参考文献

Fragile X Syndrome (FXS) is a neurological disorder caused by epigenetic silencing of the FMR1 gene. Reactivation of FMR1 is a potential therapeutic approach for FXS that would correct the root cause of the disease. Here,using a candidate-based shRNA screen,we identify nine epigenetic repressors that promote silencing of FMR1 in FXS cells (called FMR1 Silencing Factors,or FMR1- SFs). Inhibition of FMR1-SFs with shRNAs or small molecules reactivates FMR1 in cultured undifferentiated induced pluripotent stem cells,neural progenitor cells (NPCs) and post-mitotic neurons derived from FXS patients. One of the FMR1-SFs is the histone methyltransferase EZH2,for which an FDA-approved small molecule inhibitor,EPZ6438 (also known as tazemetostat),is available. We show that EPZ6438 substantially corrects the characteristic molecular and electrophysiological abnormalities of cultured FXS neurons. Unfortunately,EZH2 inhibitors do not efficiently cross the blood-brain barrier,limiting their therapeutic use for FXS. Recently,antisense oligonucleotide (ASO)-based approaches have been developed as effective treatment options for certain central nervous system disorders. We therefore derived efficacious ASOs targeting EZH2 and demonstrate that they reactivate FMR1 expression and correct molecular and electrophysiological abnormalities in cultured FXS neurons,and reactivate FMR1 expression in human FXS NPCs engrafted within the brains of mice. Collectively,our results establish EZH2 inhibition in general,and EZH2 ASOs in particular,as a therapeutic approach for FXS. View Publication

In mammals,early embryonic gastrulation process is high energy demanding. Previous studies showed that,unlike endoderm and mesoderm cells,neuroectoderm differentiated from human embryonic stem cells relied on aerobic glycolysis as the major energy metabolic process,which generates lactate as the final product. Here we explored the function of intracellular lactate during neuroectoderm differentiation. Our results revealed that the intracellular lactate level was elevated in neuroectoderm and exogenous lactate could further promote hESCs differentiation towards neuroectoderm. Changing intracellular lactate levels by sodium lactate or LDHA inhibitors had no obvious effect on BMP or WNT/?-catenin signaling during neuroectoderm differentiation. Notably,histone lactylation,especially H3K18 lactylation was significant upregulated during this process. We further performed CUT&Tag experiments and the results showed that H3K18la is highly enriched at gene promoter regions. By analyzing data from CUT&Tag and RNA-seq experiments,we further identified that four genes,including PAX6,were transcriptionally upregulated by lactate during neuroectoderm differentiation. A H3K18la modification site at PAX6 promoter was verified and exogenous lactate could also rescue the level of PAX6 after shPAX6 inhibition.Supplementary InformationThe online version contains supplementary material available at 10.1007/s00018-024-05510-x. View Publication

Pancreatic islets are densely packed cellular aggregates containing various hormonal cell types essential for blood glucose regulation. Interactions among these cells markedly affect the glucoregulatory functions of islets along with the surrounding niche and pancreatic tissue-specific geometrical organization. However,stem cell (SC)-derived islets generated in vitro often lack the three-dimensional extracellular microenvironment and peri-vasculature,which leads to the immaturity of SC-derived islets,reducing their ability to detect glucose fluctuations and insulin release. Here,we bioengineer the in vivo-like pancreatic niches by optimizing the combination of pancreatic tissue-specific extracellular matrix and basement membrane proteins and utilizing bioprinting-based geometrical guidance to recreate the spatial pattern of islet peripheries. The bioprinted islet-specific niche promotes coordinated interactions between islets and vasculature,supporting structural and functional features resembling native islets. Our strategy not only improves SC-derived islet functionality but also offers significant potential for advancing research on islet development,maturation,and diabetic disease modeling,with future implications for translational applications. The glucoregulatory functions of pancreatic islets are affected by their surrounding niche and spatial organization. Here,bioengineered stem-cell derived islet niches use bioprinting-based geometrical guidance to promote islet maturation for improved functionality and diabetes research. View Publication

Spinocerebellar ataxia type 3 (SCA3) is an autosomal dominant neurodegenerative disease caused by repeat expansion of the CAG trinucleotide within exon 10 of the ATXN3 gene. This mutation results in the production of an abnormal ataxin-3 protein containing an extended polyglutamine tract,referred to as mutant ataxin-3. In this study,we investigated the therapeutic potential of CRISPR/Cas9-mediated genome editing for SCA3. First,we designed a specific single-guide RNA targeting the ATXN3 gene and constructed the corresponding targeting vector. Induced pluripotent stem cells (iPSCs) derived from a SCA3 patient were then electroporated with the CRISPR/Cas9 components. Positive clones were screened and validated by PCR and Sanger sequencing to obtain genome-editing iPSCs (GE-iPSCs). Subsequently,the pluripotency of GE-iPSCs was confirmed,and the effects of genome editing on mutant ataxin-3 protein expression and Golgi apparatus morphology were assessed using Western blotting and immunofluorescence analyses. Our results demonstrated that targeted insertion of polyadenylation signals (PAS) upstream of the abnormal CAG repeats effectively suppressed the production of mutant ataxin-3. This intervention also reduced the formation of neuronal nuclear inclusions in differentiated neurons,restored the structural integrity of the Golgi apparatus (which exhibited a loose and enlarged morphology in SCA3 cells),and increased the expression levels of Golgi structural proteins (GM130 and GORASP2). In conclusion,our findings indicate that the targeted insertion of PAS upstream of the abnormal CAG repeats in the ATXN3 gene represents a promising therapeutic strategy for SCA3 through genome editing.Supplementary InformationThe online version contains supplementary material available at 10.1038/s41598-025-93369-8. View Publication

SUMMARYUnderstanding how human gene expression is coordinately regulated by functional units of proteins across the genome remains a major biological goal. Here,we present COMET,a high-throughput screening platform for combinatorial effector targeting for the identification of transcriptional modulators. We generate libraries of combinatorial dCas9-based fusion proteins,containing two to six effector domains,allowing us to systematically investigate more than 110,000 combinations of effector proteins at endogenous human loci for their influence on transcription. Importantly,we keep full proteins or domains intact,maintaining catalytic cores and surfaces for protein-protein interactions. We observe more than 5800 significant hits that modulate transcription,we demonstrate cell type specific transcriptional modulation,and we further investigate epistatic relationships between our effector combinations. We validate unexpected combinations as synergistic or buffering,emphasizing COMET as both a method for transcriptional effector discovery,and as a functional genomics tool for identifying novel domain interactions and directing locus-specific biochemistry. View Publication

Ribosome profiling,which is based on deep sequencing of ribosome footprints,has served as a powerful tool for elucidating the regulatory mechanism of protein synthesis. However,the current method has substantial issues: contamination by rRNAs and the lack of appropriate methods to measure ribosome numbers in transcripts. Here,we overcome these hurdles through the development of “Ribo-FilterOut”,which is based on the separation of footprints from ribosome subunits by ultrafiltration,and “Ribo-Calibration”,which relies on external spike-ins of stoichiometrically defined mRNA-ribosome complexes. A combination of these approaches estimates the number of ribosomes on a transcript,the translation initiation rate,and the overall number of translation events before its decay,all in a genome-wide manner. Moreover,our method reveals the allocation of ribosomes under heat shock stress,during aging,and across cell types. Our strategy of modified ribosome profiling measures kinetic and stoichiometric parameters of cellular translation across the transcriptome. Ribosome profiling faces issues with rRNA contamination and measurements of ribosome numbers on transcripts. Here,the authors develop Ribo-FilterOut and Ribo-Calibration,methods which can be used to estimate kinetic and stoichiometric parameters of translation under various conditions. View Publication

BackgroundCHD7 encodes an ATP-dependent chromodomain helicase DNA binding protein; mutations in this gene lead to multiple developmental disorders,including CHARGE (Coloboma,Heart defects,Atresia of the choanae,Retardation of growth and development,Genital hypoplasia,and Ear anomalies) syndrome. How the mutations cause multiple defects remains largely unclear. Embryonic definitive endoderm (DE) generates the epithelial compartment of vital organs such as the thymus,liver,pancreas,and intestine.MethodsIn this study,we used the clustered regularly interspaced short palindromic repeats (CRISPR)/Cas9 technique to delete the CHD7 gene in human embryonic stem cells (hESCs) to generate CHD7 homozygous mutant (CHD7?/?),heterozygous mutant (CHD7+/?),and control wild-type (CHD7+/+) cells. We then investigated the ability of the hESCs to develop into DE and the other two germ layers,mesoderm and ectoderm in vitro. We also compared global gene expression and chromatin accessibility among the hESC-DE cells by RNA sequencing (RNA-seq) and the assay for transposase-accessible chromatin with sequencing (ATAC-seq).ResultsWe found that deletion of CHD7 led to reduced capacity to develop into DE and mesoderm in a dose-dependent manner. Loss of CHD7 led to significant changes in the expression and chromatin accessibility of genes associated with several pathways. We identified 40 genes that were highly down-regulated in both the expression and chromatin accessibility in CHD7 deleted hESC-DE cells.ConclusionsCHD7 is critical for DE and mesodermal development from hESCs. Our results provide new insights into the mechanisms by which CHD7 mutations cause multiple congenital anomalies.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13287-025-04437-9. View Publication

The spatial organization of molecules in a cell is essential for their functions. While current methods focus on discerning tissue architecture,cell–cell interactions,and spatial expression patterns,they are limited to the multicellular scale. We present Bento,a Python toolkit that takes advantage of single-molecule information to enable spatial analysis at the subcellular scale. Bento ingests molecular coordinates and segmentation boundaries to perform three analyses: defining subcellular domains,annotating localization patterns,and quantifying gene–gene colocalization. We demonstrate MERFISH,seqFISH +,Molecular Cartography,and Xenium datasets. Bento is part of the open-source Scverse ecosystem,enabling integration with other single-cell analysis tools.Supplementary InformationThe online version contains supplementary material available at 10.1186/s13059-024-03217-7. View Publication

Huntington’s disease and other disorders of the basal ganglia create challenges for biomolecule-based medicines given the poor accessibility of these deep brain structures following intracerebral or intravascular delivery. Here,we found that low dose,low volume delivery of unbiased AAV libraries into the globus pallidus allowed recovery of novel capsids capable of broad access to key deep brain and cortical structures relevant for human therapies. One such capsid,AAV-DB-3,provided transduction of up to 45% of medium spiny neurons in the adult NHP striatum,along with substantial transduction of relevant deep layer neurons in the cortex. Notably,AAV-DB-3 behaved similarly in mice as in NHPs and potently transduced human neurons derived from induced pluripotent stem cells. Thus,AAV-DB-3 provides a unique AAV for network level brain gene therapies that translates up and down the evolutionary scale for preclinical studies and eventual clinical use. To date,brain gene therapies require high vector doses. Here,authors devised an AAV capsid screen and found variants with unprecedented potency for transduction of deep brain and cortical neurons and human iPSC-neurons with cell tropism relevant for Huntington’s and Parkinson’s disease. View Publication

AbstractBackgroundParkinson's disease (PD) is a chronic neurodegenerative disorder characterized by the loss of dopaminergic neurons in the substantia nigra,which promotes a sustained inflammatory environment in the central nervous system. Regulatory T cells (Tregs) play an important role in the control of inflammation and might play a neuroprotective role. Indeed,a decrease in Treg number and function has been reported in PD. In this context,pramipexole,a dopaminergic receptor agonist used to treat PD symptoms,has been shown to increase peripheral levels of Treg cells and improve their suppressive function. The aim of this work was to determine the effect of pramipexole on immunoregulatory Treg cells and its possible neuroprotective effect on human dopaminergic neurons differentiated from human embryonic stem cells.MethodsTreg cells were sorted from white blood cells of healthy human donors. Assays were performed with CD3/CD28?activated and non?activated Treg cells treated with pramipexole at concentrations of 2 or 200 ng/mL. These regulatory cells were co?cultured with in vitro?differentiated human dopaminergic neurons in a cytotoxicity assay with 6?hydroxydopamine (6?OHDA). The role of interleukin?10 (IL?10) was investigated by co?culturing activated IL?10?producing Treg cells with neurons. To further investigate the effect of treatment on Tregs,gene expression in pramipexole?treated,CD3/CD28?activated Treg cells was determined by Fluidigm analysis.ResultsPramipexole?treated CD3/CD28?activated Treg cells showed significant protective effects on dopaminergic neurons when challenged with 6?OHDA. Pramipexole?treated activated Treg cells showed neuroprotective capacity through mechanisms involving IL?10 release and the activation of genes associated with regulation and neuroprotection.ConclusionAnti?CD3/CD28?activated Treg cells protect dopaminergic neurons against 6?OHDA?induced damage. In addition,activated,IL?10?producing,pramipexole?treated Tregs also induced a neuroprotective effect,and the supernatants of these co?cultures promoted axonal growth. Pramipexole?treated,activated Tregs altered their gene expression in a concentration?dependent manner,and enhanced TGF??related dopamine receptor regulation and immune?related pathways. These findings open new perspectives for the development of immunomodulatory therapies for the treatment of PD. Pramipexole?treated,activated regulatory T cells protect dopaminergic neurons against 6 OHDA damage and promote primary neurite length. This could be due to the production of the regulatory cytokine IL?10 and an increased expression of genes related to regulation and neuroprotection. View Publication