Biology subjects

Chen, L.

Publications and source records attributed to Chen, L..

At least 667 records · Page 37Linked to original sources

bioRxiv2022

A toolkit of highly selective and sensitive genetically encoded neuropeptide sensors

Neuropeptides are key signaling molecules in the endocrine and nervous systems that regulate many critical physiological processes, including energy balance, sleep and circadian rhythms, stress, and social behaviors. Understanding the functions of neuropeptides in vivo requires the ability to monitor their dynamics with high specificity, sensitivity, and spatiotemporal resolution; however, this has been hindered by the lack of direct, sensitive and non-invasive tools. Here, we developed a series of GRAB (G protein-coupled receptor activation-based) sensors for detecting somatostatin (SST), cholecystokinin (CCK), corticotropin-releasing factor (CRF), neuropeptide Y (NPY), neurotensin (NTS), and vasoactive intestinal peptide (VIP). These fluorescent sensors utilize the corresponding GPCRs as the neuropeptide-sensing module with the insertion of a circular-permutated GFP as the optical reporter. This design detects the binding of specific neuropeptides at nanomolar concentration with a robust increase in fluorescence. We used these GRAB neuropeptide sensors to measure the spatiotemporal dynamics of endogenous SST release in isolated pancreatic islets and to detect the release of both CCK and CRF in acute brain slices. Moreover, we detect endogenous CRF release induced by stressful experiences in vivo using fiber photometry and 2-photon imaging in mice. Together, these new sensors establish a robust toolkit for studying the release, function, and regulation of neuropeptides under both physiological and pathophysiological conditions.

neuroscience↗

bioRxiv2022

Exploiting deep transfer learning for the prediction of functional noncoding variants using genomic sequence

MotivationThough genome-wide association studies have identified tens of thousands of variants associated with complex traits and most of them fall within the noncoding regions, they may not the causal ones. The development of high-throughput functional assays leads to the discovery of experimental validated noncoding functional variants. However, these validated variants are rare due to technical difficulty and financial cost. The small sample size of validated variants makes it less reliable to develop a supervised machine learning model for achieving a whole genome-wide prediction of noncoding causal variants. ResultsWe will exploit a deep transfer learning model, which is based on convolutional neural network, to improve the prediction for functional noncoding variants. To address the challenge of small sample size, the transfer learning model leverages both large-scale generic functional noncoding variants to improve the learning of low-level features and context-specific functional noncoding variants to learn high-level features toward the contextspecific prediction task. By evaluating the deep transfer learning model on three MPRA datasets and 16 GWAS datasets, we demonstrate that the proposed model outperforms deep learning models without pretraining or retraining. In addition, the deep transfer learning model outperforms 18 existing computational methods in both MPRA and GWAS datasets. Availabilityhttps://github.com/lichen-lab/TLVar Supplementary InformationSupplementary data are available at Bioinformatics online. Contactchen61@iu.edu

bioinformatics↗

bioRxiv2022

A novel PLS1 c.981+1G>A variant causes autosomal-dominant hereditary hearing loss in a family via up-regulation of the PI3K-Akt signaling pathway

BackgroundThe fimbrin protein family contains a variety of proteins, of which Plastin1 (PLS1) is an important member. The latest researches show that variations in the coding region of PLS1 gene is related to the development of deafness. However, it remains unknown about the molecular mechanism of deafness caused by PLS1 gene variants. Methodswhole exome sequencing was performed on the affected family members and healthy ones to identify the pathogenic variants, followed by sanger sequencing. Minigene assay was conducted to investigate the impact of the variant on PLS1 mRNA splicing. The pathogenicity of the variant was further investigated in zebrafish. RNA-sequencing (RNA-seq) was done for analyzing the dysregulated downstream signaling pathways caused by knockdown of PLS1 expression. ResultsWe identified a novel variant PLS1 c.981+1G>A in a large Chinese family with hearing loss and demonstrated that the variant is responsible for the occurrence of hearing loss by inducing Exon8 skipping or deletion of Exon8 (47bp). The variant caused abnormal inner ear phenotypes, characterized by decreased mean otolith distance, anterior otolith diameter, posterior otolith diameter, and cochlear diameter, swimming speed and distance in zebrafish. Furthermore, silencing PLS1 expression significantly upregulated genes in the PI3K-Akt signaling pathway, including Col6a3, Spp1, Itgb3 and Hgf. ConclusionsPLS1 c.981+1G>A is a novel pathogenic variant for hearing loss by inducing Exon8 skipping or deletion of Exon8 (47bp). Up-regulation of the PI3K-Akt signaling pathway plays an important role in the pathogenesis of PLS-1 gene.

genetics↗

bioRxiv2022

LANTSA: Landmark-based transferable subspace analysis for single-cell and spatial transcriptomics

Single-cell RNA sequencing (scRNA-seq) and spatial transcriptomics (ST) technologies provide new insights to understand tissue organization and biological function. Accurately capturing the relationships of samples (e.g., sequenced cells, spatial locations) will result in reliable and consistent outcomes in downstream analyses. However, this undertaking remains a challenge for large-volume or cross-platform datasets due to transcriptional heterogeneity and high computational demands. Here, we introduce landmark-based transferable subspace analysis (LANTSA) to solve such challenges for scRNA-seq and ST datasets. Specifically, LANTSA constructs a representation graph of samples for clustering and visualization based on a novel subspace model, which can learn a more accurate representation and is theoretically proven to be linearly proportional to data size in terms of the time consumption. Furthermore, LANTSA uses a dimensionality reduction technique as an integrative method to extract the discriminants underlying the representation structure, which enables label transfer from one (learning) dataset (i.e., scRNA-seq profiles) to the other (prediction) datasets (e.g., scRNA-seq or ST profiles), thus solving the massive-volume or cross-platform problem. We demonstrated the superiority of LANTSA to identify accurate data structures via clustering evaluation on benchmark datasets of various scRNA-seq protocols, 10x Visium, and Slide-seq ST platforms. Moreover, we confirmed the integration capability of LANTSA to transfer cell annotation on large-scale and cross-platform scRNA-seq datasets. Finally, we validated the effectiveness of LANTSA for the identification of multiple mouse brain areas as well as the spatial mapping of cell types within cortical layers by integrating scRNA-seq and ST data.

bioinformatics↗

bioRxiv2022

Longitudinal dynamics of microvascular recovery after acquired cortical injury

Acquired brain injuries due to trauma damage cortical vasculature, which in turn impairs blood flow to injured tissues. There are reports of vascular morphological recovery following traumatic brain injury (TBI), but the remodeling process has not been examined longitudinally in detail after injury in vivo. Understanding the dynamic processes that influence recovery is thus critically important. We evaluated the longitudinal and dynamic microvascular recovery and remodeling up to 2 months post injury using live brain miniscope and 2-photon microscopic imaging. The new imaging approaches captured dynamic morphological and functional recovery processes at high spatial and temporal resolution in vivo. Vessel painting documented the initial loss and subsequent temporal morphological vascular recovery at the injury site. Miniscopes were used to longitudinally image the temporal dynamics of vascular repair in vivo after brain injury in individual mice across each cohort. We observe near-immediate nascent growth of new vessels in and adjacent to the injury site that peaks between 14-21 days post injury. 2-photon microscopy confirms new vascular growth and further demonstrates differences between cortical layers after cortical injury: large vessels persist in deeper cortical layers (>200 m), while superficial layers exhibit a dense plexus of fine (and often non-perfused) vessels displaying regrowth. Functionally, blood flow increases mirror increasing vascular density. Filopodia development and endothelial sprouting is measurable within 3 days post injury that rapidly transforms regions devoid of vessels to dense vascular plexus in which new vessels become increasingly perfused. Within 7 days post injury, blood flow is observed in these nascent vessels. Behavioral analysis reveals improved vascular modulation after 9 days post injury, consistent with vascular regrowth. We conclude that morphological recovery events are closely linked to functional recovery of blood flow to the compromised tissues, which subsequently leads to improved behavioral outcomes.

neuroscience↗

bioRxiv2022

A sporadic Alzheimer's blood-brain barrier model for developing ultrasound-mediated delivery of Aducanumab and anti-Tau antibodies.

RationaleThe blood-brain barrier (BBB) is a major impediment to therapeutic intracranial drug delivery for the treatment of neurodegenerative diseases, including Alzheimers disease (AD). Focused ultrasound applied together with microbubbles (FUS+MB) is a novel technique to transiently open the BBB and increase drug delivery. Evidence suggests that FUS+MB is safe, however the effects of FUS+MB on human BBB cells, especially in the context of AD, remain sparsely investigated. MethodsHere we generated BBB cells (induced brain endothelial cells (iBECs) and astrocytes (iAstrocytes)) from apolipoprotein E gene allele E4 (APOE4, high AD risk) and allele E3 (APOE3, lower AD risk) carrying patient-derived induced pluripotent stem cells (iPSCs). We then developed a human sporadic AD BBB cell platform to investigate the effects of FUS+MB on BBB cells and screen for the delivery of two potentially therapeutic AD antibodies. ResultsWe utilized this robust and reproducible human BBB model to demonstrate increased delivery of therapeutic AD antibodies across the BBB following FUS+MB treatment, including an analogue of Aducanumab (AduhelmTM; anti-amyloid-{beta}) and a novel anti-Tau antibody RNF5. Our results also demonstrate the safety of FUS+MB indicated by minimal changes in the cell transcriptome as well as little or no changes in cell viability and inflammatory responses within the first 24 h post FUS+MB. Finally, we report a more physiologically relevant hydrogel-based 2.5D BBB model as a key development for FUS+MB-mediated drug delivery screening, with potentially higher translational utility. ConclusionOur results demonstrate an important translatable patient BBB cell model for identifying FUS+MB-deliverable drugs and screening for cell- and patient-specific effects of FUS+MB, accelerating the use of FUS+MB as a therapeutic modality in AD. One Sentence SummaryFocused ultrasound increases the in vitro delivery of therapeutic antibodies Aducanumab and anti-Tau in a sporadic Alzheimers disease patient-derived blood-brain barrier cell model.

neuroscience↗

bioRxiv2022

Synaptic Location Is a Determinant of the Detrimental Effects of α-Synuclein Pathology to Glutamatergic Transmission in the Basolateral Amygdala

The presynaptic protein -synuclein (Syn) has been suggested to be involved in the pathogenesis of Parkinsons disease (PD). In PD, the amygdala is prone to develop insoluble Syn aggregates, and it has been suggested that circuit dysfunction involving the amygdala contributes to the psychiatric symptoms. Yet, how Syn aggregates affect amygdala function is unknown. In this study, we examined Syn in glutamatergic axon terminals and the impact of its aggregation on glutamatergic transmission in the basolateral amygdala (BLA). We found that Syn is primarily present in the vesicular glutamate transporter 1-expressing (vGluT1+) terminals in mouse BLA, which is consistent with higher levels of Syn expression in vGluT1+ glutamatergic neurons in the cerebral cortex relative to the vGluT2+ glutamatergic neurons in the thalamus. We found that Syn aggregation selectively decreased the cortico-BLA, but not the thalamo-BLA, transmission; and that cortico-BLA synapses displayed enhanced short-term depression upon repetitive stimulation. In addition, using confocal microscopy, we found that vGluT1+ axon terminals exhibited decreased levels of soluble Syn, which suggests that lower levels of soluble Syn might underlie the enhanced short-term depression of cortico-BLA synapses. In agreement with this idea, we found that cortico-BLA synaptic depression was also enhanced in Syn knockout mice. In conclusion, both basal and dynamic cortico-BLA transmission were disrupted by abnormal aggregation of Syn and these changes might be relevant to the perturbed cortical control of the amygdala that has been suggested to play a role in psychiatric symptoms in PD.

neuroscience↗

bioRxiv2022

Systematic Evaluation of Different R-loop Mapping Methods: Achieving Consensus, Resolving Discrepancies and Uncovering Distinct Types of RNA:DNA Hybrids

R-loop, a three-stranded nucleic acid structure, has been recognized to play pivotal roles in critical physiological and pathological processes. Multiple technologies have been developed to profile R-loops genome-wide, but the existing data suffer from major discrepancies on determining genuine R-loop localization and its biological functions. Here, we experimentally and computationally evaluate eight representative R-loop mapping technologies, and reveal inherent biases and artifacts of individual technologies as key sources of discrepancies. Analyzing signals detected with different R-loop mapping strategies, we note that genuine R-loops predominately form at gene promoter regions, whereas most signals in gene body likely result from structured RNAs as part of repeat-containing transcripts. Interestingly, our analysis also uncovers two classes of R-loops: The first class consists of typical R-loops where the single-stranded DNA binding protein RPA binds both the template and non-template strands. By contrast, the second class appears independent of Pol II-mediated transcription and is characterized by RPA binding only in the template strand. These two different classes of RNA:DNA hybrids in the genome suggest distinct biochemical activities involved in their formation and regulation. In sum, our findings will guide future use of suitable technology for specific experimental purposes and the interpretation of R-loop functions.

genomics↗

bioRxiv2022

The exchange rates of amide and arginine guanidinium CEST in the mouse brain

PurposeTo develop a pipeline for measuring the exchange rates and concentrations of in vivo excgangeable protons, and to demonstrate this for the amide and arginine (Arg) guanidinium (Guan) protons in mobile proteins in the mouse brain. MethodsAn ultra-short echo (UTE) CEST sequence with a continuous wave presaturation (preRadCEST) was applied to acquire Z-spectra with robustness to motion and physiological fluctuations. AmideCEST and Arginine guanCEST (ArgCEST) were extracted and their proton concentrations and exchange rates obtained using a two-step multi-B1 Bloch fitting approach that included the semisolid macromolecular background. To minimize contamination from the amine protons from creatine and phosphocreatine, ArgCEST measurements were performed on the Guanidinoacetate N-methyltransferase deficiency (GAMT-/-) mouse characterized by low creatine and phosphocreatine concentrations in the brain. ResultsFor the amideCEST proton pool, the exchange rate and concentrations were found to be 59.6 {+/-} 9.0 s-1 and 41.7 {+/-} 7.0 mM, respectively, with the maximum signal observed at B1 = 0.8 T. For the ArgCEST proton, the guanidinium exchange these were 70.1 {+/-} 5.5 s-1 and 10.1 {+/-} 1.3 mM, respectively, with the maximum effect observed at B1 = 0.9 T. The current study suggests that the inverse pH dependence in GuanCEST of brain is led by the CrCEST component, not ArgCEST. ConclusionThe current pipeline is expected to have general use for in vivo CEST quantitation and optimization of visible CEST resonances.

biophysics↗

bioRxiv2022

Nitric oxide negatively regulates gibberellin signaling to coordinate growth and salt tolerance in Arabidopsis

In response to dynamically altered environments, plants must finely coordinate the balance between growth and stress responses for their survival. However, the underpinning regulatory mechanisms remain largely elusive. The phytohormone gibberellin promotes growth via a derepression mechanism by proteasomal degradation of the DELLA transcription repressors. Conversely, the stress-induced burst of nitric oxide (NO) enhances stress tolerance, largely relaying on NO-mediated S-nitrosylation, a redox-based posttranslational modification. Here, we show that S-nitrosylation of Cys-374 in the Arabidopsis RGA protein, a key member of DELLAs, inhibits its interaction with the F-box protein SLY1, thereby preventing its proteasomal degradation under salinity condition. The accumulation of RGA consequently retards growth but enhances salt tolerance. We propose that NO negatively regulates gibberellin signaling via S-nitrosylation of RGA to coordinate the balance of growth and stress responses when challenged by adverse environments.

plant biology↗

bioRxiv2022

Cryomilling Tethered Chromatin Conformation Capture reveal new insights into inter-chromosomal interactions

Traditional methods used to map the three-dimensional organization of chromatin in-situ generally involve chromatin conformation capture by formaldehyde crosslinking, followed by detergent solubilization and enzymatic digestion of DNA. Ligation of proximal DNA fragments followed by next generation sequencing (NGS) generates contact information that enables a global view of the chromatin conformation. Here, we explore the use of cryomilling to physically fragmentize the cells under cryogenic conditions to probe chromatin interactions in the cryomilled cell fragments by the tethered chromatin conformation capture (TCC). Our results show that cryomilling TCC (CTCC) can generate a global contact map similar to that obtained with in-situ Hi-C. This result suggests that summation of chromatin interactions mapped in individual subcellular fragments can reconstitute the global contact map of intact cells in an ensemble manner, paving the way for chromatin conformation analyses of solid tissue by CTCC. Compared with the conventional in-situ methods such as Hi-C, CTCC shows more uniform access to different subcompartments of the folded genome. On the other hand, most inter-chromosomal (trans) contacts are diminished or lost in CTCC except for a group of unique trans contacts that remain intact throughout the cryomilling and in- vitro crosslinking steps. These apparently ultra-stable trans interactions have much enhanced signal in CTCC due to the elimination of signals of most, presumably weak and transient trans interactions. Systematic and comparative analyses between CTCC and in-situ Hi-C provide further insights into the chromatin structure organization and reveal a generally unentangled chromosome interface and the existence of stable inter-chromosomal contacts that may represent intermingled inter-chromosomal interfaces.

genomics↗

bioRxiv2022

New efficient intercellular spread mode of respiratory syncytial virus contributes to neutralization escape and persistence

There is no licensed vaccine or therapeutic antibody for respiratory syncytial virus (RSV). The induction of high-titer, potent neutralizing antibodies cannot completely inhibit breakthrough infection and enhanced respiratory disease (ERD), encouraging a focus on the relationship between virus intercellular spread and neutralizing antibodies. By blocking the known intercellular spread modes and with the aid of ultrahigh-resolution imaging, we observed a new efficient mode of intercellular spread in which RSV-infected cells directly transfer viral materials (including viral replication factories) to neighboring cells through protruding open-ended microfilament-rich intercellular nanotubes. The new mode is virion-independent and antibody-insensitive, beginning as early as 3 h post infection. Furthermore, replication-defective viral genomes (DVGs) might also utilize the new mode, facilitating the establishment of latent viral infections. Therefore, our data provide a new perspective on RSV cell-to-cell spread and might help to explain the immune escape and latent persistence of paramyxoviruses.

microbiology↗

bioRxiv2022

STORM: spectral sparsification helps restore the spatial structure at single-cell resolution

Single-cell RNA sequencing thoroughly quantifies the individual cell transcriptomes but renounces the spatial structure. Conversely, recently emerged spatial transcriptomics technologies capture the cellular spatial structure but skimp cell or gene resolutions. Cell-cell affinity estimated by ligand-receptor interactions can partially reconstruct the quasi-structure of cells but falsely include the pseudo affinities between distant or indirectly interacting cells. Here, we develop a software package, STORM, to reconstruct the single-cell resolution quasi-structure from the spatial transcriptome with diminished pseudo affinities. STORM first curates the representative single-cell profiles for each spatial spot from a candidate library, then reduces the pseudo affinities in the intercellular affinity matrix by partial correlation, spectral graph sparsification, and spatial coordinates refinement. STORM embeds the estimated interactions into a low-dimensional space with the cross-entropy objective to restore the intercellular quasi-structures, which facilitates the discovery of dominant ligand-receptor pairs between neighboring cells at single-cell resolution. STORM reconstructed structures achieved shape Pearson correlations ranging from 0.91 to 0.97 on the mouse hippocampus and human organ tumor microenvironment datasets. Furthermore, STORM can solely de novo reconstruct the quasi-structures at single-cell resolution, i.e., reaching the cell-type proximity correlations 0.68 and 0.89 between reconstructed and immunohistochemistry-informed spatial structures on a human developing heart dataset and a tumor microenvironment dataset, respectively.

bioinformatics↗

bioRxiv2022

A GSVA based gene set synergizing with CD4+T cell bearing harmful factors yield risk signals in HBV related diseases via amalgamation of artificial intelligence

Genes encoding chemokines and extracellular matrix (ECM) play pivotal roles in chronic HBV infection (CHB), HBV related fibrosis (HBV-LF) and hepatocellular carcinoma (HBV-HCC). The landscape and potential of these genes in prognosis across diseases stages have not been fully and systemically understood. In this study, we defined an HBV-LF associated gene set comprised of chemokines and ECM related genes directly induced by initial HBV infection through GSVA algorithm that named as CLST (C stands for CXCL9, CXCL10, CCL19 and CCL20; L for LUM; S for SOX9 and SPP1; T for THBS1, THBS2) and evaluated its biomarker values in CHB and HBV-LF. Enrichment scores (ES) of CLST was subsequently observed synergized with activated CD4+T cells (aCD4) highly related to T helper cell 17 (TH17) associated genes and immune checkpoints and addressed as risk signals due to bearing harmful prognosis factors in tumor tissues of patients with HBV-HCC. Dual higher enrichment score (ES) of CLST and aCD4 in HBV-HCC patients exhibited worse overall survival (OS). Feature genes specific to these two gene sets showed promising clinical relevance in early-stage of HBV-HCC definition and OS prediction incorporating laboratory parameters via artificial intelligence (AI) systems. Finally, a novel mechanistic insight into the issue was proposed that PEG IFN- as an immunotherapy through modulating CLST signal in treatment responders and these immune signals down-regulation could be beneficial for HBV related diseases control and prevention. Together, our study provides GSVA and AI derived immunogenomic prognosis signatures and clinical utility of these signals will be benefit for HBV related diseases cure.

immunology↗

bioRxiv2022

Collateral cleavage of 28s rRNA by RfxCas13d causes death of mice

The CRISPR-Cas13 system is an RNA-guided RNA-targeting system, and has been widely used in transcriptome engineering with potentially important clinical applications. However, it is still controversial whether Cas13 exhibits collateral activity in mammalian cells. Here, we found that knocking down gene expression using RfxCas13d in the adult brain neurons caused death of mice, which was not resulted from the loss of target gene function or off-target effects. Mechanistically, we showed that RfxCas13d exhibited collateral activity in mammalian cells, which is positively correlated with the abundance of target RNA. The collateral activity of RfxCas13d could cleave 28s rRNA into two fragments, leading to translation attenuation and activation of the ZAK-JNK/p38-immediate early gene (IEG) pathway. These results provide new mechanistic insights into the collateral activity of RfxCas13d and warn that the biosafety of CRISPR-Cas13 system needs further evaluation before applying it to clinical treatments.

bioengineering↗

bioRxiv2022

Spinal cord homeostatic plasticity gates mechanical allodynia in chronic pain

Central sensitization caused by disinhibition of spinal cord circuits is a key mechanism of mechanical allodynia in neuropathic pain. Despite intense efforts, the molecular mechanisms that drive disinhibition and induce allodynia after peripheral nerve injury remain unclear. Using the spared-nerve injury (SNI) model of allodynia, we here demonstrate that SNI induces disinhibition of spinal nociceptive circuits by triggering homeostatic synaptic plasticity. Specifically, SNI-triggered homeostatic plasticity suppresses the inhibitory outputs of parvalbumin-positive (PV+) interneurons that form synapses on both primary afferent terminals and excitatory interneurons, causing hyperactivation of the nociceptive pathway. Using genetic manipulations, we identified the retinoic acid receptor RAR as the key mediator of the homeostatic synaptic plasticity underlying this synaptic disinhibition. Deletion of RAR in PV+ neurons blocked SNI-induced spinal disinhibition, central sensitization, and allodynia. Moreover, deletion of RAR in spinal PV+ neurons or application of an RAR antagonist in the spinal cord prevented the development of SNI-induced mechanical allodynia. Together, our results reveal a molecular mechanism of neuropathic pain whereby homeostatic plasticity causes the mis-direction of tactile information flow to ascending nociceptive pathways following peripheral nerve injury.

neuroscience↗

bioRxiv2022

Hypoxia Dynamically Regulates DBC1 Ubiquitination and Stability by SIAH2 and OTUD5 in Breast Cancer Progression

DBC1 has been characterized as a key regulator of physiological and pathophysiological activities, such as DNA damage, senescence and tumorigenesis. However, the mechanism by which the functional stability of DBC1 is regulated has yet to be elucidated. Here, we report that the ubiquitination-mediated degradation of DBC1 is dynamically regulated by the E3 ubiquitin ligase SIAH2 and deubiquitinase OTUD5 under hypoxic stress. Mechanistically, hypoxia promoted the competitive binding of SIAH2 with OTUD5 to DBC1, resulting in the ubiquitination and subsequent degradation of DBC1 through the ubiquitin-proteasome pathway. Siah2 knockout inhibited tumor cell proliferation and migration, which could be rescued by double knockout of Siah2/DBC1. Human tissue microarray analysis further revealed that the SIAH2/DBC1 axis was responsible for tumor progression under hypoxic stress. These findings define a key role of the hypoxia-mediated SIAH2-DBC1 pathway in the progression of human breast cancer and provide novel insights into the metastatic mechanism of breast cancer.

cell biology↗

bioRxiv2022

Hbxip (Lamtor5) is essential for embryogenesis and regulates embryonic stem cell differentiation through activating mTORC1

Hbxip, also named Lamtor5, has been well characterized as a transcriptional coactivator in various cancers. However, the role of Hbxip in normal development remains unexplored. Here, we demonstrated that homozygous knockout of Hbxip leads to embryonic lethality, with retarded growth around E7.5. Using Hbxip knockout embryonic stem cells (ESCs), we showed that depletion of Hbxip compromises the self-renewal of ESCs, with reduced expression of pluripotency genes, reduced cell proliferation, and decreased colony forming capacity. In addition, Hbxip-/- ESCs are defective in differentiation, particularly ectodermal and mesodermal differentiation. Consistently, Hbxip-/- epiblast fails to differentiate properly, indicated by sustained expression of Oct4 in E8.5 Hbxip-/- epiblast. Mechanistically, in ESCs, Hbxip interacts with other components of the Ragulator complex, which is required for mTORC1 activation by amino acids. Importantly, ESCs depleted of Ragulator subunits, Lamtor3 or Lamtor4, display differentiation defects similar to those of Hbxip-/- ESCs. Moreover, Hbxip-/-, p14-/-, and p18-/- mice, lacking subunits of the Ragulator complex, also share similar phenotypes, embryonic lethality and retarded growth around E7-8. Thus, we conclude that Hbxip plays a pivotal role in the development and differentiation of the epiblast, as well as the self-renewal and differentiation of ESCs, through activating mTORC1 signaling.

developmental biology↗