IgH Gene Expression Research Articles

Spt5-mediated enhancer transcription directly couples enhancer activation with physical promoter interaction.

Active enhancers are frequently transcribed, yet the regulatory role of enhancer transcription remains debated. Here, we depleted the RNA polymerase II pausing and elongation factor Spt5 in activated mouse B cells and found that approximately 50% of enhancer-gene pairs showed co-regulated transcription, consistent with a potential functional requirement for enhancer transcription. In particular, Spt5 depletion led to loss of super-enhancer-promoter physical interaction and gene expression at the immunoglobulin heavy-chain locus (Igh), abrogating antibody class switch recombination. This defect correlated strictly with loss of enhancer transcription but did not affect acetylation of histone H3 at lysine 27, chromatin accessibility and occupancy of Mediator and cohesin at the enhancer. Strikingly, CRISPRa-mediated rescue of enhancer transcription in Spt5-depleted cells restored Igh gene expression. Our work suggests that Spt5-mediated enhancer transcription underlies the physical and functional interaction between a subset of active enhancers and their target promoters.

Hydrogen peroxide modulates immunoglobulin expression by targeting the 3′Igh regulatory region through an NFκB-dependent mechanism

Reactive oxygen species such as hydrogen peroxide (H2O2) appear to play a role in signal transduction in immune cells and have been shown to be synthesized upon antigen-mediated activation and to facilitate cellular activation in B- and T-cells. However, an effect of H2O2 on B-cell function (i.e. immunoglobulin (Ig) expression) has been less well-characterized. The effects of H2O2 exposure on lymphocytes may be partly mediated by oxidative modulation of the NFκB signal transduction pathway, which also plays a role in Ig heavy chain (Igh) gene expression. Igh transcription in B lymphocytes is an essential step in antibody production and is governed through a complex interaction of several regulatory elements, including the 3′Igh regulatory region (3′IghRR). Utilizing an in vitro mouse B-cell line model, this study demonstrates that exposure to low μM concentrations of H2O2 can enhance 3′IghRR-regulated transcriptional activity and Igh gene expression, while either higher concentrations of H2O2 or the expression of a degradation resistant inhibitory κB (IκBα super-repressor) can abrogate this effect. Furthermore, suppressive H2O2 concentrations increased protein levels of the p50 NFκB sub-unit, IκBα, and an IκBα immunoreactive band which was previously characterized as an IκBα cleavage product exhibiting stronger inhibitory function than native IκBα. Taken together, these observations suggest that exposure of B lymphocytes to H2O2 can alter Igh transcriptional activity and Ig expression in a complex biphasic manner which appears to be mediated by NFκB and altered 3′IghRR activity. These results may have significant implications to disease states previously associated with the 3′IghRR.

In a model of immunoglobulin heavy‐chain (IGH)/MYCtranslocation, theIgh3′ regulatory region inducesMYCexpression at the immature stage of B cell development

Reciprocal translocations involving the immunoglobulin loci and the cellular oncogene MYC are hallmark mutations of the human postgerminal center B cell neoplasm, Burkitt's lymphoma. They are occasionally found in other B cell lymphomas, as well. Translocations involving the heavy chain locus (IGH) place the MYC gene either in cis with both the intronic enhancer Emu and the IGH 3' regulatory region (3'RR) or in cis with only the 3'RR. The result is deregulated MYC expression. Recent studies have led to some controversy as to when, during B lymphocyte development, IGH/MYC chromosome translocations take place. A related issue, relevant not only to lymphoma development but also to normal controls on IGH gene expression, is the stage, during B lymphocyte development, at which the 3'RR is capable of activating MYC expression. We have developed mice transgenic for a human MYC (hMYC) gene under control of the four core enhancers from the mouse Igh 3'RR. Unlike other transgenic mouse models where premature and inappropriate MYC expression disrupts normal B cell development, the hMYC transgene in these studies carries a mutation that prohibits MYC protein synthesis. As a result, hMYC expression can be analyzed in all of the normal B cell compartments. Our data show that hMYC is expressed almost exclusively in B-lineage cells and is induced to high levels as soon as bone marrow cells reach the immature B cell stage.

A negative regulatory element in the rabbit 3′IgH chromosomal region

Mouse and human IgH loci contain several 3'IgH enhancers. In rabbit, a single hs1,2 enhancer is located 3' of the distal germ line Calpha gene, Calpha13. We searched for additional regulatory elements in this region by using a luciferase reporter assay and nucleotide sequence analysis. Within 8 kb 3' of Calpha13, we identified a 1-kb fragment that negatively regulated the hs1,2 enhancement of the Ialpha promoter. This negative regulatory element, Calpha-NRE, contains a conserved 300-bp region that is associated with 8 of the 13 germ line Calpha genes. This conserved region contains an E box that, by electrophoretic mobility shift assay, binds an E47-like protein. At the 5' end, Calpha-NRE also includes a 270-bp region with 20-bp repeats nearly identical to those 3' of mouse and human Calpha genes, and these repeats bind unidentified nuclear protein(s). Calpha-NRE appears to be a novel regulatory element that may contribute to the regulation of IgH gene expression.

Differential Functions for the Transcription Factor E2A in Positive and Negative Gene Regulation in Pre-B Lymphocytes

The transcription factors encoded by the E2A gene have been shown to play essential roles in the initiation and progression of lymphocyte development. However, there is still a lack of comprehensive understanding of E2A downstream genes in B-cell development. We previously developed a gene tagging-based chromatin immunoprecipitation (ChIP) system to directly evaluate E2A target genes in B-cell development. Here, we have improved this ChIP strategy and used it in conjunction with microarray analysis on E2A-deficient pre-B-cell lines to determine E2A target genes in lymphocyte development. Both microarray data and ChIP studies confirmed that E2A directly controls IgH gene expression. The microarray assay also revealed genes that were significantly up-regulated after E2A disruption. ChIP analysis showed that E2A was most likely to be directly involved in repression of some of these target genes such as Nfil3 and FGFR2. An inducible E2A reconstitution system further demonstrated that E2A-mediated repression of Nfil3 and FGFR2 was reversible. Collectively, these findings indicate that E2A is a positive regulator for one set of genes and a negative regulator for another set of genes in developing B lymphocytes.

Notch signaling suppresses IgH gene expression in chicken B cells: implication in spatially restricted expression of Serrate2/Notch1 in the bursa of Fabricius.

The bursa of Fabricius is a central organ for chicken B cell development and provides an essential microenvironment for expansion of the B cell pool and for generation of a diversified B cell repertoire. We report here that genes encoding the Notch family of transmembrane proteins, key regulators of cell fate determination in development, are differentially expressed in the bursa of Fabricius: Notch1 is expressed in medullary B cells located close to the basement membrane-associated epithelium (BMAE). In contrast, a Notch ligand, Serrate2, is expressed exclusively in the BMAE, which surrounds bursal medulla. A basic helix-loop-helix-type transcription factor, Hairy1, a downstream target of Notch signaling, is expressed in the bursa coordinately with Notch1 and Serrate2 and an immature B cell line, TLT1, which expresses both Notch1 and Serrate2. Furthermore, stable expression of a constitutively active form of chicken Notch1 or Notch2 in a B cell line results in a down-regulation of surface IgM expression, which is accompanied by the reduction of IgH gene transcripts. Transient reporter assay with the human IgH gene intronic enhancer reveals that an active form of Notch1 inhibits the IgH enhancer activity in chicken B cells, suggesting that Notch-mediated signals suppress the IgH gene expression via influencing the IgH intronic enhancer. These findings raise the possibility that the local activation of Notch1 in a subset of B cells by Serrate2 expressed in BMAE may influence the cell fate decision that is involved in B cell differentiation and selection inside the bursa.

The lymphoid-specific cofactor OBF-1 is essential for the expression of a V H promoter/HS1,2 enhancer-linked transgene in late B cell development

Mice deficient for the lymphoid-specific cofactor OBF-1 display reduced levels of IgG, IgA and IgE. To examine whether the lowered immunoglobulin expression is linked to reduced activity of IgH cis-regulatory elements, OBF-1 −/− mice were crossed with mice expressing transgenes driven by a V H or β-globin promoter linked to the HS1,2 enhancer. Here we show that OBF-1 is essential for the induced expression of a V H promoter-linked transgene, in contrast to a β-globin promoter-dependent transgene, in LPS/IL-4 or CD40-stimulated splenic B cells. Furthermore, impaired transgene expression is observed in OBF-1 −/− peritoneal B cells. This deficiency may be linked to OBF-1, as peritoneal cells from normal mice express OBF-1 protein constitutively. Our data link OBF-1 to IgH gene expression in late B lymphoid development.

3′ IgH Enhancer Elements Shift Synergistic Interactions During B Cell Development

IgH gene expression is tightly controlled over the course of B cell development, B cell activation, and the subsequent differentiation of these cells into Ig-secreting plasmacytes. There are several transcriptional enhancers that map within and downstream of the IgH locus, and some of these have been clearly implicated in the developmental regulation of IgH gene assembly and expression. While some of the individual enhancers from this locus have been studied extensively, the functional interactions possible among this group of enhancers have been largely unexplored. In the present study, we have measured the transcriptional activities of combinations of enhancers introduced into B-lineage cell lines at several different developmental stages. We detected a developmental progression in which the 3' enhancers are initially inactive, then become strongly active through synergistic interactions, and finally achieve a strong level of activity with little interdependency. The relative contributions of E mu (the intron enhancer) and of the 3' enhancers also change as a function of developmental stage. We discuss these results in light of parallel studies of developmental changes in transcription factor requirements.

Temporal Control of IgH Gene Expression in Developing B Cells by the 3' Locus Control Region

The suggested roles of the downstream 3′ regions acting as a Locus Control Region (LCR), have allowed comparisons to be made between the regulation of the IgH locus with other model systems whose gene expression is governed by LCR activity. Here we summarize the importance of the IgH 3′LCR and its putative functional role in IgH gene expression and compare it with the 5′LCR regulatory region of the human β-globin locus.

Identification of 3' alpha-hs4, a novel Ig heavy chain enhancer element regulated at multiple stages of B cell differentiation.

In addition to E mu, several elements downstream of the IgH cluster, i.e. 3' of the C alpha gene, are involved in regulating IgH gene rearrangement and expression. This entire downstream regulatory region was shown to be deleted in the mutant myeloma cell line, LP1.2. The deletion encompasses approximately 34 kb and is presumably responsible for the reduced levels of IgH expression in this cell line. An additional regulatory element, included in the LP1.2 deletion, was identified by investigation of a DNase I hypersensitivity site located approximately 33 kb downstream of the alpha gene and present in pre-B and plasma cells. This novel IgH gene enhancer element, termed 3' alpha-hs4, is capable of activity throughout B cell development. Transient transfection of 3' alpha-hs4 in a CAT reporter gene construct shows transcriptional enhancement activity approximating that of E mu in S194 plasmacytoma and M12.4.1 and A-20 B cell lines; while in a pre-B cell line, 18-81, the average activity is 25% that of E mu. Enhancer activity was localized to an 800 bp fragment. The activity of 3' alpha-hs4 is orientation independent and appears to be B cell specific. Tight regulation of 3' alpha-hs4 is inferred from its variable activity in different plasmacytoma cell lines and within the pre B cell line, 18-81.

Regulation of lymphoid-specific immunoglobulin mu heavy chain gene enhancer by ETS-domain proteins.

The enhancer for the immunoglobulin mu heavy chain gene (IgH) activates a heterologous gene at the pre-B cell stage of B lymphocyte differentiation. A lymphoid-specific element, microB, is necessary for enhancer function in pre-B cells. A microB binding protein is encoded by the PU.1/Spi-1 proto-oncogene. Another sequence element, microA, was identified in the mu enhancer that binds the product of the ets-1 proto-oncogene. The microA motif was required for microB-dependent enhancer activity, which suggests that a minimal B cell-specific enhancer is composed of both the PU.1 and Ets-1 binding sites. Co-expression of both PU.1 and Ets-1 in nonlymphoid cells trans-activated reporter plasmids that contained the minimal mu enhancer. These results implicate two members of the Ets family in the activation of IgH gene expression.