Characterisation of genetic variation regulating gene expression within the MHC class III region. (360G-Wellcome-074318_Z_04_A)

Characterisation of genetic variation regulating gene expression within the MHC class III region This study aims to define DNA sequence variation that alters the way genes are regulated. The inability to identify regulatory DNA polymorphisms is a major roadblock to investigating the genetic basis of susceptibility to common multifactorial diseases. For variation resulting in a coding change in the translated protein, the consequences are amenable to functional prediction and testing. In contrast it is currently unclear how best to identify and analyse functionally important non-coding polymorphisms that modulate gene expression. I propose to define how allele-specific gene expression relates to sequence diversity in vivo across a 300kb region of the Major Histocompatibility Complex Class III region on chromosome 6. Until recently, it has been possible to analyse only a minority of genes for allele-specific expression due to the requirement for a transcribed marker polymorphism to distinguish between alleles. This study will be able to determine allele-specific expression among all combinations of variants at a given gene using a novel approach I have developed which quantifies the relative allelic loading of actively transcribing Pol II, the enzyme responsible for transcript synthesis. The endpoint of this research would be to resolve allelic differences in primary human cells in a disease state. However to attribute allelic differences to genetic variance on a given allele, heritability needs to be assessed. Therefore initial work will use a model system of lymphoblastoid B cell lines established from family pedigrees spanning three generations of individuals. Molecular mechanisms operating in vivo will be resolved by interrogating specific stages in the process of transcriptional regulation, epigenetic phenomena and protein-DNA binding. These molecular models of how genetic variants may modulate transcription will be tested by reporter gene analysis and by mutagenesis of large intact genomic regions transferred into cells. I will proceed to analyse candidate regulatory variants in other cellular contexts, notably primary human peripheral blood mononuclear cells.