Biology subjects

Huang, G.

Publications and source records attributed to Huang, G..

At least 109 records · Page 6Linked to original sources

bioRxiv2020

Single-Cell Transcriptomics Identifies Dysregulated Metabolic Programs of Aging Alveolar Progenitor Cells in Lung Fibrosis

Aging is a critical risk factor in progressive lung fibrotic diseases such as idiopathic pulmonary fibrosis (IPF). Loss of integrity of type 2 alveolar epithelial cells (AEC2s) is the main causal event in the pathogenesis of IPF. To systematically examine the genomic program changes of AEC2s with aging and lung injury, we performed unbiased single cell RNA-seq analyses of lung epithelial cells from either uninjured or bleomycin-injured young and old mice. Major lung epithelial cell types were readily identified with canonical cell markers in our dataset. Heterogenecity of AEC2s was apparent, and AEC2s were then classified into three subsets according to their gene signatures. Genes related to lipid metabolism and glycolysis were significantly altered within these three clusters of AEC2s, and also affected by aging and lung injury. Importantly, IPF AEC2s showed similar genomic programming and metabolic changes as that of AEC2s from bleomycin injured old mouse lungs relative to controls. Furthermore, perturbation of both lipid metabolism and glycolysis significantly changed progenitor renewal capacity in 3-Demensional organoid culture of AEC2s. Taken togather, this work identified metabolic defects of AEC2s in aging and during lung injury. Strategies to rectify these altered programs would promote AEC2 renewal which in turn improves lung repair. One sentence summaryMetabolic defects of alveolar progenitors in aging and during lung injury impair their renewal.

cell biology↗

bioRxiv2020

Zinc Metabolic Defect of Aging Alveolar Progenitors in Progressive Pulmonary Fibrosis

Idiopathic pulmonary fibrosis (IPF) is a fatal form of interstitial lung disease and aging has been identified as a risk factor to the disease. Alveolar type II cells (AEC2s) function as progenitor cells in the lung. Growing evidences indicate that IPF results from repeating AEC2 injury and inadequate epithelial repair. We previously reported that there was a significant loss of alveolar progenitors in the lungs of patients with IPF. In our current study, we performed single cell RNA-seq of epithelial cells from lungs of patients with IPF and healthy donors as well as epithelial cells from old and young mouse lungs with bleomycin injury. We identified a defect of zinc metabolism of AEC2s from IPF lungs and bleomycin-injured old mouse lungs. We further discovered that a specific zinc transporter ZIP8 was down regulated in IPF AEC2s and AEC2s from aged mice. Loss of ZIP8 expression is associated with impaired AEC2 renewal through sirtuin signaling in aging and IPF. Targeted deletion of Zip8 in murine AEC2 compartment led to reduced AEC2 renewal capacity, impaired AEC2 recovery, and worsened lung fibrosis after bleomycin injury. In summary, we have identified novel metabolic defects of AEC2s during aging and in IPF which contribute to the pathogenesis of lung fibrosis. Therapeutic strategies to restore critical components of these metabolic programs could improve AEC2 progenitor activity and mitigate ongoing fibrogenesis. In BriefLiang et al. performed single cell RNA-seq (scRNA-seq) of epithelial cells in IPF and in mice and discovered a zinc metabolic defect of alveolar progenitor cells (AEC2) in IPF and injured old mice characterized by down-regulation of a specific zinc transporter ZIP8. Manipulation of ZIP8 and zinc in 3D organoid culture of AEC2 in vitro and targeted deletion of Zip8 in AEC2s in vivo demonsrated a role of ZIP8 in promoting AEC2 progenitor function. HighlightsO_LIScRNA-seq revealed dysregulation of zinc metabolism in AEC2s and decreased stem cell signaling in IPF C_LIO_LIReduced SLC39A8 and related gene expression of IPF AEC2s and aged mouse AEC2s C_LIO_LIZIP8-dependent zinc metabolism is required for AEC2 renewal C_LIO_LITargeted deletion of Slc39a8 impaired AEC2 renewal and promoted lung fibrosis C_LI

cell biology↗

bioRxiv2020

Definition and Signatures of Lung Fibroblast Populations in Development and Fibrosis in Mice and Men

The heterogeneity of fibroblasts in the murine and human lung during homeostasis and disease is increasingly recognized. It remains unclear if the different phenotypes identified to date are characteristic of unique subpopulations with unique progenitors or whether they arise by a process of differentiation from common precursors. Our understanding of this ubiquitous cell type is limited by an absence of well validated, specific, markers with which to identify each cell type and a clear consensus on the distinct populations present in the lung. Here we describe single cell RNA sequencing (scRNA-seq) analysis on mesenchymal cells from the murine lung throughout embryonic (E) development (E9.5 - 17.5), at post-natal day (P1 - 15), as well as in the adult and the aged murine lungs before and after bleomycin-induced fibrosis. We carried out complementary scRNA-seq on human lung tissue from a P1 lung, a month 21 lung and lung tissue from healthy donors and patients with idiopathic pulmonary fibrosis (IPF). The murine and human data were supplemented with publicly available scRNA-seq datasets. We consistently identified lipofibroblasts, myofibroblasts, pericytes, mesothelial cells and smooth muscle cells. In addition, we identified a novel population delineated by Ebf1 (early B-cell factor 1) expression and an intermediate subtype. Comparative analysis with human mesenchymal cells revealed homologous mesenchymal subpopulations with remarkably conserved transcriptomic signatures. Comparative analysis of changes in gene expression in the fibroblast subpopulations from age matched non-fibrotic and fibrotic lungs in the mouse and human demonstrates that many of these subsets contribute to matrix gene expression in fibrotic conditions. Subtype selective transcription factors were identified and putative divergence of the clusters during development were delineated. Prospective isolation of these fibroblast subpopulations, localization of signature gene markers, and lineage-tracing each cluster are under way in the laboratory. This analysis will enhance our understanding of fibroblast heterogeneity in homeostasis and fibrotic disease conditions.

cell biology↗

bioRxiv2020

An ABA Synthesis Enzyme Allele OsNCED2 Promotes the Aerobic Adaption in Upland Rice

There are two ecotypes, upland rice and irrigate rice, during its evolution in rice. Upland rice exhibits aerobic adaptive phenotype via its stronger root system and more rapid drought responses compared with its counterpart, irrigated rice. We assessed the functional variation and applications of the aerobic adaptive allele OsNCED2T cloned from IRAT104 which is a cultivar of upland rice. OsNCED2-overexpressing transgenic rice and OsNCED2T-NILs exhibited significantly higher ABA contents at the seedling and reproductive stages, which can improve root development (RD) and drought tolerance (DT) to promote aerobic adaptation in upland rice. RNA-Seq-based expression profiling of transgenic versus wild-type rice identified OsNCED2-mediated pathways that regulate RD and DT. Meanwhile, OsNCED2-overexpressing rice exhibited significantly increased reactive oxygen species (ROS)-scavenging abilities and transcription levels of many stress- and development-related genes, which regulate RD and DT. A SNP mutation (C to T) from irrigated rice to upland rice, caused the functional variation of OsNCED2, and the enhanced RD and DT mediated by this site under aerobic conditions could promote higher yield of upland rice. These results show that OsNCED2T, through ABA synthesis, positively modulates RD and DT which confers aerobic adaptation in upland rice and might serve as a novel gene for breeding aerobic adaptive or water-saving rice.

plant biology↗

bioRxiv2020

A pan-cancer analysis of CpG Island gene regulation reveals extensive plasticity within Polycomb targets

CpG Island promoter genes make up more than half of human genes, and a subset regulated by Polycomb-Repressive Complex 2 (PRC2+-CGI) become DNA methylated and silenced in cancer. Here, we perform a systematic regulatory analysis of CGI genes across TCGA cancer types, finding that PRC2+-CGI genes are frequently prone to transcriptional upregulation as well. These upregulated PRC2+-CGI genes control important pathways such as Epithelial-Mesenchymal Transition and TNF-associated inflammatory response, and have the greatest cancer-type specificity among any class of CGI genes. Using publicly available chromatin datasets and genetic perturbations, we show that transcription factor binding sites (TFBSs) within distal enhancers underlie transcriptional activation of PRC2+-CGI genes, in contrast to PRC2-free CGI genes which are predominantly regulated by promoter TFBSs. Surprisingly, a large subset of PRC2+-CGI genes that are upregulated in one cancer type are also hypermethylated/silenced in at least one other cancer type, highlighting the high degree of regulatory plasticity likely derived from their complex regulatory patterns during normal development.

cancer biology↗

bioRxiv2020

A Connectome and Analysis of the Adult Drosophila Central Brain

The neural circuits responsible for animal behavior remain largely unknown. We summarize new methods and present the circuitry of a large fraction of the brain of the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses in, and proofread such large data sets. We define cell types, refine computational compartments, and provide an exhaustive atlas of cell examples and types, many of them novel. We provide detailed circuits consisting of neurons and their chemical synapses for most of the central brain. We make the data public and simplify access, reducing the effort needed to answer circuit questions, and provide procedures linking the neurons defined by our analysis with genetic reagents. Biologically, we examine distributions of connection strengths, neural motifs on different scales, electrical consequences of compartmentalization, and evidence that maximizing packing density is an important criterion in the evolution of the flys brain.

bioinformatics↗

bioRxiv2020

VISTA: Virtual ImmunoSTAining for pancreatic disease quantification in murine cohorts

Mechanistic studies of pancreatic disease progression using animal models require objective and quantifiable assessment of tissue changes among animal cohorts. Disease state quantification, however, relies heavily on tissue immunostaining, which can be expensive, labor- and time-intensive, and all too often produces uneven staining that is prone to variable interpretation between experts and inaccurate quantification. Here we develop a fully automated semantic segmentation tool using deep learning for the rapid and objective quantification of histologic features using hematoxylin and eosin (H&E) stained pancreatic tissue sections acquired from murine pancreatic cancer models. The tool was successfully trained to segment and quantify multiple histopathologic features of pancreatic pre-cancer evolution, including normal acinar structures, the ductal phenotype of acinar-to ductal metaplasia (ADM), dysplasia, and the expanding stromal compartment. Disease quantifications produced by our computational tool were highly correlated to the results obtained by immunostaining markers of normal and diseased tissue (DAPI, amylase, and cytokeratins; correlation score= 0.9, 0.95, and 0.91, respectively) and were able to accurately reproduce immunostain patterns. Moreover, our tool was able to distinguish ADM from dysplasia, which are not reliably distinguished by immunostaining, and avoid the pitfalls of uneven or poor-quality staining. Using this tool, we quantified the changes in histologic feature abundance for murine cohorts with oncogenic Kras-driven disease at 2 months and 5 months of age (n=12, n=13). The calculated changes in histologic feature abundance were consistent with biological expectations, showing an expansion of the stromal compartment, a reduction of normal acinar tissue, and an increase in both ADM and dysplasia as disease progresses (p= 2e-6, 6e-7, 4e-4, and 3e-5, respectively). These results demonstrate the tools efficacy for accurate and rapid quantification of multiple histologic features using an objective and automated platform. Our tool promises to rapidly accelerate and improve the quantification of altered pancreatic disease progression in animal studies.

bioinformatics↗

bioRxiv2020

Molecular architecture of the luminal ring of the Xenopus laevis nuclear pore complex

Nuclear pore complex (NPC) mediates the flow of substances between the nucleus and cytoplasm in eukaryotic cells. Here we report the cryo-electron tomography (cryo-ET) structure of the luminal ring (LR) of the NPC from Xenopus laevis oocyte. The observed key structural features of the LR are independently confirmed by single-particle cryo-electron microscopy (cryo-EM) analysis. The LR comprises eight butterfly-shaped subunits, each containing two symmetric wings. Each wing consists of four elongated, tubular protomers. Within the LR subunit, the eight protomers form a Finger domain, which directly contacts the fusion between the inner and outer nuclear membranes, and a Grid domain, which serves as a rigid base for the Finger domain. Two neighbouring LR subunits interact with each other through the lateral edges of their wings to constitute a Bumper domain, which displays two major conformations and appears to cushion neighbouring NPCs. Our study reveals previously unknown features of the LR and potentially explains the elastic property of the NPC.

biophysics↗

bioRxiv2020

Structure of the Cytoplasmic Ring of the Xenopus laevis Nuclear Pore Complex

Nuclear pore complex (NPC) exhibits structural plasticity and has only been characterized at local resolutions of up to 15 [A] for the cytoplasmic ring (CR). Here we present a single-particle cryo-electron microscopy (cryo-EM) structure of the CR from Xenopus laevis NPC at average resolutions of 5.5-7.9 [A], with local resolutions reaching 4.5 [A]. Improved resolutions allow identification and placement of secondary structural elements in the majority of the CR components. The two Y complexes in each CR subunit interact with each other and associate with those from flanking subunits, forming a circular scaffold. Within each CR subunit, the Nup358-containing region wraps around the stems of both Y complexes, likely stabilizing the scaffold. Nup205 connects the short arms of the two Y complexes and associates with the stem of a neighbouring Y complex. The Nup214-containing region uses an extended coiled-coil to link Nup85 of the two Y complexes and protrudes into the axial pore of the NPC. These previously uncharacterized structural features reveal insights into NPC assembly.

biophysics↗

bioRxiv2020

A Connectome of the Adult Drosophila Central Brain

The neural circuits responsible for behavior remain largely unknown. Previous efforts have reconstructed the complete circuits of small animals, with hundreds of neurons, and selected circuits for larger animals. Here we (the FlyEM project at Janelia and collaborators at Google) summarize new methods and present the complete circuitry of a large fraction of the brain of a much more complex animal, the fruit fly Drosophila melanogaster. Improved methods include new procedures to prepare, image, align, segment, find synapses, and proofread such large data sets; new methods that define cell types based on connectivity in addition to morphology; and new methods to simplify access to a large and evolving data set. From the resulting data we derive a better definition of computational compartments and their connections; an exhaustive atlas of cell examples and types, many of them novel; detailed circuits for most of the central brain; and exploration of the statistics and structure of different brain compartments, and the brain as a whole. We make the data public, with a web site and resources specifically designed to make it easy to explore, for all levels of expertise from the expert to the merely curious. The public availability of these data, and the simplified means to access it, dramatically reduces the effort needed to answer typical circuit questions, such as the identity of upstream and downstream neural partners, the circuitry of brain regions, and to link the neurons defined by our analysis with genetic reagents that can be used to study their functions. Note: In the next few weeks, we will release a series of papers with more involved discussions. One paper will detail the hemibrain reconstruction with more extensive analysis and interpretation made possible by this dense connectome. Another paper will explore the central complex, a brain region involved in navigation, motor control, and sleep. A final paper will present insights from the mushroom body, a center of multimodal associative learning in the fly brain.

neuroscience↗

bioRxiv2020

Discovery of peculiar cells in the peri-tumoral regions and peripheral blood of glioblastoma patients by single cell RNA-sequencing

The glioblastoma (GBM) recurrence rate is high, despite multimodal treatment including surgery, radiotherapy, chemotherapy, and immunotherapy. Most recurrences occur at the resection margin that is located outside the GBM contrast-enhancing region (C region) of magnetic resonance imaging. However, the nature of the GBM cells that lie outside the C region is unclear. We used single-cell RNA sequencing (scRNA-seq) to compare 12 samples taken from inside and outside the C region from four patients with GBM and identified a cluster of GBM cells outside the C region that exhibited fragmented CNVs in chromosomes such as 1, 10, 12 and 19 (herein termed CNV 4). A transcription factor-transcription cofactor interaction network was constructed to uncover the transcriptional regulatory mechanism of these CNV 4 GBM cells that had prognostic significance (p < 0.05). Furthermore, a sub-cluster of these CNV 4 GBM cells possessed stem cell-like properties and had tumorigenic potential. In parallel, we analyzed peripheral blood mononuclear cells (PBMCs) from four patients with GBM, three patients with epilepsy and three healthy volunteers by scRNA-seq. We identified a novel subtype of proliferative immune cells only in patients with GBM. Overall, our findings indicate that CNV 4 cells might contribute to GBM recurrence and proliferative circulating immune cells are specific to patients with GBM. This study sheds light on a neglected aspect of GBM, and opens new avenues to explore GBM recurrence and immunotherapy. SignificanceWe show that a subset of GBM cells outside the C region possess the properties of stem cell and tumorigenesis potential, which might be responsible for cancer recurrence. Besides, proliferative lymphocytes are specific to the PBMCs of patients with GBM, which could help to develop novel immunotherapy.

cancer biology↗

bioRxiv2019

Structural basis for pore blockade of the human cardiac sodium channel Nav1.5 by tetrodotoxin and quinidine

Among the nine subtypes of human voltage-gated Na+ (Nav) channels, tetrodotoxin (TTX)-resistant Nav1.5 is the primary one in heart. More than 400 point mutations have been identified in Nav1.5 that are associated with cardiac disorders exemplified by type 3 long QT syndrome and Brugada syndrome, making Nav1.5 a common target for class I antiarrythmic drugs. Here we present the cryo-EM structures of Nav1.5 bound to quinidine and TTX with resolutions at 3.2 [A] and 3.3 [A], respectively, for the pore domain. The structures allow mapping of 278 disease-related mutations in the resolved region, establishing the framework for mechanistic investigation of these pathogenic mutations. Quinidine is positioned right below the selectivity filter and coordinated by residues from repeats I, III, and IV. Pore blockade is achieved through both direct obstruction of the ion permeation path and induced rotation of a Tyr residue that leads to intracellular gate closure. Structural comparison of Nav1.5 with our previously reported Nav1.2/1.4/1.7 reveals the molecular determinant for the different susceptibility to TTX by TTX-resistant and -sensitive Nav subtypes, and highlights the functional significance of the underexplored and least conserved extracellular loops.

biophysics↗

bioRxiv2019

Senescence of alveolar stem cells drives progressive pulmonary fibrosis

Tissue fibrosis is a common pathological outcome of chronic disease that markedly impairs organ function leading to morbidity and mortality. In the lung, idiopathic pulmonary fibrosis (IPF) is an insidious and fatal interstitial lung disease associated with declining pulmonary function. Here, we show that alveolar type 2 (AT2) stem cells isolated from IPF lung tissue exhibit characteristic transcriptomic features of cellular senescence. We used conditional loss of Sin3a in adult mouse AT2 cells to initiate a program of p53-dependent cellular senescence, AT2 cell depletion, and spontaneous, progressive pulmonary fibrosis. We establish that senescence rather than loss of epithelial stem cells serves as a proximal driver of Tgf{beta} activation and progressive fibrosis and show that either genetic or pharmacologic interventions targeting p53 activation, senescence, or downstream Tgf{beta} activation, block fibrogenesis.

cell biology↗

bioRxiv2019

Sub-species niche specialization in the oral microbiome is associated with nasopharyngeal carcinoma risk in an endemic area of southern China

Oral health and changes in the oral microbiome have been associated with both local and systemic cancer. Poor oral hygiene is a known risk factor for Nasopharyngeal Carcinoma (NPC), a virally-associated head and neck cancer endemic to southern China. We explored the relationship between NPC and the oral microbiome using 16s rRNA sequencing in a study of 499 NPC patients and 495 population-based age and sex frequency-matched controls from an endemic area of Southern China. We found a significant reduction in community richness in cases compared to controls. Differences in the overall microbial community structure between cases and controls could not be explained by other potential confounders; disease status explained 5 times more variation in the Unweighted UniFrac distance than the next most explanatory variable. In feature-based analyses, we identified a pair of co-excluding Granulicatella adiacens (G. adicans) amplicon sequence variants (ASVs) which were strongly associated with NPC status and differed by a single nucleotide. The G. adicans variant an individual carried was also associated with the overall microbial community based on beta diversity. Co-occurrence analysis suggested the two G. adicans ASVs sit at the center of two co-excluding clusters of closely related organisms. Our results suggest there are differences in the oral microbiome between NPC patients and healthy controls and these may be associated with both a loss of microbial diversity and niche specialization among closely related commensals. Importance The relationship between oral health and the risk of Nasopharyngeal Carcinoma has been previously established. However, the role of oral microbiome has not been evaluated in the disease in a large epidemiological study. This paper clearly establishes a difference in the oral microbiome between NPC patients and healthy controls which cannot be explained by other confounding factors. It furthermore identifies a pair of closely related co-excluding organisms associated with the disease, highlighting the importance of modern methods for single nucleotide resolution in 16s rRNA sequence characterization. To the best of our knowledge, this is one of the first examples of cancer-associated niche-specialization of the oral microbiome.

microbiology↗

bioRxiv2019

FAP overexpression induce Epithelial-Mesenchymal Transition (EMT) in oral squamous cell carcinoma by down-regulating DPP9 gene.

FAP acts as a tumor promoter via epithelial-mesenchymal transition (EMT) in human oral squamous cell carcinoma (OSCC). The present study was designed to investigate the interaction proteins with FAP and explore the precise mechanism of FAP promoting EMT in OSCC. IP-MS analysis confirmed that DPP9 was an interacting protein of FAP. DPP9 was down-regulated in OSCC tissue samples compared with MNT using immunohistochemistry and quantitative-PCR detection. Lower DPP9 was correlated with unfavorable overall survival of patients with OSCC. Repressing DPP9 accelerates the proliferation of OSCC cells in vitro and in vivo. Mechanistically, overexpression of FAP downregulate the expression of the DPP9 and the effect of FAP on OSCC proliferation, migration, invasion and EMT could be reversed by up-regulated DPP9. Our study suggests that FAP could induce EMT and promote carcinogenesis in oral squamous cell carcinoma by down-regulating DPP9 gene. That will hint different dimension on therapy for patients with OSCC.

molecular biology↗

bioRxiv2019

In vitro and in vivo roles of glucocorticoid and vitamin D receptors in the control of cardiomyocyte proliferative potential

Cardiomyocyte (CM) proliferative potential varies considerably across species. While lower vertebrates and neonatal mammals retain robust capacities for CM proliferation, adult mammalian CMs lose proliferative potential due to cell-cycle withdrawal and polyploidization, failing to mount a proliferative response to regenerate lost CMs after cardiac injury. The decline of murine CM proliferative potential occurs in the neonatal period when the endocrine system undergoes drastic changes for adaptation to extrauterine life. We recently demonstrated that thyroid hormone (TH) signaling functions as a primary factor driving CM proliferative potential loss in vertebrates. Whether other hormonal pathways govern this process remains largely unexplored. Here we showed that agonists of glucocorticoid receptor (GR) and vitamin D receptor (VDR) suppressed neonatal CM proliferation in vitro. We next examined CM nucleation and proliferation in mutant mice lacking GR or VDR specifically in CMs, but we observed no difference between mutant and control littermates. Additionally, we generated compound mutant mice that lack GR or VDR and express dominant-negative TH receptor alpha in their CMs, and similarly observed no increase in CM proliferative potential compared to dominant-negative TH receptor alpha mice alone. Thus, although GR and VDR activation in cultured CMs is sufficient to inhibit CM proliferation, they seem to be dispensable for CM cell-cycle exit and binucleation in vivo. In addition, given the recent report that VDR activation in zebrafish promotes CM proliferation and tissue regeneration, our results suggest distinct roles of VDR in zebrafish and rodent CM cell-cycle regulation.

developmental biology↗

bioRxiv2019

Molecular basis for allosteric regulation of the type 2 ryanodine receptor channel gating by key modulators

The type-2 ryanodine receptor (RyR2) is responsible for releasing Ca2+ from the sarcoplasmic reticulum of cardiomyocytes, subsequently leading to muscle contraction. Here, we report four cryo-EM structures of porcine RyR2 bound to distinct modulators that collectively provide mechanistic insight into RyR2 regulation. Ca2+ alone induces a contraction of the Central domain that facilitates the dilation of S6 bundle, but is insufficient to open the pore. The small molecule agonist PCB95 helps Ca2+ to overcome the barrier for opening. FKBP12.6 induces a relaxation of the Central domain that decouples it from the S6 bundle, stabilizing RyR2 in a closed state. Caffeine locks the Central domain in a constitutively contracted state, while further addition of ATP opens the channel by strengthening the coupling between the U-motif and S6. Our study marks an important step towards mechanistic understanding of the complicated regulation of this key channel whose aberrant activity engenders life-threatening cardiac disorders.

biochemistry↗

bioRxiv2019

CNOT1 mediates phosphorylation via Protein kinase A on the circadian clock

At the core of the mammalian circadian feedback loop, CLOCK (NPAS2)-BMAL1 is the positive element to activate transcription of downstream genes encoding the negative elements PERs and CRYs. Here we show that CNOT1 associates with both CLOCK and BMAL1, promotes their phosphorylation and increases their protein stability, and in turn inhibits the transcriptional activity of CLOCK-BMAL1. Expression of either CLOCK, BMAL1 or CNOT1 could interact with endogenous Protein Kinase A (PKA) as assessed by co-immunoprecipitation (Co-IP) and kinase assays. PKA could phosphorylate CLOCK and BMAL1 and this was promoted by CNOT1. Genetic deletion of PKA-C by CRISPR-Cas9 results in a longer period of the circadian rhythm; while overexpression of PKA-C induces a shorter period. Furthermore, we demonstrate that CNOT1 associates with CLOCK and BMAL1 in the mouse liver and promotes their phosphorylation. PER2, but not CRY2, is also a PKA target. Our results suggest that CNOT1 and PKA play a critical role in the mammalian circadian clock, revealing a conserved function in eukaryotic circadian regulations.

developmental biology↗