Biology subjects

Zhu, Y.

Publications and source records attributed to Zhu, Y..

At least 523 records · Page 29Linked to original sources

bioRxiv2022

RBP45d is required for 5' splice site selection via binding to intronic U-rich elements and interaction with PRP39a in Arabidopsis thaliana

A large number of cryptic splice sites in eukaryotic genome are generally dormant unless activated by mutations of authentic splice sites or splicing factors. How cryptic splice sites are used remains unknown in plants. Here, we identified two cryptic splicing regulators, RBP45d and PRP39a that are homologs of yeast U1 auxiliary protein Nam8 and Prp39, respectively, via genetic screening for suppressors of the virescent sot5 mutant, which results from a point mutation at the 5 splice site (5 ss) of intron 7. PCR and DNA sequencing data showed that loss-of-function mutations in RBP45d and PRP39a significantly increase the level of a cryptically spliced mRNA that encodes a mutated but partially functional sot5 protein, rescuing sot5 to the WT phenotype. Yeast two hybrid and bimolecular fluorescence complementation assays demonstrated that RBP45d and PRP39a interact each other and also with the U1C, a core subunit of U1 small nuclear ribonucleoprotein (U1 snRNP). RNA electronic mobility shift assay showed that RBP45d directly binds to the uridine (U)-rich RNA sequence downstream of the cryptic 5 ss. Consistently, our transcriptomic analysis revealed that a set of introns with U-rich sequences are retained in rbp45d. However, we found that other RBP45/47 members do not function redundantly with RBP45d, at least in regulation of cryptic splicing. Collectively, our data suggest that RBP45d is required for 5 ss selection via binding to intronic U-rich elements and PRP39a in plants. One sentence summaryThe Arabidopsis RBP45d interacting with U1C and PRP39a is required for 5 ss selection via binding to intronic U-rich elements.

plant biology↗

bioRxiv2022

IL-12/18/21 pre-activation enhances the anti-tumor efficacy of expanded γδT cells and overcomes resistance to anti-PD-L1 treatment

{gamma}{delta}T cells are promising candidates for cellular immunotherapy due to their immune regulation through cytokine production and MHC-independent direct cytotoxicity against a broad spectrum of tumors. However, current {gamma}{delta}T cell-based cancer immunotherapy has limited efficacies and novel strategies are needed to improve its clinical outcomes. Here, we report that cytokine pre-treatment with IL-12/18, IL-12/15/18, IL-12/18/21 and IL-12/15/18/21 could effectively enhance the activation and cytotoxicity of in vitro expanded murine and human {gamma}{delta}T cells. However, only adoptive transfer of IL-12/18/21 pre-activated {gamma}{delta}T cells significantly inhibited tumor growth in a murine melanoma model and a hepatocellular carcinoma model. Both IL-12/18/21 pre-activated antibody-expanded and zoledronate-expanded human {gamma}{delta}T cells effectively controlled tumor growth in a humanized mouse model. IL-12/18/21 pre-activation promoted {gamma}{delta}T cell proliferation and cytokine production in vivo and enhanced IFN-{gamma} and TNF- production, as well as granzyme B expression by endogenous CD8+ T cells in a cell-cell contact dependent manner. Furthermore, the adoptive transfer of IL-12/18/21 pre-activated {gamma}{delta}T cells could overcome the resistance to anti-PD-L1 therapy, and the combination therapy had a synergistic effect on the therapeutic outcomes. Moreover, the enhanced anti-tumor function of adoptively transferred IL-12/18/21 pre-activated {gamma}{delta}T cells was largely diminished in the absent of endogenous CD8+T cells when administered alone or in combination with anti-PD-L1, suggesting a CD8+T cell-dependent mechanism. Taken together, IL-12/18/21 pre-activation could promote {gamma}{delta}T cell anti-tumor function and overcome the resistance to checkpoint blockade therapy, indicating an effective combinational cancer immunotherapeutic strategy. SynopsisO_LIIL-12/18/21 pre-activation enhances the anti-tumor efficacy of adoptively transferred {gamma}{delta}T cells by promoting its proliferation, activation, and cytotoxicity, as well as activating endogenous CD8+ T cells. C_LIO_LIAdoptive transfer of IL-12/18/21 pre-activated {gamma}{delta}T cells could overcome the resistance to anti-PD-L1 therapy, indicating an effective combinational cancer immunotherapeutic strategy. C_LI

immunology↗

bioRxiv2022

Development of highly potent non-covalent inhibitors of SARS-CoV-2 3CLpro

The SARS-CoV-2 virus is the causal agent of the ongoing pandemic of coronavirus disease 2019 (COVID-19). There is an urgent need for potent, specific antiviral compounds against SARS-CoV-2. The 3C-like protease (3CLpro) is an essential enzyme for the replication of SARS-CoV-2 and other coronaviruses, and thus is a target for coronavirus drug discovery. Nearly all inhibitors of coronavirus 3CLpro reported so far are covalent inhibitors. Here, we report the development of specific, non-covalent inhibitors of 3CLpro. The most potent one, WU-04, effectively blocks SARS-CoV-2 replications in human cells with EC50 values in the 10-nM range. WU-04 also inhibits the 3CLpro of SARS-CoV and MERS-CoV with high potency, indicating that it is a pan-inhibitor of coronavirus 3CLpro. WU-04 showed anti-SARS-CoV-2 activity similar to that of PF-07321332 (Nirmatrelvir) in K18-hACE2 mice when the same dose was administered orally. Thus, WU-04 is a promising drug candidate for coronavirus treatment. One-Sentence SummaryA oral non-covalent inhibitor of 3C-like protease effectively inhibits SARS-CoV-2 replication.

microbiology↗

bioRxiv2022

The role and mechanism of NRF2 in endothelial cells and smooth muscle cells of pulmonary artery

Pulmonary hypertension (PH) is a kind of fatal disease. There are no existing drugs that could reverse the diseas. NF-E2-related factor 2 (NRF2) is one of the most important moleculars in the range of cell protection. We examined the expression of NRF2 in PH models and explored the role of NRF2 in regulating abnormal phenotypes in pulmonary artery cells. We determined the expression level of NRF2 in lung tissues of PH model decreased significantly. We found that NRF2 was reduced in rat pulmonary artery endothelial cells (rPAEC) under hypoxia, while it was overexpressed in rat pulmonary artery smooth muscle cells (rPASMC) under hypoxia. Next, the results showed that knockdown NRF2 in rPAEC promoted endothelial-mesenchymal transformation and upregulated reactive oxygen species level. After the rPASMC was treated with siRNA or activator, we found that NRF2 could promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways, and accelerate cell migration by affecting MMP2/3/7. Therefore, the study suggests that the combination of NRF2 activators should be considered to eliminate the promoting effect of NRF2 activators on proliferation and migration of rPASMC. Summary statementHypoxia regulates NRF2 in PAECs and PASMCs differently. We found that NRF2 could promote cell proliferation by regulating PDGFR/ERK1/2 and mTOR/P70S6K pathways, and accelerate cell migration by affecting MMP2/3/7.

cell biology↗

bioRxiv2022

Single cell, whole embryo phenotyping of pleiotropic disorders of mammalian development

Mouse models are a critical tool for studying human diseases, particularly developmental disorders, as well as for advancing our general understanding of mammalian biology. However, it has long been suspected that conventional approaches for phenotyping are insufficiently sensitive to detect subtle defects throughout the developing mouse. Here we set out to establish single cell RNA sequencing (sc-RNA-seq) of the whole embryo as a scalable platform for the systematic molecular and cellular phenotyping of mouse genetic models. We applied combinatorial indexing-based sc-RNA-seq to profile 101 embryos of 26 genotypes at embryonic stage E13.5, altogether profiling gene expression in over 1.6M nuclei. The 26 genotypes include 22 mouse mutants representing a range of anticipated severities, from established multisystem disorders to deletions of individual enhancers, as well as the 4 wildtype backgrounds on which these mutants reside. We developed and applied several analytical frameworks for detecting differences in composition and/or gene expression across 52 cell types or trajectories. Some mutants exhibited changes in dozens of trajectories (e.g., the pleiotropic consequences of altering the Sox9 regulatory landscape) whereas others showed phenotypes affecting specific subsets of cells. We also identify differences between widely used wildtype strains, compare phenotyping of gain vs. loss of function mutants, and characterise deletions of topological associating domain (TAD) boundaries. Intriguingly, even among these 22 mutants, some changes are shared by heretofore unrelated models, suggesting that developmental pleiotropy might be "decomposable" through further scaling of this approach. Overall, our findings show how single cell profiling of whole embryos can enable the systematic molecular and cellular phenotypic characterization of mouse mutants with unprecedented breadth and resolution.

genomics↗

bioRxiv2022

New genetic tools enable dissection of a global stress response in the early-branching species Fusobacterium nucleatum

Fusobacterium nucleatum, long known as a common oral microbe, has recently garnered attention for its ability to colonize tissues and tumors elsewhere in the human body. Clinical and epidemiological research has now firmly established F. nucleatum as an oncomicrobe associated with several major cancer types. However, with the current research focus on host associations, little is known about gene regulation in F. nucleatum itself, including global stress response pathways that typically ensure the survival of bacteria outside their primary niche. This is due to the phylogenetic distance of Fusobacteriota to most model bacteria, their limited genetic tractability, and paucity of known gene functions. Here, we characterize a global transcriptional stress response network governed by the extracytoplasmic function sigma factor, {sigma}E. To this aim, we developed several new genetic tools for this anaerobic bacterium, including four different fluorescent marker proteins, inducible gene expression, scarless gene deletion, and transcriptional and translational reporter systems. Using these tools, we identified a {sigma}E response partly reminiscent of phylogenetically distant Proteobacteria but induced by exposure to oxygen. Although F. nucleatum lacks canonical RNA chaperones such as Hfq, we uncovered conservation of the non-coding arm of the {sigma}E response in form of the non-coding RNA FoxI. This regulatory small RNA (sRNA) acts as an mRNA repressor of several membrane proteins, thereby supporting the function of {sigma}E. In addition to the characterization of a global stress response in F. nucleatum, the genetic tools developed here will enable further discoveries and dissection of regulatory networks in this early-branching bacterium. SIGNIFICANCE STATEMENTFusobacterium nucleatum is an abundant member of the oral microbiome that can spread throughout the body and colonize secondary sites, including cancer tissues where it promotes tumor progression. Understanding how F. nucleatum is able to adapt to this new environment might open new therapeutic opportunities, but we currently lack basic molecular knowledge of gene regulation in this phylogenetically distinct bacterium. We developed much-needed genetic tools for use in F. nucleatum and with their aid uncovered a stress response mediated by the transcriptional activator {sigma}E and an associated small RNA. Our findings in an early-branching bacterium reveal surprising parallels to and differences from the {sigma}E response in well-characterized model bacteria and provide a framework that will accelerate research into the understudied phylum Fusobacteriota.

microbiology↗

bioRxiv2022

Self-cyclisation as a general and efficient platform for peptide and protein macrocyclisation

Macrocyclisation of proteins and peptides results in a remarkable increase in structural stability, making cyclic peptides and proteins of great interest in drug discovery--either directly as drug leads or as in the case of cyclised nanodiscs (cNDs), as tools for studies of trans-membrane receptors and membrane-active peptides. Various biological methods have been developed that are capable of yielding head-to-tail macrocyclised products. Such enzymatic methods require careful optimisation of cyclisation over polymerisation. Here, we describe the engineering of self-cyclising "autocyclase" proteins, where an intramolecular rearrangement can be triggered to yield a monomeric cyclic product in high yields. We characterise the self-cyclisation reaction mechanism and demonstrate how the unimolecular reaction path can circumvent existing challenges of enzymatic cyclisation. We use the method to produce several notable cyclic peptides and proteins, demonstrating how autocyclases offer a simple and scalable way to access a vast diversity of macrocyclic biomolecules.

biochemistry↗

bioRxiv2022

Integrating Self-Attention Transformer with Triplet Neural Networks for Protein Gene Ontology Prediction

Accurate identification of protein function is critical to elucidate life mechanism and design new drugs. We proposed a novel deep-learning method, ATGO, to predict Gene Ontology (GO) attributes of proteins through a triplet neural-network architecture embedded with pre-trained self-attention transformer models. The method was systematically tested on 1068 non-redundant benchmarking proteins and 3328 targets from the third Critical Assessment of Protein Function Annotation (CAFA) challenge. Experimental results showed that ATGO achieved a significant increase of the GO prediction accuracy compared to the state-of-the-art approaches in all aspects of molecular function, biological process, and cellular component. Detailed data analyses showed that the major advantage of ATGO lies in the utilization of attention transformer models which can extract discriminative functional pattern from the feature embeddings. Meanwhile, the proposed triplet network helps enhance the association of functional similarity with feature similarity in the sequence embedding space. In addition, it was found that the combination of the network scores with the complementary homology-based inferences could further improve the accuracy and coverage of the predicted models. These results demonstrated a new avenue for high-accuracy deep-learning function prediction that is applicable to large-scale protein function annotations from sequence alone. AvailabilityThe benchmark dataset, standalone package, and online server for ATGO are available at https://zhanggroup.org/ATGO/. Author SummaryIn the post-genome sequencing era, a major challenge in computational molecular biology is to annotate the biological functions of all gene and gene products, which have been classified, in the context of the widely used Gene Ontology (GO), into three aspects of molecular function, biological process, and cellular component. In this work, we proposed a new open-source deep-learning architecture, ATGO, to deduce GO terms of proteins from the primary amino acid sequence, through the integration of the triplet neural-network with attention transformer models. Large benchmark tests showed that, when powered with a pre-trained self-attention transformer model, ATGO achieved a significantly improved performance than other state-of-the-art approaches in all the GO aspect predictions. Following the rapid progress of self-attention neural network techniques, which have demonstrated remarkable impacts on language processing and multi-sensory data process, and most recently on protein structure prediction, this study showed the significant potential of attention transformer models on protein function annotations.

bioinformatics↗

bioRxiv2022

Utilizing artificial intelligence system to build the digital structural proteome of reef-building corals

Reef-building corals play an important role in the marine ecosystem, and analyzing their proteomes from a structural perspective will exert positive effects on exploring their biology. Here we integrated mass spectrometry with newly published ColabFold to obtain digital structural proteomes of dominant reef-building corals. 8,382 proteins co-expressed in A. muricata, M. foliosa and P. verrucosa were identified, then 8,166 of them got predicted structures after around 4,060 GPU hours of computation. The resulting dataset covers 83.6% of residues with a confident prediction, while 25.9% have very high confidence. Our work provides insight-worthy predictions for coral research, confirms the reliability of ColabFold in practice, and is expected to be a reference case in the impending high-throughput era of structural proteomics.

bioinformatics↗

bioRxiv2022

APOE deficiency impacts neural differentiation and cholesterol biosynthesis in human iPSC-derived cerebral organoids

The apolipoprotein E (APOE) gene is the strongest genetic risk factor for Alzheimers disease (AD); however, how it modulates brain homeostasis is not clear. The apoE protein is a major lipid carrier in the brain transporting lipids such as cholesterol among different brain cell types. Here, we show that APOE deficiency in human iPSC-derived cerebral organoids impacts brain lipid homeostasis by modulating multiple cellular and molecular pathways. Molecular profiling through single cell RNA-sequencing revealed that APOE deficiency leads to changes in cellular composition of isogenic cerebral organoids likely by modulating the EIF2 signaling pathway as these events were alleviated by the treatment of a pathway inhibitor ISRIB. APOE deletion also leads to activation of the Wnt/{beta}-catenin signaling pathway with concomitant decrease of SFRP1 expression in glia cells. Importantly, the critical role of apoE in cell type-specific lipid homeostasis was observed upon APOE deletion in cerebral organoids with a specific upregulation of cholesterol biosynthesis in excitatory neurons and excessive lipid accumulation in astrocytes. Relevant to human AD, APOE4 cerebral organoids show altered neurogenesis and cholesterol metabolism compared to those with APOE3. Our work demonstrates critical roles of apoE in brain homeostasis and offers critical insights into the APOE4-related pathogenic mechanisms.

cell biology↗

bioRxiv2022

Diversity and biogeography of plant leaf bacteria along a latitudinal gradient

Plant-associated microbes are essential for promoting plant well-being, maintaining biodiversity, and supporting ecosystem function. However, little is known about the geographic distribution of plant-microbe symbioses and how they are formed and change along latitudinal gradients. Here we identified leaf bacteria for 328 plant species sampled from 10 forests along a tropical to temperate gradient in China. We analyzed the diversity and composition of plant leaf-associated bacteria and quantified the contributions of hosts, habitats, and neighborhood plants to the plant-bacterial symbiosis. We found a strong latitudinal gradient in leaf bacterial diversity and composition. Bacterial assemblages on leaves were most strongly selected by host plants, and the selection pressure increased with latitude. In contrast, at low latitudes and at large geographical scales multiple factors were found to jointly regulate bacterial community composition. Our result also showed that plant-bacteria symbiotic networks were structured by network hub bacteria taxa with high co-occurrence network centrality, and the abundance of temperate hub taxa was more influenced by host plants than that in tropical forests. For the first time, we documented a previously unrecognized latitudinal gradient in plant-bacterial symbioses that was regulated by a joint effect of multiple factors at low latitudes but mostly by host selection at high latitudes, implying that leaf microbiomes are likely to respond differently to global change along the latitudinal gradient.

ecology↗

bioRxiv2022

Mouse genomic rewriting and tailoring: synthetic Trp53 and humanized ACE2

Genetically Engineered Mouse Models (GEMMs) aid in understanding human pathologies and developing new therapeutics, yet recapitulating human diseases authentically in mice is challenging to design and execute. Advances in genomics have highlighted the importance of non-coding regulatory genome sequences controlling spatiotemporal gene expression patterns and splicing to human diseases. It is thus apparent that including regulatory genomic regions during the engineering of GEMMs is highly preferable for disease modeling, with the prerequisite of large-scale genome engineering ability. Existing genome engineering methods have limits on the size and efficiency of DNA delivery, hampering routine creation of highly informative GEMMs. Here, we describe mSwAP-In (mammalian Switching Antibiotic resistance markers Progressively for Integration), a method for efficient genome rewriting in mouse embryonic stem cells. We first demonstrated the use of mSwAP-In for iterative genome rewriting of up to 115 kb of the Trp53 locus, as well as for genomic humanization of up to 180 kb ACE2 locus in response to the COVID-19 pandemic. Second, we showed the hACE2 GEMM authentically recapitulated human ACE2 expression patterns and splicing, and importantly, presented milder symptoms without mortality when challenged with SARS-CoV-2 compared to the K18-ACE2 model, thus representing a more authentic model of infection.

genomics↗

bioRxiv2022

Targeting Chromatin Effector Pygo2 to Enhance Immunotherapy in Prostate Cancer

The noninflamed microenvironment in prostate cancer represents a barrier to immunotherapy. Genetic alterations underlying cancer cell-intrinsic oncogenic signaling have emerging roles in shaping the immune landscape. Recently, we identified Pygopus 2 (PYGO2) as the driver oncogene for the amplicon at 1q21.3. Here, using transgenic mouse models of metastatic prostate adenocarcinoma, we found that Pygo2 deletion decelerated tumor progression, diminished metastases, and extended survival. Pygo2 loss augmented the infiltration of cytotoxic T lymphocytes (CTLs) and sensitized tumor cells to T cell killing. Mechanistically, Pygo2 orchestrated a p53/Sp1/Kit/Ido1 signaling network to foster a microenvironment hostile to CTLs. Genetic or pharmacological inhibition of Pygo2 enhanced the anti-tumor efficacy of immunotherapies using immune checkpoint blockade, adoptive cell transfer, or myeloid-derived suppressor cell inhibitors. In human prostate cancer samples, Pygo2 expression was inversely correlated with CD8+ T cells. Our results highlight a promising path to improving immunotherapy with targeted therapy for lethal prostate cancer.

cancer biology↗

bioRxiv2022

Identification of cancer-keeping genes as therapeutic targets by finding network control hubs

Finding cancer-driver genes - the genes whose mutations may transform normal cells into cancerous ones - remains a central theme of cancer research. We took a different perspective; instead of considering normal cells, we focused on cancerous cells and genes that maintained abnormal cell growth which we named cancer-keeping genes (CKGs). Intervention in CKGs may rectify aberrant cell growth so that they can be adopted as therapeutic targets for cancer treatment. We developed a novel approach to identifying CKGs by extending the well-established theory of network structural controllability, which aims at finding a control scheme (i.e., a minimal set of non-overlapping control paths covering all nodes) and control nodes (driver genes) that can steer the cell from any state to the designated state. Going beyond driver genes defined by one control scheme, we introduced control-hub genes located in the middle of a control path of every control scheme. Control hubs are essential for maintaining cancerous states and thus can be taken as CKGs. We applied our CKG-based approach to bladder cancer (BLCA) as a case study. All the genes on the cell cycle and p53 pathways in BLCA were identified as CKGs, showing the importance of these genes in cancer and demonstrating the power of our new method. Furthermore, sensitive CKGs that could be easily changed by structural perturbation were better suited as therapeutic targets. Six sensitive CKGs (RPS6KA3, FGFR3, N-cadherin (CDH2), EP300, caspase-1, and FN1) were subjected to small-interferencing-RNA knockdown in two BLCA cell lines to validate their cancer-suppressing effects. Knocking down RPS6KA3 in a mouse model of BLCA significantly inhibited the growth of tumor xenografts in mice. Combined, our results demonstrated the value of CKGs as therapeutic targets for cancer therapy. Key pointsO_LIFocus on genes that maintain aberrant cell growth, named cancer-keeping genes (CKGs). C_LIO_LIDevelop a novel approach for finding CKGs by extending the well-estabilished theory of network structural controllability to total network controllability. C_LIO_LIApply the new method to bladder cancer and experimentally validated the cancer-suppressing function of six CKGs in two bladder cancer cell lines and that of one CKG in bladder cancer mice. C_LI

bioinformatics↗

bioRxiv2022

Long-term ovarian cancer survivors: spatial transcriptomics depict ligand-receptor crosstalk heterogeneity at the tumor-stroma interface

Advanced high-grade serous ovarian cancer (HGSC) is an aggressive disease that accounts for 70% of all ovarian cancer deaths. Nevertheless, 15% of patients diagnosed with advanced HGSC survive more than 10 years. The identification of predictive markers associated with tumors developed from these long-term survivors (LTS) is crucial to identifying therapeutic targets for the disease, and thus improving patient survival rates. Reports to date have not fully established the stromal heterogeneity of the tumor microenvironment (TME) in ovarian cancer and its association with clinical outcomes. We used a spatial transcriptomics platform to generate spatially resolved transcript profiles in treatment naive advanced HGSC from LTS and short-term survivors (STS), and determined whether cancer-associated fibroblasts (CAFs) heterogeneity is associated with survival in patients with advanced HGSC. We integrated spatial transcriptomics with single-cell RNA sequencing data to distinguish tumor and stroma regions, and developed a method to investigate spatially resolved ligand-receptor interactions between various tumor and CAF subtypes in the TME. In addition, we used multiplex immunohistochemistry techniques to validate our findings. We found that a specific subtype of CAFs and its spatial location relative to a particular ovarian cancer cell subtype in the TME correlate with long-term survival in advanced HGSC patients. We also demonstrated that significant APOE-LRP5 crosstalk occurred at the stroma-tumor interface in tumor tissues from STS compared to LTS, suggesting that such crosstalk plays a crucial role in modulating the malignant phenotype of HGSC, and could serve as a predictive biomarker of patient survival.

cancer biology↗

bioRxiv2022

Genetic or transcranial magnetic stimulation of B-RAF-MEK signaling promotes CST axon sprouting and functional regeneration

Facilitating axon regeneration in the injured central nervous system remains a challenging task. RAF-MEK signaling plays an important role in axon elongation during nervous system development. Here we show that activation of B-RAF in mature corticospinal neurons elicited the expression of a discrete set of transcription factors previously implicated in the regeneration of zebrafish optic nerve axons. Genetic activation of B-RAF-MEK signaling promoted robust regeneration and sprouting of corticospinal tract axons after injury. Newly sprouting axon collaterals formed synaptic connections with spinal interneurons, correlating with the recovery of skilled motor function. Seeking a non-invasive way to stimulate axon regeneration, we found that suprathreshold high-frequency repetitive transcranial magnetic stimulation activates the B-RAF canonical effectors MEK1/2 and requires MEK1/2 activity to promote corticospinal axon regeneration and sprouting after injury. These data demonstrate a central role of neuron-intrinsic RAF-MEK signaling in enhancing the growth capacity of mature corticospinal neurons and propose HF-rTMS as a potential therapy for spinal cord injury. One Sentence SummaryGenetic or HF-rTMS-mediated activation of B-RAF- MEK signaling promotes CST axon sprouting and functional regeneration after a spinal cord injury.

neuroscience↗

bioRxiv2022

Uncovering Hidden Enhancers Through Unbiased In Vivo Testing

Transcriptional enhancers are a predominant class of noncoding regulatory elements that activate cell type-specific gene expression. Tissue-specific enhancer-associated chromatin signatures have proven useful to identify candidate enhancer elements at a genome-wide scale, but their sensitivity for the comprehensive detection of all enhancers active in a given tissue in vivo remains unclear. Here we show that a substantial proportion of in vivo enhancers are hidden from discovery by conventional chromatin profiling methods. In an initial comparison of over 1,200 in vivo validated tissue-specific enhancers with tissue-matched mouse developmental epigenome data, 14% (n=286) of active enhancers did not show canonical enhancer-associated chromatin signatures in the tissue in which they are active. To assess the prevalence of enhancers not detectable by conventional chromatin profiling approaches in more detail, we used a high throughput transgenic enhancer reporter assay to systematically screen over 1.3 Mb of mouse genomic sequence at two critical developmental loci, assessing a total of 281 consecutive 5kb regions for in vivo enhancer activity in mouse embryos. We observed reproducible enhancer-reporter activity in 88 tissue-specific elements, 26% of which did not show canonical enhancer-associated chromatin signatures in the corresponding tissues. Overall, we find these hidden enhancers are indistinguishable from marked enhancers based on levels of evolutionary conservation, enrichment of transcription factor families, and genomic positioning relative to putative target genes. In combination, our retrospective and prospective studies assessed only 0.1% of the mouse genome and identified 309 tissue-specific enhancers that are hidden from current chromatin-based enhancer identification approaches. Our findings suggest the existence of tens of thousands of active enhancers throughout the genome that remain undetected by current chromatin profiling approaches and are an unappreciated source of additional genome function of import in interpreting growing whole human genome sequencing data.

genomics↗

bioRxiv2022

Chemical genetic screening identifies nalacin as an inhibitor of GH3 amido synthetase for auxin conjugation

Root system is critical for plant growth and development. To develop plant growth regulators functioning in root development, we performed a phenotype-based chemical screen in Arabidopsis and identified a chemical, nalacin, that mimicked the effects of auxin on root development. Genetic, pharmacological and biochemical approaches demonstrated that nalacin exerts its auxin-like activities by inhibiting indole-3-acetic acid (IAA) conjugation that is mediated by Gretchen Hagen 3 (GH3) acyl acid amido synthetases. The crystal structure of Arabidopsis GH3.6 in complex with D4 (a derivative of nalacin) together with docking simulation analysis revealed the molecular basis of the inhibition of group II GH3 by nalacin. Sequence alignment analysis indicated broad bioactivities of nalacin and D4 as inhibitors of GH3s in vascular plants, which were confirmed, at least, in tomato and rice. In summary, our work identifies nalacin as a potent inhibitor of IAA conjugation mediated by group II GH3 that plays versatile roles in hormone-regulated plant development and has potential applications in both basic research and agriculture.

plant biology↗