Research Area:	Genetics and Genomics
Technology Exchange:	Bioinformatics, SNP typing, Statistical genetics and Transcript profiling
Scientific Themes:	Genetics & Genomics and Immunology & Infectious Disease
Keywords:	Major Histocompatibility Complex, regulatory polymorphism, infectious disease, inflammatory disease, autoimmune disease and human genome
Web Links:	Wellcome Trust website Podcast 'Genetic Variation in Inflammation and Immunity'

There is growing evidence that genetic variation plays an important role in the determination of individual susceptibility to disease. However fine mapping disease associations and resolving specific functionally important variants has proved problematic, particularly in non-coding DNA where variation may alter the way genes are regulated. This is a major bottleneck in current genetic research.

The overall aim of my research programme is to understand the genetic basis of susceptibility to common infectious, inflammatory and autoimmune diseases. We seek to do so by adopting a functional genomics approach in which we investigate how naturally occurring genetic variation between individuals modulates expression of genes critical to mounting an appropriate immune and inflammatory response. We have a particular interest in genetic diversity involving the human Major Histocompatibility Complex (MHC) on chromosome 6p21 which is the major genomic risk locus implicated in many of these diseases, notably autoimmunity.

We adopt a number of different experimental approaches to defining regulatory genetic variants including analysis of allele-specific gene expression, expression quantitative trait mapping and detailed characterisation of how sequence diversity modulates the epigenetic and genetic control of gene expression. We study both human cell lines as a model system, and primary cells from healthy volunteers and patients with specific disease phenotypes to allow the context specificity of the effects of regulatory variation to be resolved.

Name	Department	Institution	Country
Prof Adrian VS Hill	Jenner Institute	Oxford University	UK
Dr Stephen J Chapman MRCP	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Prof Gil McVean	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Dr Jean-Baptiste Cazier	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Dr Helen Lockstone		Wellcome Trust Human Gene Centre, Oxford	UK
Prof Raashid Luqmani	Nuffield Department of Orthopaedics, Rheumatology and Musculoskeletal Science	University of Oxford	UK
Dr Smita Patel	Clinical Immunology	University of Oxford	UK
Prof Paul Bowness	Experimental Medicine Division	Oxford University	UK
Emeritus Prof Helen Chapel	Experimental Medicine Division	Oxford University	UK
Emeritus Prof George Ebers	Wellcome Trust Centre for Human Genetics	Oxford University	UK
Prof Charles Hinds		Barts and The Royal London	UK

Knight JC. 2013. Genomic modulators of the immune response. Trends Genet, 29 (2), pp. 74-83. Read abstract | Read more

Our understanding of immunity has historically been informed by studying heritable mutations in both the adaptive and innate immune responses, including primary immunodeficiency and autoimmune diseases. Recent advances achieved through the application of genomic and epigenomic approaches are reshaping the study of immune dysfunction and opening up new avenues for therapeutic interventions. Moreover, applying genomic techniques to resolve functionally important genetic variation between individuals is providing new insights into immune function in health. This review describes progress in the study of rare variants and primary immunodeficiency diseases arising from whole-exome sequencing (WES), and discusses the application, success, and challenges of applying genome-wide association studies (GWAS) to disorders of immune function and how they may inform more rational use of therapeutics. In addition, the application of expression quantitative-trait mapping to immune phenotypes, progress in understanding MHC disease associations, and insights into epigenetic mechanisms at the interface of immunity and the environment are reviewed. Hide abstract

Fairfax BP, Makino S, Radhakrishnan J, Plant K, Leslie S, Dilthey A, Ellis P, Langford C, Vannberg FO, Knight JC. 2012. Genetics of gene expression in primary immune cells identifies cell type-specific master regulators and roles of HLA alleles. Nat Genet, 44 (5), pp. 502-510. Read abstract | Read more

Trans-acting genetic variants have a substantial, albeit poorly characterized, role in the heritable determination of gene expression. Using paired purified primary monocytes and B cells, we identify new predominantly cell type-specific cis and trans expression quantitative trait loci (eQTLs), including multi-locus trans associations to LYZ and KLF4 in monocytes and B cells, respectively. Additionally, we observe a B cell-specific trans association of rs11171739 at 12q13.2, a known autoimmune disease locus, with IP6K2 (P = 5.8 × 10(-15)), PRIC285 (P = 3.0 × 10(-10)) and an upstream region of CDKN1A (P = 2 × 10(-52)), suggesting roles for cell cycle regulation and peroxisome proliferator-activated receptor γ (PPARγ) signaling in autoimmune pathogenesis. We also find that specific human leukocyte antigen (HLA) alleles form trans associations with the expression of AOAH and ARHGAP24 in monocytes but not in B cells. In summary, we show that mapping gene expression in defined primary cell populations identifies new cell type-specific trans-regulated networks and provides insights into the genetic basis of disease susceptibility. Hide abstract

Vandiedonck C, Taylor MS, Lockstone HE, Plant K, Taylor JM, Durrant C, Broxholme J, Fairfax BP, Knight JC. 2011. Pervasive haplotypic variation in the spliceo-transcriptome of the human major histocompatibility complex. Genome Res, 21 (7), pp. 1042-1054. Read abstract | Read more

The human major histocompatibility complex (MHC) on chromosome 6p21 is a paradigm for genomics, showing remarkable polymorphism and striking association with immune and non-immune diseases. The complex genomic landscape of the MHC, notably strong linkage disequilibrium, has made resolving causal variants very challenging. A promising approach is to investigate gene expression levels considered as tractable intermediate phenotypes in mapping complex diseases. However, how transcription varies across the MHC, notably relative to specific haplotypes, remains unknown. Here, using an original hybrid tiling and splice junction microarray that includes alternate allele probes, we draw the first high-resolution strand-specific transcription map for three common MHC haplotypes (HLA-A1-B8-Cw7-DR3, HLA-A3-B7-Cw7-DR15, and HLA-A26-B18-Cw5-DR3-DQ2) strongly associated with autoimmune diseases including type 1 diabetes, systemic lupus erythematosus, and multiple sclerosis. We find that haplotype-specific differences in gene expression are common across the MHC, affecting 96 genes (46.4%), most significantly the zing finger protein gene ZFP57. Differentially expressed probes are correlated with polymorphisms between haplotypes, consistent with cis effects that we directly demonstrate for ZFP57 in a cohort of healthy volunteers (P = 1.2 × 10(-14)). We establish that alternative splicing is significantly more frequent in the MHC than genome-wide (72.5% vs. 62.1% of genes, P ≤ 1 × 10(-4)) and shows marked haplotypic differences. We also unmask novel and abundant intergenic transcription involving 31% of transcribed blocks identified. Our study reveals that the renowned MHC polymorphism also manifests as transcript diversity, and our novel haplotype-based approach marks a new step toward identification of regulatory variants involved in the control of MHC-associated phenotypes and diseases. Hide abstract

Fairfax BP, Davenport EE, Makino S, Hill AV, Vannberg FO, Knight JC. 2011. A common haplotype of the TNF receptor 2 gene modulates endotoxin tolerance. J Immunol, 186 (5), pp. 3058-3065. Read abstract | Read more

Endotoxin tolerance is characterized by the suppression of further TNF release upon recurrent exposure to LPS. This phenomenon is proposed to act as a homeostatic mechanism preventing uncontrolled cytokine release such as that observed in bacterial sepsis. The regulatory mechanisms and interindividual variation of endotoxin tolerance induction in man remain poorly characterized. In this paper, we describe a genetic association study of variation in endotoxin tolerance among healthy individuals. We identify a common promoter haplotype in TNFRSF1B (encoding TNFR2) to be strongly associated with reduced tolerance to LPS (p = 5.82 × 10(-6)). This identified haplotype is associated with increased expression of TNFR2 (p = 4.9 × 10(-5)), and we find basal expression of TNFR2, irrespective of genotype and unlike TNFR1, is associated with secondary TNF release (p < 0.0001). Functional studies demonstrate a positive-feedback loop via TNFR2 of LPS-induced TNF release, confirming this previously unrecognized role for TNFR2 in the modulation of LPS response. Hide abstract

Wicks K, Knight JC. 2011. Transcriptional repression and DNA looping associated with a novel regulatory element in the final exon of the lymphotoxin-Β gene Genes and Immunity, 12 (2), pp. 126-135. Read abstract | Read more

Transcriptional regulation has a critical role in the coordinate and context-specific expression of a cluster of genes encoding members of the tumour necrosis factor (TNF) superfamily found at chromosome 6p21, comprising TNF, LTA (encoding lymphotoxin-α) and LTB (encoding lymphotoxin-Β). This is important, as dysregulated expression of these genes is implicated in susceptibility to many autoimmune, inflammatory and infectious diseases. We describe here a novel regulatory element in the fourth exon of LTB, which is highly conserved, localises to the only CpG island in the locus, and is associated with a DNase I hypersensitive site and specific histone modifications. We find evidence of binding by Yin Yang 1 (YY1), cyclic AMP response element (CRE)-binding protein (CREB) and CCCTC-binding factor (CTCF) to this region in Jurkat T cells, which is associated with transcriptional repression on reporter gene analysis. Chromatin conformation capture experiments show evidence of DNA looping, involving interaction of this element with the LTB promoter, LTA promoter and TNF 3′ untranslated region (UTR). Small interfering RNA (siRNA) experiments demonstrate a functional role for YY1 and CREB in LTB expression. Our findings provide evidence of additional complexity in the transcriptional regulation of LTB with implications for coordinate expression of genes in this important genomic locus. © 2011 Macmillan Publishers Limited All rights reserved. Hide abstract

Maugeri N, Radhakrishnan J, Knight JC. 2010. Genetic determinants of HSP70 gene expression following heat shock. Hum Mol Genet, 19 (24), pp. 4939-4947. Read abstract | Read more

The regulation of heat shock protein expression is of significant physiological and pathophysiological significance. Here we show that genetic diversity is an important determinant of heat shock protein 70 expression involving local, likely cis-acting, polymorphisms. We define DNA sequence variation for the highly homologous HSPA1A and HSPA1B genes in the major histocompatibility complex on chromosome 6p21 and establish quantitative and specific assays for determining transcript abundance. We show for lymphoblastoid cell lines established from individuals of African ancestry that following heat shock, expression of HSPA1B is associated with rs400547 (P 3.88 × 10(-8)) and linked single nucleotide polymorphisms (SNPs) located 62-93 kb telomeric to HSPA1B. This association was found to explain 31 and 29% of the variance in HSPA1B expression following heat shock or in resting cells, respectively. The associated SNPs show marked variation in minor allele frequency among populations, being more common in individuals of African ancestry, and are located in a region showing population-specific haplotypic block structure. The work illustrates how analysis of a heritable induced expression phenotype can be highly informative in defining functionally important genetic variation. Hide abstract

Ramagopalan SV, Heger A, Berlanga AJ, Maugeri NJ, Lincoln MR, Burrell A, Handunnetthi L, Handel AE, Disanto G, Orton SM, Watson CT, Morahan JM, Giovannoni G, Ponting CP, Ebers GC, Knight JC et al. 2010. A ChIP-seq defined genome-wide map of vitamin D receptor binding: associations with disease and evolution. Genome Res, 20 (10), pp. 1352-1360. Read abstract | Read more

Initially thought to play a restricted role in calcium homeostasis, the pleiotropic actions of vitamin D in biology and their clinical significance are only now becoming apparent. However, the mode of action of vitamin D, through its cognate nuclear vitamin D receptor (VDR), and its contribution to diverse disorders, remain poorly understood. We determined VDR binding throughout the human genome using chromatin immunoprecipitation followed by massively parallel DNA sequencing (ChIP-seq). After calcitriol stimulation, we identified 2776 genomic positions occupied by the VDR and 229 genes with significant changes in expression in response to vitamin D. VDR binding sites were significantly enriched near autoimmune and cancer associated genes identified from genome-wide association (GWA) studies. Notable genes with VDR binding included IRF8, associated with MS, and PTPN2 associated with Crohn's disease and T1D. Furthermore, a number of single nucleotide polymorphism associations from GWA were located directly within VDR binding intervals, for example, rs13385731 associated with SLE and rs947474 associated with T1D. We also observed significant enrichment of VDR intervals within regions of positive selection among individuals of Asian and European descent. ChIP-seq determination of transcription factor binding, in combination with GWA data, provides a powerful approach to further understanding the molecular bases of complex diseases. Hide abstract

Fairfax BP, Vannberg FO, Radhakrishnan J, Hakonarson H, Keating BJ, Hill AV, Knight JC. 2010. An integrated expression phenotype mapping approach defines common variants in LEP, ALOX15 and CAPNS1 associated with induction of IL-6. Hum Mol Genet, 19 (4), pp. 720-730. Read abstract | Read more

Interleukin-6 (IL-6) is an important modulator of inflammation and immunity whose dysregulation is associated with a number of disease states. There is evidence of significant heritability in inter-individual variation in IL6 gene expression but the genetic variants responsible for this remain to be defined. We adopted a combined approach of mapping protein and expression quantitative trait loci in peripheral blood mononuclear cells using high-density single-nucleotide polymorphism (SNP) typing for approximately 2000 loci implicated in cardiovascular, metabolic and inflammatory syndromes to show that common SNP markers and haplotypes of LEP (encoding leptin) associate with a 1.7- to 2-fold higher level of lipopolysaccharide (LPS)-induced IL-6 expression. We subsequently demonstrate that basal leptin expression significantly correlates with LPS-induced IL-6 expression and that the same variants at LEP which associate with IL-6 expression are also major determinants of leptin expression in these cells. We find that variation involving two other genomic regions, CAPNS1 (encoding calpain small subunit 1) and ALOX15 (encoding arachidonate 15-lipoxygenase), show significant association with IL-6 expression. Although this may be a subset of all such trans-acting effects, we find that the same ALOX15 variants are associated with induced expression of tumour necrosis factor and IL-1beta consistent with a broader role in acute inflammation for ALOX15. This study provides evidence of novel genetic determinants of IL-6 production with implications for understanding susceptibility to inflammatory disease processes and insight into cross talk between metabolic and inflammatory pathways. It also provides proof of concept for use of an integrated expression phenotype mapping approach. Hide abstract

Ramagopalan SV, Maugeri NJ, Handunnetthi L, Lincoln MR, Orton SM, Dyment DA, Deluca GC, Herrera BM, Chao MJ, Sadovnick AD, Ebers GC, Knight JC et al. 2009. Expression of the multiple sclerosis-associated MHC class II Allele HLA-DRB1*1501 is regulated by vitamin D. PLoS Genet, 5 (2), pp. e1000369. Read abstract | Read more

Multiple sclerosis (MS) is a complex trait in which allelic variation in the MHC class II region exerts the single strongest effect on genetic risk. Epidemiological data in MS provide strong evidence that environmental factors act at a population level to influence the unusual geographical distribution of this disease. Growing evidence implicates sunlight or vitamin D as a key environmental factor in aetiology. We hypothesised that this environmental candidate might interact with inherited factors and sought responsive regulatory elements in the MHC class II region. Sequence analysis localised a single MHC vitamin D response element (VDRE) to the promoter region of HLA-DRB1. Sequencing of this promoter in greater than 1,000 chromosomes from HLA-DRB1 homozygotes showed absolute conservation of this putative VDRE on HLA-DRB1*15 haplotypes. In contrast, there was striking variation among non-MS-associated haplotypes. Electrophoretic mobility shift assays showed specific recruitment of vitamin D receptor to the VDRE in the HLA-DRB1*15 promoter, confirmed by chromatin immunoprecipitation experiments using lymphoblastoid cells homozygous for HLA-DRB1*15. Transient transfection using a luciferase reporter assay showed a functional role for this VDRE. B cells transiently transfected with the HLA-DRB1*15 gene promoter showed increased expression on stimulation with 1,25-dihydroxyvitamin D3 (P = 0.002) that was lost both on deletion of the VDRE or with the homologous "VDRE" sequence found in non-MS-associated HLA-DRB1 haplotypes. Flow cytometric analysis showed a specific increase in the cell surface expression of HLA-DRB1 upon addition of vitamin D only in HLA-DRB1*15 bearing lymphoblastoid cells. This study further implicates vitamin D as a strong environmental candidate in MS by demonstrating direct functional interaction with the major locus determining genetic susceptibility. These findings support a connection between the main epidemiological and genetic features of this disease with major practical implications for studies of disease mechanism and prevention. Hide abstract

Taylor JM, Wicks K, Vandiedonck C, Knight JC. 2008. Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements. Nucleic Acids Res, 36 (15), pp. 4845-4862. Read abstract | Read more

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. Hide abstract

Knight JC, Keating BJ, Kwiatkowski DP. 2004. Allele-specific repression of lymphotoxin-alpha by activated B cell factor-1. Nat Genet, 36 (4), pp. 394-399. Read abstract | Read more

Genetic variation at the human LTA locus, encoding lymphotoxin-alpha, is associated with susceptibility to myocardial infarction, asthma and other diseases. By detailed haplotypic analysis of the locus, we identified a single-nucleotide polymorphism (SNP) at LTA+80 as a main predictor of LTA protein production by human B cells. We found that activated B-cell factor-1 (ABF-1) binds to this site in vitro and suppresses reporter gene expression, but only in the presence of the LTA+80A allele. Using haplotype-specific chromatin immunoprecipitation, we confirmed that ABF-1 is preferentially recruited to the low-producer allele in vivo. These findings provide a molecular model of how LTA expression may be genetically regulated by allele-specific recruitment of the transcriptional repressor ABF-1. Hide abstract

Knight JC, Keating BJ, Rockett KA, Kwiatkowski DP. 2003. In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nat Genet, 33 (4), pp. 469-475. Read abstract | Read more

In vivo characterization of regulatory polymorphisms is a key requirement for next-generation human genetic analysis. Here we describe haploChIP, a method that uses chromatin immunoprecipitation (ChIP) and mass spectrometry to identify differential protein-DNA binding in vivo associated with allelic variants of a gene. We demonstrate this approach with the imprinted gene SNRPN. HaploChIP showed close correlation between the level of bound phosphorylated RNA polymerase II at the SNRPN locus and allele-specific expression. Application of the approach to the TNF/LTA locus identified functionally important haplotypes that correlate with allele-specific transcription of LTA. The haploChIP method may be useful in high-throughput screening for common DNA polymorphisms that affect gene regulation in vivo. Hide abstract

Knight JC, Udalova I, Hill AV, Greenwood BM, Peshu N, Marsh K, Kwiatkowski D. 1999. A polymorphism that affects OCT-1 binding to the TNF promoter region is associated with severe malaria. Nat Genet, 22 (2), pp. 145-150. Read abstract | Read more

Genetic variation in cytokine promoter regions is postulated to influence susceptibility to infection, but the molecular mechanisms by which such polymorphisms might affect gene regulation are unknown. Through systematic DNA footprinting of the TNF (encoding tumour necrosis factor, TNF) promoter region, we have identified a single nucleotide polymorphism (SNP) that causes the helix-turn-helix transcription factor OCT-1 to bind to a novel region of complex protein-DNA interactions and alters gene expression in human monocytes. The OCT-1-binding genotype, found in approximately 5% of Africans, is associated with fourfold increased susceptibility to cerebral malaria in large case-control studies of West African and East African populations, after correction for other known TNF polymorphisms and linked HLA alleles. Hide abstract