Bioinformatics and functional analysis of naturally occurring variation in human circadian clock proteins

Research in our laboratory focuses on the relation between genes, brain and behaviour. We are specifically interested in the molecular mechanism and evolution of the circadian clock, the endogenous pacemaker that drives daily bio-rhythms in nearly all organisms. In human, disrupted function of the circadian clock may lead to various diseases including cancer.
Our research focuses on natural variation in clock genes, trying to identify specific sites important for circadian function that are maintained by natural selection [1,2], using Drosophila as a model system. We are employing various bioinformatic tools to generate working hypotheses which are further tested by ‘wet’ lab approaches. We have recently identified natural variations in mammalian clock genes (including human genes). The proposed project will allow us to widen our research and test the functional role of natural variations that we have identified in mammalian clock genes.
Circadian clocks consist of transcriptional negative feedback loops that modulate protein expression, producing 24 hr periodicity [3]. In mammals the negative feedback is driven by clock proteins PER and CRY repressing their own transcription, by interacting with E-box binding transcription factors CLOCK and BMAL1. Mammals have two CRY paralogues (CRY1, CRY2) which may have slightly different roles. We have analysed CRY vertebrate sequences [4] and found evidence for a few specific residues important for functional divergence.
To compare the function of CRY1 and CRY2 and to test the role of specific residues we will use a cell based luciferase reporter assay [5]. An already available plasmid carrying CRY1 will be used to produce new constructs that carry either CRY2 or constructs where each of the three functional divergent sites has been mutated. Each of these CRY plasmids will be used to transfect HEK293T cells together with pGL3P-mPER1 luciferase reporter, pCMV-BMAL1 and pCMV-CLOCK. The Luciferase activity will be determined and rate of repression of the different CRY variants will be compared. We have also identified number of non-synonymous SNPs in human circadian-clock genes. The different alleles will be cloned and the same system will be used to test the rate of repression of CLOCK/CMAL1.

For more information visit our lab at : www.le.ac.uk/genetics/et22/


We are an equal opportunities employer and particularly welcome applications for Ph.D. places from women, minority ethnic and other under-represented groups.