Circadian period is compensated for repressor protein turnover rates in single cells.

Most mammalian cells have molecular circadian clocks that generate widespread rhythms in transcript and protein abundance. While circadian clocks are robust to fluctuations in the cellular environment, little is known about the mechanisms by which the circadian period compensates for fluctuating metabolic states. Here, we exploit the heterogeneity of single cells both in circadian period and a metabolic parameter-protein stability-to study their interdependence without the need for genetic manipulation. We generated cells expressing key circadian proteins (CRYPTOCHROME1/2 (CRY1/2) and PERIOD1/2 (PER1/2)) as endogenous fusions with fluorescent proteins and simultaneously monitored circadian rhythms and degradation in thousands of single cells. We found that the circadian period compensates for fluctuations in the turnover rates of circadian repressor proteins and uncovered possible mechanisms using a mathematical model. In addition, the stabilities of the repressor proteins are circadian phase dependent and correlate with the circadian period in a phase-dependent manner, in contrast to the prevailing model.

Identification of Novel Nuclear Factor of Activated T Cell (NFAT)-associated Proteins in T Cells.

Transcription factors of the nuclear factor of activated T cell (NFAT) family are essential for antigen-specific T cell activation and differentiation. Their cooperative DNA binding with other transcription factors, such as AP1 proteins (FOS, JUN, and JUNB), FOXP3, IRFs, and EGR1, dictates the gene regulatory action of NFATs. To identify as yet unknown interaction partners of NFAT, we purified biotin-tagged NFATc1/αA, NFATc1/ßC, and NFATc2/C protein complexes and analyzed their components by stable isotope labeling by amino acids in cell culture-based mass spectrometry. We revealed more than 170 NFAT-associated proteins, half of which are involved in transcriptional regulation. Among them are many hitherto unknown interaction partners of NFATc1 and NFATc2 in T cells, such as Raptor, CHEK1, CREB1, RUNX1, SATB1, Ikaros, and Helios. The association of NFATc2 with several other transcription factors is DNA-dependent, indicating cooperative DNA binding. Moreover, our computational analysis discovered that binding motifs for RUNX and CREB1 are found preferentially in the direct vicinity of NFAT-binding motifs and in a distinct orientation to them. Furthermore, we provide evidence that mTOR and CHEK1 kinase activity influence NFAT's transcriptional potency. Finally, our dataset of NFAT-associated proteins provides a good basis to further study NFAT's diverse functions and how these are modulated due to the interplay of multiple interaction partners.

A conditional Smg6 mutant mouse model reveals circadian clock regulation through the nonsense-mediated mRNA decay pathway.

Nonsense-mediated messenger RNA (mRNA) decay (NMD) has been intensively studied as a surveillance pathway that degrades erroneous transcripts arising from mutations or RNA processing errors. While additional roles in physiological control of mRNA stability have emerged, possible functions in mammalian physiology in vivo remain unclear. Here, we created a conditional mouse allele that allows converting the NMD effector nuclease SMG6 from wild-type to nuclease domain-mutant protein. We find that NMD down-regulation affects the function of the circadian clock, a system known to require rapid mRNA turnover. Specifically, we uncover strong lengthening of free-running circadian periods for liver and fibroblast clocks and direct NMD regulation of Cry2 mRNA, encoding a key transcriptional repressor within the rhythm-generating feedback loop. Transcriptome-wide changes in daily mRNA accumulation patterns in the entrained liver, as well as an altered response to food entrainment, expand the known scope of NMD regulation in mammalian gene expression and physiology.

Mutational scanning identified amino acids of the CLOCK exon 19-domain essential for circadian rhythms.

AIM: In the mammalian circadian clock, the CLOCK/BMAL1 heterodimer binds to E-box enhancer elements in the promoters of its target genes to activate transcription. The classical Clock mice, the first circadian mouse mutant discovered, are behaviourally arrhythmic. In this mutant, CLOCK lacks a 51 amino acid domain corresponding to exon 19 (CLOCKΔ19), which is required for normal transactivation. While the importance of this CLOCK domain for circadian rhythms is well established, the exact molecular mechanism is still unclear. METHODS: Using CRISPR/Cas9 technology, we created a CLOCK knockout - CLOCK rescue system in human circadian reporter cells and performed systematic mutational scanning to assess the functionality of individual amino acids within the CLOCK exon 19-domain. RESULTS: CLOCK knockout cells were arrhythmic, and circadian rhythms could be rescued by introducing wild-type CLOCK, but not CLOCKΔ19. In addition, we identified several residues, whose mutation failed to rescue rhythms in CLOCK knockout cells. Many of these are part of the hydrophobic binding interface of the predicted dimer of the CLOCK exon 19-domain. CONCLUSION: Our data not only indicate that CLOCK/BMAL1 oligomerization mediated by the exon 19-domain is important for circadian dynamics but also suggest that the exon 19-domain provides a platform for binding coactivators and repressors, which in turn is required for normal circadian rhythms.

Live-cell imaging of circadian clock protein dynamics in CRISPR-generated knock-in cells.

The cell biology of circadian clocks is still in its infancy. Here, we describe an efficient strategy for generating knock-in reporter cell lines using CRISPR technology that is particularly useful for genes expressed transiently or at low levels, such as those coding for circadian clock proteins. We generated single and double knock-in cells with endogenously expressed PER2 and CRY1 fused to fluorescent proteins allowing us to simultaneously monitor the dynamics of CRY1 and PER2 proteins in live single cells. Both proteins are highly rhythmic in the nucleus of human cells with PER2 showing a much higher amplitude than CRY1. Surprisingly, CRY1 protein is nuclear at all circadian times indicating the absence of circadian gating of nuclear import. Furthermore, in the nucleus of individual cells CRY1 abundance rhythms are phase-delayed (~5 hours), and CRY1 levels are much higher (>5 times) compared to PER2 questioning the current model of the circadian oscillator.

Recruitment of Histone Methyltransferase Ehmt1 to Foxp3 TSDR Counteracts Differentiation of Induced Regulatory T Cells.

Differentiation toward CD4+ regulatory T (Treg) cells is essentially dependent on an epigenetic program at Treg signature genes, which involves remodeling of the Treg-specific demethylated regions (TSDRs). In particular, the epigenetic status of the conserved non-coding sequence 2 of Foxp3 (Foxp3 TSDR) determines expression stability of the master transcription factor and thus Treg lineage identity. However, the molecular mechanisms controlling the epigenetic remodeling at TSDRs in Treg and conventional T cells are largely unknown. Using a combined approach of DNA pull-down and mass spectrometric analysis, we report a novel regulatory mechanism in which transcription factor Wiz recruits the histone methyltransferase Ehmt1 to Foxp3 TSDR. We show that both Wiz and Ehmt1 are crucial for shaping the region with the repressive histone modification H3K9me2 in conventional T cells. Consistently, knocking out either Ehmt1 or Wiz by CRISPR/Cas resulted in the loss of H3K9me2 and enhanced Foxp3 expression during iTreg differentiation. Moreover, the essential role of the Wiz-Ehmt1 interaction as observed at several TSDRs indicates a global function of Ehmt1 in the Treg differentiation program.