Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements

The TNF locus on chromosome 6p21 encodes a family of proteins with key roles in the immune response whose dysregulation leads to severe disease. Transcriptional regulation is important, with cell type and stimulus-specific enhancer complexes involving the proximal TNF promoter. We show how quantitative chromatin profiling across a 34 kb region spanning the TNF locus has allowed us to identify a number of novel DNase hypersensitive sites and characterize more distant regulatory elements. We demonstrate DNase hypersensitive sites corresponding to the lymphotoxin alpha (LTA) and tumour necrosis factor (TNF) promoter regions, a CpG island in exon 4 of lymphotoxin beta (LTB), the 3′ end of nuclear factor of kappa light polypeptide gene enhancer in B-cells inhibitor-like 1 (NFKBIL1) and 3.4 kb upstream of LTA. These sites co-localize to highly conserved DNA sequences and show evidence of cell type specificity when lymphoblastoid, Jurkat, U937, HeLa and HEK293T cell lines are analysed using Southern blotting. For Jurkat T cells, we define histone modifications across the locus. Peaks of acetylated histone H3 and H4, together with tri-methyl K4 of histone H3, correspond to hypersensitive sites, notably in exon 4 of LTB. We provide evidence of a functional role for an intergenic DNase I hypersensitive site distal to LTA in Jurkat cells based on reporter gene analysis, with evidence of recruitment of upstream stimulatory factors (USF) transcription factors.

[1]  Thomas D. Schmittgen,et al.  Analysis of relative gene expression data using real-time quantitative PCR and the 2(-Delta Delta C(T)) Method. , 2001, Methods.

[2]  R. Sandaltzopoulos,et al.  Solid phase DNase I footprinting: quick and versatile. , 1994, Nucleic acids research.

[3]  A. Goldfeld,et al.  T Cell-Specific Expression of the Human TNF-α Gene Involves a Functional and Highly Conserved Chromatin Signature in Intron 3 1 , 2003, The Journal of Immunology.

[4]  J. Stamatoyannopoulos,et al.  Genome-wide identification of DNaseI hypersensitive sites using active chromatin sequence libraries. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[5]  Carl Wu.  The 5′ ends of Drosophila heat shock genes in chromatin are hypersensitive to DNase I , 1980, Nature.

[6]  Joseph B. Rayman,et al.  Analysis of promoter binding by the E2F and pRB families in vivo: distinct E2F proteins mediate activation and repression. , 2000, Genes & development.

[7]  S. Corre,et al.  Upstream stimulating factors: highly versatile stress-responsive transcription factors. , 2005, Pigment cell research.

[8]  G. Ghosh,et al.  Crystal structure of p50/p65 heterodimer of transcription factor NF-κB bound to DNA , 1998, Nature.

[9]  S. Elgin,et al.  The formation and function of DNase I hypersensitive sites in the process of gene activation. , 1988, The Journal of biological chemistry.

[10]  Pei-ji Chen,et al.  An exceptionally well-preserved theropod dinosaur from the Yixian Formation of China , 1998, Nature.

[11]  M. Groudine,et al.  Chromosomal subunits in active genes have an altered conformation. , 1976, Science.

[12]  P. Baeuerle,et al.  Tumor necrosis factor β (TNF-β) induces binding of the NF-κB transcription factor to a high-affinity κB element in the TNF-β promoter , 1990 .

[13]  Hiroshi Sato,et al.  Functional SNPs in the lymphotoxin-α gene that are associated with susceptibility to myocardial infarction , 2002, Nature Genetics.

[14]  T. Wolfsberg,et al.  DNase-chip: a high-resolution method to identify DNase I hypersensitive sites using tiled microarrays , 2006, Nature Methods.

[15]  F. Macian,et al.  NFAT proteins: key regulators of T-cell development and function , 2005, Nature Reviews Immunology.

[16]  Y. Yoshikawa,et al.  Identification of I kappa BL as the second major histocompatibility complex-linked susceptibility locus for rheumatoid arthritis. , 2003, American journal of human genetics.

[17]  C V Jongeneel,et al.  Kappa B-type enhancers are involved in lipopolysaccharide-mediated transcriptional activation of the tumor necrosis factor alpha gene in primary macrophages , 1990, The Journal of experimental medicine.

[18]  A. Hamsten,et al.  Allele-specific chromatin remodeling of the tumor necrosis factor-alpha promoter. , 2006, Biochemical and biophysical research communications.

[19]  H. Inoko,et al.  A second susceptibility gene for developing rheumatoid arthritis in the human MHC is localized within a 70-kb interval telomeric of the TNF genes in the HLA class III region. , 2001, Genomics.

[20]  J. Gimble,et al.  Tumor necrosis factor-alpha mRNA accumulation in human myelomonocytic cell lines. Role of transcriptional regulation by DNA sequence motifs and mRNA stabilization. , 1991, Journal of immunology.

[21]  J. V. Falvo,et al.  Activation-dependent intrachromosomal interactions formed by the TNF gene promoter and two distal enhancers , 2007, Proceedings of the National Academy of Sciences.

[22]  L. Hood,et al.  Analysis of the gene-dense major histocompatibility complex class III region and its comparison to mouse. , 2003, Genome research.

[23]  R. Geha,et al.  CD40 ligation and IL‐4 use different mechanisms of transcriptional activation of the human lymphotoxin α promoter in B cells , 1998, European journal of immunology.

[24]  R. Reeves,et al.  A poly(dA-dT) upstream activating sequence binds high-mobility group I protein and contributes to lymphotoxin (tumor necrosis factor-beta) gene regulation , 1992, Molecular and cellular biology.

[25]  Alice Young,et al.  Identifying gene regulatory elements by genome-wide recovery of DNase hypersensitive sites. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[26]  J. V. Falvo,et al.  Stimulus-Specific Assembly of Enhancer Complexes on the Tumor Necrosis Factor Alpha Gene Promoter , 2000, Molecular and Cellular Biology.

[27]  D. Kufe,et al.  Transcriptional and posttranscriptional regulation of tumor necrosis factor gene expression in human monocytes. , 1988, The Journal of clinical investigation.

[28]  A. Goldfeld,et al.  Inducer-Specific Enhanceosome Formation Controls Tumor Necrosis Factor Alpha Gene Expression in T Lymphocytes , 2002, Molecular and Cellular Biology.

[29]  M. Lenardo,et al.  Lymphotoxin activation by human T-cell leukemia virus type I-infected cell lines: role for NF-kappa B , 1990, Journal of virology.

[30]  Francesca Chiaromonte,et al.  ESPERR: learning strong and weak signals in genomic sequence alignments to identify functional elements. , 2006, Genome research.

[31]  J. Stamatoyannopoulos,et al.  High-throughput localization of functional elements by quantitative chromatin profiling , 2004, Nature Methods.

[32]  B. M. Greenwood,et al.  TNF concentration in fatal cerebral, non-fatal cerebral, and uncomplicated Plasmodium falciparum malaria , 1990, The Lancet.

[33]  William Stafford Noble,et al.  Identification and analysis of functional elements in 1% of the human genome by the ENCODE pilot project , 2007, Nature.

[34]  Ian F. Dunn,et al.  A Lipopolysaccharide-Specific Enhancer Complex Involving Ets, Elk-1, Sp1, and CREB Binding Protein and p300 Is Recruited to the Tumor Necrosis Factor Alpha Promoter In Vivo , 2000, Molecular and Cellular Biology.

[35]  A. Hamsten,et al.  Allele-specific chromatin remodeling of the tumor necrosis factor-α promoter , 2006 .

[36]  J. V. Falvo,et al.  A stimulus-specific role for CREB-binding protein (CBP) in T cell receptor-activated tumor necrosis factor alpha gene expression. , 2000, Proceedings of the National Academy of Sciences of the United States of America.

[37]  S. Nedospasov,et al.  Functional analysis of the lymphotoxin-beta promoter. Sequence requirements for PMA activation. , 1996, Journal of immunology.

[38]  B. Beutler,et al.  Tumor Necrosis Factor: The molecules and Their Emerging Role in Medicine , 1992 .

[39]  D. Voon,et al.  TNF and phorbol esters induce lymphotoxin-beta expression through distinct pathways involving Ets and NF-kappa B family members. , 2004, Journal of Immunology.

[40]  D. Kwiatkowski,et al.  Variation in the TNF-α promoter region associated with susceptibility to cerebral malaria , 1994, Nature.

[41]  D. Kwiatkowski,et al.  Complex NF-κB Interactions at the Distal Tumor Necrosis Factor Promoter Region in Human Monocytes* , 1998, The Journal of Biological Chemistry.

[42]  F. E. Chen,et al.  Regulation of DNA binding by Rel/NF-kappaB transcription factors: structural views. , 1999, Oncogene.

[43]  A. Rao,et al.  Molecular analysis of a locus control region in the T helper 2 cytokine gene cluster: a target for STAT6 but not GATA3. , 2004, Proceedings of the National Academy of Sciences of the United States of America.

[44]  G. Ghosh,et al.  Regulation of DNA binding by Rel/NF-κB transcription factors: structural views , 1999, Oncogene.

[45]  William Stafford Noble,et al.  Genome-scale mapping of DNase I sensitivity in vivo using tiling DNA microarrays , 2006, Nature Methods.

[46]  I. Udalova,et al.  Conserved kappa B element located downstream of the tumor necrosis factor alpha gene: distinct NF-kappa B binding pattern and enhancer activity in LPS activated murine macrophages. , 1995, Oncogene.

[47]  A. Goldfeld,et al.  Transactivator of Transcription from HIV Type 1 Subtype E Selectively Inhibits TNF Gene Expression via Interference with Chromatin Remodeling of the TNF Locus1 , 2006, The Journal of Immunology.

[48]  A. Moorman,et al.  Assumption-free analysis of quantitative real-time polymerase chain reaction (PCR) data , 2003, Neuroscience Letters.

[49]  Axel Visel,et al.  Enhancer identification through comparative genomics. , 2006, Seminars in cell & developmental biology.

[50]  T. Kouzarides.  Chromatin Modifications and Their Function , 2007, Cell.

[51]  R. Campbell,et al.  A distinct bipartite motif is required for the localization of inhibitory kappaB-like (IkappaBL) protein to nuclear speckles. , 2002, The Biochemical journal.

[52]  K. Docherty,et al.  The helix-loop-helix transcription factor USF (upstream stimulating factor) binds to a regulatory sequence of the human insulin gene enhancer. , 1993, The Biochemical journal.

[53]  W. Fiers,et al.  Tumor necrosis factor : molecular and cellular biology and clinical relevance , 1993 .

[54]  D. Voon,et al.  TNF and Phorbol Esters Induce Lymphotoxin-β Expression through Distinct Pathways Involving Ets and NF-κB Family Members1 , 2004, The Journal of Immunology.

[55]  D. Kwiatkowski,et al.  Variation in the TNF-alpha promoter region associated with susceptibility to cerebral malaria. , 1994, Nature.

[56]  Shane C. Dillon,et al.  Identifying gene regulatory elements by genomic microarray mapping of DNaseI hypersensitive sites. , 2006, Genome research.

[57]  F. Christiansen,et al.  The central MHC gene IKBL carries a structural polymorphism that is associated with HLA-A3,B7,DR15 , 1999, Immunogenetics.

[58]  R. Campbell,et al.  Characterization of a novel gene in the human major histocompatibility complex that encodes a potential new member of the I kappa B family of proteins. , 1994, Human molecular genetics.

[59]  Kevin Marsh,et al.  A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria , 1999, Nature Genetics.

[60]  K. Tracey,et al.  Tumor necrosis factor, other cytokines and disease. , 1993, Annual review of cell biology.

[61]  J. Nardone,et al.  Chromatin-based regulation of cytokine transcription in Th2 cells and mast cells. , 2005, International immunology.

[62]  T. Maniatis,et al.  Human tumor necrosis factor alpha gene regulation by virus and lipopolysaccharide. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[63]  D. Haussler,et al.  Evolutionarily conserved elements in vertebrate, insect, worm, and yeast genomes. , 2005, Genome research.

[64]  F. Spertini,et al.  Transcriptional and translational control of TNF‐α gene expression in human monocytes by major histocompatibility complex class II ligands , 1996, European journal of immunology.

[65]  Sophie Palmer,et al.  Complete MHC haplotype sequencing for common disease gene mapping. , 2004, Genome research.

[66]  Vip Viprakasit,et al.  A Regulatory SNP Causes a Human Genetic Disease by Creating a New Transcriptional Promoter , 2006, Science.

[67]  M. Feldmann,et al.  INHIBITORY EFFECT OF TNFα ANTIBODIES ON SYNOVIAL CELL INTERLEUKIN-1 PRODUCTION IN RHEUMATOID ARTHRITIS , 1989, The Lancet.

[68]  Meenakshi Singh,et al.  Stepwise replication identifies a low-producing lymphotoxin-α allele as a major risk factor for early-onset leprosy , 2007, Nature Genetics.

[69]  D. Kwiatkowski,et al.  Similarities and differences between human and murine TNF promoters in their response to lipopolysaccharide. , 1999, Journal of immunology.

[70]  E. Peterhans,et al.  Chromatin structure and DNase I hypersensitivity in the transcriptionally active and inactive porcine tumor necrosis factor gene locus. , 1992, Nucleic acids research.

[71]  Michael B. Brenner,et al.  Regulation of Tumor Necrosis Factor Alpha Gene Expression by Mycobacteria Involves the Assembly of a Unique Enhanceosome Dependent on the Coactivator Proteins CBP/p300 , 2003, Molecular and Cellular Biology.

[72]  P. Baeuerle,et al.  Tumor necrosis factor beta (TNF-beta) induces binding of the NF-kappa B transcription factor to a high-affinity kappa B element in the TNF-beta promoter. , 1990, Cytokine.

[73]  P. Vyas,et al.  Cis-acting sequences regulating expression of the human α-globin cluster lie within constitutively open chromatin , 1992, Cell.

[74]  W. Schaffner,et al.  Rapid detection of octamer binding proteins with 'mini-extracts', prepared from a small number of cells. , 1989, Nucleic acids research.

[75]  G. Kollias,et al.  Predominant pathogenic role of tumor necrosis factor in experimental colitis in mice , 1997, European journal of immunology.

[76]  N. Dufour,et al.  Human Tumor Necrosis Factor-α Gene 3′ Untranslated Region Confers Inducible Toxin Responsiveness to Homologous Promoter in Monocytic THP-1 Cells* , 1999, The Journal of Biological Chemistry.

[77]  A. Nordheim,et al.  Cyclosporin A blocks the expression of lymphotoxin α, but not lymphotoxin β, in human peripheral blood mononuclear cells , 2002 .