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Chromatin structure and DNA methylation of the IL-4 gene in human TH2 cells

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Abstract

Human TH2 cell differentiation results in the selective demethylation of several specific CpG dinucleotides in the IL-4 and IL-13 genes, which are expressed in activated TH2, but not TH1, cells. This demethylation is accompanied by the appearance of six DNase I hypersensitive sites within 1.4 kb at the 5'-end of the IL-4 gene. Micrococcal nuclease (MNase) digestion revealed that in both TH1 and TH2 cells nine nucleosomes with a repeat length of 201 bp are identically positioned around the 5'-end of the IL-4 gene. However, only in TH2 cells are six out of the eight intervening linkers exposed to DNase I. This suggests that a major perturbation of the higher-order chromatin structure occurs above the level of the nucleosome in vivo. It is observed in cells that are poised for expression but which are not actively expressing the gene (i.e. resting TH2 cells). Notably, all the demethylated CpGs in TH2 cells are found in DNA that is accessible to DNase I. This may suggest that the opening of the chromatin structure allows binding of specific trans-acting factors that prevent de novo methylation.

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Abbreviations

CpG:

cysteine-guanine dinucleotide

DHS:

DNase I hypersensitive site

DNase I:

deoxyribonuclease I

FACS:

fluorescence activated cell sorting

IL-4:

interleukin-4

MNase:

micrococcal nuclease

TAE:

Tris, acetate, EDTA buffer

TH1 cells:

differentiated T-helper lymphocytes that express interferon gamma

TH2 cells:

differentiated T-helper lymphocytes that express interleukins-4, -5 and -13

References

  • Agarwal S, Rao A (1998) Modulation of chromatin structure regulates cytokine gene expression during T cell differentiation. Immunity 9:765–775

    Article  PubMed  CAS  Google Scholar 

  • Ansel KM, Djuretic I, Tanasa B, Rao A (2006) Regulation of Th2 differentiation and Il4 locus accessibility. Annu Rev Immunol 24:607–656

    Article  PubMed  CAS  Google Scholar 

  • Ashe HL, Monks J, Wijgerde M, Fraser P, Proudfoot NJ (1997) Intergenic transcription and transinduction of the human beta-globin locus. Genes Dev 11:2494–2509

    Article  PubMed  CAS  Google Scholar 

  • Avni O, Lee D, Macian F et al (2002) TH cell differentiation is accompanied by dynamic changes in histone acetylation of cytokine genes. Nat Immunol 3:643–651

    Article  PubMed  CAS  Google Scholar 

  • Baguet A, Bix M (2004) Chromatin landscape dynamics of the Il4-Il13 locus during T helper 1 and 2 development. Proc Natl Acad Sci U S A 101:11410–11415

    Article  PubMed  CAS  Google Scholar 

  • Bert AG, Johnson BV, Baxter EW, Cockerill PN (2007) A modular enhancer is differentially regulated by GATA and NFAT elements that direct different tissue-specific patterns of nucleosome positioning and inducible chromatin remodeling. Mol Cell Biol 27:2870–2885

    Article  PubMed  CAS  Google Scholar 

  • Bix M, Wang ZE, Thiel B, Schork NJ, Locksley RM (1998) Genetic regulation of commitment to interleukin 4 production by a CD4+ T cell-intrinsic mechanism. J Exp Med 188:2289–2299

    Article  PubMed  CAS  Google Scholar 

  • Boeger H, Griesenbeck J, Strattan JS, Kornberg RD (2003) Nucleosomes unfold completely at a transcriptionally active promoter. Mol Cell 11:1587–1598

    Article  PubMed  CAS  Google Scholar 

  • Bruniquel D, Schwartz RH (2003) Selective, stable demethylation of the interleukin-2 gene enhances transcription by an active process. Nat Immunol 4:235–240

    Article  PubMed  CAS  Google Scholar 

  • Bulger M, Groudine M (1999) Looping versus linking: toward a model for long-distance gene activation. Genes Dev 13:2465–2477

    Article  PubMed  CAS  Google Scholar 

  • Carmo-Fonseca M (2002) The contribution of nuclear compartmentalization to gene regulation. Cell 108:513–521

    Article  PubMed  CAS  Google Scholar 

  • Cirillo LA, Lin FR, Cuesta I et al (2002) Opening of compacted chromatin by early developmental transcription factors HNF3 (FoxA) and GATA-4. Mol Cell 9:279–289

    Article  PubMed  CAS  Google Scholar 

  • Cousins DJ, Lee TH, Staynov DZ (2002) Cytokine coexpression during human Th1/Th2 cell differentiation: direct evidence for coordinated expression of Th2 cytokines. J Immunol 169:2498–2506

    PubMed  CAS  Google Scholar 

  • Elgin SCR, Workman JL (2000) Chromatin structure and gene expression. Oxford University Press, Oxford

    Google Scholar 

  • Felsenfeld G, Groudine M (2003) Controlling the double helix. Nature 421:448–453

    Article  PubMed  CAS  Google Scholar 

  • Fields PE, Lee GR, Kim ST, Bartsevich VV, Flavell RA (2004) Th2-specific chromatin remodeling and enhancer activity in the Th2 cytokine locus control region. Immunity 21:865–876

    Article  PubMed  CAS  Google Scholar 

  • Gilbert N, Boyle S, Fiegler H et al (2004) Chromatin architecture of the human genome: gene-rich domains are enriched in open chromatin fibers. Cell 118:555–566

    Article  PubMed  CAS  Google Scholar 

  • Gribnau J, Diderich K, Pruzina S, Calzolari R, Fraser P (2000) Intergenic transcription and developmental remodeling of chromatin subdomains in the human beta-globin locus. Mol Cell 5:377–386

    Article  PubMed  CAS  Google Scholar 

  • Gross DS, Garrard WT (1988) Nuclease hypersensitive sites in chromatin. Annu Rev Biochem 57:159–197

    Article  PubMed  CAS  Google Scholar 

  • Guo L, Hu-Li J, Paul WE (2005) Probabilistic regulation in TH2 cells accounts for monoallelic expression of IL-4 and IL-13. Immunity 23:89–99

    Article  PubMed  CAS  Google Scholar 

  • Hebbes TR, Clayton AL, Thorne AW, Crane-Robinson C (1994) Core histone hyperacetylation co-maps with generalized DNase I sensitivity in the chicken beta-globin chromosomal domain. EMBO J 13:1823–1830

    PubMed  CAS  Google Scholar 

  • Higgs DR (1998) Do LCRs open chromatin domains? Cell 95:299–302

    Article  PubMed  CAS  Google Scholar 

  • Kim ST, Fields PE, Flavell RA (2007) Demethylation of a specific hypersensitive site in the Th2 locus control region. Proc Natl Acad Sci U S A 104:17052–17057

    Article  PubMed  CAS  Google Scholar 

  • Kimura T, Mills FC, Allan J, Gould H (1983) Selective unfolding of erythroid chromatin in the region of the active beta-globin gene. Nature 306:709–712

    Article  PubMed  CAS  Google Scholar 

  • Kuo CT, Leiden JM (1999) Transcriptional regulation of T lymphocyte development and function. Annu Rev Immunol 17:149–187

    Article  PubMed  CAS  Google Scholar 

  • Lee GR, Fields PE, Flavell RA (2001) Regulation of IL-4 gene expression by distal regulatory elements and GATA-3 at the chromatin level. Immunity 14:447–459

    Article  PubMed  CAS  Google Scholar 

  • Meehan RR, Stancheva I (2001) DNA methylation and control of gene expression in vertebrate development. Essays Biochem 37:59–70

    PubMed  CAS  Google Scholar 

  • Ravindra A, Weiss K, Simpson RT (1999) High-resolution structural analysis of chromatin at specific loci: Saccharomyces cerevisiae silent mating-type locus HMRa. Mol Cell Biol 19:7944–7950

    PubMed  CAS  Google Scholar 

  • Reitman M, Felsenfeld G (1990) Developmental regulation of topoisomerase II sites and DNase I-hypersensitive sites in the chicken beta-globin locus. Mol Cell Biol 10:2774–2786

    PubMed  CAS  Google Scholar 

  • Riviere I, Sunshine MJ, Littman DR (1998) Regulation of IL-4 expression by activation of individual alleles. Immunity 9:217–228

    Article  PubMed  CAS  Google Scholar 

  • Rogan DF, Cousins DJ, Staynov DZ (1999) Intergenic transcription occurs throughout the human IL-4/IL-13 gene cluster. Biochem Biophys Res Commun 255:556–561

    Article  PubMed  CAS  Google Scholar 

  • Rogan DF, Cousins DJ, Santangelo S et al (2004) Analysis of intergenic transcription in the human IL-4/IL-13 gene cluster. Proc Natl Acad Sci U S A 101:2446–2451

    Article  PubMed  CAS  Google Scholar 

  • Santangelo S (2002) Studies on the chromatin structure of the IL-4/IL-13 gene cluster in human T lymphocytes. PhD Thesis, University of London

  • Santangelo S, Cousins DJ, Winkelmann NE, Staynov DZ (2002) DNA methylation changes at human Th2 cytokine genes coincide with DNase I hypersensitive site formation during CD4+ T cell differentiation. J Immunol 169:1893–1903

    PubMed  CAS  Google Scholar 

  • Sapojnikova N, Thorne A, Myers F, Staynov D, Crane-Robinson C (2008) The chromatin of active genes is not in a permanently open conformation. J Mol Biol 386:290–299

    Article  PubMed  CAS  Google Scholar 

  • Sexton T, Schober H, Fraser P, Gasser SM (2007) Gene regulation through nuclear organization. Nat Struct Mol Biol 14:1049–1055

    Article  PubMed  CAS  Google Scholar 

  • Staynov DZ (1983) Possible nucleosome arrangements in the higher-order structure of chromatin. Int J Biol Macromol 5:3–9

    Article  CAS  Google Scholar 

  • Staynov DZ (2000) DNase I digestion reveals alternating asymmetrical protection of the nucleosome by the higher order chromatin structure. Nucleic Acids Res 28:3092–3099

    Article  PubMed  CAS  Google Scholar 

  • Staynov DZ, Crane-Robinson C (1988) Footprinting of linker histones H5 and H1 on the nucleosome. EMBO J 7:3685–3691

    PubMed  CAS  Google Scholar 

  • Staynov DZ, Proykova YG (1998) Quantitative analysis of DNase I digestion patterns of oligo- and polynucleosomes. J Mol Biol 279:59–71

    Article  PubMed  CAS  Google Scholar 

  • Staynov DZ, Proykova YG (2007) Topological constraints on the possible structures of the 30 nm chromatin fibre. Chromosoma 117:67–76

    Article  PubMed  Google Scholar 

  • Trojer P, Reinberg D (2007) Facultative heterochromatin: is there a distinctive molecular signature? Mol Cell 28:1–13

    Article  PubMed  CAS  Google Scholar 

  • van Holde KE (1988) Chromatin. Springer-Verlag, New York

    Google Scholar 

  • Vicent GP, Nacht AS, Smith CL et al (2004) DNA instructed displacement of histones H2A and H2B at an inducible promoter. Mol Cell 16:439–452

    Article  PubMed  CAS  Google Scholar 

  • Wallrath L, Elgin SCR (1995) Position effect variegation in Drosophila is associated with an altered chromatin structure. Genes Dev 9:1263–1277

    Article  PubMed  CAS  Google Scholar 

  • Wang X, Simpson RT (2001) Chromatin structure mapping in Saccharomyces cerevisiae in vivo with DNase I. Nucleic Acids Res 29:1943–1950

    Article  PubMed  CAS  Google Scholar 

  • Weintraub H, Groudine M (1976) Chromosomal subunits in active genes have an altered conformation. Science 193:848–856

    Article  PubMed  CAS  Google Scholar 

  • Wilson CB, Makar KW, Shnyreva M, Fitzpatrick DR (2005) DNA methylation and the expanding epigenetics of T cell lineage commitment. Semin Immunol 17:105–119

    Article  PubMed  CAS  Google Scholar 

  • Wolffe AP (1998) Chromatin : structure and function. Academic Press, San Diego, London

    Google Scholar 

  • Woodcock CL, Dimitrov S (2001) Higher-order structure of chromatin and chromosomes. Curr Opin Genet Dev 11:130–135

    Article  PubMed  CAS  Google Scholar 

  • Workman JL, Kingston RE (1998) Alteration of nucleosome structure as a mechanism of transcriptional regulation. Annu Rev Biochem 67:545–579

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgements

This paper is dedicated to the memory of Nicole Winkelmann, a promising young scientist who died aged 25. The work was supported by a MRC grant number G9536930.

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Authors and Affiliations

  1. Santangelo Consulting Pty Ltd., 299357, Singapore, Singapore

    Samantha Santangelo

  2. MRC & Asthma UK Centre for Allergic Mechanisms of Asthma, King’s College London, London, SE1 9RT, UK

    David J. Cousins

  3. National Heart & Lung Institute, Imperial College London, Guy Scadding Building Dovehouse Street, London, SW3 6LY, UK

    Kostas Triantaphyllopoulos & Dontcho Z. Staynov

Authors
  1. Samantha Santangelo
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  2. David J. Cousins
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  3. Kostas Triantaphyllopoulos
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  4. Dontcho Z. Staynov
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Corresponding author

Correspondence to Dontcho Z. Staynov.

Additional information

Responsible Editor: Wendy Bickmore.

Nicole Winkelmann passed away in 2004.

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Fig. S1

Analysis of chromatin structure in IL-13 by MNase and DNase I. (A) Map of IL-13 and upstream CpG-rich region showing the positions of the BclI sites and the radioactively labelled probe used. (B) Autoradiograph of Southern blot of BclI-digested MNase and DNase I digestion series of TH1 and TH2 cells. DNA marker sizes are shown in kilobases. The position of the TH2-specific DHS III is shown. The pattern of bands produced by MNase in lanes 11 and 20 were digitized and are shown as densitometric traces to the right of the MNase digestion panels (GIF 139 kb)

High resolution image (TIFF 694 kb)

Fig. S2

Analysis of chromatin structure in IL-4 by MNase. Autoradiograph of Southern blot of BclI-digested MNase digestion series of fibroblasts. DNA marker sizes are shown in kilobases. The pattern of bands produced by MNase in lane 4 was digitized and is shown as a densitometric trace to the right of the digestion panel (GIF 120 kb)

High resolution image (TIFF 578 kb)

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Santangelo, S., Cousins, D.J., Triantaphyllopoulos, K. et al. Chromatin structure and DNA methylation of the IL-4 gene in human TH2 cells. Chromosome Res 17, 485–496 (2009). https://doi.org/10.1007/s10577-009-9040-3

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  • DOI: https://doi.org/10.1007/s10577-009-9040-3

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