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Gao, S.

Publications and source records attributed to Gao, S..

At least 109 records · Page 6Linked to original sources

Identification and characterization of the de novo methyltransferases for eukaryotic N6-methyladenine (6mA)

DNA N6-adenine methylation (6mA) is rediscovered as an important epigenetic mark in eukaryotes. We previously identified the MT-A70 family member AMT1 as the maintenance methyltransferase (MTase), but the identity of the de novo MTase(s) for 6mA establishment remains unidentified. The unicellular model eukaryote Tetrahymena thermophila offers a unique opportunity for studying de novo methylation: during sexual reproduction, the 6mA-free zygotic nucleus develops into 6mA-positive new macronucleus (MAC) marked by genome-wide de novo methylation. Here we identified that TET1 and TET2 are essential for de novo 6mA in the new MAC, but not for maintenance 6mA in the somatic MAC. Knockout of TET1 and/or TET2 not only abolished methylation on most methylatable ApT positions, but also attenuated methylation on the rest. The substantial 6mA decrease in turn altered gene expression pattern and reduced the survival rate of sexual progenies, highlighting the importance of dedicated de novo MTase activity.

molecular biology↗

Bioorthogonal labeling and enrichment of histone monoaminylation reveal its accumulation and regulatory function in cancer cell chromatin

Histone monoaminylation (i.e., serotonylation and dopaminylation) is an emerging category of epigenetic mark occurring on the fifth glutamine (Q5) residue of H3 N-terminal tail, which plays significant roles in gene transcription. Current analysis of histone monoaminylation is mainly based on site-specific antibodies and mass spectrometry, which either lacks high resolution or is time-consuming. In this study, we report the development of chemical probes for bioorthogonal labeling and enrichment of histone serotonylation and dopaminylation. These probes were successfully applied for the monoaminylation analysis of in vitro biochemical assays, cells, and tissue samples. The enrichment of monoaminylated histones by the probes further confirmed the crosstalk between H3Q5 monoaminylation and H3K4 methylation. Finally, combining the ex vivo and in vitro analyses based on the developed probes, we have shown that both histone serotonylation and dopaminylation are highly enriched in tumor tissues that overexpress transglutaminase 2 (TGM2) and regulate the three-dimensional architecture of cellular chromatin. TOC O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=154 SRC="FIGDIR/small/586010v3_ufig1.gif" ALT="Figure 1"> View larger version (47K): org.highwire.dtl.DTLVardef@1bee3f7org.highwire.dtl.DTLVardef@1687013org.highwire.dtl.DTLVardef@1960bd0org.highwire.dtl.DTLVardef@1e19a04_HPS_FORMAT_FIGEXP M_FIG C_FIG

biochemistry↗

HER2 Prognostic Significance and Therapeutic Implications in Gastric Cardia Adenocarcinoma: Insights from Multi-Omics Profiling

Therapeutic targets for Gastric Cardia Adenocarcinoma (GCA), particularly for HER2- negative patients, are lacking. Here, we conducted multi-omics profiling on 128 GCA patients using mass spectrometry, whole-exome sequencing, RNA-Seq, and metabolomics. We found HER2 to be a favorable prognostic marker for GCA. Employing molecular counting, we categorized patients into HER2-high, -low, and -negative groups. We uncovered an enrichment of DNA repair features in the HER2-high group, while HER2-low and -negative groups exhibited strong inflammation. We found that tumor mutation burden may not be the distinguishing factor among these three groups. We revealed that the HER2-negative and -low groups have a tumor-suppressive immune microenvironment, and HER2 expression is associated with fatty acid metabolic profiles and inflammation in the blood of patients. Our study revealed anti-inflammatory and immune checkpoint inhibition, targeting PD-L2 and the CD47/SIRPA pair, as therapeutic strategies for HER2-negative GCA patients. These findings highlight promising avenues for personalized treatment for GCA.

cancer biology↗

Near telomere-to-telomere genome assemblies of two Chlorella species unveil the composition and evolution of centromeres in green algae

BackgroundCentromeres play a crucial and conserved role in cell division, although their composition and evolutionary history in green algae, the evolutionary ancestors of land plants, remains largely unknown. ResultsWe constructed near telomere-to-telomere (T2T) assemblies for two Trebouxiophyceae species, Chlorella sorokiniana NS4-2 and Chlorella pyrenoidosa DBH, with chromosome numbers of 12 and 13, and genome sizes of 58.11 Mb and 53.41 Mb, respectively. We identified and validated their centromere sequences using CENH3 ChIP-seq and found that, similar to humans and higher plants, the centromeric CENH3 signals of green algae display a pattern of hypomethylation. Interestingly, the centromeres of both species largely comprised transposable elements, although they differed significantly in their composition. Species within the Chlorella genus display a more diverse centromere composition, with major constituents including members of the LTR/Copia, LINE/L1, and LINE/RTEX families. This is in contrast to green algae including Chlamydomonas reinhardtii, Coccomyxa subellipsoidea, and Chromochloris zofingiensis, in which centromere composition instead has a pronounced single-element composition. Moreover, we observed significant differences in the composition and structure of centromeres among chromosomes with strong collinearity within the Chlorella genus, suggesting that centromeric sequence evolves more rapidly than sequence in non-centromeric regions. ConclusionsThis study not only provides high-quality genome data for comparative genomics of green algae but gives insight into the composition and evolutionary history of centromeres in early plants, laying an important foundation for further research on their evolution.

genomics↗

Enhanced microbiota profiling in patients with quiescent Crohn's disease through comparison with paired healthy first-degree relatives using fecal metagenomics and metabolomics

Prior studies indicate no correlation between gut microbiota of healthy first-degree relatives (HFDRs) of Crohns disease (CD) patients and development of CD. Here, we utilized HFDRs as controls to examine the microbiota and metabolome in individuals with active (CD-A) and quiescent (CD-R) CD, thereby minimizing the influence of genetic and environmental factors. Compared to non-relative controls, the use of HFDR controls identified fewer differential taxa. Faecalibacterium, Dorea, and Fusicatenibacter showed decreased abundances in CD-R, independent of inflammation, and correlated with fecal SCFAs. Validation with a large multi-center cohort confirmed decreased abundances in Faecalibacterium and other SCFA-producing genera in CD-R. Classification models based on these genera distinguished CD-R and CD-A from healthy individuals, in both the discovery and validation cohorts. Thus, the decreased presence of Faecalibacterium, Dorea, and Fusicatenibacter in CD-R likely contributed to disease relapse through reduced SCFA production, highlighting their potential as diagnostic markers and therapeutic targets for CD.

pathology↗

GhCPK28 negatively regulates the immune response by phosphorylating GhTIFY3b

The soil-borne fungus Verticillium dahliae (V. dahliae) seriously inhibits cotton (Gossypium hirsutum) growth and productivity. The immune system of cotton against this pathogen is largely unknown. Here we investigated the involvement of Ca2+-dependent protein kinases (CPKs) in this immunity. One CPK coding gene GhCPK28 had reduced expression after infection by V. dahliae in cotton. Knocking down of the GhCPK28 by virus induced gene silencing led to enhanced resistance to V. dahliae which is accompanied by a higher ROS accumulation and systemic acquired response. GhCPK28 was found to phosphorylate a TIFY family protein GhTIFY3b and reduce its stability. Silencing GhTIFY3b increased the susceptibility of cotton to V. dahliae. Together, this study indicate that GhCPK28 is a negative regulator of defense against V. dahliae infection and the GhTIFY3b might be a target protein of its activity. It sheds light on the immune system against V. dahliae and provides candidate genes for improving resistance against Verticillium wilt in cotton.

plant biology↗

Self-regulation of the DNA N6-adenine methyltransferase AMT1 in the unicellular eukaryote Tetrahymena thermophila

DNA N6-adenine methylation (6mA) is involved in gene transcription as a potential epigenetic mark in eukaryotes. Despite the reported methyltransferase (MTase) for 6mA methylation in several eukaryotes, the regulatory mechanisms that govern the activity of 6mA MTase remain elusive. Here, we exploited the 6mA MTase AMT1 to elucidate its self-regulation in the unicellular eukaryote Tetrahymena thermophila. Firstly, detailed endogenous localization of AMT1 was delineated both in vegetative and sexual stages, revealing a correlation between the 6mA reestablishment in the new MAC and the occurrence of zygotically expressed AMT1. Catalytically inactive AMT1 reduced 6mA level on the AMT1 gene and its expression level, suggesting that AMT1 modulated its own transcription via 6mA. Furthermore, AMT1-dependent 6mA regulated the transcription of its target genes thus affecting the cell fitness, as demonstrated by manipulating the dosage of AMT1 using AMT1-RNAi strains. Our findings unveil a positive feedback loop of transcriptional activation on the AMT1 gene and highlight the crucial role of AMT1-dependent 6mA for gene transcription.

genetics↗

PackDock: a Diffusion Based Side Chain Packing Model for Flexible Protein-Ligand Docking

Structure-based drug design (SBDD) relies on accurate knowledge of protein structure and ligand-binding conformations. However, most of the static conformations obtained by advanced methods such as structural biology and de novo protein folding algorithms often dont meet the needs for drug design. We introduce PackDock, a flexible docking method that combines "conformation selection" and "induced fit" mechanisms in a two-stage docking pipeline. The core module of this method is PackPocket, which uses a diffusion model to explore the side-chain conformation space in ligand binding pockets, both with or without a ligand. We evaluate our method using several tests that reflect real-world application scenarios. (1) Side-chain packing and Re-docking experiments validate the ability of PackDock to predict accurate side-chain conformations and ligand conformations. (2) Cross-docking experiments with apo and non-homologous ligand-induced holo structures align with real docking scenarios, demonstrating PackDocks practical value. (3) Docking experiments with hypothetical models show that PackPocket can potentially conduct SBDD starting from protein sequence information only. Additionally, we found that PackDock can identify key amino acid conformation changes, which may provide insights for lead compound optimization. We demonstrate PackDock can accurately predict the complex conformations in various application scenarios, by combining the conformation selection theory and the induced fit theory, and by using the ability of PackPocket to accurately predict the side chain conformations in the pocket region. We believe this method can improve the usability of existing structures, providing a new perspective for the SBDD community.

bioinformatics↗

Comprehensive genome annotation of the model ciliate Tetrahymena thermophila by in-depth epigenetic and transcriptomic profiling

The ciliate Tetrahymena thermophila is a well-established unicellular model eukaryote, contributing significantly to foundational biological discoveries. Despite its acknowledged importance, current Tetrahymena biology studies face challenges due to gene annotation inaccuracy, particularly the notable absence of untranslated regions (UTRs). To comprehensively annotate the Tetrahymena macronuclear genome, we collected extensive transcriptomic data spanning various cell stages. To ascertain transcript orientation and transcription start/end sites, we incorporated data of epigenetic marks displaying enrichment towards the 5 end of gene bodies, including H3 lysine 4 tri-methylation (H3K4me3), H2A.Z, nucleosomes, and N6-methyldeoxyadenine (6mA). Additionally, we integrated Nanopore direct sequencing (DRS), strand-specific RNA-seq, and ATAC-seq data. Using a newly-developed bioinformatic pipeline, coupled with manual curation and experimental validation, our work yielded substantial improvements to the current gene models, including the addition of 2,481 new genes, updates to 6,257 existing genes, and the incorporation of 5,917 alternatively spliced isoforms. Furthermore, novel UTR information was annotated for 26,223 high-confidence genes. Intriguingly, 16% of protein-coding genes were identified to have natural antisense transcripts (NATs) characterized by high diversity in alternative splicing, thus offering insights into understanding transcriptional regulation. Our work will enhance the utility of Tetrahymena as a robust genetic toolkit for advancing biological research.

bioinformatics↗

Characterization of RNA editing profiles in rice endosperm development

Rice (Oryza sativa L.) endosperm provides nutrients for seed germination and determines grain yield. RNA editing, a post-transcriptional modification essential for plant development, unfortunately, is not fully characterized during rice endosperm development. Here, we conduct genome re-sequencing and RNA sequencing for rice endosperms across five successive developmental stages and perform systematic analyses to characterize RNA editing profiles during rice endosperm development. We find that the majority of their editing sites are C-to-U CDS-recoding in mitochondria, leading to increased hydrophobic amino acids, and affecting structures and functions of mitochondrial proteins. Comparative analysis of RNA editing profiles across the five developmental stages reveals that CDS-recoding sites present higher editing frequencies with lower variabilities, and recoded amino acids, particularly caused by these sites with higher editing frequencies, tend to exhibit stronger evolutionary conservation across many land plants. Based on these results, we further classify mitochondrial genes into three groups that present distinct patterns in terms of editing frequency and variability of CDS-recoding sites. Besides, we identify a series of P- and PLS-class pentatricopeptide repeat (PPR) proteins with editing potential and construct PPR-RNA binding profiles, yielding candidate PPR editing factors related to rice endosperm development. Taken together, our findings provide valuable insights for deciphering fundamental mechanisms of rice endosperm development underlying RNA editing machinery. Author summaryRice endosperm development, a critical process determining quality and yield of our mankinds essential food, is regulated by RNA editing that provokes RNA base alterations by protein factors. However, our understanding of this regulation is incomplete. Hence, we systematically characterize RNA editing profiles during rice endosperm development. We find that editing sites resulting in amino acid changes, called "CDS-recoding", predominate in mitochondria, leading to increased hydrophobic amino acids and affecting structures and functions of proteins. Comparative analysis of RNA editing profiles during rice endosperm development reveals that CDS-recoding sites present higher editing frequencies with lower variabilities. Furthermore, evolutionary conservation of recoded amino acids caused by these CDS-recoding sites is positively correlated with editing frequency across many land plants. We classify mitochondrial genes into three groups that present distinct patterns in terms of editing frequency and variability of CDS-recoding sites, indicating different effects of these genes on rice endosperm development. In addition, we identify candidate protein factors associated closely with RNA editing regulation. To sum up, our findings provide valuable insights for fully understanding the role of RNA editing during rice endosperm development.

bioinformatics↗

Centromere landscapes resolved from hundreds of human genomes

High-fidelity (HiFi) sequencing has facilitated the assembly and analysis of the most repetitive region of the genome, the centromere. Nevertheless, our current understanding of human centromeres draws from a relatively small number of telomere-to-telomere assemblies, and so has not yet captured its full diversity. In this study, we investigated the genomic diversity of human centromere higher order repeats (HORs) using both HiFi reads and haplotype-resolved assemblies from hundreds of samples drawn from ongoing pangenome-sequencing projects and reprocessed using a novel HOR annotation pipeline, HiCAT-human. We use this wealth of data to provide a global survey of the centromeric HOR landscape, in particular finding that 23 HORs exhibited significant copy number variability between populations. We detected three centromere genotypes with imbalance population frequencies on each of chromosome 5, 8 and 17. An inter-assembly comparison of HOR loci further revealed that while HOR array structures are diverse, they nevertheless tend to form a number of specific landscapes, each exhibiting different levels of HOR subunit expansion and possibly reflecting a cyclical evolutionary transition from homogeneous to nested structures and back.

genomics↗

Biochemical and Structural Characterization of Fapy·dG Replication by Human DNA Polymerase β

N6-(2-deoxy-,{beta}-D-erythro-pentofuranosyl)-2,6-diamino-4-hydroxy-5-formamido-pyrimidine (Fapy*dG) is formed from a common intermediate and in comparable amounts to the well-studied mutagenic DNA lesion 8-oxo-7,8-dihydro-2-deoxyguanosine (8-OxodGuo). Fapy*dG preferentially gives rise to G [->] T transversions and G [->] A transitions. However, the molecular basis by which Fapy*dG is processed by DNA polymerases during this mutagenic process remains poorly understood. To address this we investigated how DNA polymerase {beta} (Pol {beta}), a model mammalian polymerase, bypasses a templating Fapy*dG, inserts Fapy*dGTP, and extends from Fapy*dG at the primer terminus. When Fapy*dG is present in the template, Pol {beta} incorporates TMP less efficiently than either dCMP or dAMP. Kinetic analysis revealed that Fapy*dGTP is a poor substrate but is incorporated [~]3-times more efficiently opposite dA than dC. Extension from Fapy*dG at the 3-terminus of a nascent primer is inefficient due to the primer terminus being poorly positioned for catalysis. Together these data indicate that mutagenic bypass of Fapy*dG is likely to be the source of the mutagenic effects of the lesion and not Fapy*dGTP. These experiments increase our understanding of the promutagenic effects of Fapy*dG.

biochemistry↗

Capturing Snapshots of Nucleosomal H2A K13/K15 Ubiquitination Mediated by the Monomeric E3 Ligase RNF168

AbstractsThe DNA damage repair regulatory protein RNF168, a monomeric RING-type E3 ligase, plays a crucial role in regulating cell fate and DNA repair by specific and efficient ubiquitination of the adjacent Lys13 and Lys15 sites at the H2A N-terminal tail. However, understanding how RNF168 coordinates with its cognate E2 enzyme UbcH5c to ubiquitinate H2AK13/15 site-specifically has long been hampered by the lack of high-resolution structures of RNF168 and UbcH5c[~]Ub in complex with nucleosomes. Here, we developed mechanism-based chemical trapping strategies and determined the cryo-EM structures of the RNF168/UbcH5c-Ub/NCP complex captured in transient H2AK13/15 monoubiquitination and adjacent dual-monoubiquitination reactions. Our structural analysis revealed that RNF168 stably binds to the nucleosomal H2A-H2B acidic patch through a basic helix with multiple interactions, which positions the UbcH5c active centre directly over the H2A N-terminus, providing a "helix-anchoring" mode for monomeric E3 ligase RNF168 on nucleosome in contrast to the "compass-binding" mode of dimeric E3 ligases. Furthermore, our chemically synthesized ubiquitinated histones have enabled the elucidation of the efficiency of Ub installation and the interplay between the initial and subsequent Ub modifications on the adjacent H2A K13 and K15 sites. Overall, our work not only provides structural snapshots of H2A K13/K15 site-specific monoubiquitination and adjacent dual-monoubiquitination, but also offers a near-atomic resolution structural framework for understanding how pathogenic mutations or physiological modifications affect the molecular function of RNF168 in H2A K13/15 ubiquitination.

biochemistry↗

Suppression of epileptic seizures by transcranial activation of K+-selective channelrhodopsin

Optogenetics is a valuable tool for studying the mechanisms of neurological diseases and is now being developed for therapeutic applications. In rodents and macaques, improved channelrhodopsins have been applied to achieve transcranial optogenetic stimulation. While transcranial photoexcitation of neurons has been achieved, noninvasive optogenetic inhibition for treating hyperexcitability-induced neurological disorders has remained elusive. There is a critical need for effective inhibitory optogenetic tools that are highly light-sensitive and capable of suppressing neuronal activity in deep brain tissue. In this study, we developed a highly sensitive K+-conductive channelrhodopsin (hsKCR) by molecular engineering of the recently discovered Hyphochytrium catenoides kalium (potassium) channelrhodopsin 1. Transcranial activation of hsKCR significantly prolongs the time to the first seizure, increases survival, and decreases seizure activity in several mouse epileptic models. Our approach for transcranial optogenetic inhibition of neural hyperactivity may be adapted for cell type-specific neuromodulation in both basic and preclinical settings.

neuroscience↗

Three-dimensional dipole orientation mapping with high temporal-spatial resolution using polarization modulation

Fluorescence polarization microscopy is widely used in biology for molecular orientation properties. However, due to the limited temporal resolution of single-molecule orientation localization microscopy and the limited orientation dimension of polarization modulation techniques, achieving simultaneous high temporal-spatial resolution mapping of the three-dimensional (3D) orientation of fluorescent dipoles remains an outstanding problem. Here, we present a super-resolution 3D orientation mapping (3DOM) microscope that resolves 3D orientation by extracting phase information of the six polarization modulation components in reciprocal space. 3DOM achieves an azimuthal precision of 2{degrees} and a polar precision of 3{degrees} with spatial resolution of 128 nm in the experiments. We validate that 3DOM not only reveals the heterogeneity of the milk fat globule membrane, but also elucidates the 3D structure of biological filaments, including the 3D spatial conformation of {lambda}-DNA and the structural disorder of actin filaments. Furthermore, 3DOM images the dipole dynamics of microtubules labeled with green fluorescent protein in live U2OS cells, reporting dynamic 3D orientation variations. Given its easy integration into existing wide-field microscopes, we expect the 3DOM microscope to provide a multi-view versatile strategy for investigating molecular structure and dynamics in biological macromolecules across multiple spatial and temporal scales.

molecular biology↗

A Day in the Life of Arabidopsis: 24-Hour Time-lapse Single-nucleus Transcriptomics Reveal Cell-type specific Circadian Rhythms

Functional circadian clock is fundamental to the adaptation and survival of organisms. In land plants, the comprehensive profiling of circadian gene expression at the single-cell level is largely unknown partly due to the challenges in obtaining precisely-timed single cells from plant cells embedded within cell walls. To bridge this gap, we employed single-nucleus RNA sequencing (snRNA-seq) on twelve seedling samples collected at 2-hour intervals over a 24-hour day in Arabidopsis, yielding a total of over 130,000 nuclei. From this data, we found that three cell clusters in the shoot share a coherent rhythm, while more than 3,000 genes display cell-type specific rhythmic expression. Only 19 genes are oscillating in more than ten different cell types, and the majority of them are well-documented core oscillators, suggesting the snRNA-seq circadian data could be used to identify key circadian regulators in a broad range of plant systems. Our data provides the first comprehensive resource for plant circadian rhythms at the single-cell level (hosted at https://zhailab.bio.sustech.edu.cn/sc_circadian).

plant biology↗

Application of slow-controlled release fertilizer coordinates the carbon flow in carbon-nitrogen metabolism to effect rice quality

Slow-controlled release fertilizers are experiencing a popularity in rice cultivation due to their effectiveness in yield and quality with low environmental costs. However, the underlying mechanism by which these fertilizers regulate grain quality remains inadequately understood. This study investigated the effects of five fertilizer management practices on rice quality in a two-year field experiment: CK, conventional fertilization, and four applications of slow-controlled release fertilizer (UF, urea formaldehyde; SCU, sulfur-coated urea; PCU, polymer-coated urea; BBF, controlled-release bulk blending fertilizer). In 2020 and 2021, the yields of UF and SCU groups showed significant decreases when compared to conventional fertilization, accompanied by a decline in nutritional quality. Additionally, PCU group exhibited poorer cooking and eating qualities. However, BBF group achieved increases in both yield (10.8 t hm-2 and 11.0 t hm-2) and grain quality reaching the level of CK group. The sufficient nitrogen supply in both the PCU and BBF groups during the grain-filling stage led to a greater capacity for the accumulation of proteins and amino acids in the PCU group compared to starch accumulation. Intriguingly, BBF group showed better carbon-nitrogen metabolism than that of PCU group. The optimal nitrogen supply present in BBF group suitable boosted the synthesis of amino acids involved in the glycolysis/ tricarboxylic acid cycle, thereby effectively coordinating carbon-nitrogen metabolism. The application of the new slow-controlled release fertilizer, BBF, is advantageous in regulating the carbon flow in the carbon-nitrogen metabolism to enhance rice quality.

plant biology↗

High-fidelity, high-resolution light-field reconstruction with physics-informed neural representation learning

Light-field microscopy (LFM) captures 3D biological dynamics by single 2D snapshots but suffers from limited resolution and artifacts during 2D-to-3D inversion. Here, we introduce light-field meta neural representation (LFMNR), a novel self-supervised paradigm that utilizes physics-informed light-field implicit neural representation (LFINR) and meta learning for high-quality 3D reconstruction in Fourier-LFM. By developing a physics-based hybrid-rendering model, LFINR achieves artifact-free light-field reconstruction with enhanced spatial resolution (>1.4-fold improvement). Additionally, the integration of meta-learning and progressive sampling strategies mitigates INRs intrinsic limitations in low reconstruction speed caused by scene-specific optimization, enabling a [~]100-fold acceleration in the representation of consecutive volumes and facilitating the visualization of sustained 3D dynamics. These advancements enable LFMNR to deliver superior imaging with high spatiotemporal resolution and low phototoxicity, as demonstrated by capturing instantaneous voltage signals in C. elegans at 100 volumes per second and recording 6000 time points of organelle dynamics over 25 hours.

cell biology↗