Functional & biochemical characterisation of circadian timekeeping mechanisms in mammalian cells
Lead Research Organisation:
University of Cambridge
Department Name: Clinical School
Abstract
Daily rhythms underlie the biochemistry, physiology and behavior of terrestrial life. In humans, the 24-hour cycle is inextricably linked to patterns of sleep and wake, blood cortisol levels, cognitive function and the expression of around 20% of the genome in each cell. Dysregulation of these rhythms is associated with metabolic diseases and increased cancer susceptibility. In addition, the emerging field of chronopharmacology aims to take advantage of the daily changes of physiology and metabolism in order to maximize the therapeutic effect of drugs.
Circadian rhythms at the cellular level are regulated by networks of transcriptional, translational and post-translational factors. It is widely accepted that the cellular clockwork is driven by a core transcriptional feedback mechanism; the main elements being activating transcription factors CLOCK and BMAL1, and their repressors: Period (PER) and Cryptochrome (CRY). CRY is regarded as the main repressor, although PER is required for its full function, and so CRY mutants have been used as arrhythmic animal and cellular models. However, the O'Neill lab have observed persistent, albeit less robust, circadian rhythms in cells from mice that are homozygous null for CRY1 and CRY2 - reported by PER2::LUC bioluminescence. We find that these CRY-independent PER2::LUC rhythms are regulated by post-translational mechanisms, such as phosphorylation by casein kinase , that also contribute to timekeeping in wild type cells.
This project will build upon these prior findings by identifying the key molecular components that sustain timekeeping in both wild type and CRY-deficient cells, though pharmacological manipulation and tandem mass spectrometry. Furthermore it will aim to characterize the function of these pathways using cutting edge tools for genetic manipulation such as inducible expression and CRISPR-Cas9 genome editing, in order to determine the minimal set of clock components that are necessary and sufficient to sustain cellular circadian rhythms. This knowledge will reveal novel therapeutic targets for metabolic diseases and cancers, as well as opening new avenues for chronopharmacology.
Circadian rhythms at the cellular level are regulated by networks of transcriptional, translational and post-translational factors. It is widely accepted that the cellular clockwork is driven by a core transcriptional feedback mechanism; the main elements being activating transcription factors CLOCK and BMAL1, and their repressors: Period (PER) and Cryptochrome (CRY). CRY is regarded as the main repressor, although PER is required for its full function, and so CRY mutants have been used as arrhythmic animal and cellular models. However, the O'Neill lab have observed persistent, albeit less robust, circadian rhythms in cells from mice that are homozygous null for CRY1 and CRY2 - reported by PER2::LUC bioluminescence. We find that these CRY-independent PER2::LUC rhythms are regulated by post-translational mechanisms, such as phosphorylation by casein kinase , that also contribute to timekeeping in wild type cells.
This project will build upon these prior findings by identifying the key molecular components that sustain timekeeping in both wild type and CRY-deficient cells, though pharmacological manipulation and tandem mass spectrometry. Furthermore it will aim to characterize the function of these pathways using cutting edge tools for genetic manipulation such as inducible expression and CRISPR-Cas9 genome editing, in order to determine the minimal set of clock components that are necessary and sufficient to sustain cellular circadian rhythms. This knowledge will reveal novel therapeutic targets for metabolic diseases and cancers, as well as opening new avenues for chronopharmacology.
Organisations
People |
ORCID iD |
| John O'Neill (Primary Supervisor) | |
| David Wong (Student) |
Studentship Projects
| Project Reference | Relationship | Related To | Start | End | Student Name |
|---|---|---|---|---|---|
| MR/N013433/1 | 01/10/2016 | 30/04/2026 | |||
| 1857414 | Studentship | MR/N013433/1 | 01/10/2016 | 30/09/2019 | David Wong |
| Title | Circadian proteomics analysis |
| Description | This algorithm is designed to analyse data obtained from a circadian TMT quantitative proteomics and phosphoproteomics experiment. It was written in the R programming language, and is tailored to a specific experiment. The steps include cleaning the data, sample loading normalisation and internal reference scaling. The data is then analysed using established statistical methods to determine the rhythmicity of proteins and phosphopeptides. |
| Type Of Material | Computer model/algorithm |
| Year Produced | 2019 |
| Provided To Others? | No |
| Impact | This allowed the analysis of the circadian proteome and phosphoproteome in cultured mammalian cells. As a result, the circadian regulation of ion transport and protein content was elucidated. This is a significant contribution to the understanding of the cell-autonomous generation of circadian rhythms, and the data is in preparation for publication. This work also generated a useful database for other researchers to use, containing the identities of many proteins under circadian regulation that can be explored further in the future. |
| Description | Cell Biology Symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Professional Practitioners |
| Results and Impact | Annual symposium at the Laboratory of Molecular Biology where I have given talks. |
| Year(s) Of Engagement Activity | 2017,2018 |
| Description | Chronobiology Symposium at the Institute of Life Sciences, Pisa, Italy |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Undergraduate students |
| Results and Impact | I gave an oral presentation of my work about the circadian control of the mammalian cellular proteome. This reached around 70-80 people, and the audience consisted of researchers, medical students, doctors and academics. This sparked many questions and interesting discussions afterwards, and generated many ideas to take forward in our own research, as well as possible collaborations in the future. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Gordon Research Seminar and Conference, Barcelona, Spain |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | I gave a poster presentation about my work on the circadian organisation of the mammalian cellular proteome. There were around 500 conference attendees, including postgraduate research students, postdoctoral researchers and group leaders. My work generated a lot of discussion and ideas about further experiments and analyses as well as possible future collaborations. Many attendees reported that my work had changed their way of thinking about the subject. |
| Year(s) Of Engagement Activity | 2019 |
| Description | Graduate student talks at St John's College Cambridge |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Postgraduate students |
| Results and Impact | Around 50 postgraduate students from all subjects attended a symposium consisting of a few talks from a broad range of topics from molecular biology to medieval history. This started interesting conversations and ideas. |
| Year(s) Of Engagement Activity | 2017 |
| Description | LMB Open Day |
| Form Of Engagement Activity | Participation in an open day or visit at my research institution |
| Part Of Official Scheme? | No |
| Geographic Reach | Regional |
| Primary Audience | Public/other audiences |
| Results and Impact | Open day at the Laboratory of Molecular Biology, participating in activities for members of the public telling them about our research. |
| Year(s) Of Engagement Activity | 2017 |
| Description | Society for Research on Biological Rhythms |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | International |
| Primary Audience | Professional Practitioners |
| Results and Impact | International conference for researchers studying circadian rhythms. I presented a poster about my work, and this generated some interest |
| Year(s) Of Engagement Activity | 2018 |
| URL | https://srbr.org/meetings/previous-meetings/ |
| Description | Trinity College Science Society Annual Symposium |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | Local |
| Primary Audience | Undergraduate students |
| Results and Impact | Science society symposium consisting of several talks over a whole day, followed by a dinner for attendees. Broad range of topics from physics to biology. |
| Year(s) Of Engagement Activity | 2018 |
| Description | UK Clock Club, Manchester |
| Form Of Engagement Activity | A talk or presentation |
| Part Of Official Scheme? | No |
| Geographic Reach | National |
| Primary Audience | Professional Practitioners |
| Results and Impact | I gave an oral presentation about my work on circadian regulation of cellular ion transport and protein content to around 200 postgraduate students, postdoctoral researchers and group leaders from all over the UK in the Chronobiology field. This generated questions and discussions afterwards, including many ideas for future experiments and collaborations. |
| Year(s) Of Engagement Activity | 2018 |