The mechanisms by which polymorphic domains in the 5HTT gene potentially correlated with behavioural disorders modulate gene expression

Our behaviour is modulated by molecules, neurotransmitters, in our brain that act as messengers. When, where and how much of these neurotransmitters are produced is controlled by inputs from various sources. This allows input from a number of stimuli that an individual is exposed to, e.g. drugs and alcohol, to affect behaviour by changing the levels of these neurotransmitters for a specific length of time. An analogy would be that these neurotransmitters act like the electrical wiring of a lighting circuit. These circuits can not only be controlled by an off/on switch, but also by dimmer switches which alter the level of light and are used to set the mood in a room. Similarly altering levels of neurotransmitters would alter the 'mood' of the brain. If we set the dimmer high, the room is very bright and this helps to stimulate activity in the room, if the dimmer is set low, it is more relaxing. In the same way neurotransmitters may not be controlled solely by on/off switches but also by the equivalent of dimmer switches that modulate the amount or concentration of these proteins in the brain so setting our mood. We have found regions of DNA that might act as these dimmer switches to control how much of a neurotransmitter is produced in specific areas of the brain. Importantly these regions can respond to the physiological and psychological stresses as well as drugs that alter our behaviour. The interesting thing is that the DNA structure of these regulators is not the same in all of us; hence different individuals have dimmer switches potentially set to different levels. If these regulatory dimmer switches malfunction in people then it's very hard to control the amount of molecule that should be made for the correct 'mood', e.g. the wrong level of light in the room for activities such as reading or indeed the light might be on all the time or not switched on at all. In some people these control regions might not work correctly because their DNA structure is different from what would be considered 'normal' and in these cases these people may have behavioural disorders. Such genetic differences in the population are called polymorphisms and can result in a genetic predisposition to a behavioural disorder. We propose is to find out more about how these regulatory domains work so that can figure out how to fix them when they don't work properly or how to reset them for the correct mood. These DNA regulatory domains are controlled in turn by proteins that bind to them, there are many different proteins that may bind and the dimmer switch effect is controlled by how many and what kind of protein is binding. We want to take the whole 'switch' apart to identify all the functional components and then put it back together to find out what each bit or protein does; e.g. which ones are responsible for turning the 'dimmer switch' up and down. The only successful way to do this is to try and find out what is functional when the electricity is turned on. We call the process of taking the unit apart an in vitro approach and finding out how it functions an in vivo experiment, because the system is working and intact when we do it. We will use both approaches in our proposal. The regulatory domains we will study regulate how much of a protein called the serotonin transporter (5HTT) is produced. The 5HTT protein is a target for serotonin specific reuptake inhibitors (SSRIs) a popular class of antidepressants, therefore it would be expected that modulation of the amount or distribution of this protein in the brain would have affects on behaviour. There is a growing awareness of environmental factors in the progression of psychiatric and other neurological disorders. The mechanism by which these genetic domains bring about changes in concentrations of neurotransmitters demonstrates a mechanism by which environment can directly interact with genetic predisposition in the progression of a disorder.

A genetic predisposition to a number of neurological disorders including bipolar disorders has been attributed to the human serotonin transporter gene (5HTT). In particular, a variable number tandem repeat (VNTR) domain within intron 2 and a polymorphic 5' promoter variant of 5HTT have been hypothesized as predisposing factors for such conditions. We and others have demonstrated their action as transcriptional regulatory domains. We hypothesis that both domains in part bind similar transcription factors and a synergistic interaction between the two domains may be important for the final transcriptional response of a specific allele in both cell physiology and in behavioural disorders. In this proposal we address which factors bind to the individual domains in both the 5' promoter L/S and intron 2 VNTR domains under specific stimuli and we aim to determine the functional significance of binding for transcriptional regulation of the 5HTT gene. We will include analysis of: 1.Binding of factors to distinct VNTR domains in response to stimuli. 2.Differential interactions of the factors themselves in response to stimulus prior to binding to the domain. 3.Modulation of gene expression mediated by alteration of DNA structure via single/double stranded conformational change or methylation. 4.Classical modulation of transcription via activation or repression domains acting on the minimal promoter. 5.Interactions between related factors bound at both 5'promoter and intron 2 domains in cis. Importantly the differential allelic binding of factors to both VNTRs suggests that if different regulatory polymorphisms in the same gene were associated with predisposition to a disease, they would have to be analysed for their potential to act in Cis on the same allele. This has implications for the significance of clinical correlations with the importance of such domains, and may improve the understanding of psychiatric disorders and enable the development of novel therapies.