Biology subjects

Cheng, T.

Publications and source records attributed to Cheng, T..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

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↗

bioRxiv2024

EDC-3 and EDC-4 Regulate Embryonic mRNA Clearance and Biomolecular Condensate Specialization

Animal development is dictated by the selective and timely decay of mRNAs in developmental transitions, but the impact of mRNA decapping scaffold proteins in development is unknown. This study unveils the roles and interactions of the DCAP-2 decapping scaffolds EDC-3 and EDC-4 in the embryonic development of C. elegans. EDC-3 facilitates the timely removal of specific embryonic mRNAs, including cgh-1, car-1, and ifet-1 by reducing their expression, and preventing excessive accumulation of DCAP-2 condensates in somatic cells. We further uncover a novel role for EDC-3 in defining the boundaries between P-bodies, germ granules, and stress granules. Lastly, we show that EDC-4 counteracts EDC-3 and engenders the assembly of DCAP-2 with the GID (CTLH) complex, a ubiquitin ligase involved in maternal-to-zygotic transition (MZT). Our findings support a model wherein multiple RNA decay mechanisms temporally partake in the clearance of maternal and zygotic mRNAs throughout embryonic development.

molecular biology↗

bioRxiv2023

DLG1 functions upstream of SDCCAG3 and IFT20 to control targeting of polycystin-2 to the primary cilium

Polarized vesicular trafficking directs specific receptors and ion channels to cilia, but the underlying mechanisms are poorly understood. Here we describe a role for DLG1, a core component of the Scribble polarity complex, in regulating ciliary protein trafficking in kidney epithelial cells. Conditional knockout of Dlg1 in mouse kidney caused ciliary elongation and cystogenesis, and cell-based proximity labelling proteomics and fluorescence microscopy showed alterations in the ciliary proteome upon loss of DLG1. Specifically, the retromer-associated protein SDCCAG3, IFT20 and polycystin-2 (PC2) were reduced in cilia of DLG1 deficient cells compared to control cells. This phenotype was recapitulated in vivo and rescuable by re-expression of wildtype DLG1, but not a Congenital Anomalies of the Kidney and Urinary Tract (CAKUT)-associated DLG1 variant, p.T489R. Finally, biochemical approaches and Alpha Fold modelling suggested that SDCCAG3 and IFT20 form a complex that associates, at least indirectly, with DLG1. Our work identifies a key role for DLG1 in regulating ciliary protein composition and suggests that ciliary dysfunction of the p.T489R DLG1 variant may contribute to CAKUT.

cell biology↗

bioRxiv2023

Unravelling ATP processing by the AAA+ protein p97 at the atomic level

The human enzyme p97 regulates various cellular pathways by unfolding hundreds of protein substrates in an ATP-dependent manner, making it an essential component of homeostasis and impactful pharmacological target. The hexameric complex undergoes substantial conformational changes in the course of its catalytic cycle. Here, we elucidate the molecular motions that occur at the active site in the temporal window immediately before and after ATP hydrolysis by merging cryo-EM, NMR spectroscopy and MD simulations. p97 populates a metastable reaction intermediate, the ADP.Pi state, which is poised between hydrolysis and product release. Detailed snapshots reveal that the active site is finely tuned to trap and eventually discharge the cleaved phosphate. Signalling pathways originating at the active site coordinate the action of the hexamer subunits and couple hydrolysis with allosteric conformational changes. Our multidisciplinary approach enables a glimpse into the sophisticated spatial and temporal orchestration of ATP handling by a prototype AAA+ protein.

biophysics↗

bioRxiv2023

Dissection of anterior mesendoderm segregation at single cell level in zebrafish

During gastrulation, the mesendoderm is firstly specified by morphogens such as Nodal, and then segregates into endoderm and mesoderm in a Nodal concentration-dependent manner. However, the mechanism underlying the segregation and crosstalk of different sub-groups within the meso- and endoderm lineages remains unclear. Here, taking zebrafish prechordal plate (PP) and anterior endoderm (Endo) as research model, using single-cell multi-omics and live imaging analyses, we show that anterior Endo progenitors originate directly from PP progenitors. A single-cell transcriptomic trajectory analysis of wild-type, ndr1 knockdown and lft1 knockout Nodal explants confirms the diversification of anterior Endo fate from PP progenitors. Gene Ontology (GO) enrichment analysis identifies that the change of chromatin organization potentiates the segregation of anterior endodermal cell fate from PP progenitors. Single-cell ATAC & RNA sequencing further reveals that two transcriptional regulators, gsc and ripply1, exhibit varied activation patterns in PP and anterior Endo cell trajectories at both the chromatin and RNA expression levels. We further demonstrate that Ripply1 functions coordinately with Gsc to repress anterior endodermal cell fate by directly binding to the cis-elements of sox32. Modulating the expression levels of these regulators tilts the cell fate decision between the PP and anterior Endo.

developmental biology↗

bioRxiv2023

Impaired centrosome biogenesis in kidney stromal progenitors reduces abundance of interstitial lineages and accelerates injury-induced fibrosis

Defective centrosome function can disrupt embryonic kidney development, by causing changes to the renal interstitium that leads to fibrocystic disease pathologies. Yet, it remains unknown how mutations in centrosome genes impact kidney interstitial cells. Here, we examined the consequences of defective centrosome biogenesis on stromal progenitor cell growth, differentiation and fate. Conditional deletion of Cep120, a ciliopathy gene essential for centrosome duplication, in the stromal mesenchyme resulted in reduced abundance of pericytes, interstitial fibroblasts and mesangial cells. This was due to delayed mitosis, increased apoptosis, and changes in Wnt and Hedgehog signaling essential for differentiation of stromal lineages. Cep120 ablation resulted in hypoplastic kidneys with medullary atrophy and delayed nephron maturation. Finally, centrosome loss in the interstitium sensitized kidneys of adult mice, causing rapid fibrosis via enhanced TGF-{beta}/Smad3-Gli2 signaling after renal injury. Our study defines the cellular and developmental defects caused by centrosome dysfunction in embryonic kidney stroma. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=139 SRC="FIGDIR/small/535583v1_ufig1.gif" ALT="Figure 1"> View larger version (31K): org.highwire.dtl.DTLVardef@a3b059org.highwire.dtl.DTLVardef@8ed18corg.highwire.dtl.DTLVardef@5f573borg.highwire.dtl.DTLVardef@1581c20_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsO_LIDefective centrosome biogenesis in kidney stroma causes: C_LIO_LIReduced abundance of stromal progenitors, interstitial and mesangial cell populations C_LIO_LIDefects in cell-autonomous and paracrine signaling C_LIO_LIAbnormal/delayed nephrogenesis and tubular dilations C_LIO_LIAccelerates injury-induced fibrosis via defective TGF-{beta}/Smad3-Gli2 signaling axis C_LI

developmental biology↗

bioRxiv2023

Aberrant centrosome biogenesis disrupts nephron progenitor cell renewal and fate resulting in fibrocystic kidney disease

Mutations that disrupt centrosome structure or function cause congenital kidney developmental defects and fibrocystic pathologies. Yet, it remains unclear how mutations in proteins essential for centrosome biogenesis impact embryonic kidney development. Here, we examined the consequences of conditional deletion of a ciliopathy gene, Cep120, in the two nephron progenitor niches of the embryonic kidney. Cep120 loss led to reduced abundance of both metanephric mesenchyme and ureteric bud progenitor populations. This was due to a combination of delayed mitosis, increased apoptosis, and premature differentiation of progenitor cells. These defects resulted in dysplastic kidneys at birth, which rapidly formed cysts, displayed increased interstitial fibrosis, and decline in filtration function. RNA sequencing of embryonic and postnatal kidneys from Cep120-null mice identified changes in pathways essential for branching morphogenesis, cystogenesis and fibrosis. Our study defines the cellular and developmental defects caused by centrosome dysfunction during kidney development, and identifies new therapeutic targets for renal centrosomopathies. Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=190 SRC="FIGDIR/small/535568v1_ufig1.gif" ALT="Figure 1"> View larger version (39K): org.highwire.dtl.DTLVardef@198c93forg.highwire.dtl.DTLVardef@1c4750dorg.highwire.dtl.DTLVardef@e0c829org.highwire.dtl.DTLVardef@17512c3_HPS_FORMAT_FIGEXP M_FIG C_FIG HighlightsDefective centrosome biogenesis in nephron progenitors causes: O_LIReduced abundance of metanephric mesenchyme and premature differentiation into tubular structures C_LIO_LIAbnormal branching morphogenesis leading to reduced nephron endowment and smaller kidneys C_LIO_LIChanges in cell-autonomous and paracrine signaling that drive cystogenesis and fibrosis C_LIO_LIUnique cellular and developmental defects when compared to Pkd1 knockout models C_LI

developmental biology↗

bioRxiv2023

Clots reveal anomalous elastic behavior of fiber networks

The mechanical properties of many soft natural and synthetic biological materials are relevant to their function. The emergence of these properties from the collective response of the structural components of the material to external stress as well as to intrinsic cell traction, remains poorly understood. Here, we examine the nonlinear elastic behavior of blood clots by combining microscopy and rheological measurements with an elastic network model that accounts for the stretching, bending, and buckling of constituent fibrin fibers. We show that the inhibition of fibrin crosslinking reduces fiber bending stiffness and introduces an atypical fiber buckling-induced softening regime at intermediate shear, before the well-characterized stiffening regime. We also show that crosslinking and platelet contraction significantly alter force propagation in the network in a strain-dependent manner. Our mechanics-based model, supported by experiments, provides a framework to understand the origins of characteristic and anomalous regimes of non-linear elastic response not only in blood clots, but also more generally in active biopolymer networks.

biophysics↗

bioRxiv2023

RNA Sensing and Innate Immunity Constitutes a Barrier for Interspecies Chimerism

Interspecies chimera formation with human pluripotent stem cells (PSCs) holds great promise to generate humanized animal models and provide donor organs for transplant. However, the approach is currently limited by low levels of human cells ultimately represented in chimeric embryos. Different strategies have been developed to improve chimerism by genetically editing donor human PSCs. To date, however, it remains unexplored if human chimerism can be enhanced in animals through modifying the host embryos. Leveraging the interspecies PSC competition model, here we discovered retinoic acid-inducible gene I (RIG-I)-like receptor (RLR) signaling, an RNA sensor, in "winner" cells plays an important role in the competitive interactions between co-cultured mouse and human PSCs. We found that genetic inactivation of Ddx58/Ifih1-Mavs-Irf7 axis compromised the "winner" status of mouse PSCs and their ability to outcompete PSCs from evolutionarily distant species during co-culture. Furthermore, by using Mavs-deficient mouse embryos we substantially improved unmodified donor human cell survival. Comparative transcriptome analyses based on species-specific sequences suggest contact-dependent human-to-mouse transfer of RNAs likely plays a part in mediating the cross-species interactions. Taken together, these findings establish a previously unrecognized role of RNA sensing and innate immunity in "winner" cells during cell competition and provides a proof-of-concept for modifying host embryos, rather than donor PSCs, to enhance interspecies chimerism.

cell biology↗

bioRxiv2023

Compressed phenotypic screens for complex multicellular models and high-content assays

High-throughput phenotypic screens leveraging biochemical perturbations, high-content readouts, and complex multicellular models could advance therapeutic discovery yet remain constrained by limitations of scale. To address this, we establish a method for compressing screens by pooling perturbations followed by computational deconvolution. Conducting controlled benchmarks with a highly bioactive small molecule library and a high-content imaging readout, we demonstrate increased efficiency for compressed experimental designs compared to conventional approaches. To prove generalizability, we apply compressed screening to examine transcriptional responses of patient-derived pancreatic cancer organoids to a library of tumor-microenvironment (TME)-nominated recombinant protein ligands. Using single-cell RNA-seq as a readout, we uncover reproducible phenotypic shifts induced by ligands that correlate with clinical features in larger datasets and are distinct from reference signatures available in public databases. In sum, our approach enables phenotypic screens that interrogate complex multicellular models with rich phenotypic readouts to advance translatable drug discovery as well as basic biology.

systems biology↗

bioRxiv2022

GEMC1 and MCIDAS interactions with SWI/SNF complexes regulate the multiciliated cell-specific transcriptional program

Multiciliated cells (MCCs) project dozens to hundreds of motile cilia from their apical surface to promote the movement of fluids or gametes in the mammalian brain, airway or reproductive organs. Differentiation of MCCs requires the sequential action of the Geminin family transcriptional activators, GEMC1 and MCIDAS, that both interact with E2F4/5-DP1. How these factors activate transcription and the extent to which they play redundant functions remains poorly understood. Here, we demonstrate that the transcriptional targets and proximal proteomes of GEMC1 and MCIDAS are highly similar. However, we identified distinct interactions with SWI/SNF subcomplexes; GEMC1 interacts primarily with the ARID1A containing BAF complex while MCIDAS interacts primarily with BRD9 containing ncBAF complexes. Treatment with a BRD9 inhibitor impaired MCIDAS-mediated activation of several target genes and compromised the MCC differentiation program in multiple cell based models. Our data suggest that the differential engagement of distinct SWI/SNF subcomplexes by GEMC1 and MCIDAS is required for MCC-specific transcriptional regulation and mediated by their distinct C-terminal domains.

cell biology↗

bioRxiv2022

Inhibition of Centrosome Clustering Reduces Cystogenesis and Improves Kidney Function in Autosomal Dominant Polycystic Kidney Disease

Autosomal Dominant Polycystic Kidney Disease (ADPKD) is an inherited monogenic disorder accounting for [~]5% of patients with renal failure. Yet, therapeutics for the treatment of ADPKD remain limited. ADPKD tissues display defects in the biogenesis of the centrosome which causes genome instability, aberrant ciliary signaling, and secretion of pro-inflammatory factors that drive cyst growth and fibrosis. Cystic cells form excess centrosomes via a process termed centrosome amplification (CA), which often causes abnormal multipolar spindle configurations, mitotic catastrophe, and reduced cell viability. However, cells with CA can suppress multipolarity via "centrosome clustering", a key mechanism by which cells circumvent apoptosis. Here, we demonstrate that inhibiting centrosome clustering can counteract the proliferation of renal cystic cells with high incidences of CA. Using ADPKD human cells and mouse models, we show that blocking centrosome clustering with two inhibitors, CCB02 and PJ34, blocks cyst initiation and growth in vitro and in vivo. Inhibition of centrosome clustering activates a p53-mediated mitotic surveillance mechanism leading to apoptosis, reduced cyst expansion, interstitial fibrosis, and improved kidney function. Transcriptional analysis of kidneys from treated mice identified pro-inflammatory signaling pathways implicated in CA-mediated cystogenesis and fibrosis. Our results provide the first evidence that centrosome clustering is a cyst-selective target for the improvement of renal morphology and function in ADPKD.

developmental biology↗

bioRxiv2022

Mycn regulates intestinal development through ribosomal biogenesis in a zebrafish model of Feingold syndrome 1

Feingold syndrome type 1, caused by loss-of-function of MYCN, is characterized by varied phenotypes including esophageal and duodenal atresia. However, no adequate model exists for studying the syndromes pathological or molecular mechanisms, nor is there a treatment strategy. Here, we developed a zebrafish Feingold syndrome type 1 model with nonfunctional mycn, which had severe intestinal atresia. Single-cell RNA-seq identified a subcluster of intestinal cells was highly sensitive to Mycn, and impaired cell proliferation decreased the overall number of intestinal cells in the mycn mutant fish. Bulk RNA-seq and metabolomic analysis showed that expression of ribosomal genes was downregulated and amino acid metabolism was abnormal. Ribosomal profiling analysis showed decreases in free 40S, 60S, and 80S ribosome particles, which led to impaired translation in the mutant. Further, both L-leucine and Rheb, which can elevate translation via TOR pathway, rescued the intestinal phenotype of mycn mutant. In summary, by this zebrafish Feingold syndrome type 1 model, we found that disturbance of ribosomal biogenesis and blockage of protein synthesis during development are primary causes of the intestinal defect in Feingold syndrome type 1. Importantly, our work suggests that leucine supplementation may be a feasible and easy treatment option for this disease.

developmental biology↗

bioRxiv2022

Rock inhibitors target SRSF2 leukemia by disrupting cell mitosis and nuclear morphology

Spliceosome machinery mutations are common early mutations in myeloid malignancies, however effective targeted therapies against them are still lacking. In the current study, we used an in vitro high-throughput drug screen among four different isogenic cell lines and identified ROCK inhibitors (ROCKi) as selective inhibitors of SRSF2 mutants. ROCKi targeted SRSF2 Mut primary human samples in a xenografts model and were not toxic to mice nor human cells. ROCKi induced mitotic catastrophe through their apparent effects on microtubules and nuclear organization. Transmission electron microscopy revealed that SRSF2 mutations induce deep nuclear indentation and segmentation, driven by microtubule-rich cytoplasmic intrusions, which were exacerbated by ROCKi. The severe nuclear deformation driven by the combination of SRSF2 Mut and ROCKi prevent cells from completing mitosis. These findings shed light on new ways to target SRSF2 and on the role of the microtubule system in SRSF2 Mut cells.

cancer biology↗

bioRxiv2022

Lineage-mosaic and mutation-patched spike proteins for broad-spectrum COVID-19 vaccine

The widespread SARS-CoV-2 in humans results in the continuous emergence of new variants. Recently emerged Omicron variant with multiple spike mutations sharply increases the risk of breakthrough infection or reinfection, highlighting the urgent need for new vaccines with broad-spectrum antigenic coverage. Using inter-lineage chimera and mutation patch strategies, we engineered a recombinant monomeric spike variant (STFK1628x), which showed high immunogenicity and mutually complementary antigenicity to its prototypic form (STFK). In hamsters, a bivalent vaccine comprised of STFK and STFK1628x elicited high titers of broad-spectrum antibodies to neutralize all 14 circulating SARS-CoV-2 variants, including Omicron; and fully protected vaccinees from intranasal SARS-CoV-2 challenges of either the ancestral strain or immune-evasive Beta variant. Strikingly, the vaccination of hamsters with the bivalent vaccine completely blocked the within-cage virus transmission to unvaccinated sentinels, for either the ancestral SARS-CoV-2 or Beta variant. Thus, our study provides new insights and antigen candidates for developing next-generation COVID-19 vaccines.

microbiology↗

bioRxiv2022

Whole-brain in vivo base editing reverses autistic-like behaviors in mice

Autism spectrum disorder (ASD) is a highly heritable neurodevelopmental disorder associated with deficits in social communication and stereotypical behaviors. Numerous ASD-related genetic mutations have been identified and genome editing methods have been developed but successful genome editing in the whole-brain scale to alleviate autistic-like behaviors in animal models has not been achieved. Here we report the development of a new CRISPR-mediated cytidine base editor (CBE) system, which converts C{middle dot}G base pairs to T{middle dot}A. We demonstrate the effectiveness of this system by targeting an ASD-associated de novo mutation in the MEF2C gene (c.104T>C, p.L35P). We constructed a Mef2c L35P knock-in mouse and observed that Mef2c L35P heterozygous mice displayed autistic-like behaviors, including deficits in social behaviors and repetitive behaviors. We programmed the CBE to edit the C{middle dot}G base pairs of the mutated Mef2c gene (c.104T>C, p.L35P) to T{middle dot}A base pairs and delivered it via a single dose intravenous injection of blood brain barrier (BBB)-crossing AAV-PHP.eB vector into the mouse brain. This treatment restored MEF2C protein levels and reversed impairments in social interactions and repetitive behaviors in Mef2c L35P heterozygous mice. Together, this work presents an in vivo gene editing strategy in which correcting a single nucleotide mutation in the whole-brain scale could be successfully achieved, further providing a new therapeutic framework for neurodevelopmental disorders.

neuroscience↗

bioRxiv2021

GREPore-seq: A Robust Workflow to Detect Changes after Gene Editing through Long-range PCR and Nanopore Sequencing

To achieve the enormous potential of gene-editing technology in clinical therapies, both the on-target and unintended editing consequences need to be thoroughly evaluated. However, there is a lack of a comprehensive, pipelined, large-scale and economical workflow for detecting genome editing outcomes, in particular insertion or deletion of a large fragment. Here, we describe an approach for efficient and accurate detection of multiple genetic changes after CRISPR-Cas9 editing by pooled nanopore sequencing of barcoded long-range PCR products. To overcome the high error rates and indels of nanopore sequencing, we developed a pipeline to capture the barcoded sequences by grepping reads of nanopore amplicon sequencing (GREPore-seq). GREPore-seq can detect NHEJ-mediated double-stranded oligodeoxynucleotide (dsODN) insertions with comparable accuracy to Illumina next-generation sequencing (NGS). GREPore-seq also identifies HDR-mediated large gene knock-in, which excellently correlates with FACS analysis data. Low-level plasmid backbone insertion after HDR editing was also detected. We have established a practical workflow to identify genetic changes, including quantifying dsODN insertions, knock-ins, plasmid backbone insertions, and large fragment deletions after CRISPR editing. This toolkit for nanopore sequencing of pooled long amplicons should have broad applications in assessing on-target HDR editing and inadvertent large indels of over 1 kb. GREPore-seq is freely available at GitHub (https://github.com/lisiang/GREPore-seq).

bioinformatics↗

bioRxiv2021

Inhibition of infection-induced vascular permeability modulates host leukocyte recruitment to Mycobacterium marinum granulomas in zebrafish

Mycobacterial granuloma formation involves significant stromal remodeling and the growth of leaky, granuloma-associated vasculature. These permeable blood vessels aid mycobacterial growth, as anti-angiogenic or vascular normalizing therapies are beneficial host-directed therapies in pre-clinical models of tuberculosis. Here we demonstrate that vascular normalization through vascular endothelial-protein tyrosine phosphatase inhibition decreases granuloma hypoxia, the opposite effect of hypoxia-inducing anti-angiogenic therapy. Vascular normalization leads to increased T cell and decreased neutrophil recruitment to granulomas, correlates of a protective immune response against mycobacterial infection.

immunology↗