J recombination, extends into the V-D region for more than 30 kb in B cells before, during, and after V(D)J recombination and in T cells but terminates 40 kb from the first V gene. Thus, subsequent V antisense transcription before V to DJ recombination is actively prevented and must be independently activated. To find cis-acting elements that regulate this differential chromatin opening, we identified six DNase I-hypersensitive sites (HSs) in the V-D region. One conserved HS upstream of the first D gene locally regulates D genes. Two further conserved HSs near the D region mark a sharp decrease in antisense transcription, and both HSs bind CTCF in vivo. Further, they both possess enhancer-blocking activity in vivo. Thus, we propose that they are enhancer-blocking insulators preventing Emu-dependent chromatin opening extending into the V region. Thus, they are the first elements identified that may control ordered V(D)J recombination and correct assembly of antibody genes.

Replacement of Iμ-Cμ intron by NeoR gene alters Iμ germ-line expression but has no effect on V(D)J recombination

The NeoR gene has often been used to unravel the mechanisms underlying long-range interactions between promoters and enhancers during V(D)J assembly and class switch recombination (CSR) in the immunoglobulin heavy chain (IgH) locus. This approach led to the notion that CSR is regulated through competition of germ-line (GL) promoters for activities displayed by the 3′ regulatory region (3′RR). This polarized long-range effect of the 3′RR is disturbed upon insertion of NeoR gene in the IgH constant (CH) region, where only GL transcription derived from upstream GL promoters is impaired. In the context of V(D)J recombination, replacement of Eμ enhancer or Eμ core enhancer (cEμ) by NeoR gene fully blocked V(D)J recombination and μ0 GL transcription which originates 5′ of DQ52 and severely diminished Iμ GL transcription derived from Eμ/Iμ promoter, suggesting a critical role for cEμ in the regulation of V(D)J recombination and of μ0 and Iμ expression. Here we focus on the effect of NeoR gene on μ0 and Iμ GL transcription in a mouse line in which the Iμ-Cμ intron was replaced by a NeoR gene in the sense-orientation. B cell development was characterized by a marked but incomplete block at the pro-B cell stage. However, V(D)J recombination was unaffected in sorted pro-B and pre-B cells excluding an interference with the accessibility control function of Eμ. μ0 GL transcription initiation was relatively normal but the maturation step seemed to be affected most likely through premature termination at NeoR polyadenylation sites. In contrast, Iμ transcription initiation was impaired suggesting an interference of NeoR gene with the IgH enhancers that control Iμ expression. Surprisingly, in stark contrast with the NeoR effect in the CH region, LPS-induced NeoR expression restored Iμ transcript levels to normal. The data suggest that Eμ enhancer may be the master control element that counteracts the down-regulatory “Neo effect” on Iμ expression upon LPS stimulation. More importantly, they reveal a complex and developmentally regulated interplay between IgH enhancers in the control of Iμ expression.

Flavones suppress type I IL-4 receptor signaling by down-regulating the expression of common gamma chain

Immunoglobulin E (IgE) production is induced by interleukin (IL)-4 signaling mediated by type I IL-4 receptor (IL-4R) in B cells. We found that flavones inhibited IL-4-induced ε germline transcription which is essential for IgE class switching, and the phosphorylation of signal transducer and activator of transcription 6, janus kinase 3, and IL-4Rα, whereas IL-4 signaling mediated through type II IL-4R was unaffected by flavones. Furthermore, flavones reduced the expression of common gamma chain, a characteristic constituent subunit of type I IL-4R, suggesting that flavones suppress type I IL-4R signaling.