This project will aim to understand the genetic basis of individual variation in the innate immune response by adopting a functional genomic approach. The longer term aim of this work is to define specific genetic variants which are functionally important in order to complement ongoing genome-wide disease association studies of common disease, many of which involve dysregulation of the immune and inflammatory response. The project will focus on characterising diversity within a model system, lymphoblastoid B cell lines established from different individuals as part of the International HapMap Project, and in primary peripheral blood leukocytes from a cohort of healthy volunteers. Cis- and trans-acting genetic variants will be mapped using an expression quantitative trait mapping approach based on genome-wide gene expression profiling and SNP genotyping together with high throughput DNA sequencing. Cell type and stimulus specificity will be investigated together with the role of epigenetic mechanisms. Detailed characterisation of specific regulatory variants will involve a variety of molecular biology techniques to characterise protein-DNA interactions and gene expression.
This project will offer a comprehensive training programme in molecular biology and human genetics in an internationally recognised genetics research institute with state of the art facilities. This will include cell culture and isolation methodologies, DNA and RNA extraction and manipulation, quantitative PCR, gene expression profiling using microarrays, sequencing and genotyping technologies, protein-DNA binding assays including chromatin immunoprecipitation, reporter gene analysis and comprehensive bioinformatic analysis involving genotyping, sequencing and microarray data. Students will be encouraged to present and publish their work, attend weekly lab meetings and journal clubs together with departmental seminars and training courses.
Project reference number: 187
Funding and admissions information
| Name: | Dr Julian C Knight |
|---|---|
| Email: | julian.knight@well.ox.ac.uk |
| Location: | Henry Wellcome Building of Genomic Medicine |
| Research Area: | Genetics and Genomics |
| Technology Exchange: | SNP typing, Transcript profiling and Bioinformatics |
2009. The human Major Histocompatibility Complex as a paradigm in genomics research. Briefings in functional genomics & proteomics, 8 (5), pp. 379-94. Read abstract | View on PubMed
Since its discovery more than 50 years ago, the human Major Histocompatibility Complex (MHC) on chromosome 6p21.3 has been at the forefront of human genetic research. Here, we review from a historical perspective the major advances in our understanding of the nature and consequences of genetic variation which have involved the MHC, as well as highlighting likely future directions. As a consequence of its particular genomic structure, its remarkable polymorphism and its early implication in numerous diseases, the MHC has been considered as a model region for genomics, being the first substantial region to be sequenced and establishing fundamental concepts of linkage disequilibrium, haplotypic structure and meiotic recombination. Recently, the MHC became the first genomic region to be entirely re-sequenced for common haplotypes, while studies mapping gene expression phenotypes across the genome have strongly implicated variation in the MHC. This review shows how the MHC continues to provide new insights and remains in the vanguard of contemporary research in human genomics. Hide abstract
2008. Chromatin profiling across the human tumour necrosis factor gene locus reveals a complex, cell type-specific landscape with novel regulatory elements. Nucleic acids research, 36 (15), pp. 4845-62. Read abstract | View on PubMed
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
2004. Allele-specific repression of lymphotoxin-alpha by activated B cell factor-1. Nature genetics, 36 (4), pp. 394-9. Read abstract | View on PubMed
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
2003. In vivo characterization of regulatory polymorphisms by allele-specific quantification of RNA polymerase loading. Nature genetics, 33 (4), pp. 469-75. Read abstract | View on PubMed
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
2008. Genetic mapping in human disease. Science (New York, N.Y.), 322 (5903), pp. 881-8. Read abstract | View on PubMed
Genetic mapping provides a powerful approach to identify genes and biological processes underlying any trait influenced by inheritance, including human diseases. We discuss the intellectual foundations of genetic mapping of Mendelian and complex traits in humans, examine lessons emerging from linkage analysis of Mendelian diseases and genome-wide association studies of common diseases, and discuss questions and challenges that lie ahead. Hide abstract
2009. Mapping complex disease traits with global gene expression. Nature reviews. Genetics, 10 (3), pp. 184-94. Read abstract | View on PubMed
Variation in gene expression is an important mechanism underlying susceptibility to complex disease. The simultaneous genome-wide assay of gene expression and genetic variation allows the mapping of the genetic factors that underpin individual differences in quantitative levels of expression (expression QTLs; eQTLs). The availability of systematically generated eQTL information could provide immediate insight into a biological basis for disease associations identified through genome-wide association (GWA) studies, and can help to identify networks of genes involved in disease pathogenesis. Although there are limitations to current eQTL maps, understanding of disease will be enhanced with novel technologies and international efforts that extend to a wide range of new samples and tissues. Hide abstract