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1.
Cell ; 185(26): 4954-4970.e20, 2022 12 22.
Article in English | MEDLINE | ID: mdl-36493774

ABSTRACT

Nuclear pore complexes (NPCs) are channels for nucleocytoplasmic transport of proteins and RNAs. However, it remains unclear whether composition, structure, and permeability of NPCs dynamically change during the cleavage period of vertebrate embryos and affect embryonic development. Here, we report that the comprehensive NPC maturity (CNM) controls the onset of zygotic genome activation (ZGA) during zebrafish early embryogenesis. We show that more nucleoporin proteins are recruited to and assembled into NPCs with development, resulting in progressive increase of NPCs in size and complexity. Maternal transcription factors (TFs) transport into nuclei more efficiently with increasing CNM. Deficiency or dysfunction of Nup133 or Ahctf1/Elys impairs NPC assembly, maternal TFs nuclear transport, and ZGA onset, while nup133 overexpression promotes these processes. Therefore, CNM may act as a molecular timer for ZGA by controlling nuclear transport of maternal TFs that reach nuclear concentration thresholds at a given time to initiate ZGA.


Subject(s)
Nuclear Pore , Zebrafish , Animals , Embryonic Development/genetics , Gene Expression Regulation, Developmental , Nuclear Pore/metabolism , Nuclear Pore Complex Proteins/genetics , Nuclear Pore Complex Proteins/metabolism , Transcription Factors/metabolism , Zebrafish/metabolism , Zygote/metabolism , Genome
2.
Cell ; 174(4): 818-830.e11, 2018 08 09.
Article in English | MEDLINE | ID: mdl-30057113

ABSTRACT

Rtt109 is a unique histone acetyltransferase acetylating histone H3 lysine 56 (H3K56), a modification critical for DNA replication-coupled nucleosome assembly and genome stability. In cells, histone chaperone Asf1 is essential for H3K56 acetylation, yet the mechanisms for H3K56 specificity and Asf1 requirement remain unknown. We have determined the crystal structure of the Rtt109-Asf1-H3-H4 complex and found that unwinding of histone H3 αN, where K56 is normally located, and stabilization of the very C-terminal Ɵ strand of histone H4 by Asf1 are prerequisites for H3K56 acetylation. Unexpectedly, an interaction between Rtt109 and the central helix of histone H3 is also required. The observed multiprotein, multisite substrate recognition mechanism among histone modification enzymes provides mechanistic understandings of Rtt109 and Asf1 in H3K56 acetylation, as well as valuable insights intoĀ substrate recognition by histone modification enzymes in general.


Subject(s)
Aspergillus fumigatus/metabolism , Histone Acetyltransferases/metabolism , Histones/chemistry , Lysine/metabolism , Molecular Chaperones/metabolism , Acetylation , Amino Acid Sequence , Histone Acetyltransferases/chemistry , Histones/metabolism , Lysine/chemistry , Molecular Chaperones/chemistry , Protein Conformation , Saccharomyces cerevisiae/growth & development , Saccharomyces cerevisiae/metabolism , Sequence Homology , Substrate Specificity
3.
Cell ; 174(1): 72-87.e32, 2018 06 28.
Article in English | MEDLINE | ID: mdl-29861175

ABSTRACT

Recent reports indicate that hypoxia influences the circadian clock through the transcriptional activities of hypoxia-inducible factors (HIFs) at clock genes. Unexpectedly, we uncover a profound disruption of the circadian clock and diurnal transcriptome when hypoxic cells are permitted to acidify to recapitulate the tumor microenvironment. Buffering against acidification or inhibiting lactic acid production fully rescues circadian oscillation. Acidification of several human and murine cell lines, as well as primary murine TĀ cells, suppresses mechanistic target of rapamycin complex 1 (mTORC1) signaling, a key regulator of translation in response to metabolic status. We find that acid drives peripheral redistribution of normally perinuclear lysosomes away from perinuclear RHEB, thereby inhibiting the activity of lysosome-bound mTOR. Restoring mTORC1 signaling and the translation it governs rescues clock oscillation. Our findings thus reveal a model in whichĀ acid produced during the cellular metabolic response to hypoxia suppresses the circadian clock through diminished translation of clock constituents.


Subject(s)
Cell Hypoxia , Circadian Clocks , Mechanistic Target of Rapamycin Complex 1/metabolism , Adaptor Proteins, Signal Transducing , Amino Acids, Dicarboxylic/pharmacology , Animals , CLOCK Proteins/metabolism , Carrier Proteins/antagonists & inhibitors , Carrier Proteins/genetics , Carrier Proteins/metabolism , Cell Cycle Proteins , Cells, Cultured , Circadian Clocks/drug effects , Culture Media/chemistry , Eukaryotic Initiation Factors , Hydrogen-Ion Concentration , Hypoxia-Inducible Factor 1, alpha Subunit/antagonists & inhibitors , Hypoxia-Inducible Factor 1, alpha Subunit/genetics , Hypoxia-Inducible Factor 1, alpha Subunit/metabolism , Lysosomes/metabolism , Mechanistic Target of Rapamycin Complex 1/antagonists & inhibitors , Mice , Phosphoproteins/antagonists & inhibitors , Phosphoproteins/genetics , Phosphoproteins/metabolism , RNA Interference , RNA, Small Interfering/metabolism , Ras Homolog Enriched in Brain Protein/metabolism , Signal Transduction/drug effects , T-Lymphocytes/cytology , T-Lymphocytes/metabolism , Transcriptome/drug effects , Tuberous Sclerosis Complex 2 Protein/deficiency , Tuberous Sclerosis Complex 2 Protein/genetics
4.
Immunity ; 56(5): 1013-1026.e6, 2023 05 09.
Article in English | MEDLINE | ID: mdl-36944334

ABSTRACT

Sepsis is a dysregulated inflammatory consequence of systemic infection. As a result, excessive platelet activation leads to thrombosis and coagulopathy, but we currently lack sufficient understanding of these processes. Here, using the cecal ligation and puncture (CLP) model of sepsis, we observed septic thrombosis and neutrophil extracellular trap formation (NETosis) within the mouse vasculature by intravital microscopy. STING activation in platelets was a critical driver of sepsis-induced pathology. Platelet-specific STING deficiency suppressed platelet activation and granule secretion, which alleviated sepsis-induced intravascular thrombosis and NETosis in mice. Mechanistically, sepsis-derived cGAMP promoted the binding of STING to STXBP2, the assembly of SNARE complex, granule secretion, and subsequent septic thrombosis, which probably depended on the palmitoylation of STING. We generated a peptide, C-ST5, to block STING binding to STXBP2. Septic mice treated with C-ST5 showed reduced thrombosis. Overall, platelet activation via STING reveals a potential strategy for limiting life-threatening sepsis-mediated coagulopathy.


Subject(s)
Extracellular Traps , Sepsis , Thrombosis , Animals , Mice , Blood Platelets/metabolism , Extracellular Traps/metabolism , Mice, Inbred C57BL , Munc18 Proteins/metabolism , Platelet Activation , Sepsis/metabolism , Thrombosis/metabolism
5.
Genes Dev ; 36(7-8): 408-413, 2022 04 01.
Article in English | MEDLINE | ID: mdl-35393344

ABSTRACT

Chaperones influence histone conformation and intermolecular interaction in multiprotein complexes, and the structures obtained with full-length histones often provide more accurate and comprehensive views. Here, our structure of the Hat1-Hat2 acetyltransferase complex bound to Asf1-H3-H4 shows that the core domains of H3 and H4 are involved in binding Hat1 and Hat2, and the N-terminal tail of H3 makes extensive interaction with Hat2. These findings expand the knowledge about histone-protein interaction and implicate a function of Hat2/RbAp46/48, which is a versatile histone chaperone found in many chromatin-associated complexes, in the passing of histones between chaperones.


Subject(s)
Histone Acetyltransferases , Histones , Acetylation , Cell Cycle Proteins/metabolism , Chromatin , Histone Acetyltransferases/metabolism , Histone Chaperones/metabolism , Histones/metabolism , Molecular Chaperones/genetics
6.
Nature ; 615(7952): 526-534, 2023 03.
Article in English | MEDLINE | ID: mdl-36890225

ABSTRACT

The nucleolus is the most prominent membraneless condensate in the nucleus. It comprises hundreds of proteins with distinct roles in the rapid transcription of ribosomal RNA (rRNA) and efficient processing within units comprising a fibrillar centre and a dense fibrillar component and ribosome assembly in a granular component1. The precise localization of most nucleolar proteins and whether their specific localization contributes to the radial flux of pre-rRNA processing have remained unknown owing to insufficient resolution in imaging studies2-5. Therefore, how these nucleolar proteins are functionally coordinated with stepwise pre-rRNA processing requires further investigation. Here we screened 200 candidate nucleolar proteins using high-resolution live-cell microscopy and identified 12 proteins that are enriched towards the periphery of the dense fibrillar component (PDFC). Among these proteins, unhealthy ribosome biogenesis 1 (URB1) is a static, nucleolar protein that ensures 3' end pre-rRNA anchoring and folding for U8 small nucleolar RNA recognition and the subsequent removal of the 3' external transcribed spacer (ETS) at the dense fibrillar component-PDFC boundary. URB1 depletion leads to a disrupted PDFC, uncontrolled pre-rRNA movement, altered pre-rRNA conformation and retention of the 3' ETS. These aberrant 3' ETS-attached pre-rRNA intermediates activate exosome-dependent nucleolar surveillance, resulting in decreased 28S rRNA production, head malformations in zebrafish and delayed embryonic development in mice. This study provides insight into functional sub-nucleolar organization and identifies a physiologically essential step in rRNA maturation that requires the static protein URB1 in the phase-separated nucleolus.


Subject(s)
Cell Nucleolus , Exosomes , RNA Precursors , RNA Processing, Post-Transcriptional , RNA, Ribosomal , Zebrafish , Animals , Mice , Cell Nucleolus/metabolism , Embryonic Development , Exosomes/metabolism , Head/abnormalities , Microscopy , Nuclear Proteins/metabolism , RNA Precursors/metabolism , RNA, Ribosomal/genetics , RNA, Ribosomal/metabolism , RNA, Ribosomal, 28S/metabolism , Zebrafish/genetics , Zebrafish/metabolism
7.
Nature ; 621(7980): 840-848, 2023 Sep.
Article in English | MEDLINE | ID: mdl-37674084

ABSTRACT

In both cancer and infections, diseased cells are presented to human VƎĀ³9VƎĀ“2 T cells through an 'inside out' signalling process whereby structurally diverse phosphoantigen (pAg) molecules are sensed by the intracellular domain of butyrophilin BTN3A11-4. Here we show how-in both humans and alpaca-multiple pAgs function as 'molecular glues' to promote heteromeric association between the intracellular domains of BTN3A1 and the structurally similar butyrophilin BTN2A1. X-ray crystallography studies visualized that engagement ofĀ BTN3A1 with pAgs forms a composite interface for direct binding to BTN2A1, with various pAg molecules each positioned at the centre of the interface and gluing the butyrophilins with distinct affinities. Our structural insights guided mutagenesis experiments that led to disruption of the intracellular BTN3A1-BTN2A1 association, abolishing pAg-mediated VƎĀ³9VƎĀ“2 T cell activation. Analyses using structure-based molecular-dynamics simulations, 19F-NMR investigations, chimeric receptor engineering and direct measurement of intercellular binding force revealed how pAg-mediated BTN2A1 association drives BTN3A1 intracellular fluctuations outwards in a thermodynamically favourable manner, thereby enabling BTN3A1 to push off from the BTN2A1 ectodomain to initiate T cell receptor-mediated ƎĀ³ĆŽĀ“ T cell activation. Practically, we harnessed the molecular-glue model for immunotherapeutics design, demonstrating chemical principles for developing both small-molecule activators and inhibitors of human ƎĀ³ĆŽĀ“ T cell function.


Subject(s)
Butyrophilins , Lymphocyte Activation , Phosphoproteins , Receptors, Antigen, T-Cell, gamma-delta , T-Lymphocytes , Animals , Humans , Antigens, CD/immunology , Antigens, CD/metabolism , Butyrophilins/immunology , Butyrophilins/metabolism , Camelids, New World/immunology , Molecular Dynamics Simulation , Phosphoproteins/immunology , Phosphoproteins/metabolism , Receptors, Antigen, T-Cell, gamma-delta/immunology , Receptors, Antigen, T-Cell, gamma-delta/metabolism , T-Lymphocytes/cytology , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , Crystallography, X-Ray , Nuclear Magnetic Resonance, Biomolecular , Thermodynamics
8.
Nature ; 608(7923): 626-631, 2022 08.
Article in English | MEDLINE | ID: mdl-35896743

ABSTRACT

Emissions of the critical ozone-depleting and greenhouse gas nitrous oxide (N2O) from soils and industrial processes have increased considerably over the last decades1-3. As the final step of bacterial denitrification, N2O is reduced to chemically inert N2 (refs. 1,4) in a reaction that is catalysed by the copper-dependent nitrous oxide reductase (N2OR) (ref. 5). The assembly of its unique [4Cu:2S] active site cluster CuZ requires both the ATP-binding-cassette (ABC) complex NosDFY and the membrane-anchored copper chaperone NosL (refs. 4,6). Here we report cryo-electron microscopy structures of Pseudomonas stutzeri NosDFY and its complexes with NosL and N2OR, respectively. We find that the periplasmic NosD protein contains a binding site for a Cu+ ion and interacts specifically with NosL in its nucleotide-free state, whereas its binding to N2OR requires a conformational change that is triggered by ATP binding. Mutually exclusive structures of NosDFY in complex with NosL and with N2OR reveal a sequential metal-trafficking and assembly pathway for a highly complex copper site. Within this pathway, NosDFY acts as a mechanical energy transducer rather than as a transporter. It links ATP hydrolysis in the cytoplasm to a conformational transition of the NosD subunit in the periplasm, which is required for NosDFY to switch its interaction partner so that copper ions are handed over from the chaperone NosL to the enzyme N2OR.


Subject(s)
Bacterial Proteins , Cryoelectron Microscopy , Nitrous Oxide , Oxidoreductases , Pseudomonas stutzeri , Adenosine Triphosphate/metabolism , Bacterial Proteins/chemistry , Bacterial Proteins/metabolism , Bacterial Proteins/ultrastructure , Binding Sites , Copper/chemistry , Copper/metabolism , Cytoplasm/enzymology , Molecular Chaperones/metabolism , Nitrous Oxide/metabolism , Oxidoreductases/chemistry , Oxidoreductases/metabolism , Oxidoreductases/ultrastructure , Periplasm/enzymology , Protein Binding , Protein Conformation , Pseudomonas stutzeri/cytology , Pseudomonas stutzeri/enzymology
9.
Nature ; 601(7892): 245-251, 2022 01.
Article in English | MEDLINE | ID: mdl-34912119

ABSTRACT

Pattern-triggered immunity (PTI) and effector-triggered immunity (ETI) in plants enable them to respond to pathogens by activating the production of defence metabolites that orchestrate immune responses1-4. How the production of defence metabolites is promoted by immune receptors and coordinated with broad-spectrum resistance remains elusive. Here we identify the deubiquitinase PICI1 as an immunity hub for PTI and ETI in rice (Oryza sativa). PICI1 deubiquitinates and stabilizes methionine synthetases to activate methionine-mediated immunity principally through biosynthesis of the phytohormone ethylene. PICI1 is targeted for degradation by blast fungal effectors, including AvrPi9, to dampen PTI. Nucleotide-binding domain, leucine-rich-repeat-containing receptors (NLRs) in the plant immune system, such as PigmR, protect PICI1 from effector-mediated degradation to reboot the methionine-ethylene cascade. Natural variation in the PICI1 gene contributes to divergence in basal blast resistance between the rice subspecies indica and japonica. Thus, NLRs govern an arms race with effectors, using a competitive mode that hinges on a critical defence metabolic pathway to synchronize PTI with ETI and ensure broad-spectrum resistance.


Subject(s)
Oryza , Plant Diseases , Methionine , Oryza/genetics , Oryza/microbiology , Plant Diseases/microbiology , Plant Immunity/genetics , Plants , Signal Transduction/genetics
10.
Plant Cell ; 36(2): 383-403, 2024 Jan 30.
Article in English | MEDLINE | ID: mdl-37847118

ABSTRACT

The Casparian strip (CS) is a ring-like lignin structure deposited between endodermal cells that forms an apoplastic barrier to control the selective uptake of nutrients in vascular plants. However, the molecular mechanism of CS formation in rice (Oryza sativa), which possesses one CS each in the endodermis and exodermis, is relatively unknown. Here, we functionally characterized CS INTEGRITY FACTOR1 (OsCIF1a, OsCIF1b), OsCIF2, and SCHENGEN3 (OsSGN3a, OsSGN3b) in rice. OsCIF1s and OsCIF2 were mainly expressed in the stele, while OsSGN3s localized around the CS at the endodermis. Knockout of all three OsCIFs or both OsSGN3s resulted in a discontinuous CS and a dramatic reduction in compensatory (less localized) lignification and suberization at the endodermis. By contrast, ectopic overexpression of OsCIF1 or OsCIF2 induced CS formation as well as overlignification and oversuberization at single or double cortical cell layers adjacent to the endodermis. Ectopic co-overexpression of OsCIF1 and SHORTROOT1 (OsSHR1) induced the formation of more CS-like structures at multiple cortical cell layers. Transcriptome analysis identified 112 downstream genes modulated by the OsCIF1/2-OsSGN3 signaling pathway, which is involved in CS formation and activation of the compensatory machinery in native endodermis and nonnative endodermis-like cell layers. Our results provide important insights into the molecular mechanism of CIF-mediated CS formation at the root endodermal and nonendodermal cell layers.


Subject(s)
Arabidopsis , Oryza , Arabidopsis/genetics , Oryza/genetics , Plant Roots/metabolism , Cell Wall/metabolism , Peptides/metabolism , Signal Transduction/genetics
11.
Plant Cell ; 36(10): 4143-4167, 2024 Oct 03.
Article in English | MEDLINE | ID: mdl-38963884

ABSTRACT

As an essential intrinsic component of photosystem II (PSII) in all oxygenic photosynthetic organisms, heme-bridged heterodimer cytochrome b559 (Cyt b559) plays critical roles in the protection and assembly of PSII. However, the underlying mechanisms of Cyt b559 assembly are largely unclear. Here, we characterized the Arabidopsis (Arabidopsis thaliana) rph1 (resistance to Phytophthora1) mutant, which was previously shown to be susceptible to the oomycete pathogen Phytophthora brassicae. Loss of RPH1 leads to a drastic reduction in PSII accumulation, which can be primarily attributed to the defective formation of Cyt b559. Spectroscopic analyses showed that the heme level in PSII supercomplexes isolated from rph1 is significantly reduced, suggesting that RPH1 facilitates proper heme assembly in Cyt b559. Due to the loss of RPH1-mediated processes, a covalently bound PsbE-PsbF heterodimer is formed during the biogenesis of PSII. In addition, rph1 is highly photosensitive and accumulates elevated levels of reactive oxygen species under photoinhibitory-light conditions. RPH1 is a conserved intrinsic thylakoid protein present in green algae and terrestrial plants, but absent in Synechocystis, and it directly interacts with the subunits of Cyt b559. Thus, our data demonstrate that RPH1 represents a chloroplast acquisition specifically promoting the efficient assembly of Cyt b559, probably by mediating proper heme insertion into the apo-Cyt b559 during the initial phase of PSII biogenesis.


Subject(s)
Arabidopsis Proteins , Arabidopsis , Cytochrome b Group , Photosystem II Protein Complex , Phytophthora , Arabidopsis/genetics , Arabidopsis/metabolism , Photosystem II Protein Complex/metabolism , Photosystem II Protein Complex/genetics , Arabidopsis Proteins/metabolism , Arabidopsis Proteins/genetics , Cytochrome b Group/metabolism , Cytochrome b Group/genetics , Plant Diseases/microbiology , Reactive Oxygen Species/metabolism , Heme/metabolism , Mutation/genetics , Thylakoids/metabolism
12.
Mol Cell ; 74(5): 996-1009.e7, 2019 06 06.
Article in English | MEDLINE | ID: mdl-30975460

ABSTRACT

Nucleotide-binding site leucine-rich repeat (NLR) receptors perceive pathogen effectors and trigger plant immunity. However, the mechanisms underlying NLR-triggered defense responses remain obscure. The recently discovered Pigm locus in rice encodes a cluster of NLRs, including PigmR, which confers broad-spectrum resistance to blast fungus. Here, we identify PIBP1 (PigmR-INTERACTING and BLAST RESISTANCE PROTEIN 1), an RRM (RNA-recognition motif) protein that specifically interacts with PigmR and other similar NLRs to trigger blast resistance. PigmR-promoted nuclear accumulation of PIBP1 ensures full blast resistance. We find that PIBP1 and a homolog, Os06 g02240, bind DNA and function as unconventional transcription factors at the promoters of the defense genes OsWAK14 and OsPAL1, activating their expression. Knockout of PIBP1 and Os06 g02240 greatly attenuated blast resistance. Collectively, our study discovers previously unappreciated RRM transcription factors that directly interact with NLRs to activate plant defense, establishing a direct link between transcriptional activation of immune responses with NLR-mediated pathogen perception.


Subject(s)
Disease Resistance/genetics , NLR Proteins/genetics , Oryza/genetics , Plant Diseases/genetics , Plant Proteins/genetics , Transcription Factors/genetics , Binding Sites , Fungi/pathogenicity , Gene Expression Regulation, Plant , Oryza/microbiology , Plant Diseases/microbiology , Plant Immunity/genetics , Promoter Regions, Genetic , Protein Binding/genetics , Signal Transduction/genetics
13.
Proc Natl Acad Sci U S A ; 121(16): e2313820121, 2024 Apr 16.
Article in English | MEDLINE | ID: mdl-38598343

ABSTRACT

In primates, high-acuity vision is mediated by the fovea, a small specialized central region of the retina. The fovea, unique to the anthropoid lineage among mammals, undergoes notable neuronal morphological changes during postnatal maturation. However, the extent of cellular similarity across anthropoid foveas and the molecular underpinnings of foveal maturation remain unclear. Here, we used high-throughput single-cell RNA sequencing to profile retinal cells of the common marmoset (Callithrix jacchus), an early divergent in anthropoid evolution from humans, apes, and macaques. We generated atlases of the marmoset fovea and peripheral retina for both neonates and adults. Our comparative analysis revealed that marmosets share almost all their foveal types with both humans and macaques, highlighting a conserved cellular structure among primate foveas. Furthermore, by tracing the developmental trajectory of cell types in the foveal and peripheral retina, we found distinct maturation paths for each. In-depth analysis of gene expression differences demonstrated that cone photoreceptors and MĆ¼ller glia (MG), among others, show the greatest molecular divergence between these two regions. Utilizing single-cell ATAC-seq and gene-regulatory network inference, we uncovered distinct transcriptional regulations differentiating foveal cones from their peripheral counterparts. Further analysis of predicted ligand-receptor interactions suggested a potential role for MG in supporting the maturation of foveal cones. Together, these results provide valuable insights into foveal development, structure, and evolution.


Subject(s)
Callithrix , Retina , Humans , Animals , Infant, Newborn , Callithrix/anatomy & histology , Retina/metabolism , Fovea Centralis/physiology , Retinal Cone Photoreceptor Cells , Macaca , Mammals
14.
Proc Natl Acad Sci U S A ; 121(34): e2404199121, 2024 Aug 20.
Article in English | MEDLINE | ID: mdl-39136985

ABSTRACT

Low phosphate (Pi) availability decreases photosynthesis, with phosphate limitation of photosynthesis occurring particularly during grain filling of cereal crops; however, effective genetic solutions remain to be established. We previously discovered that rice phosphate transporter OsPHO1;2 controls seed (sink) development through Pi reallocation during grain filling. Here, we find that OsPHO1;2 regulates Pi homeostasis and thus photosynthesis in leaves (source). Loss-of-function of OsPHO1;2 decreased Pi levels in leaves, leading to decreased photosynthetic electron transport activity, CO2 assimilation rate, and early occurrence of phosphate-limited photosynthesis. Interestingly, ectopic expression of OsPHO1;2 greatly increased Pi availability, and thereby, increased photosynthetic rate in leaves during grain filling, contributing to increased yield. This was supported by the effect of foliar Pi application. Moreover, analysis of core rice germplasm resources revealed that higher OsPHO1;2 expression was associated with enhanced photosynthesis and yield potential compared to those with lower expression. These findings reveal that phosphate-limitation of photosynthesis can be relieved via a genetic approach, and the OsPHO1;2 gene can be employed to reinforce crop breeding strategies for achieving higher photosynthetic efficiency.


Subject(s)
Oryza , Phosphates , Photosynthesis , Oryza/genetics , Oryza/metabolism , Oryza/growth & development , Phosphates/metabolism , Plant Leaves/metabolism , Plant Leaves/genetics , Plant Proteins/genetics , Plant Proteins/metabolism , Gene Expression Regulation, Plant , Phosphate Transport Proteins/genetics , Phosphate Transport Proteins/metabolism , Plants, Genetically Modified
15.
Genome Res ; 33(10): 1818-1832, 2023 10.
Article in English | MEDLINE | ID: mdl-37730437

ABSTRACT

The subventricular zone (SVZ) is a neurogenic niche that contributes to homeostasis and repair after brain injury. However, the effects of mild traumatic brain injury (mTBI) on the divergence of the regulatory DNA landscape within the SVZ and its link to functional alterations remain unexplored. In this study, we mapped the transcriptome atlas of murine SVZ and its responses to mTBI at the single-cell level. We observed cell-specific gene expression changes following mTBI and unveiled diverse cell-to-cell interaction networks that influence a wide array of cellular processes. Moreover, we report novel neurogenesis lineage trajectories and related key transcription factors, which we validate through loss-of-function experiments. Specifically, we validate the role of Tcf7l1, a cell cycle gene regulator, in promoting neural stem cell differentiation toward the neuronal lineage after mTBI, providing a potential target for regenerative medicine. Overall, our study profiles an SVZ transcriptome reference map, which underlies the differential cellular behavior in response to mTBI. The identified key genes and pathways that may ameliorate brain damage or facilitate neural repair serve as a comprehensive resource for drug discovery in the context of mTBI.


Subject(s)
Brain Injuries, Traumatic , Neural Stem Cells , Animals , Mice , Transcriptome , Neural Stem Cells/metabolism , Neurons , Cell Differentiation , Neurogenesis/physiology , Brain Injuries, Traumatic/genetics , Brain Injuries, Traumatic/metabolism
16.
Genome Res ; 33(10): 1690-1707, 2023 10.
Article in English | MEDLINE | ID: mdl-37884341

ABSTRACT

The rumen undergoes developmental changes during maturation. To characterize this understudied dynamic process, we profiled single-cell transcriptomes of about 308,000 cells from the rumen tissues of sheep and goats at 17 time points. We built comprehensive transcriptome and metagenome atlases from early embryonic to rumination stages, and recapitulated histomorphometric and transcriptional features of the rumen, revealing key transitional signatures associated with the development of ruminal cells, microbiota, and core transcriptional regulatory networks. In addition, we identified and validated potential cross-talk between host cells and microbiomes and revealed their roles in modulating the spatiotemporal expression of key genes in ruminal cells. Cross-species analyses revealed convergent developmental patterns of cellular heterogeneity, gene expression, and cell-cell and microbiome-cell interactions. Finally, we uncovered how the interactions can act upon the symbiotic rumen system to modify the processes of fermentation, fiber digestion, and immune defense. These results significantly enhance understanding of the genetic basis of the unique roles of rumen.


Subject(s)
Metagenome , Microbiota , Sheep/genetics , Animals , Transcriptome , Rumen , Ruminants/genetics
17.
Brief Bioinform ; 25(5)2024 Jul 25.
Article in English | MEDLINE | ID: mdl-39167797

ABSTRACT

Immunotherapy with immune checkpoint inhibitors (ICIs) is increasingly used to treat various tumor types. Determining patient responses to ICIs presents a significant clinical challenge. Although components of the tumor microenvironment (TME) are used to predict patient outcomes, comprehensive assessments of the TME are frequently overlooked. Using a top-down approach, the TME was divided into five layers-outcome, immune role, cell, cellular component, and gene. Using this structure, a neural network called TME-NET was developed to predict responses to ICIs. Model parameter weights and cell ablation studies were used to investigate the influence of TME components. The model was developed and evaluated using a pan-cancer cohort of 948 patients across four cancer types, with Area Under the Curve (AUC) and accuracy as performance metrics. Results show that TME-NET surpasses established models such as support vector machine and k-nearest neighbors in AUC and accuracy. Visualization of model parameter weights showed that at the cellular layer, Th1 cells enhance immune responses, whereas myeloid-derived suppressor cells and M2 macrophages show strong immunosuppressive effects. Cell ablation studies further confirmed the impact of these cells. At the gene layer, the transcription factors STAT4 in Th1 cells and IRF4 in M2 macrophages significantly affect TME dynamics. Additionally, the cytokine-encoding genes IFNG from Th1 cells and ARG1 from M2 macrophages are crucial for modulating immune responses within the TME. Survival data from immunotherapy cohorts confirmed the prognostic ability of these markers, with p-values <0.01. In summary, TME-NET performs well in predicting immunotherapy responses and offers interpretable insights into the immunotherapy process. It can be customized at https://immbal.shinyapps.io/TME-NET.


Subject(s)
Immune Checkpoint Inhibitors , Neoplasms , Tumor Microenvironment , Humans , Immune Checkpoint Inhibitors/therapeutic use , Immune Checkpoint Inhibitors/pharmacology , Tumor Microenvironment/immunology , Neoplasms/immunology , Neural Networks, Computer , Immunotherapy
18.
Nat Chem Biol ; 20(8): 1078-1085, 2024 Aug.
Article in English | MEDLINE | ID: mdl-38890433

ABSTRACT

Biological nitrogen fixation requires substantial metabolic energy in form of ATP as well as low-potential electrons that must derive from central metabolism. During aerobic growth, the free-living soil diazotroph Azotobacter vinelandii transfers electrons from the key metabolite NADH to the low-potential ferredoxin FdxA that serves as a direct electron donor to the dinitrogenase reductases. This process is mediated by the RNF complex that exploits the proton motive force over the cytoplasmic membrane to lower the midpoint potential of the transferred electron. Here we report the cryogenic electron microscopy structure of the nitrogenase-associated RNF complex of A. vinelandii, a seven-subunit membrane protein assembly that contains four flavin cofactors and six iron-sulfur centers. Its function requires the strict coupling of electron and proton transfer but also involves major conformational changes within the assembly that can be traced with a combination of electron microscopy and modeling.


Subject(s)
Azotobacter vinelandii , Cryoelectron Microscopy , Nitrogen Fixation , Azotobacter vinelandii/metabolism , Azotobacter vinelandii/enzymology , Models, Molecular , Protein Conformation , Bacterial Proteins/metabolism , Bacterial Proteins/chemistry , Oxidoreductases/metabolism , Oxidoreductases/chemistry
19.
Nucleic Acids Res ; 52(7): 3886-3895, 2024 Apr 24.
Article in English | MEDLINE | ID: mdl-38324471

ABSTRACT

The eukaryotic epigenetic modifications 5-methyldeoxycytosine (5mC) and N6-methyldeoxyadenine (6mA) have indispensable regulatory roles in gene expression and embryonic development. We recently identified an atypical bifunctional dioxygenase CcTet from Coprinopsis cinerea that works on both 5mC and 6mA demethylation. The nonconserved residues Gly331 and Asp337 of CcTet facilitate 6mA accommodation, while D337F unexpectedly abolishes 5mC oxidation activity without interfering 6mA demethylation, indicating a prominent distinct but unclear 5mC oxidation mechanism to the conventional Tet enzymes. Here, we assessed the molecular mechanism of CcTet in catalyzing 5mC oxidation by representing the crystal structure of CcTet-5mC-dsDNA complex. We identified the distinct mechanism by which CcTet recognizes 5mC-dsDNA compared to 6mA-dsDNA substrate. Moreover, Asp337 was found to have a central role in compensating for the loss of a critical 5mC-stablizing H-bond observed in conventional Tet enzymes, and stabilizes 5mC and subsequent intermediates through an H-bond with the N4 atom of the substrates. These findings improve our understanding of Tet enzyme functions in the dsDNA 5mC and 6mA demethylation pathways, and provide useful information for future discovery of small molecular probes targeting Tet enzymes in DNA active demethylation processes.


Subject(s)
Agaricales , Dioxygenases , 5-Methylcytosine/metabolism , Crystallography, X-Ray , Dioxygenases/chemistry , Dioxygenases/genetics , Dioxygenases/metabolism , DNA Demethylation , DNA Methylation , Fungal Proteins/metabolism , Fungal Proteins/genetics , Fungal Proteins/chemistry , Hydrogen Bonding , Models, Molecular , Oxidation-Reduction , Substrate Specificity , Adenosine/analogs & derivatives , Agaricales/enzymology
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