Analysis of Modulation of Immunoglobulin Gene Expression

Courtney E. W. Sulentic1

1 Wright State University Boonshoft School of Medicine, Dayton, Ohio
Publication Name:  Current Protocols in Toxicology
Unit Number:  Unit 18.14
DOI:  10.1002/0471140856.tx1814s36
Online Posting Date:  May, 2008
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Abstract

Immunoglobulins (Ig) are critical in maintaining host immunity to a variety of pathogens. Regulation of Ig expression is a complex process involving transcriptional regulation of different Ig gene loci by many transcription factors and transcriptional regulatory regions. This complexity suggests many possible molecular targets for immunotoxicants. Therefore, thorough evaluation of chemical‐induced modulation of Ig expression may necessitate multiple experimental approaches evaluating: (1) number of B cells secreting antibodies by antibody‐forming cell response or plaque assay; (2) concentration of total secreted antibodies by enzyme‐linked immunosorbent assay (ELISA); (3) cellular proliferation and viability by cell count measurements, [3H] thymidine incorporation, and trypan blue exclusion; (4) Ig mRNA expression by quantitative real‐time reverse transcriptase–polymerase chain reaction (RT‐PCR); (5) transcriptional activity of specific Ig regulatory regions by reporter gene analysis; and (6) transcription factor binding to specific Ig regulatory regions by electrophoretic mobility shift (EMSA) and chromatin immunoprecipitation (ChIP). These experimental approaches are discussed in the unit, with detailed description of EMSA, EMSA‐western analysis, and isolation of nuclear protein. Curr. Protoc. Toxicol. 36:18.14.1‐18.14.21. © 2008 by John Wiley & Sons, Inc.

Keywords: EMSA analysis; EMSA‐western analysis; supershift EMSA analysis; immunoglobulin gene; transcription factors; regulatory sequences

     
 
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Table of Contents

  • Introduction
  • Basic Protocol 1: Electrophoretic Mobility Shift Assay (EMSA)
  • Alternate Protocol 1: EMSA‐Western Blot Analysis
  • Support Protocol 1: Nuclear Protein Isolation
  • Reagents and Solutions
  • Commentary
  • Literature Cited
  • Figures
     
 
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Materials

Basic Protocol 1: Electrophoretic Mobility Shift Assay (EMSA)

  Materials
  • Single‐stranded oligonucleotides containing binding site of interest, and appropriate control DNA
  • HED buffer (see recipe)
  • T4 polynucleotide kinase and corresponding buffer
  • [γ‐32P]ATP (sp. act. 3000 µCi/mmol)
  • HEDG buffer (see recipe)
  • Reagents for nondenaturing gel electrophoresis (also see Gallagher, ) including:
    • 10× TAE buffer (see recipe)
    • 40% (w/v) 29:1 acrylamide:bisacrylamide solution (Accugel 29:1; National Diagnostics)
    • 10% ammonium persulfate
    • TEMED
    • 5× loading dye (see recipe)
  • Nuclear protein extract (see protocol 3) or purified protein of interest, and appropriate control DNA‐binding protein
  • HEDK buffer (see recipe)
  • Protease inhibitor cocktail (e.g., ready‐to‐use cocktails or tablets; Pierce, Sigma Roche)
  • 1 M dithiothreitol (DTT)
  • Nonspecific carrier DNA: poly(dI‐dC)
  • 88°, 65°, and 37°C heat blocks
  • Centrifuge with swinging‐bucket rotor and adaptor for microcentrifuge tubes
  • STE Select‐D, G‐25 spin columns (5 Prime → 3 Prime, Inc.)
  • Equipment for gel electrophoresis (e.g., Owl Separation Systems or Bio‐Rad; also see Gallagher, ) including:
    • 20 × 20–cm glass plates, 1/8‐in. thick
    • 1.5‐mm spacers
    • Binder clips
    • Large vertical electrophoresis apparatus
    • Power source
  • Metal spatula
  • Whatman 3MM filter paper, cut to size of gel
  • Additional reagents and equipment for nondenaturing gel electrophoresis (Gallagher, ) and autoradiography ( appendix 3D)

Alternate Protocol 1: EMSA‐Western Blot Analysis

  • 2.5 pmol/µl cold, unlabeled DNA probe (identical in sequence to radiolabeled probe; see protocol 1)
  • Transfer buffer (see recipe)
  • 100% methanol
  • TBS‐T: 1× TBS (see recipe for 10× stock) containing 0.1% (v/v) Tween 20
  • Blocking buffer: 1% nonfat dry milk in TBS‐T
  • Primary antibody specific for DNA‐binding protein of interest
  • Secondary antibody, conjugated with horseradish peroxidase (HRP)
  • Detection reagent (e.g., ECL or SuperSignal from Pierce)
  • Mini western blotting apparatus (9 × 9 cm transfer area; e.g., Owl Separation Systems or Bio‐Rad)
  • Rectangular containers of dimensions slightly larger than gel/membrane, for incubations
  • Nitrocellulose or polyvinylidene fluoride (PVDF) membrane
  • Black marker
  • Autoradiography cassettes and intensifying screens (described in appendix 3D) or chemiluminescence imager (e.g., Fuji LAS 3000)

Support Protocol 1: Nuclear Protein Isolation

  Materials
  • Cultured cells or primary lymphocytes
  • Complete Mini Protease Inhibitor Cocktail (tablets from Roche)
  • 1 M dithiothreitol (DTT)
  • Phosphate‐buffered saline (PBS; appendix 2A)
  • 10 mM HEPES (pH 7.5) or HB buffer (see recipe), chilled
  • MDH buffer (see recipe), chilled
  • MDHK buffer (see recipe), chilled
  • HEDK buffer (see recipe), chilled
  • Centrifuge with swinging‐bucket rotor and adaptor for microcentrifuge tubes
  • Dounce homogenizer with tight‐fitting (type B) pestle
  • Platform rocker
  • Additional reagents and equipment for bicinchoninic acid (BCA) protein determination assay ( appendix 3I)
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Figures

  •   FigureFigure 18.14.1 Structure of the basic immunoglobulin molecule.
  •   FigureFigure 18.14.2 Organization of the immunoglobulin light and heavy chain gene loci and somatic recombination.
  •   FigureFigure 18.14.3 Organization of the immunoglobulin heavy chain gene showing the regulatory regions and the heavy chain constant regions µ, δ, γ, ɛ and α that encode for the heavy chain proteins of IgM, IgD, IgG, IgE, and IgA, respectively.
  •   FigureFigure 18.14.4 Schematic representation of EMSA and EMSA‐western results.

Videos

Literature Cited

Literature Cited
   Aparicio, O., Geisberg, J.V., and Struhl, K. 2004. Chromatin immunoprecipitation for determining the association of proteins with specific genomic sequences in vivo. Curr. Protoc. Cell Biol. 23: 17.7.1‐17.7.23.
   Asanuma, K., Tsuji, N., Endoh, T., Yagihashi, A., and Watanabe, N. 2004. Survivin enhances Fas ligand expression via up‐regulation of specificity protein 1‐mediated gene transcription in colon cancer cells. J. Immunol. 172: 3922‐3929.
   Chauveau, C., Pinaud, E., and Cogne, M. 1998. Synergies between regulatory elements of the immunoglobulin heavy chain locus and its palindromic 3′ locus control region. Eur. J. Immunol. 28: 3048‐3056.
   Gallagher, S.E. 1999. One‐dimensional gel electrophoresis using nondenaturing conditions. Curr. Protoc. Mol. Biol. 47: 10.2B.1‐10.2B.11.
   Garret, F.E., Emelyanov, A.V., Sepulveda, M.A., Flanagan, P., Volpi, S., Li, F., Loukinov, D., Eckhardt, L.A., Lobanenkov, V.V., and Birshtein, B.K. 2005. Chromatin architecture near a potential 3′ end of the Igh locus involves modular regulation of histone modifications during B‐ cell development and in vivo occupancy at CTCF sites. Mol. Cell. Biol. 25: 1511‐1525.
   Hardy, R.R., Carmack, C.E., Shinton, S.A., Kemp, J.D., and Hayakawa, K. 1991. Resolution and characterization of pro‐B and pre‐pro‐B cell stages in normal mouse bone marrow. J. Exp. Med. 173: 1213‐1225.
   Novak, U. and Paradiso, L. 1995. Identification of proteins in DNA‐protein complexes after blotting of EMSA gels. Biotechniques 19: 54‐55.
   Pawlitzky, I., Angeles, C.V., Siegel, A.M., Stanton, M.L., Riblet, R., and Brodeur, P.H. 2006. Identification of a candidate regulatory element within the 5′ flanking region of the mouse IgH locus defined by pro‐B cell‐specific hypersensitivity associated with binding of PU.1, Pax5, and E2A. J. Immunol. 176: 6839‐6851.
   Rolink, A. and Melchers, F. 1996. B‐cell development in the mouse. Immunol. Lett. 54: 157‐161.
   Rolink, A., Grawunder, U., Winkler, T.H., Karasuyama, H., and Melchers, F. 1994. IL‐2 receptor alpha chain (CD25, TAC) expression defines a crucial stage in pre‐B cell development. Int. Immunol. 6: 1257‐1264.
   Rolink, A., ten Boekel, E., Melchers, F., Fearon, D.T., Krop, I., and Andersson, J. 1996. A subpopulation of B220+ cells in murine bone marrow does not express CD19 and contains natural killer cell progenitors. J. Exp. Med. 183: 187‐194.
   Stevens, S., Ong, J., Kim, U., Eckhardt, L.A., and Roeder, R.G. 2000. Role of OCA‐B in 3′‐IgH enhancer function. J. Immunol. 164: 5306‐5312.
   Sulentic, C.E., Holsapple, M.P., and Kaminski, N.E. 2000. Putative link between transcriptional regulation of IgM expression by 2,3,7,8‐tetrachlorodibenzo‐p‐dioxin and the aryl hydrocarbon receptor/dioxin‐responsive enhancer signaling pathway. J. Pharmacol. Exp. Ther. 295: 705‐716.
   Zhang, B., Alaie‐Petrillo, A., Kon, M., Li, F., and Eckhardt, L.A. 2007. Transcription of a productively rearranged Ig VDJC{alpha} does not require the presence of HS4 in the IgH 3′ regulatory region. J. Immunol. 178: 6297‐6306.
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