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Mechanistic insights into the evolution of circadian gene regulation

Supervisor: Eva Wolf

Co-Supervisor: Susanne Foitzik, Miguel Andrade


Scientific Background:
Circadian clocks enable organisms to synchronize their physiology and behavior to the 24 h environmental light-dark cycle. The ancestral circadian clock of the migratory monarch butterfly (Danaus plexippus) combines molecular features of the mammalian- and Drosophila circadian clock and therefore provides an interesting model system to study circadian clock evolution.
The aim of this project is to understand, how the regulation of the circadian BMAL1/CLOCK transcription factors changes in evolution to maintain the circadian (~ 24 h) period length in different species and environments. In mammals, repressive Cryptochromes (CRY) and the activating histone acetyltransferase CBP competitively bind to a transactivation domain (TAD) of BMAL1 to regulate circadian gene expression (Czarna et al, 2011/2013; Garg et al, 2019). Sequence variations in the BMAL1-TAD may therefore tune circadian clock evolution by affecting repressive BMAL1-CRY- and activating BMAL1-CBP interactions. CRY transcriptional repressor activity is further modulated by interactions with PERIOD (PER) clock proteins (Schmalen et al, 2014).


PhD project: Studying mechanistic insights into the evolution of circadian gene regulation
To investigate the rewiring of animal circadian clocks over evolutionary time scales, the PhD student will take a highly interdisciplinary and collaborative approach.
To understand the basic mechanism of the ancestral circadian clock of the monarch butterfly, the PhD student will i) perform structure-function analyses of monarch clock protein complexes, ii) analyse circadian gene expression and chromatin modifications in a monarch cell line using NGS techniques, iii) perform pulldown-mass spectrometry to identify new clock protein interaction partners.
In parallel, a broader search for species specific changes in diverse organisms will be performed, which will be inspired by our mechanistic studies. Initially, the correlation of BMAL1-TAD sequence variations with changes in repressive CRYs and their co-evolution with PER proteins will be explored using bioinformatics approaches. Interesting candidates will be experimentally validated by analysing the impact of sequence variations on protein interaction affinities, as well as on BMAL1/CLOCK repression and circadian rhythmicity. Co-evolving interaction partners will be structurally analysed by 3D modelling or X-ray crystallography.

The PhD student will apply a wide range of techniques including biochemical and biophysical protein interaction studies, X-ray crystallography, bioinformatics, recombinant protein expression, protein purification, molecular biology and functional cell-based assays. Thebioinformatics- and cell-based analyses will be supported by M. Andrade, P. Baumann, S. Foitzik and H. May-Simera within the Genevo network. Candidates with a strong interest in protein structure-function relationship, protein biochemistry and evolutionary biology and previous exposure to these research areas are encouraged to apply.

Publications relevant to this project
Garg A, Orru R, Ye W, Distler U, Chojnacki JE, Köhn M, Tenzer S, Sönnichsen C and Wolf E (2019) Structural and mechanistic insights into the interaction of the circadian transcription factor BMAL1 with the KIX domain of the CREB-binding protein. JBC 294:16604–16619
Schmalen I, Reischl S, Wallach T, Klemz R, Grudziecki A, Prabu JR, Benda C, Kramer A and Wolf E (2014) Interaction of circadian clock proteins CRY1 and PER2 is modulated by zinc binding and disulfide bond formation. Cell 157:1203–1215
Czarna A, Berndt A, Singh HR, Grudziecki A, Ladurner AG, Timinszky G, Kramer A and Wolf E (2013) Structures of Drosophila cryptochrome and mouse cryptochrome1 provide insight into circadian function. Cell 153:1394–1405
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