Copyright (c) 2009 All rights reserved. http://works.bepress.com/stavnezerj Recent documents in Janet M Stavnezer en-us Sun, 31 May 2009 12:15:27 PDT 3600 http://works.bepress.com/stavnezerj/89 http://works.bepress.com/stavnezerj/89 Tue, 01 Apr 2008 10:37:17 PDT TGF-beta1 directs class switching to IgA by splenic B cells and by the surface IgM+ B cell line, I.29mu, by inducing germline (GL) Ig alpha transcripts. The promoter segment between -130 and +46, relative to the first initiation site for mouse GL alpha transcripts, is sufficient for expression and TGF-beta1 inducibility of a reporter gene in B cell lines. Within this segment resides a TGF-beta1-responsive element (TbetaRE) that is required for induction of the promoter by TGF-beta1 and, when multimerized, is sufficient to transfer TGF-beta1 inducibility to another promoter. In this report we show that a TGF-beta1-inducible complex binds the TbetaRE and contains the transcription factor core-binding factor (CBF; also known as acute myeloid leukemia, AML). Although all three CBF alpha family members activate the GL alpha promoter, only CBF alpha3 (AML-2) is induced by TGF-beta1 in splenic B and I.29mu cells. The TbetaRE contains two CBF binding sites. Mutation of both sites reduces but does not eliminate induction of the GL alpha promoter by TGF-beta1 or by overexpression of CBF, possibly due to the presence of an additional CBF site in the promoter. In addition, the TbetaRE contains two copies of another sequence motif. Mutation of these motifs eliminates TGF-beta1 induction of the GL alpha promoter. Together the data indicate that TGF-beta1 induction of the alpha promoter involves induction of CBF alpha3, which binds to the TbetaRE of the promoter along with one or more proteins. M. J. Shi Animals B-Lymphocytes Core Binding Factors DNA-Binding Proteins Gene Expression Regulation *Genes, Immunoglobulin Genes, Reporter Immunoglobulin A Immunoglobulin Class Switching Immunoglobulin M Lymphoma, B-Cell Macromolecular Substances Mice *Neoplasm Proteins *Promoter Regions (Genetics) Receptors, Antigen, B-Cell Spleen Transcription Factors Transforming Growth Factor beta Tumor Cells, Cultured http://works.bepress.com/stavnezerj/88 http://works.bepress.com/stavnezerj/88 Tue, 01 Apr 2008 10:37:12 PDT A large body of data indicate that antibody class switching is directed by cytokines by inducing or repressing transcription from unrearranged, or germline, CH genes. Interleukin 4 (IL-4) induces transcription of the germline C epsilon genes in activated B cells and subsequently, cells in this population will undergo switch recombination to immunoglobulin E. Furthermore, the data suggest that transcription of germline C epsilon genes is required for class switching. In this paper we define DNA elements required for induction of transcription of the germline C epsilon genes by IL-4. To do this, segments of DNA from the 5' flank of the initiation sites for germline epsilon RNA were ligated to a luciferase reporter gene and transfected into two mouse B cell lines, one of which can be induced to switch to IgE. By analysis of a series of 5' deletion constructs and linker-scanning mutations, we demonstrate that a 46-bp segment (residing at -126/-79 relative to the first RNA initiation site) contains an IL-4 responsive region. By electrophoretic mobility shift assays, we find that this segment binds three transcription factors: the recently described NF-IL4, one or more members of the C/EBP family of transcription factors, and NF-kappa B/p50. Mutation of any of the binding sites for these three factors abolishes or reduces IL-4 inducibility of the epsilon promoter. A 27-bp segment within this IL-4 response region containing binding sites for NF-IL4 and a C/EBP factor is sufficient to transfer IL-4 inducibility to a minimal c-fos promoter. S. Delphin Animals Base Sequence Binding Sites CCAAT-Enhancer-Binding Proteins Consensus Sequence DNA-Binding Proteins *Genes, Immunoglobulin Humans Immunoglobulin epsilon-Chains Interleukin-4 Mice Molecular Sequence Data NF-kappa B Nuclear Proteins Oligodeoxyribonucleotides *Promoter Regions (Genetics) Sequence Alignment Sequence Homology, Amino Acid Structure-Activity Relationship Transcription Factors Transcription, Genetic http://works.bepress.com/stavnezerj/87 http://works.bepress.com/stavnezerj/87 Tue, 01 Apr 2008 10:37:07 PDT Antibody class switching is regulated by transcription of unrearranged C(H) genes to produce germline (GL) transcripts which direct the choice of isotype and are required for switching. However, their role is unknown. GL transcripts are initiated at the I exons located upstream of each switch region. Although deletion of the I exon by gene targeting prevents switch recombination to that CH gene, the Ialpha exon can be replaced by an entirely different DNA segment, a minigene driven by the phosphoglycerate kinase (PGK) promoter and encoding hypoxanthine phosphoribosyl transferase (HPRT), oriented in the sense direction, without reducing antibody class switching to IgA. To understand why HPRT substitution of the Ialpha exon does not disrupt switch recombination, we have analyzed the structure of the transcript from the targeted allele in these mice. We identify a spliced transcript in which the HPRT exons are spliced to the C(alpha) gene segments, resulting in a structure similar to normal GL transcripts. The abundance of this transcript is similar to that of the normal alpha GL RNA. We also demonstrate that switching to the four IgG subclasses in B cells from these mice is reduced in comparison to wild-type mice. We discuss the possibility that the strong PGK promoter inserted at the Ig alpha locus may interfere with interaction of the promoters for gamma GL transcripts with the 3' IgH enhancer. G. Qiu Animals Exons Gene Expression Regulation Germ Cells Hypoxanthine Phosphoribosyltransferase Immunoglobulin A *Immunoglobulin Class Switching Immunoglobulin G Immunoglobulin Heavy Chains Mice Mice, Knockout *RNA Splicing Recombination, Genetic Sequence Analysis, DNA http://works.bepress.com/stavnezerj/86 http://works.bepress.com/stavnezerj/86 Tue, 01 Apr 2008 10:37:02 PDT Ig heavy chain class switch recombination (CSR) determines the expression of Ig isotypes. The molecular mechanism of CSR and the factors regulating this process have remained elusive. Recombination occurs primarily within switch (S) regions, located upstream of each heavy chain gene (except Cdelta). These repetitive sequences contain consensus DNA-binding sites for the DNA-binding protein late SV40 factor (LSF) (CP2/leader-binding protein-1c). In this study, we demonstrate by EMSA that purified rLSF, as well as LSF within B cell extracts, directly binds both Smu and Salpha sequences. To determine whether LSF is involved in regulating CSR, two different LSF dominant negative variants were stably expressed in the mouse B cell line I.29 mu, which can be induced to switch from IgM to IgA. Overexpression of these dominant negative LSF proteins results in decreased levels of endogenous LSF DNA-binding activity and an increase in cells undergoing CSR. Thus, LSF represses class switching to IgA. In agreement, LSF DNA-binding activity was found to decrease in whole cell extracts from splenic B cells induced to undergo class switching. To elucidate the mechanism of CSR regulation by LSF, the interactions of LSF with proteins involved in chromatin modification were tested in vitro. LSF interacts with both histone deacetylases and the corepressor Sin3A. We propose that LSF represses CSR by histone deacetylation of chromatin within S regions, thereby limiting accessibility to the switch recombination machinery. Elise E. Drouin Amino Acid Substitution Animals Base Sequence *Binding Sites, Antibody Cell Line Chromatin DNA-Binding Proteins Gene Silencing Glutamic Acid Glutamine Humans Immunoglobulin A *Immunoglobulin Class Switching *Immunoglobulin Switch Region Immunoglobulin alpha-Chains Immunoglobulin mu-Chains Leucine Lymphoma, B-Cell Lysine Mice Mice, Inbred C57BL Molecular Sequence Data RNA-Binding Proteins Repressor Proteins Spleen Transcription Factors Transcription, Genetic Tumor Cells, Cultured http://works.bepress.com/stavnezerj/85 http://works.bepress.com/stavnezerj/85 Tue, 01 Apr 2008 10:36:57 PDT No abstract provided. W. Dunnick Animals B-Lymphocytes Gene Expression Regulation *Immunoglobulin Switch Region Mice Mice, Transgenic Recombination, Genetic Transcription, Genetic http://works.bepress.com/stavnezerj/84 http://works.bepress.com/stavnezerj/84 Tue, 01 Apr 2008 10:36:53 PDT No abstract provided. Janet Stavnezer Animals B-Lymphocytes Gene Expression Regulation *Genes, Regulator *Genes, Switch Immunoglobulin A Immunoglobulin E Immunoglobulin G Immunoglobulin Heavy Chains Mice Transcription, Genetic Tumor Cells, Cultured http://works.bepress.com/stavnezerj/83 http://works.bepress.com/stavnezerj/83 Tue, 01 Apr 2008 10:36:49 PDT No abstract provided. E. Severinson Animals B-Lymphocytes Cells, Cultured Enzyme-Linked Immunosorbent Assay Humans Immunoglobulin E Interleukin-4 Interleukin-5 Lipopolysaccharides Mice RNA, Messenger T-Lymphocytes http://works.bepress.com/stavnezerj/82 http://works.bepress.com/stavnezerj/82 Tue, 01 Apr 2008 10:36:46 PDT Mismatch repair proteins participate in antibody class switch recombination, although their roles are unknown. Previous nucleotide sequence analyses of switch recombination junctions indicated that the roles of Msh2 and the MutL homologues, Mlh1 and Pms2, differ. We now asked if Msh2 and Mlh1 function in the same pathway during switch recombination. Splenic B cells from mice deficient in both these proteins were induced to undergo switching in culture. The frequency of switching is reduced, similarly to that of B cells singly deficient in Msh2 or Mlh1. However, the nucleotide sequences of the Smu-Sgamma3 junctions resemble junctions from Mlh1- but not from Msh2-deficient cells, suggesting Mlh1 functions either independently of or before Msh2. The substitution mutations within S regions that are known to accompany switch recombination are increased in Msh2- and Mlh1 single-deficient cells and further increased in the double-deficient cells, again suggesting these proteins function independently in class switch recombination. The finding that MMR functions to reduce mutations in switch regions is unexpected since MMR proteins have been shown to contribute to somatic hypermutation of antibody variable region genes. Carol E. Schrader Adenosine Triphosphatases Animals Base Pair Mismatch Base Sequence Carrier Proteins DNA Repair *DNA Repair Enzymes DNA-Binding Proteins Immunoglobulin Class Switching Mice Molecular Sequence Data MutS Homolog 2 Protein Neoplasm Proteins Nuclear Proteins Proto-Oncogene Proteins *Recombination, Genetic http://works.bepress.com/stavnezerj/81 http://works.bepress.com/stavnezerj/81 Tue, 01 Apr 2008 10:36:40 PDT B cell-specific activator protein (BSAP)/Pax-5 is a paired domain DNA-binding protein expressed in the developing nervous system, testis, and in all B lineage cells, except terminally differentiated plasma cells. BSAP regulates transcription of several genes expressed in B cells and also the activity of the 3' IgH enhancer. As it has binding sites within or 5' to the switch regions of nearly all Ig heavy chain C region genes and also is known to increase transcription of the germline epsilon RNA, BSAP has been hypothesized to be involved in regulation of Ab class switch recombination. To directly examine the effects of BSAP on isotype switching, we use a tetracycline-regulated expression system to overexpress BSAP in the surface IgM+ I.29 mu B cell line, a mouse cell line that can be induced to undergo class switch recombination. We find that overexpression of BSAP inhibits switching to IgA in I.29 mu cells stimulated with LPS + TGF-beta 1 + nicotinamide, but enhances switching to IgE in cells stimulated with LPS + IL-4 + nicotinamide. Parallel to its effects on switching, overexpression of BSAP inhibits germline alpha RNA expression and the transcriptional activity of the germline alpha promoter, while enhancing activity of the germline epsilon promoter. Proliferation of I.29 mu cells is not affected in this system. The possible mechanisms and significance of the effect of BSAP on isotype switching are discussed. G. Qiu Adjuvants, Immunologic Animals B-Cell-Specific Activator Protein B-Lymphocytes Base Sequence Binding Sites DNA-Binding Proteins Gene Expression Regulation Immunoglobulin A Immunoglobulin Class Switching Immunoglobulin E Immunoglobulin alpha-Chains Immunoglobulin epsilon-Chains Immunoglobulin mu-Chains Immunosuppressive Agents Lymphoma, B-Cell Mice Molecular Sequence Data Nuclear Proteins Promoter Regions (Genetics) Tetracycline *Transcription Factors Transfection Tumor Cells, Cultured http://works.bepress.com/stavnezerj/80 http://works.bepress.com/stavnezerj/80 Tue, 01 Apr 2008 10:36:35 PDT Signal transducer and activator of transcription 6 (Stat6) and NF-kappaB are widely distributed transcription factors which are induced by different stimuli and bind to distinct DNA sequence motifs. Interleukin-4 (IL-4), which activates Stat6, synergizes with activators of NF-kappaB to induce IL-4-responsive genes, but the molecular mechanism of this synergy is poorly understood. Using glutathione S-transferase pulldown assays and coimmunoprecipitation techniques, we find that NF-kappaB and tyrosine-phosphorylated Stat6 can directly bind each other in vitro and in vivo. An IL-4-inducible reporter gene containing both cognate binding sites in the promoter is synergistically activated in the presence of IL-4 when Stat6 and NF-kappaB proteins are coexpressed in human embryonic kidney 293 (HEK 293) cells. The same IL-4-inducible reporter gene is also synergistically activated by the endogenous Stat6 and NF-kappaB proteins in IL-4-stimulated I.29mu B lymphoma cells. Furthermore, Stat6 and NF-kappaB bind cooperatively to a DNA probe containing both sites, and the presence of a complex formed by their cooperative binding correlates with the synergistic activation of the promoter by Stat6 and NF-kappaB. We conclude that the direct interaction between Stat6 and NF-kappaB may provide a basis for synergistic activation of transcription by IL-4 and activators of NF-kappaB. Ching-Hung Shen Binding Sites Cell Line DNA Drug Synergism Glutathione Transferase Humans Interleukin-4 NF-kappa B NF-kappa B p50 Subunit Protein Binding Recombinant Fusion Proteins STAT6 Transcription Factor Signal Transduction Trans-Activators Transcription Factor RelA *Transcription, Genetic Tumor Cells, Cultured