Quantitative Analyses of Cryptochrome-mBMAL1 Interactions

Abstract

The mammalian cryptochromes mCRY1 and mCRY2 act as transcriptional repressors within the 24-h transcription-translational feedback loop of the circadian clock. The C-terminal tail and a preceding predicted coiled coil (CC) of the mCRYs as well as the C-terminal region of the transcription factor mBMAL1 are involved in transcriptional feedback repression. Here we show by fluorescence polarization and isothermal titration calorimetry that purified mCRY1/2CCtail proteins form stable heterodimeric complexes with two C-terminal mBMAL1 fragments. The longer mBMAL1 fragment (BMAL490) includes Lys-537, which is rhythmically acetylated by mCLOCK in vivo. mCRY1 (but not mCRY2) has a lower affinity to BMAL490 than to the shorter mBMAL1 fragment (BMAL577) and a K537Q mutant version of BMAL490. Using peptide scan analysis we identify two mBMAL1 binding epitopes within the coiled coil and tail regions of mCRY1/2 and document the importance of positively charged mCRY1 residues for mBMAL1 binding. A synthetic mCRY coiled coil peptide binds equally well to the short and to the long (wild-type and K537Q mutant) mBMAL1 fragments. In contrast, a peptide including the mCRY1 tail epitope shows a lower affinity to BMAL490 compared with BMAL577 and BMAL490(K537Q). We propose that Lys-537 mBMAL1 acetylation enhances mCRY1 binding by affecting electrostatic interactions predominantly with the mCRY1 tail. Our data reveal different molecular interactions of the mCRY1/2 tails with mBMAL1, which may contribute to the non-redundant clock functions of mCRY1 and mCRY2. Moreover, our study suggests the design of peptidic inhibitors targeting the interaction of the mCRY1 tail with mBMAL1.In mammals many physiological processes are regulated in a day-time-dependent manner. These circadian (24 h) rhythms are generated by circadian clocks, which are operated by transcriptional and translational feedback loops. In the central feedback loop, the bHLH-PAS (basic Helix-Loop-Helix-PER-ARNT-SIM) transcription factors mBMAL1 (brain and muscle ARNT-like protein) and mCLOCK (circadian locomotor output cycle kaput) activate the transcription of three period genes (mper1,2,3) and two cryptochromes (mcry1,2) (1). The mPER proteins and (even more potently) the mCRY proteins feedback-repress their own transcription by regulating the activity of mBMAL1 and mCLOCK (2, 3). Notably, the mBMAL1-mCLOCK transcription factor complex not only regulates the mper and mcry genes but also a large number of clock controlled genes, including genes involved in cell cycle regulation, cellular detoxification, and metabolism (4). Hence, the regulation of these transcription factors is of relevance for many body functions and associated diseases (e.g. sleep and depressive disorders, metabolic syndrome, cardiovascular diseases, and tumor formation) that are under the control of the circadian clock (5). The importance of mBMAL1 for clock function is clearly demonstrated by the fact that mBMAL1 Ϫ/Ϫ knock-out mice show an immediate and complete loss of circadian rhythmicity at a behavio...

Mutation of the Human Circadian Clock Gene CRY1 in Familial Delayed Sleep Phase Disorder

“…The Cry1 tail region represents the most poorly conserved and least functionally and structurally characterized region of the protein. It has been shown to affect Cry1 nuclear translocation, to interact with the Bmal1 transactivation domain possibly in an acetylation-dependent fashion and to be phosphorylated in a manner that involves regulation by DNA-PK (Chaves et al, 2006; Czarna et al, 2011; Gao et al, 2013; Hirayama et al, 2007; Xu et al, 2015). Interestingly, the tail is not essential to Cry1’s ability to restore circadian cycling to arrhythmic DKO MEFs, but does modulate the period length and amplitude of the resulting oscillation (Khan et al, 2012; Li et al, 2016).…”

“…Western Blotting was performed according to standard procedures using the above described antibodies as well as anti-Per2 (rabbit polyclonal, Alpha Diagnostics) anti-TATA binding protein TBP (mouse monoclonal, Abcam) and anti-α-Tubulin (mouse monoclonal, Sigma). Note on K358 Acetyl-Bmal1 antibody: Due to differences in residue numbering between Bmal1 splice variants, K538 in the reference sequence used by the vendor (Uniprot O00327-2) corresponds to the K537 residue for which acetylation has been described (Czarna et al, 2011; Hirayama et al, 2007). Blots were quantified using Image J and statistical significance was tested using paired t-tests in Prism 5 (GraphPad Software).…”

Molecular architecture of the mammalian circadian clock

“…The PAS domains of CLOCK and BMAL1 have been implicated in PER binding [47, 63], potentially through direct interaction with the tandem PAS homodimers of PER [64, 65]. Moreover, an overlapping region on CLOCK PAS-B has been implicated in direct binding of CRY1 [66], as has a region at the C-terminus of BMAL1 [63, 67], suggesting that the architecture of the core clock protein complex may remodel throughout the day to control transcriptional output. The unstructured C-terminal regions of CLOCK and BMAL1 exhibit structural plasticity, allowing them to interact with several transcriptional regulators that control genomic targeting through chromatin remodeling and the recruitment of transcriptional machinery [66, 68, 69].…”

Just-So Stories and Origin Myths: Phosphorylation and Structural Disorder in Circadian Clock Proteins

“…Additionally, FRQ has been shown by a variety of methods to be an ID protein that must be stabilized by another protein, a so-called ‘Nanny,’ in order for it to function in the clock (Hurley et al, 2013). Circular dichroism demonstrated ID regions in the C-terminal signaling regions of CRY and BMAL (Czarna et al, 2011), and more recently Xu et al demonstrated the importance of disorder in the TAD of BMAL1 for interaction with CRY1 (Xu et al, 2015). In this study, the Partch lab showed that CRY1 is able to repress BMAL1 transactivation by competing with CBP (p300) for binding to the intrinsically unstructured C-terminal TAD of BMAL1, with binding eliciting dynamic conformational rearrangements.…”