Wolf/Andrade/Foitzik
Mechanistic insights into the evolution of circadian gene regulation
Supervisor: Eva Wolf
Co-Supervisor: Miguel Andrade, Susanne Foitzik
Scientific Background
Circadian clocks allow organisms to adapt their physiology to 24 h light-dark cycles. We study the evolution of circadian gene regulation in animals, asking how clock-components and their molecular interactions evolved to maintain the 24 h rhythm across species.
In mammals, the bHLH-PAS CLOCK/BMAL1 transcription factors activate the expression of two Period (mPER1,2) and two Cryptochrome (mCRY1,2) clock proteins, which feedback repress CLOCK/BMAL1 (Czarna et al, 2013; Schmalen et al, 2014). The migratory monarch butterfly Danaus plexippus (dp) has an ancestral circadian clock including single repressor CRY and PER homologues (dpPER, dpCRY2) that repress dpCLOCK/BMAL1 (Yuan et al, 2007). mCRY1/2 and dpCRY2 repress CLOCK/BMAL1 by binding to a BMAL1 transactivation domain (TAD) and to the CLOCK PAS-B domain (Czarna et al, 2011; Zhang et al, 2017).
We discovered that sequence variations in the BMAL1-TAD and competing PER:CRY interactions differentially modulate the affinity of the repressive BMAL-TAD:CRY interaction in insects and mammals. While CRY has a lower affinity to the monarch TAD than to the mammalian TAD, dpPER competes less potently with dpTAD: CRY interactions than mPER1/2. Hence, we hypothesize that co-evolution of repressor CRYs, the BMAL1-TAD and PER proteins tune the stability of the repressive CRY:BMAL1-TAD complex and thereby transcriptional activity of CLOCK/BMAL1.
Project description
The prospective PhD student will explore the roles of repressor CRY and PER proteins in CLOCK/BMAL regulation in non-model organisms, such as the mosquito Anopheles gambiae (Ag) and the honey bee Apis mellifera (Am), which have a single CRY/PER clock architecture (Yuan et al, 2007). Our purified Ag- and Am repressor CRY2 proteins showed similar BMAL1-TAD sequence preferences as dpCRY2 and mCRY1/2. However the variable C-terminal tail regions of CRY appear to impact CRY:BMAL1-TAD affinity in a species-specific manner. In future we will i) investigate how the CRY tailsand PER proteins in Ag and Am impact the repressive CRY:BMAL1-TAD and CRY:CLOCK-PAS-B interactions as well as CLOCK/BMAL1 activity. ii) We will pick up additional organisms with interesting clock architectures or interesting sequence variations in their repressor CRYs, CRY-binding PER region, BMAL1-TAD or CLOCK-PAS-B for more detailed molecular mechanistic and functional studies. iii) We will explore additional clock components (e.g. co-activators, co-repressors) that regulate CLOCK/BMAL1 together with CRY and PER in different organisms.
These studies will provide molecular mechanistic insights into the co-evolution of PER, CRY, BMAL1 and CLOCK, and help us understand how this interaction network evolved to tune CLOCK/BMAL1 activity and circadian rhythmicity in a species-specific manner.
The project will integrate multi-species sequence analyses (to explore clock evolution across many species) with 3D-structural- and biochemical protein interaction studies as well as cell-based functional analyses.
What you will learn
You will learn how to employ protein biochemistry, structural biology, biophysics and bioinformatics approaches to address evolutionary questions. You will apply a broad range of techniques, including cloning, recombinant protein expression, chromatographic FPLC-based protein purification, protein interaction studies (e.g. SEC, pulldown, native PAGE, fluorescence polarization, ITC, SPR), structural studies using X-ray crystallography, Cryo-EM and AlphaFold predictions, and protein sequence analyses. Cell-based studies (e.g. reporter gene assays, circadian oscillation assays) will be done in collaborations.
Your qualifications
We encourage applications of PhD candidates with a strong interest in structural biology, protein biochemistry, biophysics, structure-function relationship and evolutionary biology and a significant previous exposure to these research areas during their master education. Practical experience with FPLC-based protein purification and some techniques involved in the biochemical, biophysical or 3D-structural analyses of purified proteins is expected.
Publications relevant to this project
Schmalen I, Reischl S, Wallach T, Klemz R, Grudziecki A, Prabu JR, Benda C, Kramer A, Wolf E (2014) Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. Cell. 157(5):1203-15. doi.org/10.1016/j.cell.2014.03.057
Czarna A, Berndt A, Singh HR, Grudziecki A, Ladurner AG, Timinszky G, Kramer A, Wolf E (2013) Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function. Cell. 153(6):1394-405. doi.org/10.1016/j.cell.2013.05.011
Czarna A, Breitkreuz H, Mahrenholz CC, Arens J, Strauss HM, Wolf E (2011) Quantitative analyses of Cryptochrome-mBMAL1 interactions: mechanistic insights into the transcriptional regulation of the mammalian circadian clock. JBC 286:22414-25. doi.org/10.1074/jbc.m111.244749
Yuan Q, Metterville D, Briscoe AD, Reppert SM (2007) Insect cryptochromes: gene duplication and loss define diverse ways to construct insect circadian clocks. Mol Biol Evol. 24(4):948-55. doi.org/10.1093/molbev/msm011
Zhang, Y., Markert, M.J., Groves, S.C., Hardin, P.E., and Merlin, C. (2017) Vertebrate-like CRYPTOCHROME 2 from monarch regulates circadian transcription via independent repression of CLOCK and BMAL1 activity PNAS114, E7516‐E7525. doi.org/10.1073/pnas.1702014114