Abstract Mapping holistic cell behaviors sculpting mammalian heart has been a goal, but so far only successes in transparent invertebrates and lower vertebrates. Using a live-imaging system comprising a customized vertical light-sheet microscope equipped with a culture module, a heartbeat-gated imaging strategy, and a digital image processing framework, we realized imaging of developing mouse hearts with uninterrupted cell lineages for up to 1.5 days. Four-dimensional landscapes of cell behaviors revealed a blueprint for ventricle chamber formation in which biased outward migration of outermost cardiomyocytes coupled with cell intercalation and horizontal division. The trabeculae, an inner muscle architecture, was developed through early fate segregation and transmural cell arrangement involving both oriented cell division and directional migration. Thus, live-imaging reconstruction affords a transformative means for deciphering mammalian organogenesis.
Significance We developed reagents and approaches to pull down ribosome-associated RNAs from Cre-labeled cells. We show that this strategy is useful to probe cell type-specific gene expression and the extent of transcript binding to ribosomes.
Studies of molecular and cellular functions of small-molecule inhibitors in cancer treatment, eliciting effects by targeting genome and epigenome associated proteins, requires measurement of drug-target engagement in single-cell resolution. Here we present EpiChem for in situ single-cell joint mapping of small molecules and multimodal epigenomic landscape. We demonstrate single-cell co-assays of three small molecules together with histone modifications, chromatin accessibility or target proteins in human colorectal cancer (CRC) organoids. Integrated multimodal analysis reveals diverse drug interactions in the context of chromatin states within heterogeneous CRC organoids. We further reveal drug genomic binding dynamics and adaptive epigenome across cell types after small-molecule drug treatment in CRC organoids. This method provides a unique tool to exploit the mechanisms of cell type-specific drug actions.
The cytoplasmic coat protein complex-II (COPII) is evolutionarily conserved machinery that is essential for efficient trafficking of protein and lipid cargos. How the COPII machinery is regulated to meet the metabolic demand in response to alterations of the nutritional state remains largely unexplored, however. Here, we show that dynamic changes of COPII vesicle trafficking parallel the activation of transcription factor X-box binding protein 1 (XBP1s), a critical transcription factor in handling cellular endoplasmic reticulum (ER) stress in both live cells and mouse livers upon physiological fluctuations of nutrient availability. Using live-cell imaging approaches, we demonstrate that XBP1s is sufficient to promote COPII-dependent trafficking, mediating the nutrient stimulatory effects. Chromatin immunoprecipitation (ChIP) coupled with high-throughput DNA sequencing (ChIP-seq) and RNA-sequencing analyses reveal that nutritional signals induce dynamic XBP1s occupancy of promoters of COPII traffic-related genes, thereby driving the COPII-mediated trafficking process. Liver-specific disruption of the inositol-requiring enzyme 1α (IRE1α)-XBP1s signaling branch results in diminished COPII vesicle trafficking. Reactivation of XBP1s in mice lacking hepatic IRE1α restores COPII-mediated lipoprotein secretion and reverses the fatty liver and hypolipidemia phenotypes. Thus, our results demonstrate a previously unappreciated mechanism in the metabolic control of liver protein and lipid trafficking: The IRE1α-XBP1s axis functions as a nutrient-sensing regulatory nexus that integrates nutritional states and the COPII vesicle trafficking.
Transcriptional networks orchestrate complex developmental processes, and such networks are commonly instigated by master regulators for development. By now, considerable progress has been made in elucidating GATA factor-dependent genetic networks that control red blood cell development. Here we reported that GATA-1 and GATA-2 co-regulated the expression of two microRNA genes, microRNA-27a and microRNA-24, with critical roles in regulating erythroid differentiation. In general, GATA-2 occupied the miR-27a∼24 promoter and repressed their transcription in immature erythroid progenitor cells. As erythropoiesis proceeded, GATA-1 directly activated miR-27a∼24 transcription, and this involved a GATA-1-mediated displacement of GATA-2 from chromatin, a process termed 'GATA switch'. Furthermore, the mature miR-27a and miR-24 cooperatively inhibited GATA-2 translation and favoured the occupancy switch from GATA-2 to GATA-1, thus completing a positive feedback loop to promote erythroid maturation. In line with the essential role of GATA factors, ectopic expression of miR-27a or miR-24 promoted erythropoiesis in human primary CD34+ haematopoietic progenitor cells and mice, whereas attenuated miR-27 or miR-24 level led to impaired erythroid phenotypes in haematopoietic progenitor cells and zebrafish. Taken together, these data integrated micro RNA expression and function into GATA factor coordinated networks and provided mechanistic insight into a regulatory circuit that comprised GATA1/2 switch and miR-27a/24 in erythropoiesis.
The eukaryotic genome is folded into higher-order conformation accompanied with constrained dynamics for coordinated genome functions. However, the molecular machinery underlying these hierarchically organized three-dimensional (3D) chromatin architecture and dynamics remains poorly understood. Here by combining imaging and sequencing, we studied the role of lamin B1 in chromatin architecture and dynamics. We found that lamin B1 depletion leads to detachment of lamina-associated domains (LADs) from the nuclear periphery accompanied with global chromatin redistribution and decompaction. Consequently, the inter-chromosomal as well as inter-compartment interactions are increased, but the structure of topologically associating domains (TADs) is not affected. Using live-cell genomic loci tracking, we further proved that depletion of lamin B1 leads to increased chromatin dynamics, owing to chromatin decompaction and redistribution toward nucleoplasm. Taken together, our data suggest that lamin B1 and chromatin interactions at the nuclear periphery promote LAD maintenance, chromatin compaction, genomic compartmentalization into chromosome territories and A/B compartments and confine chromatin dynamics, supporting their crucial roles in chromatin higher-order structure and chromatin dynamics.
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