bims-ciryme 2024-06-02 papers

Proc Natl Acad Sci U S A. 2024 Jun 04. 121(23): e2316858121

Phosphorylation of DNA-binding domains of CLOCK-BMAL1 complex for PER-dependent inhibition in circadian clock of mammalian cells.

Yuta Otobe, Eui Min Jeong, Shunsuke Ito, Yuta Shinohara, Nobuhiro Kurabayashi, Atsu Aiba, Yoshitaka Fukada, Jae Kyoung Kim, Hikari Yoshitane.

In mammals, CLOCK and BMAL1 proteins form a heterodimer that binds to E-box sequences and activates transcription of target genes, including Period (Per). Translated PER proteins then bind to the CLOCK-BMAL1 complex to inhibit its transcriptional activity. However, the molecular mechanism and the impact of this PER-dependent inhibition on the circadian clock oscillation remain elusive. We previously identified Ser38 and Ser42 in a DNA-binding domain of CLOCK as phosphorylation sites at the PER-dependent inhibition phase. In this study, knockout rescue experiments showed that nonphosphorylatable (Ala) mutations at these sites shortened circadian period, whereas their constitutive-phospho-mimetic (Asp) mutations completely abolished the circadian rhythms. Similarly, we found that nonphosphorylatable (Ala) and constitutive-phospho-mimetic (Glu) mutations at Ser78 in a DNA-binding domain of BMAL1 also shortened the circadian period and abolished the rhythms, respectively. The mathematical modeling predicted that these constitutive-phospho-mimetic mutations weaken the DNA binding of the CLOCK-BMAL1 complex and that the nonphosphorylatable mutations inhibit the PER-dependent displacement (reduction of DNA-binding ability) of the CLOCK-BMAL1 complex from DNA. Biochemical experiments supported the importance of these phosphorylation sites for displacement of the complex in the PER2-dependent inhibition. Our results provide direct evidence that phosphorylation of CLOCK-Ser38/Ser42 and BMAL1-Ser78 plays a crucial role in the PER-dependent inhibition and the determination of the circadian period.

Keywords: CRISPR-Cas9; DNA binding; circadian rhythm; mathematical modeling; phosphorylation

DOI: https://doi.org/10.1073/pnas.2316858121

PLoS Genet. 2024 May 28. 20(5): e1011278

Clock-dependent chromatin accessibility rhythms regulate circadian transcription.

Ye Yuan, Qianqian Chen, Margarita Brovkina, E Josephine Clowney, Swathi Yadlapalli.

Chromatin organization plays a crucial role in gene regulation by controlling the accessibility of DNA to transcription machinery. While significant progress has been made in understanding the regulatory role of clock proteins in circadian rhythms, how chromatin organization affects circadian rhythms remains poorly understood. Here, we employed ATAC-seq (Assay for Transposase-Accessible Chromatin with Sequencing) on FAC-sorted Drosophila clock neurons to assess genome-wide chromatin accessibility at dawn and dusk over the circadian cycle. We observed significant oscillations in chromatin accessibility at promoter and enhancer regions of hundreds of genes, with enhanced accessibility either at dusk or dawn, which correlated with their peak transcriptional activity. Notably, genes with enhanced accessibility at dusk were enriched with E-box motifs, while those more accessible at dawn were enriched with VRI/PDP1-box motifs, indicating that they are regulated by the core circadian feedback loops, PER/CLK and VRI/PDP1, respectively. Further, we observed a complete loss of chromatin accessibility rhythms in per01 null mutants, with chromatin consistently accessible at both dawn and dusk, underscoring the critical role of Period protein in driving chromatin compaction during the repression phase at dawn. Together, this study demonstrates the significant role of chromatin organization in circadian regulation, revealing how the interplay between clock proteins and chromatin structure orchestrates the precise timing of biological processes throughout the day. This work further implies that variations in chromatin accessibility might play a central role in the generation of diverse circadian gene expression patterns in clock neurons.

DOI: https://doi.org/10.1371/journal.pgen.1011278

Nat Immunol. 2024 May 28.

Circadian control of tumor immunosuppression affects efficacy of immune checkpoint blockade.

Bridget M Fortin, Shannon M Pfeiffer, Jacob Insua-Rodríguez, Hamad Alshetaiwi, Alexander Moshensky, Wei A Song, Alisa L Mahieu, Sung Kook Chun, Amber N Lewis, Alex Hsu, Isam Adam, Oliver S Eng, Nicholas R Pannunzio, Marcus M Seldin, Ivan Marazzi, Francesco Marangoni, Devon A Lawson, Kai Kessenbrock, Selma Masri.

The circadian clock is a critical regulator of immunity, and this circadian control of immune modulation has an essential function in host defense and tumor immunosurveillance. Here we use a single-cell RNA sequencing approach and a genetic model of colorectal cancer to identify clock-dependent changes to the immune landscape that control the abundance of immunosuppressive cells and consequent suppression of cytotoxic CD8+ T cells. Of these immunosuppressive cell types, PD-L1-expressing myeloid-derived suppressor cells (MDSCs) peak in abundance in a rhythmic manner. Disruption of the epithelial cell clock regulates the secretion of cytokines that promote heightened inflammation, recruitment of neutrophils and the subsequent development of MDSCs. We also show that time-of-day anti-PD-L1 delivery is most effective when synchronized with the abundance of immunosuppressive MDSCs. Collectively, these data indicate that circadian gating of tumor immunosuppression informs the timing and efficacy of immune checkpoint inhibitors.

DOI: https://doi.org/10.1038/s41590-024-01859-0

Eur J Neurosci. 2024 May 27.

Loss of temporal coherence in the circadian metabolome across multiple tissues during ageing in mice.

M Renate Buijink, Michel van Weeghel, Amy Harms, Devika S Murli, Johanna H Meijer, Thomas Hankemeier, Stephan Michel, Laura Kervezee.

Circadian clock function declines with ageing, which can aggravate ageing-related diseases such as type 2 diabetes and neurodegenerative disorders. Understanding age-related changes in the circadian system at a systemic level can contribute to the development of strategies to promote healthy ageing. The goal of this study was to investigate the impact of ageing on 24-h rhythms in amine metabolites across four tissues in young (2 months of age) and old (22-25 months of age) mice using a targeted metabolomics approach. Liver, plasma, the suprachiasmatic nucleus (SCN; the location of the central circadian clock in the hypothalamus) and the paraventricular nucleus (PVN; a downstream target of the SCN) were collected from young and old mice every 4 h during a 24-h period (n = 6-7 mice per group). Differential rhythmicity analysis revealed that ageing impacts 24-h rhythms in the amine metabolome in a tissue-specific manner. Most profound changes were observed in the liver, in which rhythmicity was lost in 60% of the metabolites in aged mice. Furthermore, we found strong correlations in metabolite levels between the liver and plasma and between the SCN and the PVN in young mice. These correlations were almost completely abolished in old mice. These results indicate that ageing is accompanied by a severe loss of the circadian coordination between tissues and by disturbed rhythmicity of metabolic processes. The tissue-specific impact of ageing may help to differentiate mechanisms of ageing-related disorders in the brain versus peripheral tissues and thereby contribute to the development of potential therapies for these disorders.

Keywords: ageing; amine metabolism; chronobiology; circadian rhythms; hypothalamus; liver; metabolomics; plasma

DOI: https://doi.org/10.1111/ejn.16428