Introduction. Obesity represents a major contributing factor in type-2 diabetes, cardiovascular disease and cancer and is already the world’s 5th biggest cause of premature death. Most susceptibility to obesity is genetic. In order to avoid obesity and to aid in its treatment it is essential that we understand its genetics.
Genome wide association studies (GWAS) to determine the causes of obesity have identified polymorphisms (SNPs) in a gene called BDNF which is expressed in the appetite controlling parts of the brain (hypothalamus) and is known to modulate appetite. Using our leading expertise in bioinformatics, molecular biology, primary cell analysis and mouse genome editing we have had previous success in identifying and analysing a number of novel tissue specific enhancer elements whose properties were altered by polymorphic variation and epigenetics (1).
Preliminary data. In order to understand the role of BDNF regulation in obesity we used bioinformatics, molecular biology and neuronal cell culture to show that different alleles of one of the GWAS associated obesity SNPs (rs10767664; p=4.69x10-26) (2) have different effects the activity of regulatory regions that modulate the expression of the BDNF gene in primary hypothalamic neurones. Recognition of the effects of this SNP on the regulation of BDNF in hypothalamic cells is already an important finding for understanding the causes of obesity and provides a strong basis for the current studentship proposal.
Methods. Dr MacKenzie has already successfully used CRISPR genome editing to rapidly generate enhancer knockouts in mice (funded by the BBSRC and in preparation for submission to Nature Medicine). In addition, we have recently been funded (MRC discovery award; £24k) to produce mice in which obesity associated BDNF regulatory elements have been deleted. This will provide the student with the relevant mouse models for analysis by the start of the studentship. The student then test the effects of removing these regulatory regions on the expression of the BDNF gene in the hypothalamus using in-situ hybridisation (to be supervised by Dr. Perry Barratt, an expert in in-situ hybridisation and feeding studies) and QrtPCR. The student will also test the feeding behaviour and metabolism of the enhancer KO mice (supervised by Prof. Mirela Delibegovic an expert in mouse models of obesity and diabetes
(3)). Later in the project the student will use CRISPR genome editing to reproduce the human allelic variants of obesity associated SNPs in these BDNF regulatory regions in mice that will be tested for resulting changes in gene expression, feeding behaviour and metabolism. Identification of the regulatory effects of these SNPs on BDNF gene expression and mouse feeding behaviour and metabolism will lead to several high impact factor papers and will make a major contribution to understanding the genetic causes of obesity thus accelerating the development of personalised therapies
Training offered. This studentship will provide the successful candidate with a unique training experience that combines genome bioinformatics with molecular biology, CRISPR genome editing, RNA expression analysis (QrtPCR and In-situ hybridisation) and in-vivo testing of food intake energy expenditure, glucose and lipid metabolism. Because the majority (>90%) of disease causing genetic variations in the human population are found within the non-coding regulatory genome this combination of genomic analysis, molecular and in-vivo skills will be highly sought after by future employers and funders.
1. Hing, B. et al (2012) . Biol Psychiatry, 71, 618-626 (Impact factor 10).
2. Speliotes, et al., (2010). Nat Genet, 42, 937-948.
3. Bence, K.K.,et al, (2006) Nat Med, 12, 917-924.(impact factor 30.3)