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The timing and duration of sleep results from the interaction between a homeostatic sleep–wake-driven process and a periodic circadian process, and involves changes in gene regulation and expression. Unraveling the contributions of both processes and their interaction to transcriptional and epigenomic regulatory dynamics requires sampling over time under conditions of unperturbed and perturbed sleep. We profiled mRNA expression and chromatin accessibility in the cerebral cortex of mice over a 3-d period, including a 6-h sleep deprivation (SD) on day 2. We used mathematical modeling to integrate time series of mRNA expression data with sleep–wake history, which established that a large proportion of rhythmic genes are governed by the homeostatic process with varying degrees of interaction with the circadian process, sometimes working in opposition. Remarkably, SD caused long-term effects on gene-expression dynamics, outlasting phenotypic recovery, most strikingly illustrated by a damped oscillation of most core clock genes, including Arntl/Bmal1, suggesting that enforced wakefulness directly impacts the molecular clock machinery. Chromatin accessibility proved highly plastic and dynamically affected by SD. Dynamics in distal regions, rather than promoters, correlated with mRNA expression, implying that changes in expression result from constitutively accessible promoters under the influence of enhancers or repressors. Serum response factor (SRF) was predicted as a transcriptional regulator driving immediate response, suggesting that SRF activity mirrors the build-up and release of sleep pressure. Our results demonstrate that a single, short SD has long-term aftereffects at the genomic regulatory level and highlights the importance of the sleep–wake distribution to diurnal rhythmicity and circadian processes.

The circadian clock drives extensive temporal gene expression programs controlling daily changes in behavior and physiology. In mouse liver, transcription factors dynamics, chromatin modifications, and RNA Polymerase II (PolII) activity oscillate throughout the 24-hour (24h) day, regulating the rhythmic synthesis of thousands of transcripts. Also, 24h rhythms in gene promoter-enhancer chromatin looping accompany rhythmic mRNA synthesis. However, how chromatin organization impinges on temporal transcription and liver physiology remains unclear. Here, we applied time-resolved chromosome conformation capture (4C-seq) in livers of WT and arrhythmic Bmal1 knockout mice. In WT, we observed 24h oscillations in promoter-enhancer loops at multiple loci including the core-clock genes Period1 , Period2 and Bmal1 . In addition, we detected rhythmic PolII activity, chromatin modifications and transcription involving stable chromatin loops at clock-output gene promoters representing key liver function such as glucose metabolism and detoxification. Intriguingly, these contacts persisted in clock-impaired mice in which both PolII activity and chromatin marks no longer oscillated. Finally, we observed chromatin interaction hubs connecting neighbouring genes showing coherent transcription regulation across genotypes. Thus, both clock-controlled and clock-independent chromatin topology underlie rhythmic regulation of liver physiology.

The mammalian circadian clock coordinates physiology with environmental cycles through the regulation of daily oscillations of gene expression. Thousands of transcripts exhibit rhythmic accumulations across mouse tissues, as determined by the balance of their synthesis and degradation. While diurnally rhythmic transcription regulation is well studied and often thought to be the main factor generating rhythmic mRNA accumulation, the extent of rhythmic posttranscriptional regulation is debated, and the kinetic parameters (e.g., half-lives), as well as the underlying regulators (e.g., mRNA-binding proteins) are relatively unexplored. Here, we developed a quantitative model for cyclic accumulations of pre-mRNA and mRNA from total RNA-seq data, and applied it to mouse liver. This allowed us to identify that about 20% of mRNA rhythms were driven by rhythmic mRNA degradation, and another 15% of mRNAs regulated by both rhythmic transcription and mRNA degradation. The method could also estimate mRNA half-lives and processing times in intact mouse liver. We then showed that, depending on mRNA half-life, rhythmic mRNA degradation can either amplify or tune phases of mRNA rhythms. By comparing mRNA rhythms in wild-type and Bmal1 −/− animals, we found that the rhythmic degradation of many transcripts did not depend on a functional BMAL1. Interestingly clock-dependent and -independent degradation rhythms peaked at distinct times of day. We further predicted mRNA-binding proteins (mRBPs) that were implicated in the posttranscriptional regulation of mRNAs, either through stabilizing or destabilizing activities. Together, our results demonstrate how posttranscriptional regulation temporally shapes rhythmic mRNA accumulation in mouse liver.

The circadian clock is an endogenous and self‐sustained oscillator that anticipates daily environmental cycles. While rhythmic gene expression of circadian genes is well‐described in populations of cells, the single‐cell mRNA dynamics of multiple core clock genes remain largely unknown. Here we use single‐molecule fluorescence in situ hybridisation (smFISH) at multiple time points to measure pairs of core clock transcripts, Rev‐erbα ( Nr1d1 ), Cry1 and Bmal1 , in mouse fibroblasts. The mean mRNA level oscillates over 24 h for all three genes, but mRNA numbers show considerable spread between cells. We develop a probabilistic model for multivariate mRNA counts using mixtures of negative binomials, which accounts for transcriptional bursting, circadian time and cell‐to‐cell heterogeneity, notably in cell size. Decomposing the mRNA variability into distinct noise sources shows that clock time contributes a small fraction of the total variability in mRNA number between cells. Thus, our results highlight the intrinsic biological challenges in estimating circadian phase from single‐cell mRNA counts and suggest that circadian phase in single cells is encoded post‐transcriptionally. SYNOPSIS Single‐molecule imaging of transcripts is combined with mathematical modelling to investigate how the mRNA distributions of core‐clock circadian genes evolve over the circadian cycle while considering multiple sources of intrinsic and extrinsic variability. Single molecule fluorescence in‐situ hybridization (smFISH) of core‐clock genes at multiple time points over the circadian cycle shows that the mean mRNA level oscillates but there is significant variability in mRNA counts between cells. Bayesian model selection identifies a mixture model of negative binomials as the preferred model of the mRNA counts. Noise decomposition of the preferred model shows that a small percentage of measured variation in core clock transcript number is attributable to circadian time. Graphical Abstract Single‐molecule imaging of transcripts is combined with mathematical modelling to investigate how the mRNA distributions of core‐clock circadian genes evolve over the circadian cycle while considering multiple sources of intrinsic and extrinsic variability.

High‐risk acute pulmonary embolism (PE) is associated with significant in‐hospital mortality. Large‐Bore Mechanical Thrombectomy (LBMT) is a treatment option for acute PE, but data on its efficacy in high‐risk PE was limited. This prospective case series reported the outcomes of 14 patients with high‐risk PE treated using a novel multipurpose mechanical aspiration system (MMAS). Most patients were in hemodynamic decompensation, requiring inotropic or mechanical circulatory support. The mean procedural time was 73.5 ± 39.2 min. Complete procedural success was achieved in 78.6% of the cases, while two patients required bailout therapies. The mean pulmonary arterial pressure decreased by 27.2%, and the right‐ventricle/left‐ventricle ratio normalized in 85.7% of patients. The primary endpoint—in‐hospital mortality—was 0%, while the major bleeding rate was 27.2%. These findings suggest that MMAS is a safe and effective intervention for acute high‐risk PE.

Post-translational histone modifications modulate chromatin activity to affect gene expression. How chromatin states underlie lineage choice in single cells is relatively unexplored. We develop sort-assisted single-cell chromatin immunocleavage (sortChIC) and map active (H3K4me1 and H3K4me3) and repressive (H3K27me3 and H3K9me3) histone modifications in the mouse bone marrow. During differentiation, hematopoietic stem and progenitor cells (HSPCs) acquire active chromatin states mediated by cell-type-specifying transcription factors, which are unique for each lineage. By contrast, most alterations in repressive marks during differentiation occur independent of the final cell type. Chromatin trajectory analysis shows that lineage choice at the chromatin level occurs at the progenitor stage. Joint profiling of H3K4me1 and H3K9me3 demonstrates that cell types within the myeloid lineage have distinct active chromatin but share similar myeloid-specific heterochromatin states. This implies a hierarchical regulation of chromatin during hematopoiesis: heterochromatin dynamics distinguish differentiation trajectories and lineages, while euchromatin dynamics reflect cell types within lineages. Sort-assisted single-cell chromatin immunocleavage (sortChIC) combines single-cell histone modification profiling with fluorescence-activated cell sorting (FACS), enabling the study of rare cell populations. H3K4me1/H3K4me3, H3K9me3 and H3K27me3 profiling of blood suggest a model of lineage-shared repressive and cell type-specific active chromatin.

Regulation of chromatin states involves the dynamic interplay between different histone modifications to control gene expression. Recent advances have enabled mapping of histone marks in single cells, but most methods are constrained to profile only one histone mark per cell. Here, we present an integrated experimental and computational framework, scChIX-seq (single-cell chromatin immunocleavage and unmixing sequencing), to map several histone marks in single cells. scChIX-seq multiplexes two histone marks together in single cells, then computationally deconvolves the signal using training data from respective histone mark profiles. This framework learns the cell-type-specific correlation structure between histone marks, and therefore does not require a priori assumptions of their genomic distributions. Using scChIX-seq, we demonstrate multimodal analysis of histone marks in single cells across a range of mark combinations. Modeling dynamics of in vitro macrophage differentiation enables integrated analysis of chromatin velocity. Overall, scChIX-seq unlocks systematic interrogation of the interplay between histone modifications in single cells. Analysis of two histone marks in single cells reveals interplay between modifications.

Background: Single-cell pooled CRISPR screens (Perturb-seq) are a powerful tool in functional genomics. A key preprocessing step is to determine which cells received which perturbations based on possibly noisy sequencing counts of the guide RNA library. Many existing approaches to this problem require fitting probabilistic models which may be computationally expensive on large screens with 10s of thousands of cells and guides. Results: We propose to view the guide count matrix as a contingency table and use Fisher's Exact Test to test for associations between cell and guide barcodes. This approach is fast, normalizes for both cell and guide-specific size factors, and provides a p-value for each cell-guide pair. Our method further uses a multiple testing correction approach that accounts for the correlation structure between the tests, and a correction for Simpson's paradox that arises due to hidden confounding. Additionally, to facilitate the development and benchmarking of guide assignment methods, we propose a framework for simulating guide counts with a realistic model of sequencing noise. Conclusions: We find that our method compares favorably to existing methods in both accuracy and runtime on simulated and real datasets. We provide our method in an easy to use R package, fishash, available at https://github.com/jackkamm/fishash. Additionally, the code to reproduce the results of this manuscript is available at https://github.com/jackkamm/fishash_analysis.Competing Interest StatementThe authors are employees of Genentech, Inc., a subsidiary of F. Hoffmann-La Roche AG.Footnotes* https://github.com/jackkamm/fishash* https://github.com/jackkamm/fishash_analysis* https://www.ncbi.nlm.nih.gov/geo/query/acc.cgi?acc=GSE272457

Vertebrates display remarkable diversity of sensorimotor behaviors, each adapted to distinct ecological and survival demands. This diversity raises fundamental questions about the evolutionary origin of motor control: do conserved spinal circuits underlie these behaviors, and how have they diverged across species. Recent studies detail spinal cell-type architecture in mammals but comparable, high-resolution atlases of the non-mammalian spinal cord are lacking. Here, we compare spinal cord cell types between fish, frogs, mice and humans, spanning ~450 million years of evolution. Across species, we define highly conserved programs of cell type specification that segregate spinal neurons into nearly identical cardinal classes during development. This contrasts with adult stages, when spinal cell-type composition selectively diverges for excitatory neuron subpopulations. Using spatial transcriptomics, we localize this species divergence to the superficial, dorsal spinal cord, where variant neuropeptide expression defines mammalian-specific cell types. The most dorsal spinal cord thus emerges as a recently evolved hub for sensory integration in mammals, a neospinal cord analogous to the neocortex.

Anesthesia recovery is critical for resuming normal physiological and neuronal functions; however, the mechanisms involved remain elusive. Here, we identify a female-selective corticosterone-mediated microglia-neuron interaction in vivo during ketamine anesthesia recovery, absent in males. This microglia-neuron interaction induces plastic and functional neuronal changes, as evidenced by increased spine density and mEPSC frequency, which is occluded upon microglia depletion. We show that this process is driven through upregulation of the stress-responsive co-chaperone Fkbp5 mRNA and its protein, FKBP51, in female microglia. Fkbp5/FKBP51 is a key intermediary in a corticosteroid-induced stress response, and its involvement points towards a critical interface between endocrine signaling and microglia. Thus, to counteract the observed KXA-mediated corticosterone increase in the blood, we remove the primary source of corticosterone through adrenalectomy. Close microglia-neuron interaction was absent, but was reinstated after corticosterone injection. Our findings offer a new mechanism of microglia-mediated neuronal plasticity during anesthesia recovery, which is mediated through corticosterone, enhancing our understanding of sex differences in brain function.