技术资料

The processes that govern human haematopoietic stem cell (HSC) self-renewal and engraftment are poorly understood and challenging to recapitulate in culture to reliably expand functional HSCs 1 – 3 . Here we identify MYC target 1 (MYCT1; also known as MTLC) as a crucial human HSC regulator that moderates endocytosis and environmental sensing in HSCs. MYCT1 is selectively expressed in undifferentiated human haematopoietic stem and progenitor cells (HSPCs) and endothelial cells but becomes markedly downregulated during HSC culture. Lentivirus-mediated knockdown of MYCT1 prevented human fetal liver and cord blood (CB) HSPC expansion and engraftment. By contrast,restoring MYCT1 expression improved the expansion and engraftment of cultured CB HSPCs. Single-cell RNA sequencing of human CB HSPCs in which MYCT1 was knocked down or overexpressed revealed that MYCT1 governs important regulatory programmes and cellular properties essential for HSC stemness,such as ETS factor expression and low mitochondrial activity. MYCT1 is localized in the endosomal membrane in HSPCs and interacts with vesicle trafficking regulators and signalling machinery. MYCT1 loss in HSPCs led to excessive endocytosis and hyperactive signalling responses,whereas restoring MYCT1 expression balanced culture-induced endocytosis and dysregulated signalling. Moreover,sorting cultured CB HSPCs on the basis of lowest endocytosis rate identified HSPCs with preserved MYCT1 expression and MYCT1-regulated HSC stemness programmes. Our work identifies MYCT1-moderated endocytosis and environmental sensing as essential regulatory mechanisms required to preserve human HSC stemness. Our data also pinpoint silencing of MYCT1 as a cell-culture-induced vulnerability that compromises human HSC expansion. Subject terms: Haematopoietic stem cells,Self-renewal,Stem-cell niche,Endocytosis,Growth factor signalling View Publication

Which isoforms of apolipoprotein E (apoE) we inherit determine our risk of developing late-onset Alzheimer’s Disease (AD),but the mechanism underlying this link is poorly understood. In particular,the relevance of direct interactions between apoE and amyloid-β (Aβ) remains controversial. Here,single-molecule imaging shows that all isoforms of apoE associate with Aβ in the early stages of aggregation and then fall away as fibrillation happens. ApoE-Aβ co-aggregates account for ~50% of the mass of diffusible Aβ aggregates detected in the frontal cortices of homozygotes with the higher-risk APOE4 gene. We show how dynamic interactions between apoE and Aβ tune disease-related functions of Aβ aggregates throughout the course of aggregation. Our results connect inherited APOE genotype with the risk of developing AD by demonstrating how,in an isoform- and lipidation-specific way,apoE modulates the aggregation,clearance and toxicity of Aβ. Selectively removing non-lipidated apoE4-Aβ co-aggregates enhances clearance of toxic Aβ by glial cells,and reduces secretion of inflammatory markers and membrane damage,demonstrating a clear path to AD therapeutics. Subject terms: Protein aggregation,Nanoscale biophysics View Publication

Adult T-cell leukemia/lymphoma (ATL) occurs after human T-cell leukemia virus type-1 (HTLV-1) infection with a long latency period exceeding several decades. This implies the presence of immune evasion mechanisms for HTLV-1-infected T cells. Although ATL cells have a CD4 + CD25 + phenotype similar to that of regulatory T cells (Tregs),they do not always possess the immunosuppressive functions of Tregs. Factors that impart effective immunosuppressive functions to HTLV-1-infected cells may exist. A previous study identified a new CD13 + Treg subpopulation with enhanced immunosuppressive activity. We,herein,describe the paired CD13 − (designated as MT-50.1) and CD13 + (MT-50.4) HTLV-1-infected T-cell lines with Treg-like phenotype,derived from the peripheral blood of a single patient with lymphoma-type ATL. The cell lines were found to be derived from HTLV-1-infected non-leukemic cells. MT-50.4 cells secreted higher levels of immunosuppressive cytokines,IL-10 and TGF-β,expressed higher levels of Foxp3,and showed stronger suppression of CD4 + CD25 − T cell proliferation than MT-50.1 cells. Furthermore,the CD13 inhibitor bestatin significantly attenuated MT-50.4 cell growth,while it did not for MT-50.1 cells. These findings suggest that CD13 expression may be involved in the increased Treg-like activity of MT-50.4 cells. Hence,MT-50.4 cells will be useful for in-depth studies of CD13 + Foxp3 + HTLV-1-infected cells. Subject terms: Cancer,Microbiology,Oncology View Publication

Dynamic regulation of gene expression is fundamental for cellular adaptation to exogenous stressors. P-TEFb-mediated pause-release of RNA polymerase II (Pol II) is a conserved regulatory mechanism for synchronous transcriptional induction in response to heat shock,but this pro-survival role has not been examined in the applied context of cancer therapy. Using model systems of pediatric high-grade glioma,we show that rapid genome-wide reorganization of active chromatin facilitates P-TEFb-mediated nascent transcriptional induction within hours of exposure to therapeutic ionizing radiation. Concurrent inhibition of P-TEFb disrupts this chromatin reorganization and blunts transcriptional induction,abrogating key adaptive programs such as DNA damage repair and cell cycle regulation. This combination demonstrates a potent,synergistic therapeutic potential agnostic of glioma subtype,leading to a marked induction of tumor cell apoptosis and prolongation of xenograft survival. These studies reveal a central role for P-TEFb underpinning the early adaptive response to radiotherapy,opening avenues for combinatorial treatment in these lethal malignancies. Subject terms: CNS cancer,Paediatric cancer,Radiotherapy View Publication

Malaria parasite invasion to host erythrocytes is mediated by multiple interactions between merozoite ligands and erythrocyte receptors that contribute toward the development of disease pathology. Here,we report a novel antigen Plasmodium prohibitin “ Pf PHB2” and identify its cognate partner “Hsp70A1A” in host erythrocyte that plays a crucial role in mediating host-parasite interaction during merozoite invasion. Using small interfering RNA (siRNA)- and glucosamine-6-phosphate riboswitch (glmS) ribozyme-mediated approach,we show that loss of Hsp70A1A in red blood cells (RBCs) or Pf PHB2 in infected red blood cells (iRBCs),respectively,inhibit Pf PHB2-Hsp70A1A interaction leading to invasion inhibition. Antibodies targeting Pf PHB2 and monoclonal antibody therapeutics against Hsp70A1A efficiently block parasite invasion. Recombinant Pf PHB2 binds to RBCs which is inhibited by anti- Pf PHB2 antibody and monoclonal antibody against Hsp70A1A. The validation of Pf PHB2 to serve as antigen is further supported by detection of anti- Pf PHB2 antibody in patient sera. Overall,this study proposes Pf PHB2 as vaccine candidate and highlights the use of monoclonal antibody therapeutics for future malaria treatment. Subject areas: biochemistry,molecular biology,immunology View Publication

Cohesin plays a crucial role in the organization of topologically-associated domains (TADs),which influence gene expression and DNA replication timing. Whether epigenetic regulators may affect TADs via cohesin to mediate DNA replication remains elusive. Here,we discover that the histone demethylase PHF2 associates with RAD21,a core subunit of cohesin,to regulate DNA replication in mouse neural stem cells (NSC). PHF2 loss impairs DNA replication due to the activation of dormant replication origins in NSC. Notably,the PHF2/RAD21 co-bound genomic regions are characterized by CTCF enrichment and epigenomic features that resemble efficient,active replication origins,and can act as boundaries to separate adjacent domains. Accordingly,PHF2 loss weakens TADs and chromatin loops at the co-bound loci due to reduced RAD21 occupancy. The observed topological and DNA replication defects in PHF2 KO NSC support a cohesin-dependent mechanism. Furthermore,we demonstrate that the PHF2/RAD21 complex exerts little effect on gene regulation,and that PHF2’s histone-demethylase activity is dispensable for normal DNA replication and proliferation of NSC. We propose that PHF2 may serve as a topological accessory to cohesin for cohesin localization to TADs and chromatin loops,where cohesin represses dormant replication origins directly or indirectly,to sustain DNA replication in NSC. View Publication

Interference with microtubule dynamics in mitosis activates the spindle assembly checkpoint (SAC) to prevent chromosome segregation errors. The SAC induces mitotic arrest by inhibiting the anaphase-promoting complex (APC) via the mitotic checkpoint complex (MCC). The MCC component MAD2 neutralizes the critical APC cofactor,CDC20,preventing exit from mitosis. Extended mitotic arrest can promote mitochondrial apoptosis and caspase activation. However,the impact of mitotic cell death on tissue homeostasis in vivo is ill-defined. By conditional MAD2 overexpression,we observe that chronic SAC activation triggers bone marrow aplasia and intestinal atrophy in mice. While myelosuppression can be compensated for,gastrointestinal atrophy is detrimental. Remarkably,deletion of pro-apoptotic Bim/Bcl2l11 prevents gastrointestinal syndrome,while neither loss of Noxa/Pmaip or co-deletion of Bid and Puma/Bbc3 has such a protective effect,identifying BIM as rate-limiting apoptosis effector in mitotic cell death of the gastrointestinal epithelium. In contrast,only overexpression of anti-apoptotic BCL2,but none of the BH3-only protein deficiencies mentioned above,can mitigate myelosuppression. Our findings highlight tissue and cell-type-specific survival dependencies in response to SAC perturbation in vivo. View Publication

Differentiation of human pluripotent stem cells (hPSCs) into subtype-specific neurons holds substantial potential for disease modeling in vitro . For successful differentiation,a detailed understanding of the transcriptional networks regulating cell fate decisions is critical. The heterochronic nature of neurodevelopment,during which distinct cells in the brain and during in vitro differentiation acquire their fates in an unsynchronized manner,hinders pooled transcriptional comparisons. One approach is to “translate” chronologic time into linear developmental and maturational time. Simple binary promotor-driven fluorescent proteins (FPs) to pool similar cells are unable to achieve this goal,due to asynchronous promotor onset in individual cells. We tested five fluorescent timer (FT) molecules expressed from the endogenous paired box 6 (PAX6) promoter in 293T and human hPSCs. Each of these FT systems faithfully reported chronologic time in 293T cells,but none of the FT constructs followed the same fluorescence kinetics in human neural progenitor cells. Subject areas: Natural sciences,Biological sciences,Biochemistry,Molecular biology,Neuroscience,Cellular neuroscience,Cell biology View Publication

Establishing robust models of human myelinating Schwann cells is critical for studying peripheral nerve injury and disease. Stem cell differentiation has emerged as a key human cell model and disease motivating development of Schwann cell differentiation protocols. Human embryonic stem cells (hESCs) are considered the ideal pluripotent cell but ethical concerns regarding their use have propelled the popularity of human induced pluripotent stem cells (hiPSCs). Given that the equivalence of hESCs and hiPSCs remains controversial,we sought to compare the molecular and functional equivalence of hESC- and hiPSC-derived Schwann cells generated with our previously reported protocol. We identified only modest transcriptome differences by RNA sequencing and insignificant proteome differences by antibody array. Additionally,both cell types comparably improved nerve regeneration and function in a chronic denervation and regeneration animal model. Our findings demonstrate that Schwann cells derived from hESCs and hiPSCs with our protocol are molecularly comparable and functionally equivalent. Subject areas: Neuroscience,Cell biology,Stem cells research,Transcriptomics View Publication

Neural–tumor interactions drive glioma growth as evidenced in preclinical models,but clinical validation is limited. We present an epigenetically defined neural signature of glioblastoma that independently predicts patients’ survival. We use reference signatures of neural cells to deconvolve tumor DNA and classify samples into low- or high-neural tumors. High-neural glioblastomas exhibit hypomethylated CpG sites and upregulation of genes associated with synaptic integration. Single-cell transcriptomic analysis reveals a high abundance of malignant stemcell-like cells in high-neural glioblastoma,primarily of the neural lineage. These cells are further classified as neural-progenitor-cell-like,astrocyte-like and oligodendrocyte-progenitor-like,alongside oligodendrocytes and excitatory neurons. In line with these findings,high-neural glioblastoma cells engender neuron-to-glioma synapse formation in vitro and in vivo and show an unfavorable survival after xenografting. In patients,a high-neural signature is associated with decreased overall and progression-free survival. High-neural tumors also exhibit increased functional connectivity in magnetencephalography and resting-state magnet resonance imaging and can be detected via DNA analytes and brain-derived neurotrophic factor in patients’ plasma. The prognostic importance of the neural signature was further validated in patients diagnosed with diffuse midline glioma. Our study presents an epigenetically defined malignant neural signature in high-grade gliomas that is prognostically relevant. High-neural gliomas likely require a maximized surgical resection approach for improved outcomes. Subject terms: Translational research,CNS cancer,DNA methylation View Publication

STAT3 is constitutively activated in many cancer types,including lung cancer,and can induce cancer cell proliferation and cancer stem cell (CSC) maintenance. STAT3 is activated by tyrosine kinases,such as JAK and SRC,but the mechanism by which STAT3 maintains its activated state in cancer cells remains unclear. Here,we show that PRMT5 directly methylates STAT3 and enhances its activated tyrosine phosphorylation in non-small cell lung cancer (NSCLC) cells. PRMT5 expression is also induced by STAT3,suggesting the presence of a positive feedback loop in cancer cells. Furthermore,methylation of STAT3 at arginine 609 by PRMT5 is important for its transcriptional activity and support of tumour growth and CSC maintenance. Indeed,NSCLC cells expressing the STAT3 mutant which R609 was replaced to alanine (R609K) show significantly impaired tumour growth in nude mice. Overall,our study reveals a mechanism by which STAT3 remains activated in NSCLC and provides a new target for cancer therapeutic approaches. Subject terms: Oncogenes,Non-small-cell lung cancer,Growth factor signalling View Publication

Hemangiosarcoma and angiosarcoma are soft-tissue sarcomas of blood vessel–forming cells in dogs and humans,respectively. These vasoformative sarcomas are aggressive and highly metastatic,with disorganized,irregular blood-filled vascular spaces. Our objective was to define molecular programs which support the niche that enables progression of canine hemangiosarcoma and human angiosarcoma. Dog-in-mouse hemangiosarcoma xenografts recapitulated the vasoformative and highly angiogenic morphology and molecular characteristics of primary tumors. Blood vessels in the tumors were complex and disorganized,and they were lined by both donor and host cells. In a series of xenografts,we observed that the transplanted hemangiosarcoma cells created exuberant myeloid hyperplasia and gave rise to lymphoproliferative tumors of mouse origin. Our functional analyses indicate that hemangiosarcoma cells generate a microenvironment that supports expansion and differentiation of hematopoietic progenitor populations. Furthermore,gene expression profiling data revealed hemangiosarcoma cells expressed a repertoire of hematopoietic cytokines capable of regulating the surrounding stromal cells. We conclude that canine hemangiosarcomas,and possibly human angiosarcomas,maintain molecular properties that provide hematopoietic support and facilitate stromal reactions,suggesting their potential involvement in promoting the growth of hematopoietic tumors. We demonstrate that hemangiosarcomas regulate molecular programs supporting hematopoietic expansion and differentiation,providing insights into their potential roles in creating a permissive stromal-immune environment for tumor progression. View Publication