The regulatory role of heme in circadian control

The 24 hour circadian clock is the fundamental internal timekeeping system that generates daily rhythms of behaviour, physiology and metabolism in nearly all higher organisms, including some bacteria. When the clock is disrupted, as in chronic shift work, the deterioation of human health is significant, and elevated levels of depression, sleep disorders, cardiovascular problems and even cancer, have been reported. Consequently, understanding how the clock works at the molecular level will have significant benefits for human and animal health because it will provide opportunities to design novel therapeutic interventions to ameliorate these negative side-effects.

In insects and vertebrates, the clock depends on two critical transcription factors, CLOCK and BMAL1 that bind to the DNA of clock target genes and activate their expression. Their role is crucial and there is convincing evidence emerging in the literature that the metallic element iron - in the form of heme iron - binds to CLOCK. Heme is a small, organic molecule with iron at the centre with an ability to bind ligands such as oxygen (O2), carbon monoxide (CO) and nitric oxide (NO). These ligands are recognised as important molecular signals so that, for example, the metabolic state of the organism (via changes in the concentrations of these ligands) might signal to heme, altering the properties of CLOCK in such a way that the regulation of downstream clock genes is changed. However, the mechanisms of heme-mediated regulation of the clock have yet to be established.

We will explore heme and gas binding to the human CLOCK transcription factor and determine the structure for the heme-CLOCK interaction. We will also study how heme affects the way in which CLOCK binds to its partner BMAL1, and also how heme is important for the physical interaction of this CLOCK-BMAL1 dimer with its DNA target sequence. Finally, we shall use fly cells to study how heme is involved in the way CLOCK-BMAL1 stimulates transcription of target clock genes using a simple luminescence readout.

The circadian clock is the internal timekeeping system that generates a daily rhythm in physiology, biochemistry and behaviour of almost all higher organisms and some prokaryotes. In mammals, the transcription factors CLOCK and BMAL1 activate transcription of period (per) and cryptochrome (cry) genes, whose products feed back and negatively regulate their own transcription. There is an accumulating body of evidence that indicates that heme iron affects circadian control, through binding to various circadian proteins, but the mechanisms of regulation are largely unknown. Our preliminary work has identified human CLOCK as target for heme binding. In this work we will examine, for the first time, the interaction of heme and heme ligands with human CLOCK, the effect of heme on formation of the CLOCK/BMAL1 dimer and DNA binding. Using mutants of human CLOCK, we will also examine the functional consequences on E-box mediated transcription, and whether these human CLOCK mutants also affect the negative regulation of transcription via PER and CRY interactions. In this way we will be able to obtain a functional readout of the role of heme in both the positive and negative limbs of the circadian molecular feedback loop.

There is a wider appreciation beginning to emerge that heme has an important regulatory role in the cell, and this application positions itself within a broader framework aimed at understanding the cell biology of heme.

WHO WILL BENEFIT FROM THE RESEARCH?

There are numerous beneficiaries.

1. The immediate existing personnel working with the PI and CI will benefit directly, through interactions with the project and the personnel hired on the project. This comes in the form of expertise exchanged between personnel, shared working habits, group meetings, shared learning, future collaborations between personnel once they have left the project etc.

2. The Departments involved, plus the University, also benefit. This comes through building new collaborations from outside, bringing new ideas, new ways of working, new skills, etc. The simple exchange of people across departments should not be under estimated: without it an organization becomes static, with no new input of ideas year after year. This movement of personnel is a great benefit to UK science and UK plc.

3. The wider community, who benefit in terms of seeing how the work develops and it being a stimulus for other projects, providing ideas and a source of discussion that filters in and out of Leicester and elsewhere. Funding of new projects encourages a dialogue with other users/interested parties, which sparks new ideas and innovation elsewhere, and new collaborations (e.g. with Edinburgh).

4. First destination employers, who benefit by picking up highly-skilled staff trained in the investigators' laboratories.

5. The wider biological community, in this country and abroad who will be interested in the results (through citations etc).

6. Heme enzymes (P450s, NO synthase for example) are a mainstay of pharmaceutical research, and this sector depends on fundamental, molecular level information emerging from academic groups around the world to prosecute their drug discovery campaigns. Our work thus feeds directly into UK plc and the contribution of molecular-level, fundamental studies of this kind should not be underestimated. Clearly, this has an impact on 'quality of life', since all biotech/pharma is concerned with improvement in quality of life and some of them have a direct interest in the role of heme in circadian control.

HOW WILL THEY BENEFIT FROM THIS RESEARCH?
There are various routes through which this can be achieved.

Obviously, publication in open-access journals is one important way of publicizing information, plus attendance at national and international meetings, for which we have requested appropriate resource. We will be in regular contact with other stake-holders in the UK and abroad, and the PI is involved in organization of various events as on-going activities, such as mini-symposia, conferences etc. This serves to publicise our work to the widest possible audience. We also routinely send our students and PDRA onto training workshops arranged by other organizations to provide training and to disseminate our work further. We are in the habit of sending PDRAs and students to smaller meetings which the PIs and CIs cannot attend, often giving talks at these events. We also have regular seminars and small meetings/conferences at Leicester, so that the ideas are publicized informally through these channels.

The University has a Business Development Office, for encouraging engagement with industry (the PI and CI have on-going links in this area).

See also Impact Statement (separate attachment).