技术资料

The ability to analyze protein function in a native context is central to understanding cellular physiology. This study explores whether tagging endogenous proteins with a reporter is a scalable strategy for generating cell models that accurately quantitate protein dynamics. Specifically,it investigates whether CRISPR-mediated integration of the HiBiT luminescent peptide tag can easily be accomplished on a large-scale and whether integrated reporter faithfully represents target biology. For this purpose,a large set of proteins representing diverse structures and functions,some of which are known or potential drug targets,were targeted for tagging with HiBiT in multiple cell lines. Successful insertion was detected for 86{\%} of the targets,as determined by luminescence-based plate assays,blotting,and imaging. In order to determine whether endogenously tagged proteins yield more representative models,cells expressing HiBiT protein fusions either from endogenous loci or plasmids were directly compared in functional assays. In the tested cases,only the edited lines were capable of accurately reproducing the anticipated biology. This study provides evidence that cell lines expressing HiBiT fusions from endogenous loci can be rapidly generated for many different proteins and that these cellular models provide insight into protein function that may be unobtainable using overexpression-based approaches. View Publication

The initiation of an intestinal tumour is a probabilistic process that depends on the competition between mutant and normal epithelial stem cells in crypts1. Intestinal stem cells are closely associated with a diverse but poorly characterized network of mesenchymal cell types2,3. However,whether the physiological mesenchymal microenvironment of mutant stem cells affects tumour initiation remains unknown. Here we provide in vivo evidence that the mesenchymal niche controls tumour initiation in trans. By characterizing the heterogeneity of the intestinal mesenchyme using single-cell RNA-sequencing analysis,we identified a population of rare pericryptal Ptgs2-expressing fibroblasts that constitutively process arachidonic acid into highly labile prostaglandin E2 (PGE2). Specific ablation of Ptgs2 in fibroblasts was sufficient to prevent tumour initiation in two different models of sporadic,autochthonous tumorigenesis. Mechanistically,single-cell RNA-sequencing analyses of a mesenchymal niche model showed that fibroblast-derived PGE2 drives the expansion οf a population of Sca-1+ reserve-like stem cells. These express a strong regenerative/tumorigenic program,driven by the Hippo pathway effector Yap. In vivo,Yap is indispensable for Sca-1+ cell expansion and early tumour initiation and displays a nuclear localization in both mouse and human adenomas. Using organoid experiments,we identified a molecular mechanism whereby PGE2 promotes Yap dephosphorylation,nuclear translocation and transcriptional activity by signalling through the receptor Ptger4. Epithelial-specific ablation of Ptger4 misdirected the regenerative reprogramming of stem cells and prevented Sca-1+ cell expansion and sporadic tumour initiation in mutant mice,thereby demonstrating the robust paracrine control of tumour-initiating stem cells by PGE2-Ptger4. Analyses of patient-derived organoids established that PGE2-PTGER4 also regulates stem-cell function in humans. Our study demonstrates that initiation of colorectal cancer is orchestrated by the mesenchymal niche and reveals a mechanism by which rare pericryptal Ptgs2-expressing fibroblasts exert paracrine control over tumour-initiating stem cells via the druggable PGE2-Ptger4-Yap signalling axis. View Publication

CRISPR genome engineering has become a powerful tool to functionally investigate the complex mechanisms of immune system regulation. While decades of work have aimed to genetically reprogram innate immunity,the utility of current approaches is restricted by poor knockout efficiencies or limited specificity for mature cell lineages in vivo. Here,we describe an optimized strategy for non-viral CRISPR-Cas9 ribonucleoprotein (cRNP) genomic editing of mature primary mouse innate lymphocyte cells (ILCs) and myeloid lineage cells that results in an almost complete loss of single or double target gene expression from a single electroporation. Furthermore,we describe in vivo adoptive transfer mouse models that can be utilized to screen for gene function during viral infection using cRNP-edited naive natural killer (NK) cells and bone-marrow-derived conventional dendritic cell precursors (cDCPs). This resource will enhance target gene discovery and offer a specific and simplified approach to gene editing in the mouse innate immune system. View Publication

PURPOSE To assess the potential for CUE-101,a novel therapeutic fusion protein,to selectively activate and expand HPV16 E711-20-specific CD8+ T cells as an off-the shelf therapy for the treatment of HPV16-driven tumors,including head and neck squamous cell carcinoma (HNSCC),cervical,and anal cancers. EXPERIMENTAL DESIGN CUE-101 is an Fc fusion protein composed of a human leukocyte antigen (HLA) complex,an HPV16 E7 peptide epitope,reduced affinity human IL2 molecules,and an effector attenuated human IgG1 Fc domain. Human E7-specific T cells and human peripheral blood mononuclear cells (PBMC) were tested to demonstrate cellular activity and specificity of CUE-101,whereas in vivo activity of CUE-101 was assessed in HLA-A2 transgenic mice. Antitumor efficacy with a murine surrogate (mCUE-101) was tested in the TC-1 syngeneic tumor model. RESULTS CUE-101 demonstrates selective binding,activation,and expansion of HPV16 E711-20-specific CD8+ T cells from PBMCs relative to nontarget cells. Intravenous administration of CUE-101 induced selective expansion of HPV16 E711-20-specific CD8+ T cells in HLA-A2 (AAD) transgenic mice,and anticancer efficacy and immunologic memory was demonstrated in TC-1 tumor-bearing mice treated with mCUE-101. Combination therapy with anti-PD-1 checkpoint blockade further enhanced the observed efficacy. CONCLUSIONS Consistent with its design,CUE-101 demonstrates selective expansion of an HPV16 E711-20-specific population of cytotoxic CD8+ T cells,a favorable safety profile,and in vitro and in vivo evidence supporting its potential for clinical efficacy in an ongoing phase I trial (NCT03978689). View Publication

Autoantibodies to transglutaminase 2 (TG2) are hallmarks of celiac disease. To address B cell tolerance and autoantibody formation to TG2,we generated immunoglobulin knock-in (Ig KI) mice that express a prototypical celiac patient-derived anti-TG2 B cell receptor equally reactive to human and mouse TG2. We studied B cell development in the presence/absence of autoantigen by crossing the Ig KI mice to Tgm2-/- mice. Autoreactive B cells in Tgm2+/+ mice were indistinguishable from their naive counterparts in Tgm2-/- mice with no signs of clonal deletion,receptor editing,or B cell anergy. The autoreactive B cells appeared ignorant to their antigen,and they produced autoantibodies when provided T cell help. The findings lend credence to a model of celiac disease where gluten-reactive T cells provide help to autoreactive TG2-specific B cells by involvement of gluten-TG2 complexes,and they outline a general mechanism of autoimmunity with autoantibodies being produced by ignorant B cells on provision of T cell help. View Publication

The combination of in vitro multi-electrode arrays (MEAs) and the neuronal differentiation of stem cells offers the capability to study human neuronal networks from patient or engineered human cell lines. Here,we use MEA-based assays to probe synaptic function and network interactions of hiPSC-derived neurons. Neuronal network behaviour first emerges at approximately 30 days of culture and is driven by glutamate neurotransmission. Over a further 30 days,inhibitory GABAergic signalling shapes network behaviour into a synchronous regular pattern of burst firing activity and low activity periods. Gene mutations in L-type voltage gated calcium channel subunit genes are strongly implicated as genetic risk factors for the development of schizophrenia and bipolar disorder. We find that,although basal neuronal firing rate is unaffected,there is a dose-dependent effect of L-type voltage gated calcium channel inhibitors on synchronous firing patterns of our hiPSC-derived neural networks. This demonstrates that MEA assays have sufficient sensitivity to detect changes in patterns of neuronal interaction that may arise from hypo-function of psychiatric risk genes. Our study highlights the utility of in vitro MEA based platforms for the study of hiPSC neural network activity and their potential use in novel compound screening. View Publication

Developmental and/or epileptic encephalopathies (DEEs) are a group of devastating genetic disorders,resulting in early-onset,therapy-resistant seizures and developmental delay. Here we report on 22 individuals from 15 families presenting with a severe form of intractable epilepsy,severe developmental delay,progressive microcephaly,visual disturbance and similar minor dysmorphisms. Whole exome sequencing identified a recurrent,homozygous variant (chr2:64083454A {\textgreater} G) in the essential UDP-glucose pyrophosphorylase (UGP2) gene in all probands. This rare variant results in a tolerable Met12Val missense change of the longer UGP2 protein isoform but causes a disruption of the start codon of the shorter isoform,which is predominant in brain. We show that the absence of the shorter isoform leads to a reduction of functional UGP2 enzyme in neural stem cells,leading to altered glycogen metabolism,upregulated unfolded protein response and premature neuronal differentiation,as modeled during pluripotent stem cell differentiation in vitro. In contrast,the complete lack of all UGP2 isoforms leads to differentiation defects in multiple lineages in human cells. Reduced expression of Ugp2a/Ugp2b in vivo in zebrafish mimics visual disturbance and mutant animals show a behavioral phenotype. Our study identifies a recurrent start codon mutation in UGP2 as a cause of a novel autosomal recessive DEE syndrome. Importantly,it also shows that isoform-specific start-loss mutations causing expression loss of a tissue-relevant isoform of an essential protein can cause a genetic disease,even when an organism-wide protein absence is incompatible with life. We provide additional examples where a similar disease mechanism applies. View Publication

Cerebrospinal fluid (CSF) is a vital liquid,providing nutrients and signaling molecules and clearing out toxic by-products from the brain. The CSF is produced by the choroid plexus (ChP),a protective epithelial barrier that also prevents free entry of toxic molecules or drugs from the blood. Here,we establish human ChP organoids with a selective barrier and CSF-like fluid secretion in self-contained compartments. We show that this in vitro barrier exhibits the same selectivity to small molecules as the ChP in vivo and that ChP-CSF organoids can predict central nervous system (CNS) permeability of new compounds. The transcriptomic and proteomic signatures of ChP-CSF organoids reveal a high degree of similarity to the ChP in vivo. Finally,the intersection of single-cell transcriptomics and proteomic analysis uncovers key human CSF components produced by previously unidentified specialized epithelial subtypes. View Publication

Enteroids are cultured primary intestinal epithelial cells that recapitulate epithelial lineage development allowing for a more complex and physiologically relevant model for scientific study. The large presence of intestinal stem cells (ISC) in these enteroids allows for the study of metabolite effects on cellular processes and resulting progeny cells. Short-chain fatty acids (SCFA) such as butyrate (BUT) are bacterial metabolites produced in the gastrointestinal tract that are considered to be beneficial to host cells. Therefore,the objective was to study the effects of SCFAs on biomarkers of ISC activity,differentiation,barrier function and epithelial defense in the intestine using mouse and human enteroid models. Enteroids were treated with two concentrations of acetate (ACET),propionate (PROP),or BUT for 24 h. Enteroids treated with BUT or PROP showed a decrease in proliferation via EdU uptake relative to the controls in both mouse and human models. Gene expression of Lgr5 was shown to decrease with BUT and PROP treatments,but increased with ACET. As a result of BUT and PROP treatments,there was an increase in differentiation markers for enterocyte,Paneth,goblet,and enteroendocrine cells. Gene expression of antimicrobial proteins Reg3$\beta$,Reg3$\gamma$,and Defb1 were stimulated by BUT and PROP,but not by ACET which had a greater effect on expression of tight junction genes Cldn3 and Ocln in 3D enteroids. Similar results were obtained with human enteroids treated with 10 mM SCFAs and grown in either 3D or Transwell™ model cultures,although tight junctions were influenced by BUT and PROP,but not ACET in monolayer format. Furthermore,BUT and PROP treatments increased transepithelial electrical resistance after 24 h compared to ACET or control. Overall,individual SCFAs are potent stimulators of cellular gene expression,however,PROP and especially BUT show great efficacy for driving cell differentiation and gene expression. View Publication

Site-1 protease (S1P) ablation in the osterix-lineage in mice drastically reduces bone development and downregulates bone marrow-derived skeletal stem cells. Here we show that these mice also suffer from spina bifida occulta with a characteristic lack of bone fusion in the posterior neural arches. Molecular analysis of bone marrow-derived non-red blood cell cells,via single-cell RNA-Seq and protein mass spectrometry,demonstrate that these mice have a much-altered bone marrow with a significant increase in neutrophils and Ly6C-expressing leukocytes. The molecular composition of bone marrow neutrophils is also different as they express more and additional members of the stefin A (Stfa) family of proteins. In vitro,recombinant Stfa1 and Stfa2 proteins have the ability to drastically inhibit osteogenic differentiation of bone marrow stromal cells,with no effect on adipogenic differentiation. FACS analysis of hematopoietic stem cells show that despite a decrease in hematopoietic stem cells,S1P ablation results in an increased production of granulocyte-macrophage progenitors,the precursors to neutrophils. These observations indicate that S1P has a role in the lineage specification of hematopoietic stem cells and/or their progenitors for development of a normal hematopoietic niche. Our study designates a fundamental requirement of S1P for maintaining a balanced regenerative capacity of the bone marrow niche. View Publication

The intestinal stem cell (ISC) marker LGR5 is a receptor for R-spondin (RSPO) that functions to potentiate Wnt signalling in the proliferating crypt. It has been recently shown that Wnt plays a priming role for ISC self-renewal by inducing RSPO receptor LGR5 expression. Despite its pivotal role in homeostasis,regeneration and cancer,little is known about the post-translational regulation of LGR5. Here,we show that the HECT-domain E3 ligases NEDD4 and NEDD4L are expressed in the crypt stem cell regions and regulate ISC priming by degrading LGR receptors. Loss of Nedd4 and Nedd4l enhances ISC proliferation,increases sensitivity to RSPO stimulation and accelerates tumour development in Apcmin mice with increased numbers of high-grade adenomas. Mechanistically,we find that both NEDD4 and NEDD4L negatively regulate Wnt/$\beta$-catenin signalling by targeting LGR5 receptor and DVL2 for proteasomal and lysosomal degradation. Our findings unveil the previously unreported post-translational control of LGR receptors via NEDD4/NEDD4L to regulate ISC priming. Inactivation of NEDD4 and NEDD4L increases Wnt activation and ISC numbers,which subsequently enhances tumour predisposition and progression. View Publication

The network topology of a protein interactome is shaped by the function of each protein,making it a resource of functional knowledge in tissues and in single cells. Today,this resource is underused,as complete network topology characterization has proved difficult for large protein interactomes. We apply a matrix visualization and decoding approach to a physical protein interactome of a dendritic cell,thereby characterizing its topology with no prior assumptions of structure. We discover 294 proteins,each forming topological motifs called bow-ties" that tie together the majority of observed protein complexes. The central proteins of these bow-ties have unique network properties display multifunctional capabilities are enriched for essential proteins and are widely expressed in other cells and tissues. Collectively the bow-tie motifs are a pervasive and previously unnoted topological trend in cellular interactomes. As such these results provide fundamental knowledge on how intracellular protein connectivity is organized and operates." View Publication