Immunoglobulin Heavy Chain mRNA Research Articles

The Ig heavy chain protein but not its message controls early B cell development

Development of progenitor B cells (ProB cells) into precursor B cells (PreB cells) is dictated by immunoglobulin heavy chain checkpoint (IgHCC), where the IgHC encoded by a productively rearranged Igh allele assembles into a PreB cell receptor complex (PreBCR) to generate signals to initiate this transition and suppressing antigen receptor gene recombination, ensuring that only one productive Igh allele is expressed, a phenomenon known as Igh allelic exclusion. In contrast to a productively rearranged Igh allele, the Igh messenger RNA (mRNA) (IgHR) from a nonproductively rearranged Igh allele is degraded by nonsense-mediated decay (NMD). This fact prohibited firm conclusions regarding the contribution of stable IgHR to the molecular and developmental changes associated with the IgHCC. This point was addressed by generating the IghTer5H∆TM mouse model from IghTer5H mice having a premature termination codon at position +5 in leader exon of IghTer5H allele. This prohibited NMD, and the lack of a transmembrane region (∆TM) prevented the formation of any signaling-competent PreBCR complexes that may arise as a result of read-through translation across premature Ter5 stop codon. A highly sensitive sandwich Western blot revealed read-through translation of IghTer5H message, indicating that previous conclusions regarding a role of IgHR in establishing allelic exclusion requires further exploration. As determined by RNA sequencing (RNA-Seq), this low amount of IgHC sufficed to initiate PreB cell markers normally associated with PreBCR signaling. In contrast, the IghTer5H∆TM knock-in allele, which generated stable IgHR but no detectable IgHC, failed to induce PreB development. Our data indicate that the IgHCC is controlled at the level of IgHC and not IgHR expression.

Cytoplasmic poly(A)-binding protein 1 (PABPC1) interacts with the RNA-binding protein hnRNPLL and thereby regulates immunoglobulin secretion in plasma cells

Increasing evidence indicates that alternative processing of mRNA, including alternative splicing, 3' alternative polyadenylation, and regulation of mRNA stability/translation, represents a major mechanism contributing to protein diversification. For example, in alternative polyadenylation, the 3' end of the immunoglobulin heavy chain mRNA is processed during B cell differentiation, and this processing involves RNA-binding proteins. hnRNPLL (heterogeneous nuclear ribonucleoprotein L-like protein) is an RNA-binding protein expressed in terminally differentiated lymphocytes, such as memory T cells and plasma cells. hnRNPLL regulates various processes of RNA metabolism, including alternative pre-mRNA splicing and RNA stability. In plasma cells, hnRNPLL also regulates the transition from the membrane isoform of the immunoglobulin heavy-chain (mIgH) to the secreted isoform (sIgH), but the precise mechanism remains to be identified. In this study, we report that hnRNPLL specifically associates with cytoplasmic PABPC1 (poly(A)-binding protein 1) in both T cells and plasma cells. We found that although PABPC1 is not required for the alternative splicing of CD45, a primary target of hnRNPLL in lymphocytes, PABPC1 does promote the binding of hnRNPLL to the immunoglobulin mRNA and regulates switching from mIgH to sIgH in plasma cells. Given the recently identified role of PABPC1 in mRNA alternative polyadenylation, our findings suggest that PABPC1 recruits hnRNPLL to the 3'-end of RNA and regulates the transition from membrane Ig to secreted Ig through mRNA alternative polyadenylation. In conclusion, our study has revealed a mechanism that regulates immunoglobulin secretion in B cells via cooperation between a plasma cell-specific RBP (hnRNPLL) and a universally expressed RBP (PABPC1).

Inter- and intratumoral heterogeneity of BCL2 correlates with IgH expression and prognosis in follicular lymphoma.

Most follicular lymphomas (FLs) are genetically defined by the t(14;18)(q32;q21) translocation that juxtaposes the BCL2 gene to the immunoglobulin heavy chain (IgH) 3' regulatory regions (IgH-3'RRs). Despite this recurrent translocation, FL cases are heterogeneous in terms of intratumoral clonal diversity for acquired mutations and variations in the tumor microenvironment. Here we describe an additional mechanism that contributes to inter- and intratumoral heterogeneity in FLs. By applying a novel single-molecule RNA fluorescence-based in situ hybridization (FISH) technique to detect mRNA molecules of BCL2 and IgH in single cells, we found marked heterogeneity in the number of BCL2 mRNA transcripts within individual lymphoma cells. Moreover, BCL2 mRNA molecules correlated with IgH mRNA molecules in individual cells both in t(14;18) lymphoma cell lines and in patient samples. Consistently, a strong correlation between BCL2 and IgH protein levels was found in a series of 205 primary FL cases by flow cytometry and immunohistochemistry. Inter- and intratumoral heterogeneity of BCL2 expression determined resistance to drugs commonly used in FL treatment and affected overall survival of FL patients. These data demonstrate that BCL2 and IgH expressions are heterogeneous and coregulated in t(14;18)-translocated cells, and determine the response to therapy in FL patients.

The Eleven-Nineteen Lysine-rich Leukemia Gene (ELL2) Influences the Histone H3 Protein Modifications Accompanying the Shift to Secretory Immunoglobulin Heavy Chain mRNA Production

In plasma cells, immunoglobulin heavy chain (IgH) secretory-specific mRNA is made in high abundance as a result of both increased promoter proximal poly(A) site choice and weak splice-site skipping. Ell2, the eleven-nineteen lysine rich leukemia gene, is a transcription elongation factor that is induced ∼6-fold in plasma cells and has been shown to drive secretory-specific mRNA production. Reducing ELL2 by siRNA, which reduced processing to the secretion-specific poly(A) site, also influenced the methylations of histone H3K4 and H3K79 on the IgH gene and impacted positive transcription factor b (pTEFb), Ser-2 carboxyl-terminal phosphorylation, and polyadenylation factor additions to RNA polymerase II. The multiple lineage leukemia gene (MLL) and Dot1L associations with the IgH gene were also impaired in the absence of ELL2. To investigate the link between histone modifications, transcription elongation, and alternative RNA processing in IgH mRNA production, we performed chromatin immunoprecipitation on cultured mouse B and plasma cells bearing the identical IgH γ2a gene. In the plasma cells, as compared with the B cells, the H3K4 and H3K79 methylations extended farther downstream, past the IgH enhancer to the end of the transcribed region. Thus the downstream H3K4 and H3K79 methylation of the IgH associated chromatin in plasma cells is associated with increased polyadenylation and exon skipping, resulting from the actions of ELL2 transcription elongation factor.

Immunoglobulin heavy chain gene regulation through polyadenylation and splicing competition

The immunoglobulin heavy chain (IgH) genes, which encode one of the two chains of antibody molecules, were the first cellular genes shown to undergo developmentally regulated alternative RNA processing. These genes produce two different mRNAs from a single primary transcript. One mRNA is cleaved and polyadenylated at an upstream poly(A) signal while the other mRNA removes this poly(A) signal by RNA splicing and is cleaved and polyadenylated at a downstream poly(A) site. A broad range of studies have been performed to understand the mechanism of IgH RNA processing regulation during B lymphocyte development. The model that has emerged is much more complex than envisioned by the earliest view of regulation through poly(A) signal choice. Regulation requires that the IgH gene contain competing splice and cleavage-polyadenylation reactions with balanced efficiencies. Because non-IgH genes with these structural features also can be regulated, IgH gene-specific sequence elements are not required for regulation. Changes in cleavage-polyadenylation and RNA splicing, as well as pol II elongation, all contribute to IgH developmental RNA processing regulation. Multiple factors are likely involved in the regulation during B lymphocyte maturation. Additional biologically relevant factors that contribute to IgH regulation remain to be identified and incorporated into a mechanistic model for regulation. Much of the work to date confirms the complex nature of IgH mRNA regulation and suggests that a thorough understanding of this control will remain a challenge. However, it is also likely that such understanding will help elucidate novel mechanisms of RNA processing regulation.

Loading and phosphorylation of RNA polymerase II influence production of secretory-specific Ig heavy chain mRNA in plasma cells. (35.33)

Abstract A crucial step in the development of B cells into plasma cells is the increased production of secreted immunoglobulin which occurs by differential selection of the secretory-specific polyA site. Factors involved in polyadenylation can be coupled with transcription through phosphorylation of the carboxyl-terminal domain (CTD) of RNA pol II. We analyzed loading and phosphorylation patterns of RNA Polymerase II along an identical immunoglobulin heavy chain gene expressed either in B- or plasma cells by chromatin immunoprecipitation using various antibodies. Data indicate that the switch to secreted immunoglobulin heavy chain mRNA production is influenced by increased RNA Polymerase II accumulation and CTD phosphorylation at the promoter in plasma cells. Loading of transcription factors is increased in the plasma cells. This allows for increased access of specific processing factors to the polymerase and nascent RNA. Supported by NIH-CA-86433 to C.M.

Antagonism of aryl hydrocarbon receptor-dependent induction of CYP1A1 and inhibition of IgM expression by di- ortho-substituted polychlorinated biphenyls

Halogenated aromatic hydrocarbons (HAHs) are ubiquitous environment contaminants that produce many of their toxic effects by binding to the aryl hydrocarbon receptor (AhR). However, several investigations have demonstrated that certain polychlorinated biphenyl (PCB) congeners, principally di- ortho-chlorinated PCB congeners, or mixtures containing multiple di- ortho-chlorinated PCBs, inhibit AhR-mediated responses induced by other toxic HAHs. Most relevant to the present study are past reports demonstrating antagonism by these uniquely acting PCB congeners on AhR agonist-mediated inhibition of humoral immune responses. The mechanism responsible for antagonism of AhR agonists by certain PCBs is presently unknown. The present study evaluated the antagonist activity of several di- ortho-substituted PCB congeners [PCB47 (2,2′,4,4′), PCB52 (2,2′,5,5′), PCB128 (2,2′,3,3′,4,4′), and PCB153 (2,2′,4,4′,5,5′)] when present in combination with AhR agonists [TCDD (2,3,7,8,-tetrachlorodibenzo- p-dioxin), PCB126 (3,3′,4,4′,5), and PCB77 (3,3′,4,4′)] on CYP1A1 induction and inhibition of lipopolysaccharide (LPS)-induced immunoglobulin production in the CH12.LX B cell line. In contrast to non- ortho-substituted PCB (PCB77), which showed additive effects on CYP1A1 induction in combination with TCDD, all of the di- ortho-substituted PCBs examined produced antagonism. Di- ortho-substituted PCB (PCB52) also antagonized TCDD- or PCB126- mediated inhibition of IgM secretion and immunoglobulin heavy chain mRNA expression in the LPS-activated B cells. In addition, PCB52 inhibited TCDD-induced AhR DNA binding to a dioxin-responsive element. Collectively, these results suggest that the mechanism responsible for antagonism by di- ortho-substituted PCB congeners of AhR agonist-mediated CYP1A1 induction and inhibition of antibody responses in B cells occurs through interference with agonist activation of the cytosolic AhR complex.

Ontogeny of lymphoid organs and immunoglobulin producing cells in atlantic cod (Gadus morhua L.)

The ontogeny of lymphoid organs and the development of cells expressing immunoglobulin heavy chain (IgH) mRNA as well as cells containing immunoglobulin (IgM) were studied in Atlantic cod ( Gadus morhua L.), a marine teleost. Head kidney and spleen appeared as the first lymphoid organs, present at the time of hatching, whereas thymus was observed in 9 mm larvae. Fully developed lymphoid organs were not achieved until after metamorphosis. Cells expressing IgH mRNA were detected in paraffin sections of larvae and juveniles by in situ hybridization. Positive cells were not detected in fish smaller than 33 mm (58 days after hatching). IgH mRNA expression coincided with the first appearance of immunoglobulin-positive cells as revealed by immunohistochemistry in the same animals.

Localisation of Ig heavy chain mRNA positive cells in Atlantic cod (Gadus morhuaL.) tissues; identified byin situhybridisation

Localisation of immunoglobulin heavy chain (IgH) producing cells was determined in sections from head kidney, spleen, thymus, gills, gut, skin, heart and liver from the Atlantic cod. In general, IgH mRNA positive cells were detected in all organs examined and were mainly located to the connective tissue surrounding the vascular system in these organs. In the head kidney and spleen, IgH mRNA positive cells appeared as single distributed cells or as dense clusters, whereas in the thymus only single distributed positive cells were observed. The percentage of Ig heavy chain mRNA positive (plasma) cells in the head kidney, spleen and thymus was estimated at about 1% of the total cell mass. The number of IgH mRNA positive cells was lower than this in all the other organs examined.

Cross-linking of OX40 ligand, a member of the TNF/NGF cytokine family, induces proliferation and differentiation in murine splenic B cells

OX40 is a member of the TNF/NGF-receptor family expressed on activated T cells, whose ligand is found on activated T and B cells. In the present study, we show that cross-linking of OX40L on CD40L-stimulated B cells, αlgD dextran-stimulated B cells, or both results in a significantly enhanced proliferative response with no change In the cell survival rate. Furthermore, OX40 stimulation increases Immunoglobulin heavy chain mRNA levels and Immunoglobulin secretion, which could not be blocked by anti-cytokine antibodies. In additional molecular studies, we show that OX40L cross-linking results In the down-regulation of the transcription factor BSAP. This, In turn, leads to a change In the In vivo binding pattern of the imunoglobulin heavy chain gene 3′ α enhancer, suggesting its activation. This effect may thus be one mechanism for OX40-induced Increase In Immunoglobulin secretion. In conclusion, our data suggest that the OX40-OX40L interaction is a novel pathway in T cell-dependent B cell proliferation and differentiation.

Minimal membrane and secreted μ poly(A) signals specify developmentally-regulated immunoglobulin heavy chain mRNA ratios without RNA splicing

Minimal membrane and secreted μ poly(A) signals specify developmentally-regulated immunoglobulin heavy chain mRNA ratios without RNA splicing

The half-life of immunoglobulin mRNA increases during B-cell differentiation: a possible role for targeting to membrane-bound polysomes.

The increase in abundance of immunoglobulin heavy-chain (IgH) mRNA that accompanies development of a B cell into a plasma cell is mainly due to post-transcriptional events. By constructing mu genes, whose expression is under inducible control, we determined the half-life of pulsed mu transcripts to be approximately 20 hr in plasmacytoma hosts and approximately 3 hr in B-cell lymphomas. Interestingly, a mu gene with a mutated signal sequence that is found on free (rather than membrane-bound) polysomes decays in the plasmacytoma host with a shortened half-life of 3 hr. Thus, a change in the turnover rate of IgH mRNA plays an important role in the cell-type specificity of immunoglobulin gene expression; this change may be a consequence of the fact that IgH mRNA is located in the membrane-bound polysome fraction.

A limited number of B cell lineages generates the heterogeneity of a secondary immune response.

We have studied the cellular basis for heterogeneity in the secondary immune response by creating a large set of B cell hybridomas from a single C57BL/6 mouse immunized with the hapten (4-hydroxy-3-nitrophenyl)acetyl coupled to chicken gamma-globulin, and searching among the cells for all possible sets of clonally related lines. Among 28 independent cell lines from a single animal we find that 21 of them fall into seven small families of from two to five members; only seven lines have no matches among the set. A statistical analysis determines that the number of genetically distinct B cell precursors whose progeny were isolated during this secondary response is 18, with 95% confidence limits of 14 and 26. The distribution of family sizes implies that each activated clone has proliferated to approximately the same extent. Complete sequencing of the variable region of immunoglobulin heavy chain mRNA from 19 of the cell lines reveals many shared somatic mutations among related lines, implying a pronounced founder effect has occurred to eliminate most of the progeny of each precursor B cell. This may be the result either of a high frequency of debilitating somatic mutation or from selection against cells still expressing the idiotype of primary anti-(4-hydroxy-3-nitrophenyl)acetyl antibodies.

Mutational analysis of the immunoglobulin heavy chain promoter region.

Complete immunoglobulin heavy chain (IgH) genes (gamma and mu) containing the intronic IgH enhancer and mutations in the upstream promoter region were constructed in vitro and introduced into murine J558L myeloma cells by protoplast fusion. S1-nuclease mapping experiments demonstrated that IgH gene expression was extremely sensitive to mutation in an upstream region containing the octanucleotide sequence ATGCAAAT. Significant IgH mRNA levels were detected in RNA from cells transfected with IgH gene constructs in which all upstream sequences on the 5' proximal side of this element were deleted. Similar results were obtained using the precise inverse of the IgH octamer, which is found in the upstream promoter region of immunoglobulin light chain genes. Deletion of the IgH octamer, or point mutation of adenine to guanine at position 6, resulted in the loss of correctly initiated IgH mRNA. A DNA binding factor from J558L nuclear extracts was identified that appeared to recognize the octamer on the basis of differential binding to homologous restriction fragments containing the various mutations and that bound preferentially with octamer DNA fragments derived from functional relative to nonfunctional IgH constructs. Collectively, these data suggest that the octamer element contains residues that are critical to accurate immunoglobulin gene transcription and that may serve as part of a recognition locus for nuclear factors important to B-cell-specific immunoglobulin expression.

DNA Rearrangement Causes a High Rate of Spontaneous Mutation at the Immunoglobulin Heavy-Chain Locus of a Mouse Myeloma Cell Line

The spontaneous mutation rate of immunoglobulin genes expressed in myeloma cells is well above that of other genes expressed in these or in other cell types. The nature of such mutations in one myeloma cell line, MPC11, was explored at the molecular level. Included in this study were MPC11 variants representing 24 independent and spontaneous mutations affecting immunoglobulin secretion. Of the mutants studied, 19 had ceased immunoglobulin heavy chain (IgH) production (nonproducers), and 5 produced from as little as 1/1,000 to as much as 1/10 the amount of immunoglobulin produced by MPC11 (low producers). Only one of the MPC11 mutants (a nonproducer) showed no evidence of DNA rearrangement in or near the expressed IgH gene. The formerly expressed gamma 2b gene had been deleted in 18 of the 19 nonproducers. All of the low producers had undergone DNA rearrangement in or near the expressed IgH gene, and three of them produced immunoglobulin of a new heavy chain class. The cause for reduced heavy-chain synthesis in the low producers is not yet known. However, in several of these mutants, the defect appeared to be posttranscriptional. In these cell lines, steady-state IgH mRNA levels were much lower than in the parent cell line, while the heavy-chain gene transcription rate remained unchanged.

DNA rearrangement causes a high rate of spontaneous mutation at the immunoglobulin heavy-chain locus of a mouse myeloma cell line.

The spontaneous mutation rate of immunoglobulin genes expressed in myeloma cells is well above that of other genes expressed in these or in other cell types. The nature of such mutations in one myeloma cell line, MPC11, was explored at the molecular level. Included in this study were MPC11 variants representing 24 independent and spontaneous mutations affecting immunoglobulin secretion. Of the mutants studied, 19 had ceased immunoglobulin heavy chain (IgH) production (nonproducers), and 5 produced from as little as 1/1,000 to as much as 1/10 the amount of immunoglobulin produced by MPC11 (low producers). Only one of the MPC11 mutants (a nonproducer) showed no evidence of DNA rearrangement in or near the expressed IgH gene. The formerly expressed gamma 2b gene had been deleted in 18 of the 19 nonproducers. All of the low producers had undergone DNA rearrangement in or near the expressed IgH gene, and three of them produced immunoglobulin of a new heavy chain class. The cause for reduced heavy-chain synthesis in the low producers is not yet known. However, in several of these mutants, the defect appeared to be posttranscriptional. In these cell lines, steady-state IgH mRNA levels were much lower than in the parent cell line, while the heavy-chain gene transcription rate remained unchanged.

Cloning and structural analysis of cDNAs for bcl-2 and a hybrid bcl-2/immunoglobulin transcript resulting from the t(14;18) translocation

cDNAs for the bcl-2 mRNA were cloned from a human common acute lymphoblastic leukemia cell line. Nucleotide sequence analyses showed that the 6 kb bcl-2 mRNA potentially encodes a 26 kd protein that is homologous to a predicted Epstein-Barr virus protein. Most t(14;18) translocation breakpoints cluster within a narrow region of a 5.4 kb exon that contains a long 3′-untranslated region of the bcl-2 mRNA. As a result of t(14;18) translocation, hybrid bcl-2/immunoglobulin heavy chain transcripts are produced that consist of the 5′ half of the bcl-2 mRNA fused to a “decapitated” immunoglobulin heavy chain mRNA. Nucleotide sequence analyses confirmed that the hybrid transcripts continue to encode a normal bcl-2 protein. Our results suggest that t(14;18) translocations alter expression of the bcl-2 gene both by transcriptional activation and by abnormal posttranscriptional regulation of bcl-2 mRNA.

During B-cell differentiation enhancer activity and transcription rate of immunoglobulin heavy chain genes are high before mRNA accumulation

During differentiation of B lymphocytes, a low level of immunoglobulin heavy chain gene transcripts is apparent at the pre-B-cell stage, and a dramatic increase in immunoglobulin mRNA level is seen after stimulated B cells have matured into immunoglobulin-secreting plasma cells. We have measured the transcription rate of endogenous heavy chain genes using cell lines representative of various stages of murine B-lymphocyte differentiation. We observe a good correlation between RNA polymerase density, as determined by nuclear run-on transcription experiments, and the activity of the heavy chain gene enhancer, as assayed by transfection experiments. Both enhancer activity and heavy chain gene transcription are very high in pre-B-cell lines. Thus we conclude that the increased accumulation of immunoglobulin heavy chain mRNA in immunoglobulin-secreting plasma cells is regulated mainly by posttranscriptional processes.

Mouse immunoglobulin genes: a bacterial plasmid containing the entire coding sequence for a pre-gamma 2a heavy chain.

A DNA sequence complementary to the entire coding part of a mouse gamma 2a immunoglobulin heavy chain mRNA isolated from a myeloma producing a levan binding protein (UPC 10), has been cloned in the PstI site of pBR 322. Transformants containing sequences complementary to purified gamma 2a heavy chain mRNA were selected. One transformant, pG2a-10-21, containing a 1750 nucleotide insert, has been characterized by hybrid-arrested translation and purification of gamma 2a heavy chain mRNA on DNA-DBM cellulose filters. Restriction enzyme analysis and partial sequencing demonstrate that the pG2a-10-21 contains the complete structural sequence for the gamma 2a heavy chain and predicts the sequence of a 18 amino acid hydrophobic amino terminal extra piece segment.

Translation of human immunoglobulin heavy chain mRNA in vitro

MOUSE immunoglobulin (Ig) mRNAs have been the subject of intensive study. Purification of Ig light chain mRNAs from myeloma or plasmacytoma cells has been monitored by translation in vitro, and studies have been directed towards sequence determination and gene titration Moreover, partial purification of Ig heavy chain mRNA has also been achieved1–5. No comparable work on the Ig mRNA of any other species has yet been published. We report here the preparation, fractionation and translation in vitro of mRNA from cells of a cloned normal human B lymphocytic cell line. RPMI 1788 (ref. 6). grown in tissue culture. Human Ig μ chains were identified in the translation product by specific immunoprecipitation and electrophoretic mobility in sodium dodecyl sulphate (SDS)-urea polyacrylamide gels.