Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms

Cells and tissues have the ability to respond to different stresses and maintain their homeostasis. The b-Zip transcription factor Xrp1 was found to play a pivotal role in this context in Drosophila. Xrp1 is required for the response to irradiation, in genetic conditions of ribosomal protein (Rp) deficiency leading to cell competition, and more generally in mediating the Integrated Stress Response (ISR). However, how these diverse stresses converge to and activate Xrp1 is still unclear. Our results demonstrate the existence of three separate mechanisms of Xrp1 induction. In response to Rp stress, a short isoform of Xrp1 is produced and requires the function of the ribosomal protein RpS12. In response to irradiation, a specific mRNA isoform is transcriptionally induced, which encodes the Xrp1-short protein isoform, in a p53-dependent manner. In response to the ISR, the long isoform of Xrp1 is translated by the derepression of a uORF present on the Xrp1 5'UTR, a mechanism that requires eIF2 phosphorylation, RpS12, and a transcriptional induction by ATF4. Therefore, our work provides a comprehensive understanding of the role of Xrp1 in different situations of cellular stress, illustrating the complex level of integration of the mechanisms controlling this central gene.

Minor introns are rare but evolutionarily conserved introns in the human genome. Defects in minor spliceosome components have been reported to cause several human diseases. Mutations in ZRSR2 are frequently observed in myelodysplastic syndrome (MDS) patients. However, the precise molecular mechanism by which these mutations cause a loss of ZRSR2 function in minor intron splicing remain elusive. In this study, we characterized the impact of MDS-causing missense mutations in ZRSR2. Most of the mutant ZRSR2 proteins we tested showed different subnuclear localization pattern from that of wild-type ZRSR2 protein. Co-immunoprecipitation (co-IP) analysis revealed that UHM missense mutations disrupted the interaction between ZRSR2 and SF3B complex. We also demonstrated that ZRSR2 preferentially immunoprecipitated minor spliceosome components over major spliceosome components and it directly interacts with SF3B1. Furthermore, co-IP analysis utilizing a variety of ZRSR2 mutants revealed that mutations in the UHM and Zn2 domains significantly attenuated the interaction with SF3B complex. Finally, UHM and Zn2 mutations markedly disrupted the supporting activity of ZRSR2 for minor intron splicing. Our results indicate that disruption of the physical interaction with SF3B complex is one of the hallmarks of MDS-causing mutations of ZRSR2, which results in aberrant splicing of minor introns.

The CRISPR/Cas12a system has emerged as a highly useful tool for genomic editing due to its ability to process multiple guide RNAs (gRNAs) from a single promoter. In this study, we explored the potential of combinatorial CRISPR/Cas12a to excise targeted loci in the mammalian genome by inducing double-strand breaks (DSBs) upstream and downstream of targeted exons. We designed a library of gRNA pairs targeting introns flanking 54 exons of 18 essential genes and 17 exons of 10 nonessential genes, aiming to assess the efficiency of Cas12 in excising targeted loci. While single gRNAs directly targeting essential exons produced expected fitness defects in cells, pairs of guides targeting flanking intronic regions resulted in only trivial viability effects, suggesting that excision rarely occurred. Further analysis showed no meaningful correlation between gRNA efficiency, orientation, GC content, or cut site geometry and the success of locus excision. These findings indicate that, under the conditions tested, the Cas12a system does not yield locus excision at the efficiency required for sensitive negative selection assays.

Chlorella sorokiniana is an industrially important microalga, but its genetic manipulation remains limited by poor transformation efficiency. Although several barriers have been proposed, the role of intracellular nuclease activity has received little attention. Here, we compared the degradation of exogenous double-stranded DNA in whole-cell lysates from transformation-recalcitrant (Chlorella sorokiniana and Tetradesmus obliquus) and transformation-proficient (Phaeodactylum tricornutum, Microchloropsis gaditana, and Nannochloropsis oceanica) microalgae. C. sorokiniana rapidly degraded both linear and plasmid DNA, while T. obliquus exhibited intermediate activity. In contrast, no detectable degradation was observed in the transformation-proficient species. DNA degradation in C. sorokiniana occurred within minutes, and nuclease activity in both recalcitrant species was inhibited by EDTA and Zn2+, consistent with a metal ion-dependent endonuclease. These findings suggest that intracellular nuclease activity may represent an underexplored barrier to exogenous DNA stability during transformation and provide a potential target for improving genetic transformation of recalcitrant microalgae.

Many RNA-binding proteins play pleiotropic roles in the cell, often through disparate domains that can be decoupled. Here we undertook a mutational analysis of yeast RNA-binding protein Ssd1 to better understand domains and processes required for tolerating extra chromosomes, a special class of aneuploidy. Ssd1 is important for cellular fitness when cells carry extra chromosomes, for reasons that are not clear. Ssd1 has multiple distinct domains, many of which are poorly characterized, including multiple RNA binding folds, nuclear import and export signals, an intrinsically disordered domain, an RNA polymerase II-interacting region, and many phosphorylation sites. We measured multiple phenotypes, including the ability to tolerate an extra copy of yeast chromosome XII as a representative aneuploid. Mutating RNA binding domains disrupts fitness in aneuploid, but not euploid, cells and ablates binding of a representative Ssd1 target. However, mutations affecting phase separation or interaction with the kinase Cbk1, which regulates Ssd1 granule formation during the cell cycle, did not affect aneuploidy tolerance. Evaluating correlations across these and other growth phenotypes informed on functional relationships among domains. We discuss implications for Ssd1 function and its role in aneuploidy tolerance in yeast.

Human Ca2+/calmodulin (CaM)-dependent protein kinase II alpha (CaMKII) is a highly abundant and conserved protein in the brain. It is a multifunctional serine/threonine kinase that phosphorylates cellular substrates across subcellular compartments, activating signaling pathways essential for long-term memory storage. However, a detailed understanding of protein structure and enzyme-substrate interaction mechanisms require a method to produce active, stable, and pure proteins at scale. Since phosphorylation can alter catalytic activity and substrate affinity, examining the non-phosphorylated state enables an unbiased characterization of basal enzymatic function. Therefore, producing the non-phosphorylated form of the kinase is important for biochemical and structural studies aimed at understanding kinase activation mechanisms. We describe a detailed protocol for the expression and high-yield purification of full-length, non-phosphorylated human CaMKII in bacterial cells. CaMKII was co-expressed with {lambda}-phosphatase to maintain it in a non-phosphorylated state. Protein expression was induced by autoinduction at low temperature to enhance the stability and solubility of CaMKII. Recombinant CaMKII was purified by Ni-NTA affinity chromatography, followed by gel filtration chromatography. Although CaMKII has been considered difficult to purify in its non-phosphorylated form, we obtained it in high yield, and the protein was predominantly non-phosphorylated. Our results show that combining co-expression with autoinduction improves both the solubility and yield of the homogeneous non-phosphorylated form of CaMKII, which could be suitable for subsequent biochemical and structural studies.

Cajal bodies (CBs) are nuclear condensates known for their role in sn- and snoRNA maturation in animal, but their function in plant stress responses remains poorly understood. We compared wild-type (WT) Arabidopsis thaliana with the CB-lacking ncb-1 mutant during hypoxia and reoxygenation, focusing on nuclear retention of transcripts. In both genotypes, hypoxia caused nuclear accumulation of poly(A+) RNA and extensive reprogramming of gene expression. However, the transcriptional response, including induction of hypoxia-responsive genes, was considerably weaker in ncb-1, both during stress and recovery. In nuclear RNA, introns with low (intron retention index, IRX < 10%) and high (IRX > 50%) retention showed opposite, genotype-dependent dynamics. During hypoxia, efficiently spliced introns (IRX < 10%) accounted for over 70% of introns detected in nuclear RNA. Relative to normoxia, this proportion increased two- to threefold in WT but only moderately in the mutant. Together with poly(A+) RNA accumulation, this suggests that largely spliced transcripts, including those of genes involved in protein transport and ribosome biogenesis, are held in the nucleus during stress. Conversely, strongly retained introns, often terminal and located in genes related to hypoxia and other abiotic stresses, were most abundant in WT nuclear RNA under normoxia. Their proportion dropped sharply during hypoxia, and much less so in ncb-1. This indicate that WT plants maintain a nuclear reserve of incompletely spliced stress-related transcripts that are processed upon stress. The mutant also showed reduced interaction between U2 snRNA and U2B'', which leads to splicing defects. Our results indicate that CBs regulate which transcripts are held in the nucleus and how they are processed, contributing to plant tolerance of oxygen deprivation and recovery.

Whether the homologous parallel triplex (HP-triplex, formed by dsDNA and a homologous ssDNA) acts as an intermediate prior to DNA recombination remains controversy, owing to the intrinsic instability and lake of experimental data. Here, we report a HP-triplex that is stable at least at 35 oC under protein-free conditions. Two complementary circular ssDNAs were first hybridized at a 1:1 ratio to form an LR-chimera containing both Z-DNA and B-DNA. Then the circular homologous ssDNA (T-rich) was found to easily hybridize with this LR-chimera to form the HP-triplex (strand ratio of 2:1), involving both left- and right-handed triplex parts under the topological constraint. The third strand locates in the major groove and forms hydrogen bonds with both bases in each base pair of dsDNA without obvious strand exchange. Our data indicate that the third-strand ssDNA may first bind to the B-DNA region, followed by association with the Z-DNA region to assemble the full triplex structure. Structural simulations and analyses reveal that topological constraint imposed by the circular architecture raises the energy barrier for strand displacement, thereby stabilizing this triplex. In summary, the dynamic HP triplex can be stabilized via a Z B junction structure that mimics the in vivo one formed by negative supercoiling. These findings demonstrate that HP-triplex may act as an intermediate for DNA recombination, anti-gene strategy, and other interactions between dsDNA and homologous ssDNA or RNA.

Translation initiation is a critical stage of protein synthesis in prokaryotes, where ribosomes assemble on mRNA to begin decoding genetic information. The interactions between ribosomal RNA (rRNA) and mRNA significantly influence this process, with features like ribosomal binding site (RBS) affecting ribosomal dynamics. However, direct experimental validation of these types of interaction is yet to be provided. In this study, we developed an optimized small sub-unit sequencing (SSU-seq) protocol for Escherichia coli, enabling the detailed examination of ribosomal complexes and subunits dynamics. Unlike Ribo-seq, which primarily maps fully assembled ribosomes, SSU-seq captures both ribosomal subunits and intact ribosomes, offering a comprehensive view of translation initiation and elongation events. By integrating experimental data with predictive models, our study advances the understanding of translation regulation in prokaryotes and underscores the utility of SSU-seq in decoding the complexities of ribosome-mRNA interactions. Our analysis reveals distinct patterns of small subunit distribution across the entire mRNAs with higher density near positions with stronger rRNA-mRNA hybridization. The results may suggest that rRNA-mRNA hybridization positions along the entire transcript facilitate efficient start codon recognition by maintaining proximity between the small subunit and the mRNA.

In eukaryotic cells, the positive transcription elongation factor b (P-TEFb) plays a critical role in the transition of RNAPII from the paused state to actively transcribing mode. Although three different cyclin (Cyc) T (CycT1, T2a, and T2b) interact with CDK9 to form functional P-TEFb, a majority of P-TEFb complexes are comprised of the CycT1 and CDK9. To analyze the role of the CycT1 subunit of P-TEFb on cellular transcription, we established HEK293T cells lacking CycT1 proteins (CycT1-KO cells). While no apparent growth defects were observed with CYcT1-KO cells, the level of CDK9 was decreased and no compensatory over-expression of CycT2 was observed, suggesting that the reduced level of functional P-TEFb complexes is sufficient for the normal cell growth. Reporter gene assays indicate that NF-kappaB-dependent transcription induced by PMA was impaired in CycT1-KO cells although the function of NF-kappaB (P65) per se was not affected. On the other hand, AP1-dependent transactivation induced by PMA was unaffected in CycT1-KO cells. Moreover, transcription stimulated by JQ1, a strong P-TEFb inducer, was reduced in CycT1-KO cells. Data of biochemical analysis indicate that P-TEFb with CycT1 (P-TEFb (CDK9:CycT1)) was efficiently released from 7SKsnRNP by JQ1 while P-TEFb (CDK9:CycT2) was not affected by JQ1. Transcriptome analysis indicated that the lack of CycT1 had a minor effect on the steady-state transcription. However, expression of JQ1-dependent genes was severely reduced in CycT1-KO cells. Interestingly, referring to gene expression profiles of 33 different types of cancer revealed that 11 out of 13 top JQ1-dependent genes were also highly upregulated in Pancreatic Adenocarcinoma. From these results, we conclude that although many genes can be regulated by both P-TEFb (CDK9:CycT1) and P-TEFb (CDK9:CycT2), CycT1 plays a critical role in regulating highly inducible genes, which are also aberrantly regulated in a particular type of cancer.

Objectives: The bile acid nuclear receptor Farnesoid X Receptor (FXR, NR1H4) is a major regulator of metabolism and energy homeostasis in peripheral organs. It modulates bile acid, glucose, and lipid metabolism, as well as fat mass and body weight. However, FXR is also expressed in the brain, particularly in the hypothalamus, a key center for the regulation of energy homeostasis. Although one study has demonstrated a role for brain FXR activation in energy balance, its specific hypothalamic role is still unknown. Here, we examined the role of FXR in the mediobasal hypothalamus in the regulation of energy balance. Methods: We used a genetic approach combined with metabolic phenotyping to determine the effect of FXR invalidation in the mediobasal hypothalamus on metabolic parameters involved in the central regulation of energy homeostasis. Results: Our results demonstrate that hypothalamic FXR deficiency induces a positive energy balance, resulting in a reduction in energy expenditure due to alterations in glucose metabolism accompanied by structural changes in white adipose tissues. Conclusion: This study uncovers a previously unrecognized role for hypothalamic FXR in the central homeostatic control of energy balance, providing new insights into its contribution to peripheral glucose metabolism and adipose tissue structural remodeling.

Chromosome was "self-assembled" into a complex three-dimensional topology in the nucleus, which provided the structural basis for important biological processes such as gene expression regulation and cell differentiation. The current understanding of the three-dimensional organization of the genome is based on high-throughput sequencing and imaging technologies to visualize the conformation of chromatin in its native environment. This study proposes a new research perspective and technical means to use fusion genes to determine the interaction information and three-dimensional structure of chromosomes. This three-dimensional information provided by the fusion gene spectrum not only shows the universality of the spatial layout of chromosomes, but also increases the interpretability of the occurrence of fusion genes. The chromatin conformation capture data of multiple tissues and periods is significantly correlated with the strength of fusion genes. For example, the frequency of trans-interaction between chromosomes 11 and 17 was the highest, and the frequency of fusion gene occurrence corresponding to KMT2A on chromosome 11 was the highest. Therefore, we infer that this structural universality is a common feature of the human genome, which provides a theoretical basis and data for predicting chromosome fusion events.

Shortening of the poly(A) tail initiates mRNA decay, and the human CCR4-NOT is the principal cytoplasmic deadenylase. Although its subunits have been defined, their functions have not been comprehensively evaluated in cells. We depleted each CCR4-NOT subunit and measured transcriptome-wide effects. CCR4-NOT regulated transcripts from thousands of genes, many of which associated with the complex in cells. Distinct groups of transcripts responded according to the modular organization of the complex, while subsets exhibited unique responses to specific subunits. Poly(A) tail analysis revealed that the CNOT7 and CNOT8 deadenylases are the predominant regulators of poly(A) tail length in these cells. CCR4-NOT targets were enriched in regulatory pathways including cell proliferation, apoptosis, stress responses, and cancer-related processes. Bioinformatic analysis identified sequence determinants and candidate recruitment factors associated with CCR4-NOT regulation. The results define the breadth and specificity of CCR4-NOT-mediated mRNA decay and provide a resource for dissecting regulatory mechanisms and biological roles.

Dysfunctions in transcription cause severe human diseases. In eukaryotes, transcription initiation by RNA polymerase (Pol) II requires the formation of the pre-initiation complex (PIC) composed of general transcription factors (GTFs), Pol II, and an essential multiprotein coactivator, Mediator. In vitro experiments with GTFs and Pol II have depicted a linear sequence of PIC assembly. However, PIC assembly paths in vivo at the genomic scale and Mediator's role in these paths remain poorly understood. One strategy characterizes ChIP-seq occupancy alterations in Mediator mutants, but analysis of these data still lacks a formalized interpretation framework. In this study, we address this need by developing a quantitative framework that scores candidate assembly paths according to their ability to explain these alterations. This framework combines a model of PIC assembly based on a system of differential equations with parameter optimization based on a penalized criterion. We applied this new framework to datasets from two yeast Mediator mutants, in the Med17 and Med10 subunits. Our results show that the best-supported PIC assembly paths differ from those inferred in vitro. In particular, we show that it is important to distinguish the core TFIIH module from the TFIIK kinase module, and that similar paths starting with Mediator or TBP receive different levels of support. Furthermore, although multiple paths fit the data equally well, they differ markedly for specific gene groups. Taken together, the results show that the path can depend on the gene, and are also compatible with more than one path being operational in vivo for a single gene. This work improves our understanding of PIC assembly paths and transcription initiation, and opens perspectives for future modeling and experimental studies.

Ultraviolet-C (UV-C) radiation is a potent environmental stressor that induces DNA damage, accumulation of reactive oxygen species (ROS), and cellular dysfunction. This also initiates coordinated molecular responses to regulate cellular adaptation and recovery. Small extracellular vesicles (sEVs) have emerged as key mediators of intercellular communication through the selective incorporation of microRNAs (miRNAs), proteins, and lipids. However, the temporal changes of sEV-associated cargo during the progression from UV-C-induced damage to recovery remain uncharacterized. We hypothesised that UV-C exposure induces stage-specific changes in sEV cargo (miRNA and proteins) that promote the transition of cells from damage to the response and recovery phases. To examine this, a time-resolved UV-C exposure model was established using Caenorhabditis elegans (C. elegans). In this paper, we studied 2.5 hours of UV-C exposure, including cellular damage, intracellular ROS levels, and DNA integrity, to define the damage and repair state. Based on these temporal variations, samples were collected: i) immediately after exposure, ii) 6 hours post-exposure, and iii) 16 hours post-exposure were designated as damage, response, and recovery conditions, respectively. sEVs isolated from whole-worm preparations were characterised using biophysical and molecular approaches, followed by miRNA sequencing and proteomic analyses. Integrative analysis further identified miRNA-protein associations and associated them with pathways involved in stress adaptation, metabolism, signal transduction, and cellular homeostasis. Collectively, these findings provide a temporal molecular framework of sEV cargo associated with UV-C-induced damage, response, and recovery in C. elegans. Overall, this study identified candidate molecular signatures and miRNA-protein associations that underlie the temporally selective packaging of sEV-associated cargo following UV-C-induced damage, providing a basis for future tissue-specific and mechanistic investigations.

Autism Spectrum Disorder (ASD) is a neurodevelopmental disorder characterized by repetitive behavior and impaired social interactions. Recent reports from human postmortem brain samples of ASD show transcriptome-wide dysregulations, including changes in differential gene expression and alternative splicing. An important mechanism regulating transcriptome function is the editing of double-stranded RNA by adenosine deaminases acting on RNA (ADARs), which play regulatory roles in neurodevelopment and innate immunity. We explore the ADAR editing changes associated with the use of Selective Serotonin Reuptake Inhibitors (SSRIs) in Arid1b haploinsufficient mouse models. Our results show dynamic changes in site-specific editing rates in autistic mice. We further demonstrate the transcriptome-wide effects of differential ADAR editing in terms of changes in miRNA-mRNA interactions. Moreover, our findings highlight a site-specific change in editing rates for evolutionarily conserved sites in mammals and ASD associated risk genes, such as Gria2. Our analysis of significant biological processes shows the association of differentially edited genes with processes vital for neurotransmission. Overall, our findings imply that the use of SSRIs can induce distinct ADAR editing signatures, potentially contributing to ASD pathology.

In the germline, small RNA-mediated gene regulatory pathways are associated with phase separated condensates known as germ granules across species. Germ granules can be challenging to purify based on their biophysical properties, therefore we used an in vivo proximity labeling approach, TurboID, to define the proximal interaction networks of the germ granule-localized Argonaute proteins: CSR-1, PRG-1/Piwi, PPW-2/WAGO-3, WAGO-1 and WAGO-4, and the nuclear-enriched Argonaute HRDE-1 as a comparator, in C. elegans. We found that although CSR-1 and WAGO-4 bind to the same set of 22G- small RNAs and target an overlapping set of germline genes, they possess distinct proximal interaction networks. Displacement of WAGO-4 from the germ granules highlights distinct interactors involved in chromosome biology and points to a potential nuclear role for WAGO-4. PRG-1/Piwi, WAGO-1, and PPW-2/WAGO-3, act in a common pathway and share an interaction network, while HRDE-1 displays a distinct interaction network that highlights its potential to influence splicing, rather than its widely accepted role in histone modification. Performing TurboID on other sRNA pathway factors, including the RdRP RRF-1 led us to uncover a potential nuclear role for this protein, and overlapping our TurboID data with published proximity labeling data enabled us to identify a core set of eleven mostly uncharacterized proteins that play roles in fertility, germ granule organization, and transgenerational epigenetic inheritance. One of these factors, D2005.4, appears to be a heme-binding germ granule-localized protein with a Tetratricopeptide Repeat Domain that interacts with all germ granule AGOs and may play scaffolding or enzymatic roles in germ granules. Collectively, our work highlights the utility of proximity labelling to uncover insights into AGO and sRNA pathway factor function and identify new factors that could be of importance in germ cell biology.

JAK-STAT signalling pathway activation relies mostly on the transcriptional regulation of its ligands. The pathway, conserved in vertebrates and invertebrates, is required for the development and homeostasis of various organs. In contrast to the complexity of the vertebrate pathway, with a multiplicity of ligands, receptors, kinases and transcription factors, the simplicity of the Drosophila pathway with only three ligands (Upd1, Upd2 and Upd3), a single receptor (Dome), a JAK kinase (Hop) and a STAT transcription factor (STAT92E) facilitates the genetic study of its multiple functions. To discover how, when and where the Upd1 ligand performs each organogenetic function, we have searched for the different enhancers controlling its dynamic spatio-temporal regulation and used them to generate minigenes replicating specific stages of Upd1 expression. The combination of these minigenes with mutations deleting all three Upd ligands, allowed us to identify the timing when the various embryonic functions are mediated, including head involution, tracheae, corpora allata and prothoracic gland specification, posterior spiracle formation and hindgut elongation. We uncover most of the enhancers controlling STAT activation during embryonic organogenesis and those driving activation in the adult testis and the ovary. As expected, we identified redundant shadow enhancers but, interestingly, we found a different type of redundant enhancers that mediate identical developmental functions although driving completely different expression patterns. We name this new class of functionally redundant enhancers driving different expression as shade enhancers. We propose shade enhancers provide evolutionary plasticity as they could allow the pattern of gene expression to diversify without altering the underlying body plan.

MicroRNAs have been likened to the "dark matter" of eukaryotic genomes, reflecting their pervasive regulatory influence. MicroRNAs were first identified through genetic studies of developmental timing in the nematode Caenorhabditis elegans. Let-7 was the first microRNA recognized to be broadly conserved. The principal target of Let-7 in the developmental timing pathway is the TRIM-NHL RNA-binding protein LIN-41. During the L4 larval stage, Let-7 represses lin-41 translation by binding to two Let-7 complementary sites in the lin-41 3'UTR. Despite the importance of microRNA-based translational regulation, the underlying molecular mechanisms are incompletely understood. Through genetic analysis, we discovered an unrecognized feature of the mechanism by which Let-7 controls lin-41 translation. This mechanism requires a 5'-regulatory exon containing a seven-amino acid upstream open reading frame (uORF) and conserved sequence elements. Genome editing indicates that the specific uORF amino acid sequence itself is not important. Our data suggest that uORF translation and 5'UTR structure limit initiation at the downstream lin-41 start codon, enabling tight control by Let-7. Without this mechanism, the Let-7 microRNA is unable to properly regulate lin-41 to enable proper development.

Obesity is a major health concern worldwide and is caused by an excessive fat accumulation in the body due to adipogenesis. Recent studies suggest that excessive stress or disruption of circadian rhythms can enhance adipogenic commitment and lead to obesity. Moreover, this adipogenic commitment occurs at a specific time of day, highlighting a correlation with the circadian rhythm. However, the dynamical nature of this kind of stress-induced adipogenic differentiation remains an unresolved puzzle. Herein, we proposed a mathematical model centred on the key transcription factor PPAR{gamma}, associated with adipogenic differentiation, and demonstrated that adipogenic commitment is a bistable, irreversible differentiation process in which slow positive feedback regulation of PPAR{gamma} primarily controls the differentiation commitment threshold. The model reproduced the experimental observations regarding the extent of adipogenesis induced by constant and pulsatile stress input signals and explained the correlation between adipogenic differentiation time and circadian phase. It further predicts how to control the adipogenic commitment even under stressful conditions. Overall, our study elucidates how stress signalling dynamically governs adipogenesis.