Phase separation and biorhythms control biological processes in the spatial and temporal dimensions, respectively, but mechanisms of four-dimensional integration remain elusive. Here, we identified an evolutionarily conserved XBP1s-SON axis that establishes a cell-autonomous mammalian 12-hour ultradian rhythm of nuclear speckle liquid-liquid phase separation (LLPS) dynamics, separate from both the 24-hour circadian clock and the cell cycle. Higher expression of nuclear speckle scaffolding protein SON, observed at early morning/early afternoon, generates diffuse and fluid nuclear speckles, increases their interactions with chromatin proactively, transcriptionally amplifies the unfolded protein response, and protects against proteome stress, whereas the opposites are observed following reduced SON level at early evening/late morning. Correlative Son and proteostasis gene expression dynamics are further observed across the entire mouse life span. Our results suggest that by modulating the temporal dynamics of proteostasis, the nuclear speckle LLPS may represent a previously unidentified (chrono)therapeutic target for pathologies associated with dysregulated proteostasis.

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RNA in situ hybridization (ISH) is used to visualize spatio-temporal gene expression patterns with broad applications in biology and biomedicine. Here we provide a protocol for mRNA ISH in developing pupal wings and abdomens for model and non-model Drosophila species. We describe best practices in pupal staging, tissue preparation, probe design and synthesis, imaging of gene expression patterns, and image-editing techniques. This protocol has been successfully used to investigate the roles of genes underlying the evolution of novel color patterns in non-model Drosophila species.

13To understand how novel animal patterning emerged, one needs to ask how the 14 development of color patterns has changed among diverging species. Here we examine 15 three species of fruit flies -Drosophila guttifera (D. guttifera), Drosophila palustris (D. 16 palustris), and Drosophila subpalustris (D. subpalustris) -displaying a varying number 17 of abdominal spot rows that were either gained or lost throughout evolutionary time. 18 Through in situ hybridization, we examine the mRNA expression patterns for the 19 pigmentation genes Dopa decarboxylase (Ddc), tan (t), and yellow (y) during pupal 20 development. Our results show that Ddc, t, and y are co-expressed in identical patterns, 21 each foreshadowing the adult abdominal spots in D. guttifera, D. palustris, and D. 22 subpalustris. 23

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While circadian rhythms are entrained to the once daily light-dark cycle of the sun, many marine organisms exhibit ~12h ultradian rhythms corresponding to the twice daily movement of the tides. Although human ancestors emerged from circatidal environment millions of years ago, direct evidence of the existence of ~12h ultradian rhythms in humans are lacking. Here, we performed prospective, temporal transcriptome profiling of peripheral white blood cells and identified robust ~12h transcriptional rhythms from three healthy subjects. Pathway analysis implicated ~12h rhythms effecting RNA and protein metabolism, with strong homology to the circatidal gene programs previously identified in Cnidarian marine species. We further observed ~12h rhythms of intron retention events of genes involved in MHC class I antigen presentation in all three subjects, synchronized to those of mRNA splicing gene expressions in each individual. Gene regulatory network inference revealed XBP1, GABPA and KLF7 as putative transcriptional regulators of human ~12h rhythms. These results thus establish human ~12h biological rhythms have a primordial evolutionary origin and are likely to have far-reaching implications in human health and disease.

Background. The study of aging and its mechanisms, such as cellular senescence, has provided valuable insights into age-related pathologies, thus contributing to their prevention and treatment. The current abundance of high throughput data combined with the surge of robust analysis algorithms has facilitated novel ways of identifying underlying pathways that may drive these pathologies. Methods. With the focus on identifying key regulators of lung aging, we performed comparative analyses of transcriptional profiles of aged versus young human subjects and mice, focusing on the common age-related changes in the transcriptional regulation in lung macrophages, T cells, and B immune cells. Importantly, we validated our findings in cell culture assays and human lung samples. Results. We identified Lymphoid Enhancer Binding Factor 1 (LEF1) as an important age- associated regulator of gene expression in all three cell types across different tissues and species. Follow-up experiments showed that the differential expression of long and short LEF1 isoforms is a key regulatory mechanism of cellular senescence. Further examination of lung tissue from patients with Idiopathic Pulmonary Fibrosis (IPF), an age-related disease with strong ties to cellular senescence, we demonstrated a stark dysregulation of LEF1. Conclusions. Collectively, our results suggest that the LEF1 is a key factor of aging, and its differential regulation is associated with human and murine cellular senescence.