DAY a novel signalling partner of Drosophila melanogaster CRYPTOCHROME

Lead Research Organisation: University of Leicester
Department Name: Genetics

Abstract

The 24 hour circadian clock controls every facet of an organism's physiology and behaviour. In humans, dysfunction of the clock not only generates temporal disorientation and sleep problems, but is also involved in pathologies such as obesity, mental illness, cardiovascular disease and cancer. The fruitfly Drosophila melanogaster is an ideal model organism for the study of circadian rhythms because the clock mechanism shares the same design and molecular components with mammals. The clock is an endogenous and self-sustained process driven by the interplay of many proteins. Among these there are molecules whose function is to receive and integrate environmental cues, and pass on this information to other clock proteins. The blue-light photopigment CRYPTOCHROME (CRY) is one such molecule, but in some cells, CRY also performs as a core clock protein independent from light. We have been working with CRY for several years and recently have discovered that another protein, we (ironically) call DAY, is able to interact with CRY, but only at night. DAY contains four PDZ domains, which are regions involved in protein-protein interactions. PDZ proteins can bring together membrane bound and cytosolic components to promote certain signalling pathways. Our preliminary work has shown that day mRNA cycles and is present in the lateral brain, most likely in a subset of circadian neurons where CRY functions exclusively as a photopigment. We did not detect the expression of day in the dorsal brain, nor in peripheral cells such as the eyes or the Malpighian tubules, where CRY also provides core clock function, suggesting that DAY may inhibit this role. In this proposal we aim to test this hypothesis as follows. We will characterise the expression of DAY and extend our work on day mRNA. We will provide additional evidence for the interaction between DAY and CRY. We will investigate the molecular, cellular and behavioural phenotypes arising from several combinations of up- and down-regulation of day and cry in central clock neurons and in peripheral tissues. These studies will elucidate the role played by this novel protein involved in the regulation of the circadian clock and will contribute to the understanding of the mode of action of CRY. Moreover, we believe that our work will add some mechanistic information to the catalogue of discrepancies reported for the different classes of central clock neurons.

Technical Summary

A large scale yeast-two-hybrid screen has identified the PDZ protein CG15803-PA as producing a highly reliable interaction with Drosophila CRYPTOCHROME (CRY). Interestingly, CRY contains PDZ binding motifs in its C-terminus. We further characterised the interaction and found that CG15803-PA binds to CRY in darkness but not in light, thus we renamed this protein as DARK-ACTIVE-IN YEAST (DAY). Previously we had shown that CRY binds to the clock proteins PERIOD (PER) and TIMELESS (TIM) in a light-dependent manner, although, in Drosophila S2 cells, CRY co-immunoprecipitates with PER and TIM also in the dark. We therefore speculate that, at night, DAY may antagonise the interaction of CRY with other clock proteins, thereby limiting CRY's role to light signalling. It is currently believed that CRY operates exclusively as a photopigment in pacemaker neurons, whereas in peripheral clock cells, or, as we have recently observed, in Dorsal Neurons (a subset of 'pacemaker neurons') CRY additionally functions as a light-independent clock component. Thus, we examined day expression by in situ hybridisation and detected staining in a lateral region of the brain, corresponding to the position of the circadian Lateral Neurons, but not in Dorsal Neurons or in peripheral clocks. We also observed circadian cycling in the abundance of day mRNA. Thus wherever CRY is restricted to a photoreceptor role, DAY is co-expressed, and wherever CRY also plays a clock-related role, DAY is absent, supporting our hypothesis. In this proposal we seek to consolidate and extend these findings and to test our hypothesis on the significance of DAY/CRY interaction. We also propose to investigate the role of day in other behaviours such as geotaxis, following CRY involvement in it, and sexual behaviour, due to the presence of day mRNA in the male accessory glands and to sexual dimorphism in the level of expression in the brain.
 
Description In the fruitfly Drosophila melanogaster the circadian clock is constituted by two interlocked negative transcription/translation feedback loops. Although the current molecular model portrays the clock as cell autonomous, evidence has shown that cross-talk among neurons is an essential part of its functioning. However, our understanding of the functional organisation of the neuronal network is still limited. For many years we have been studying the blue-light detecting protein CRYPTOCHROME (CRY), whose function is to receive light information and pass it on to other clock proteins. The starting point of this work has been the discovery of a novel protein, DAY, seemingly able to interact with CRY. DAY contains four PDZ domains, regions involved in protein-protein interactions that are found in proteins promoting signalling pathways. Our first findings suggested that DAY might be present in the Lateral Neurons but not in peripheral clock cells. The former are clock cells were CRY has only light-dependent activities, in the latter CRY seems able to operate also independently from light. Our hypothesis was that DAY might be responsible for the different functions that CRY exerts in different cell types. During this project we realised that DAY is not found in the canonical Lateral Neurons, although it is present in cells very close by. After many experiments we obtained evidence that DAY is not directly involved in the circadian clock, but modulates sleep homeostasis. Likewise, CRY, which we have confirmed interacts with DAY in vivo, is involved in the regulation of sleep. We have also shown that misregulation of DAY increases sexual arousal. Males start courting females earlier and are more effective than controls. Potentially this might be caused by two mechanisms. 1. A generalised increased in arousal, as evinced by sleep reduction. 2. DAY is expressed in olfactory glomeruli involved in detection of male pheromone, which acts as a courtship inhibitor.

During our studies we came across a puzzling result. We obtained a mild increase in the length of the period of locomotor activity by driving mis-expression of DAY in th Lateral Neurons. Instead, applying the same manipulation to all clock neurons did not result in any period change. This suggested that different clock neurons might have opposing roles in the circadian network, which goes against the current cell-autonomous model of the clock. To test this hypothesis, we turned to CRY_, a form of CRY with a 20 amino acid deletion in its C_terminus, which we had previously shown to produce robust changes in the period of locomotor activity. Using this and other manipulations we were able to confirm our findings and to draw a functional map connecting the neurons of the circadian network.

In conclusion, although we have proven our original predictions wrong, this work led to some exciting and unexpected results. We have identified a new synaptic role for CRY, we have drawn a map of the functional connections among clock neurons and we have clarified the role of DAY as a protein involved in arousal and likely localised in central synapses.
Exploitation Route Exploiting Drosophila for finding out how the circadian system works can have implication for human and animal health and wellbeing. Restricting our interest to the peculiarities of insects might have applications in agriculture and pest control. We had presented two ideas to the Enterprise and Business Development Office of the University of Leicester for patent application. These ideas did not seem viable in their original form. We are now in the process (March 2012) of applying for a Follow-on-Funding Pathfinder stemming from our work
Sectors Environment,Healthcare,Pharmaceuticals and Medical Biotechnology

 
Description We led the development of an international consortium INsecTIME, which has applied for a Marie Curie Initial Training Network. Our research is internationally recognised. We led the development of a consortium, INsecTIME, which spans seven EU countries plus Israel and brings together leading Universities and three successful SMES. INsecTIME's strategic objective is to advance the current knowledge on insect timing with the simultaneous development of a new breed of young researchers that have extensive inter-disciplinary skills for molecular genetic intervention and manipulation of economically important insects.
Sector Education,Healthcare
Impact Types Cultural,Economic

 
Description BBSRC responsive mode
Amount £440,000 (GBP)
Funding ID BB/P010121/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 04/2017 
End 03/2020
 
Description MOLECULAR AND CELLULAR DISSECTION OF MORNING AND EVENING CHRONOTYPES IN DROSOPHILA MELANOGASTER
Amount £629,938 (GBP)
Funding ID BB/H018093/1 
Organisation Biotechnology and Biological Sciences Research Council (BBSRC) 
Sector Public
Country United Kingdom
Start 02/2011 
End 01/2014
 
Title CRY C-terminus mutant lines 
Description Transgenic (UAS-) fly lines to express mutated forms of the C-terminal domain of cryptochrome . In particular we produced the following mutations, S(526)D, E(530)P, V(531)K. We also produced (UAS-) fly lines expressing a C-terminal region of CRY (starting from residue 492 to the end at 542) fused to GFP 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
 
Title CRY C-terminus mutants 
Description plasmids containing mutated forms of the C-terminal domain of cryptochrome. In particular we produced the following mutations, S(526)D, E(530)P, V(531)K. We also produced a plasmid expressing the C-terminal region of CRY (starting from residue 492 to the end at 542) fused to GFP 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Title DAY overexpression plasmids 
Description Several plasmids containing the full length coding sequence of the day gene, untagged and tagged with Strep II and/or hemagglutinin. These plasmids are for expression in bacterial, yeast and Drosophila cells. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Title DAY-GAL4 
Description Promoter-GAL4 plasmids comprising 1, 1.5, 3, 4.5 and 6 kb upstream of day coding sequence 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Title DAY-GAL4 transgenics 
Description transgenic fly lines comprising 1, 1.5, 3, 4.5 and 6 kb upstream of day coding sequence 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
 
Title Day overexpression 
Description transgenic (UAS-) fly lines that can overexpress HA and HA+ StrepII tagged forms of DAY when crossed with appropriate (GAL4) driver lines. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
 
Title Minos mobilisation 
Description Fly lines resulting from the mobilisation of the transposable Minos element Mi{ET1}MB07628 to generate day-null alleles via imprecise excision 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
 
Title anti-DAY antibody 
Description A rabbit polyclonal antibody (serum + affinity purified) against 2 synthetic peptides from DAY protein 
Type Of Material Antibody 
Provided To Others? No  
 
Title day[mini-w+]day donor plasmid 
Description plasmid necessary for the production of a day-null mutant by homologous recombination. The construct we generated carries 3+3 kb of day genomic DNA flanking the coding region of day, which has been replaced by the marker gene miniwhite+. 
Type Of Material Model of mechanisms or symptoms - in vitro 
Provided To Others? No  
 
Title day[miniw+]day transgenics 
Description Transgenic fly lines corresponding to the knock-out of day by homologous recombination. 
Type Of Material Model of mechanisms or symptoms - non-mammalian in vivo 
Provided To Others? No  
 
Description Cinchron 
Organisation Academy of Sciences of the Czech Republic
Department Institute of Entomology
Country Czech Republic 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation BioFly
Country Israel 
Sector Private 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation Hebrew University of Jerusalem
Country Israel 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation Institute of Neurobiology Alfred-Fessard
Country France 
Sector Charity/Non Profit 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation Jagiellonian University
Department Department of Biology and Cell Imaging
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation Oxitec Ltd
Country United Kingdom 
Sector Private 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation University of Groningen
Country Netherlands 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation University of Münster
Country Germany 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation University of Padova
Department Department of Biology
Country Italy 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation University of Valencia
Country Spain 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description Cinchron 
Organisation University of Wurzburg
Department Biozentrum
Country Germany 
Sector Academic/University 
PI Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017. Coordinator: Prof Kyriacou, Leicester.
Collaborator Contribution Application to a MARIE SKLODOWSKA-CURIE ACTIONS Innovative Training Networks (ITN), January 2017
Impact Application, no outputs yet
Start Year 2017
 
Description INsecTIME 
Organisation BioFly
Country Israel 
Sector Private 
PI Contribution The work developed during the tenure of this grant has been instrumental in creating the background conditions for developing a collaboration with Oxitec (http://www.oxitec.com/) and Bio-Fly (http://www.bio-bee.com/site/in_page.asp?pa_id=155&page_id=214), companies involved in biological insect control. With these companies we are collaborators in a current bid to the European Union for a Marie Curie Initial training award.
Start Year 2011
 
Description INsecTIME 
Organisation Oxitec Ltd
Country United Kingdom 
Sector Private 
PI Contribution The work developed during the tenure of this grant has been instrumental in creating the background conditions for developing a collaboration with Oxitec (http://www.oxitec.com/) and Bio-Fly (http://www.bio-bee.com/site/in_page.asp?pa_id=155&page_id=214), companies involved in biological insect control. With these companies we are collaborators in a current bid to the European Union for a Marie Curie Initial training award.
Start Year 2011
 
Description An Introduction to biological clocks 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Big Bang at Leicester Grammar School, Leicester Grammar School, 25 February 2017, Leicester, UK
Year(s) Of Engagement Activity 2017
 
Description Behavioural Genetics 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach International
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Big Bang at Leicester Grammar School 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact presentation on the circadian clock and its impact on humans and the natural world. People who attended understood the importance of the circadian clock, its impact on health and wellbeing and how to look for sign of the clock in every day life
Year(s) Of Engagement Activity 2017
 
Description Big Bang event at Leicester Grammar School 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact The hands onb activity we presented sparked lots of interests on Genetics and use of fruit fly for medical or fundamental research from parents and pupils

We were asked to take part the following year
Year(s) Of Engagement Activity 2015
 
Description Drosophila as a model organism 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach local
Primary Audience Schools
Results and Impact Dynamic DNA day, organised by Genie, 13 Sept 2012, presentation and hands on activities to year 9 students. powerpoint presentation

leaflets

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Dynamic DNA Day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? Yes
Geographic Reach Regional
Primary Audience Schools
Results and Impact every year 600 pupils and their teachers experience a Genetic Journey that many will always remember.

every year more school ask to take part that we can possibly accomodate
Year(s) Of Engagement Activity 2006,2007,2008,2009,2011
URL http://www2.le.ac.uk/departments/genetics/genie/outreach/dynamic-dna
 
Description Genes networks and rhythms 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach international
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar, Department of Cytology and Histology, Institute of Zoology Jagiellonian University, Krakow, Poland

no actual impacts realised to date
Year(s) Of Engagement Activity 2009
 
Description Leicester Grammar Junior School visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? Yes
Geographic Reach Local
Primary Audience Schools
Results and Impact We presented a talk on circadian rhythms and the use of Drosophila as a model system to about 61 year 5 & 6 pupils. We showed the flies and how we measure circadian rhythms in activity/rest. We talked about chronotypes and how we can assess them in humans. We had a drawing competition, a 'fly race' competition and a discussion at the end

The visit was considered positive by 15 and very positive by 31 pupils. Instead 15 pupils did not enjoy it. The science teacher was very impressed and we werer asked to contribute another project in March/April 2015
Year(s) Of Engagement Activity 2014
 
Description Loughborough Grammar school visit 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact About 50-60 years 11-13s pupil attended the lecture which was very well received

increase interest from pupils
Year(s) Of Engagement Activity 2014
 
Description Measuring circadian rhythms 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact stand with multiple acitivities and examples on how to measure circadian rhythms. Very good turnout of people who asked many questions about the circadian clock, sleep and how improve their sleep/wake cycle
Year(s) Of Engagement Activity 2016
URL https://nearme.thebigbangfair.co.uk/Event/?e=2520
 
Description Molecular analysis of the circadian clock 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Participants in your research or patient groups
Results and Impact Invited seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description School visit (Oundle) 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact About 60-70 sixth form students attended a lecture to inform them on the circadian clock

There was great interest about the subject and the talk was followed by an interview to be broadcasted on the school radio
Year(s) Of Engagement Activity 2006,2014
 
Description The clock within us 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Schools
Results and Impact Public Lecture: The clock within us. Uppingham School, 15 November 2017, Uppingham, UK
Year(s) Of Engagement Activity 2017
 
Description The neurogenetics of the circadian clock 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Primary Audience Participants in your research or patient groups
Results and Impact Invited Seminar

no actual impacts realised to date
Year(s) Of Engagement Activity 2012
 
Description Wurzburg open day 
Form Of Engagement Activity Participation in an activity, workshop or similar
Part Of Official Scheme? No
Geographic Reach Local
Primary Audience Public/other audiences
Results and Impact Open day in Wurzburg, Germany together with our European collaborators. The workshop showed the importance of rhythmicity and photoperiodism in humans and the natural world. The attendees demonstrated increased interest and awareness after attending our activities.
Year(s) Of Engagement Activity 2017
 
Description York Festival of Ideas 
Form Of Engagement Activity A talk or presentation
Part Of Official Scheme? No
Geographic Reach Regional
Primary Audience Public/other audiences
Results and Impact Presented a lecture on the importance of our body clock for health and wellbeing. This was in the context of a symposium on sleep and the body clock. The participants left with increased awareness about the subject.
Year(s) Of Engagement Activity 2016