Biology subjects

Gao, D.

Publications and source records attributed to Gao, D..

At least 37 records · Page 2Linked to original sources

bioRxiv2024

Elevated levels of intracellular RNA lariats suppress the antiviral response

Recent studies report the genetic loss of the lariat debranching enzyme (DBR1) activity increases susceptibility to viral infection. Here, we show that more than 25% of human introns contain large hairpin structures created by the folding of two Alu elements inserted in opposite orientation. In wildtype cells, this large reservoir of endogenous dsRNA is efficiently degraded. In DBR1-null cells, lariats accumulate in the cytosol and dsRNA becomes enriched. We demonstrate how the chronic exposure to these lariats attenuates the dsRNA sensors, reducing the response of the MDA5, RIG-I, RNase L and PKR sensing pathways. We observe evidence for both attenuation and endogenous dsRNA in anti-viral response and viral evasion. Lariats are transiently elevated during infection (e.g. HSV-1, influenza, KSHV). The HSV-1 genome expresses multiple, stable lariats that may attenuate dsRNA sensors during latency. HIGHLIGHTSO_LIIntronic inverted repeat Alu elements constitute largest source of endogenous dsRNA. C_LIO_LIIn the absence of DBR1, lariats accumulate in the cytoplasm and form dsRNA. C_LIO_LIChronic exposure to endogenous dsRNA in a DBR1-depleted environment desensitizes the dsRNA sensing pathway. C_LIO_LIICP0 intron 1 of HSV-1 has a highly-structured stable lariat. C_LI GRAPHICAL ABSTRACT O_FIG O_LINKSMALLFIG WIDTH=196 HEIGHT=200 SRC="FIGDIR/small/627371v1_ufig1.gif" ALT="Figure 1"> View larger version (49K): org.highwire.dtl.DTLVardef@3698bborg.highwire.dtl.DTLVardef@654727org.highwire.dtl.DTLVardef@1229245org.highwire.dtl.DTLVardef@b10622_HPS_FORMAT_FIGEXP M_FIG C_FIG

molecular biology↗

bioRxiv2024

Finite element model of the non-keratinized buccal tissue under the impact of negative pressure

The buccal mucosa is a highly interesting site for non-invasive drug delivery due to its relatively permeable epithelium and good accessibility. Recently, device-based systems have enabled the delivery of macromolecular drugs by leveraging mechanical stretching forces on the tissue to assist drug diffusion. Despite the successful exploitation of the buccal route with such systems, the biomechanics of buccal tissue are still poorly characterized and understood due to a lack of adequate characterization methods. Therefore, we propose a combination of physiological tissue modeling with simple suction experiments as a tool for characterizing and understanding the buccal tissue under the impact of negative pressure. Here, we present an initial step towards a multiphasic and poroelastic model specifically designed for the non-keratinized buccal tissue under the impact of negative pressure. A validated finite element model (FEM) for human skin was adapted to represent the histological structure of porcine buccal tissue. We performed suction experiments using the NIMBLE device, specifically developed for measuring skin stiffness, to characterize its mechanical behavior and train the FEM model. The resulting simulation tracks essential physiological parameters and allows the prediction of measurable changes in the tissue, such as the thinning of the epithelium and single-cell stretching. The FEM simulation was validated through histochemically stained tissue sections at the NIMBLE application site. A good correlation was demonstrated between predicted and experimentally observed changes. This work serves as a first step towards a computational representation of buccal tissue under the impact of negative pressure.

bioengineering↗

bioRxiv2024

Modeling Glioma Intratumoral Heterogeneity with Primary Human Neural Stem and Progenitor Cells

Glioblastoma multiforme (GBM) is a deadly form of glioma notable for its significant intratumoral heterogeneity, which is believed to drive therapy resistance. GBM has been observed to mimic a neural stem cell hierarchy reminiscent of normal brain development. However, it is still unclear how cell-of-origin shapes intratumoral heterogeneity. Here, we develop a model of glioma initiation using neural stem and progenitor cells (NSPCs) purified from fetal human brain tissue. We previously described a method to prospectively isolate and culture tripotent neural stem cells (NSCs), bipotent glial progenitor cells (GPCs), and unipotent oligodendrocyte precursor cells (OPCs). We transduced these isogenic lines with dominant-negative TP53R175H and NF1 knockdown, a commonly-used genetic model of GBM in mice. These reprogrammed lines robustly engrafted when transplanted into the brains of immunodeficient mice, and showed significant expansion over time. Engrafted cells were reextracted from the mouse brain for single cell RNA sequencing (scRNA-seq), in order to quantify how the cell-of-origin modulates the cellular subtypes found in the resulting tumor. This result revealed the strong influence the cell-of-origin plays in glioma heterogeneity. Our platform is highly adaptable and allows for modular and systematic interrogation of how cell-of-origin shape the tumor landscape.

cancer biology↗

bioRxiv2024

Predicting the Impact of Dialyzer Choice and Binder Dialysate Flow Rate on Bilirubin Removal

Liver failure is the 12th leading cause of death worldwide. Protein bound toxins such as bilirubin are responsible for many complications of the disease. Binder dialysis systems use albumin dialysate and detoxifying sorbent columns to remove these toxins. Systems like the Molecular Adsorbent Recirculating System (MARS) and BioLogic-DT have existed since the 1990s, but survival benefit in randomized controlled trials have not been consistent. Thus, a new generation of binder dialysis systems, including Open Albumin Dialysis (OPAL) and the Advanced Multi-Organ Replacement System (AMOR) are being developed. Optimal conditions for binder dialysis have not been established. We developed and validated a computational model of bound solute dialysis using established thermodynamic theories. Our objective is to improve AMOR therapy. We confirmed our models validity by predicting the impact of changing between two benchtop dialysis setups using different polysulfone dialyzers (F3 and F6HPS). We then applied it to predict the impact of varying dialysate flow rate on toxin removal. We found that bilirubin removal is independent of dialysate flow rate within the clinically relevant range (20 mL/min - 800 mL/min), matching our models predictions. At very low dialysate flow rates (2 mL/min), bilirubin removal declines, deviating from the thermodynamic model. This model may be useful to achieving optimal clinical outcomes by setting optimal dialyzer and flow rate conditions. Further improvement is possible by accounting for toxin adsorption onto the dialyzer membrane.

bioengineering↗

bioRxiv2024

AMH protects the ovary from doxorubicin by regulating cell fate and the response to DNA damage

Anti-Mullerian hormone (AMH) protects the ovarian reserve from chemotherapy, and this effect is most pronounced with Doxorubicin (DOX). However, the mechanisms of DOX toxicity and AMH rescue in the ovary remain unclear. Herein, we characterize these mechanisms in various ovarian cell types using scRNAseq. In the mesenchyme, DOX activates the intrinsic apoptotic signaling pathway through p53 class mediators, particularly affecting theca progenitors, while co-treament with AMH halts theca differentiation and reduces apoptotic gene expression. In preantral granulosa cells, DOX upregulates the cell cycle inhibitor Cdkn1a and dysregulates Wnt signaling, which are ameliorated by AMH co-treatment. Finally, in follicles, AMH induces Id3, a protein involved in DNA repair, which is necessary to prevent the accumulation of DNA lesions marked by {gamma}-H2AX in granulosa cells. Altogether this study characterizes cell, and follicle stage-specific mechanisms of AMH protection of the ovary, offering promising new avenues for fertility preservation in cancer patients undergoing chemotherapy. HighlightsO_LIDoxorubicin treatment induces DNA damage that activates the p53 pathway in stromal and follicular cells of the ovary. C_LIO_LIAMH inhibits the proliferation and differentiation of theca and granulosa cells and promotes follicle survival following Doxorubicin insult. C_LIO_LIAMH treatment mitigates Doxorubicin-induced DNA damage in the ovary by preventing the accumulation of {gamma}-H2AX-positive unresolved foci, through increased expression of ID3, a protein involved in DNA repair. C_LI Graphical Abstract O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=189 SRC="FIGDIR/small/595356v1_ufig1.gif" ALT="Figure 1"> View larger version (42K): org.highwire.dtl.DTLVardef@19f6752org.highwire.dtl.DTLVardef@383e74org.highwire.dtl.DTLVardef@8c38f7org.highwire.dtl.DTLVardef@1efe797_HPS_FORMAT_FIGEXP M_FIG C_FIG

developmental biology↗

bioRxiv2024

Charting Single Cell Lineage Dynamics and Mutation Networks via Homing CRISPR

Single cell lineage tracing, essential for unraveling cellular dynamics in disease evolution is critical for developing targeted therapies. CRISPR-Cas9, known for inducing permanent and cumulative mutations, is a cornerstone in lineage tracing. The novel homing guide RNA (hgRNA) technology enhances this by enabling dynamic retargeting and facilitating ongoing genetic modifications. Charting these mutations, especially through successive hgRNA edits, poses a significant challenge. Our solution, LINEMAP, is a computational framework designed to trace and map these mutations with precision. LINEMAP meticulously discerns mutation alleles at single-cell resolution and maps their complex interrelationships through a mutation evolution network. By utilizing a Markov Process model, we can predict mutation transition probabilities, revealing potential mutational routes and pathways. Our reconstruction algorithm, anchored in the Markov models attributes, reconstructs cellular lineage pathways, shedding light on the cells evolutionary journey to the minutiae of single-cell division. Our findings reveal an intricate network of mutation evolution paired with a predictive Markov model, advancing our capability to reconstruct single-cell lineage via hgRNA. This has substantial implications for advancing our understanding of biological mechanisms and propelling medical research forward.

bioinformatics↗

bioRxiv2023

Bacillus velezensis HBXN2020 alleviates Salmonella Typhimurium-induced colitis by improving intestinal barrier integrity and reducing inflammation

Bacillus velezensis is a species of Bacillus that has been widely investigated because of its broad-spectrum antimicrobial activity. However, most studies on Bacillus velezensis have focused on the biocontrol of plant diseases, with few reports on antagonizing Salmonella Typhimurium infections. In this investigation, it was discovered that Bacillus velezensis HBXN2020, which was isolated from healthy black pigs, possessed strong anti-stress and broad-spectrum antibacterial activity. Importantly, Bacillus velezensis HBXN2020 did not cause any adverse side effects in mice when administered at various doses (1 x 107, 1 x 108, and 1 x 109 CFU) for 14 d. Supplementing Bacillus velezensis HBXN2020 spores, either as a curative or preventive measure, dramatically reduced the levels of Salmonella Typhimurium ATCC14028 in the mices feces, ileum, cecum, and colon, as well as the disease activity index (DAI), in a model of infection caused by this pathogen in mice. Additionally, supplementing Bacillus velezensis HBXN2020 spores significantly regulated cytokine levels (TNF-, IL-1{beta}, IL-6, and IL-10) and maintained the expression of tight junction proteins and mucin protein. Most importantly, adding Bacillus velezensis HBXN2020 spores to the colonic microbiota improved its stability and increased the amount of beneficial bacteria (Lactobacillus and Akkermansia). All together, Bacillus velezensis HBXN2020 can improve intestinal microbiota stability and barrier integrity and reduce inflammation to help treat infection by Salmonella Typhimurium.

microbiology↗

bioRxiv2023

Adeno-to-squamous transition drives resistance to KRAS inhibition in LKB1 mutant lung cancer

KRASG12C inhibitors including adagrasib and sortorasib have shown clinical promise in targeting KRASG12C-mutated lung cancers, however, most patients eventually develop drug resistance. In lung adenocarcinoma patients with co-occurring KRASG12C and STK11/LKB1 mutations, we found a high squamous gene signature at baseline significantly correlated with poor adagrasib response. Through integrative studies of Lkb1-deficient KRASG12Cand KrasG12D lung cancer mouse models and/or organoids treated with KRAS inhibitors, we found tumor cells invoked a lineage plasticity program: adeno-to-squamous transition (AST) that mediated resistance to KRAS inhibition. Transcriptomic and epigenomic analyses revealed {Delta}Np63 drives AST and modulates response to KRAS inhibition. We identified an intermediate high-plasticity cell state with distinct gene expression program marked by Krt6a upregulation. Notably, higher KRT6A expression at baseline correlated with shorter overall survival in KRAS-mutant patients receiving adagrasib. These data support the role of AST in KRAS inhibitor resistance and provide predictive biomarker for KRAS-targeted therapies in lung cancer.

cancer biology↗

bioRxiv2023

TLR2 Regulates Hair Follicle Cycle and Regeneration via BMP Signaling.

The etiology of hair loss remains enigmatic, and current remedies remain inadequate. Transcriptome analysis of aging hair follicles uncovered changes in immune pathways, including Toll-like receptors (TLRs). Our findings demonstrate that the maintenance of hair follicle homeostasis and the regeneration capacity after damage depends on TLR2 in hair follicle stem cells (HFSCs). In healthy hair follicles, TLR2 is expressed in a cycle-dependent manner and governs HFSCs activation by countering inhibitory BMP signaling. Hair follicles in aging and obesity exhibit a decrease in both TLR2 and its endogenous ligand carboxyethylpyrrole (CEP), a metabolite of polyunsaturated fatty acids. Administration of CEP stimulates hair regeneration through a TLR2-dependent mechanism. These results establish a novel connection between TLR2-mediated innate immunity and HFSC activation, which is pivotal to hair follicle health and the prevention of hair loss and provide new avenues for therapeutic intervention. SummaryHair follicle stem cells TLR2 is required for hair homeostasis and regeneration. While TLR2 stimulation by endogenous ligand promotes hair growth, reduction in TLR2 and its ligand in aging and obesity may diminish hair growth.

immunology↗

bioRxiv2023

PMS1 as a target for splice modulation to prevent somatic CAG repeat expansion in Huntington's disease

Huntingtons disease (HD) is a dominantly inherited neurodegenerative disorder whose motor, cognitive, and behavioral manifestations are caused by an expanded, somatically unstable CAG repeat in the first exon of HTT that lengthens a polyglutamine tract in huntingtin. Genome-wide association studies (GWAS) have revealed DNA repair genes that influence the age-at-onset of HD and implicate somatic CAG repeat expansion as the primary driver of disease timing. To prevent the consequent neuronal damage, small molecule splice modulators (e.g., branaplam) that target HTT to reduce the levels of huntingtin are being investigated as potential HD therapeutics. We found that the effectiveness of the splice modulators can be influenced by genetic variants, both at HTT and other genes where they promote pseudoexon inclusion. Surprisingly, in a novel hTERT-immortalized retinal pigment epithelial cell (RPE1) model for assessing CAG repeat instability, these drugs also reduced the rate of HTT CAG expansion. We determined that the splice modulators also affect the expression of the mismatch repair gene PMS1, a known modifier of HD age-at-onset. Genome editing at specific HTT and PMS1 sequences using CRISPR-Cas9 nuclease confirmed that branaplam suppresses CAG expansion by promoting the inclusion of a pseudoexon in PMS1, making splice modulation of PMS1 a potential strategy for delaying HD onset. Comparison with another splice modulator, risdiplam, suggests that other genes affected by these splice modulators also influence CAG instability and might provide additional therapeutic targets.

genetics↗

bioRxiv2023

Concurrent profiling of multiscale 3D genome organization and gene expression in single mammalian cells

The organization of mammalian genomes within the nucleus features a complex, multiscale three-dimensional (3D) architecture. The functional significance of these 3D genome features, however, remains largely elusive due to limited single-cell technologies that can concurrently profile genome organization and transcriptional activities. Here, we report GAGE-seq, a highly scalable, robust single-cell co-assay that simultaneously measures 3D genome structure and transcriptome within the same cell. Employing GAGE-seq on mouse brain cortex and human bone marrow CD34+ cells, we comprehensively characterized the intricate relationships between 3D genome and gene expression. We found that these multiscale 3D genome features collectively inform cell type-specific gene expressions, hence contributing to defining cell identity at the single-cell level. Integration of GAGE-seq data with spatial transcriptomic data revealed in situ variations of the 3D genome in mouse cortex. Moreover, our observations of lineage commitment in normal human hematopoiesis unveiled notable discordant changes between 3D genome organization and gene expression, underscoring a complex, temporal interplay at the single-cell level that is more nuanced than previously appreciated. Together, GAGE-seq provides a powerful, cost-effective approach for interrogating genome structure and gene expression relationships at the single-cell level across diverse biological contexts.

genomics↗

bioRxiv2023

BNIP3-mediated mitophagy boosts the competitive dominant growth of lenvatinib resistant cells via reprogramming energy metabolism in HCC

Although increasing studies has demonstrated that cell competition widely involved in the growth and homeostasis of multicellular organisms is closely linked to tumorigenesis and development, the mechanistic contributions to the association between tumor cell competition-driven heterogeneity and drug resistance remains ill-defined. In our study, lenvitinib-resistant hepatocellular carcinoma (HCC) cells display obviously competitive growth dominance against sensitive cells through reprogramming energy metabolism. Mechanistically, when BCL2 interacting protein3 (BNIP3) overexpression activates mitophagy activity in lenvatinib-resistant HCC cells, energy imbalance signal caused by reduced mitochondrial oxidative phosphorylation levels provokes the phosphorylation of AMP-activated protein kinase (AMPK) sensor; subsequently, enabled AMPK specifically targets enolase 2 (ENO2) to enhance glycolysis and eventually promots the competitive capacity and dominant growth. Of note, BNIP3 deficiency shows certain inhibition of cell competition outcome. Our findings emphasize a vital role for BNIP3-AMPK-ENO2 signaling in maintaining the competitive outcome of lenvitinib-resistant HCC cells via regulating energy metabolism; meanwhile this work recognaizes BNIP3 as a promising target to overcome HCC drug resistance.

cell biology↗

bioRxiv2023

Targeting Ribosome Biogenesis as a Novel Therapeutic Approach to Overcome EMT-related Chemoresistance in Breast Cancer.

Epithelial-to-mesenchymal transition (EMT) contributes significantly to chemotherapy resistance and remains a critical challenge in treating advanced breast cancer. The complexity of EMT, involving redundant pro-EMT signaling pathways and its paradox reversal process, mesenchymal-to-epithelial transition (MET), has hindered the development of effective treatments. In this study, we utilized a Tri-PyMT EMT lineage-tracing model and single-cell RNA sequencing (scRNA-seq) to comprehensively analyze the EMT status of tumor cells. Our findings revealed elevated ribosome biogenesis (RiBi) during the transitioning phases of both EMT and MET processes. RiBi and its subsequent nascent protein synthesis mediated by ERK and mTOR signalings are essential for EMT/MET completion. Importantly, inhibiting excessive RiBi genetically or pharmacologically impaired the EMT/MET capability of tumor cells. Combining RiBi inhibition with chemotherapy drugs synergistically reduced metastatic outgrowth of epithelial and mesenchymal tumor cells under chemotherapies. Our study suggests that targeting the RiBi pathway presents a promising strategy for treating patients with advanced breast cancer. SignificanceThis study uncovers the crucial involvement of ribosome biogenesis (RiBi) in the regulation of epithelial and mesenchymal state oscillations in breast cancer cells, which plays a major role in the development of chemoresistant metastasis. By proposing a novel therapeutic strategy targeting the RiBi pathway, the study offers significant potential to enhance treatment efficacy and outcomes for patients with advanced breast cancer. This approach could help overcome the limitations of current chemotherapy options and address the complex challenges posed by EMT-mediated chemoresistance.

cancer biology↗

bioRxiv2023

Haplotype-resolve genome assembly and resequencing provide insights into the origin and domestication of modern rose

Modern rose (Rosa hybrida) is a recently formed interspecific hybrid and has become one of the most important and widely cultivated ornamentals. Here, we report the haplotype-resolved chromosome-scale genome assembly of the tetraploid R. hybrida Samantha and a genome variation map of 233 Rosa accessions involving various wild species, and old and modern cultivars. The two subgenomes of Samantha show no significant global bias in gene loss but substantial expression bias, and homoeologous exchanges are frequently observed between subgenomes. Population genomic and genomic composition analyses reveal the contributions of wild Rosa species to modern roses, and highlight that R. odorata and its derived cultivars contribute more to modern roses than R. chinensis Old Blush. Furthermore, selective sweeps during modern rose breeding associated with major agronomic traits, including continuous flowering, floral organ identity, flower senescence, and disease resistance, are identified. This study provides insights into the genetic basis of modern rose origin and breeding history, and offers unprecedented genomic resources for rose improvement.

genomics↗

bioRxiv2023

The Evidence of Cough Contagion in Human Beings

There is the evidence for cough contagion in human beings caused by empathy rather than physiology.Due to limited study on cough contagion,researchers have doubted whether cough was contagious from emtional contagion like yawn contagion.To deal with the doubts,we observed coughs from 34 adults in Ocean University of China in real time and recorded individual s gender and local time.Then we developed a mathematical model to divide the cough process into several bouts and set a threshold for contagion to construct a response graph.With the graph,we first saw a strong effect of contagion for around 30 seconds no matter how long the bin(< 30s) was.Afterwards with mutiple measures,we extracted seven features(e.g.,duration) to describe the transmission chain and then found that there s no time difference in cough contagion.Moreover,we also discovered tthe gender difference that males were more likely to be induced by triggers.Thus,cough contagion seems to be a normal phenomenon in human beings providing support to the experimental studies of empathy theory.

animal behavior and cognition↗

bioRxiv2023

Intermediate basal cell population in prostate homeostasis and cancer initiation

Many glandular epithelia are mainly composed of basal cells and luminal cells, including the prostate gland. Adult prostate basal and luminal cells are independently self-sustained by unipotent stem cells that can reactivate multipotency under prostate inflammation and carcinogenesis contexts. However, the defined basal stem cell populations responsible for prostate regeneration and their cell fates in prostate homeostasis, inflammation and carcinogenesis remain unclear. Using a genetic proliferation tracer (ProTracer) system, we found that basal cells exhibited extensive cell loss and proliferation during androgen-mediated prostate regression and regeneration, respectively. A rare intermediate basal cell population that expresses luminal cell markers (Nkx3.1 and Pbsn) (termed Basal-B) and a large basal cell population (termed Basal-A) were identified in mouse prostates by single-cell RNA sequencing. Basal-B cells exhibited a greater capacity for organoid formation and luminal cell differentiation in vitro. Genetic lineage tracing using dual recombinases showed that prostate homeostasis and regeneration are not driven by specific basal cell types. Fate-mapping results showed that Basal-B cells had a greater tendency to generate luminal cells under bacteria-induced prostate inflammation. Deletion of Pten in basal cells resulted in Basal-A-to-Basal-B-to-luminal transition and prostatic intraepithelial neoplasia. Moreover, the human Basal-B-cell population was significantly increased in human benign prostate hyperplasia and prostatic intraepithelial neoplasia samples compared with normal prostate samples. This study identifies intermediate Basal-B cells as a potential stem cell population and provides genetic evidence of prostate basal cell lineage plasticity under physiological and pathological contexts.

developmental biology↗

bioRxiv2022

USP10 strikes down Wnt/β-catenin signaling by dual-wielding deubiquitinase activity and phase transition potential

Wnt/{beta}-catenin signaling is a conserved pathway crucially governing development, homeostasis and oncogenesis. Discovery of novel regulators holds great values in both basic and translational research. Through screening, we identified a deubiquitinase (DUB) USP10 as a novel and critical modulator of {beta}-catenin. Mechanistically, USP10 binds to key scaffold Axin1 via conserved motifs and stabilizes Axin1 through K48-linked deubiquitination, and surprisingly, tethers Axin1 and {beta}-catenin physically while promoting phase separation for {beta}-catenin suppression regardless of its enzymatic activity. Functionally, USP10 prominently regulates embryonic development and intestinal homeostasis by antagonizing {beta}-catenin via DUB activity. In colorectal cancer, USP10 substantially represses cancer growth mainly through physical binding compensation and phase separation promotion and correlates with Wnt/{beta}-catenin magnitude clinically. Collectively, we discovered USP10 functioning in multiple biological processes against {beta}-catenin and unearthed a novel enzyme-dependent and -independent "dual-regulating" mechanism by which USP10 utilizes parallelly and context-dependently. USP10 inhibitor was suggested in treating certain Wnt-related diseases.

cell biology↗

bioRxiv2022

Tissue and cell-type specific molecular and functional signatures of 16p11.2 reciprocal genomic disorder across mouse brain and human neuronal models

Recurrent deletion and duplication of [~]743 kilobases of unique genomic sequence and segmental duplications at chromosome 16p11.2 underlie a reciprocal genomic disorder (RGD; OMIM 611913 and 614671) associated with neurodevelopmental and psychiatric phenotypes, including intellectual disability, autism spectrum disorder (ASD), and schizophrenia (SCZ). To define molecular alterations associated with the 16p11.2 RGD, we performed transcriptome analyses of mice with reciprocal copy number variants (CNVs) of the syntenic chromosome 7qF3 region and human neuronal models derived from isogenic human induced pluripotent stem cells (hiPSCs) carrying CRISPR-engineered CNVs at 16p11.2. Analysis of differentially expressed genes (DEGs) in mouse cortex, striatum, cerebellum and three non-brain tissues, as well as in human neural stem cells and induced glutamatergic neurons revealed that the strongest and most consistent effects occurred within the CNV sequence, with notable instances of differential expression of genes in the immediate vicinity that could reflect position effect. While differential expression of genes outside of chromosome 16p11.2 was largely region, tissue, and cell type-specific, a small but significant minority of such DEGs was shared between brain regions or human cell types. Gene Ontology (GO) enrichment analyses to identify cellular processes dysregulated due to these CNVs found support in select circumstances for terms related to energy metabolism, RNA metabolism, and translation but did not reveal a single universally affected process. Weighted gene co-expression network analysis identified modules that showed significant correlation with reciprocal or individual CNV genotype and better captured shared effects, indicating that energy metabolism, RNA metabolism, translation and protein targeting were disrupted across all three brain regions. The first two of these processes also emerged in the human neural stem cell (NSC) data. A subset of co-expression modules that correlated with CNV genotype revealed significant enrichments for known neurodevelopmental disorder genes, loss-of-function constrained genes, FMRP targets, and chromatin modifiers. Intriguingly, neuronal differentiation of the hiPSCs revealed that both the deletion and duplication CNV resulted in similar deficits in neurite extension and branching and alterations in electrical activity. Finally, generation of cerebral organoid derivatives indicated that the CNVs reciprocally altered the ratio of excitatory and inhibitory GABAergic neurons generated during in vitro neurodevelopment, consistent with a major mechanistic hypothesis for ASD. Collectively, our data suggest that the 16p11.2 RGD involves disruption of multiple biological processes, with a relative impact that is context-specific. Perturbation of individual and multiple genes within the CNV region will be required to dissect single-gene effects, uncover regulatory interactions, and define how each contributes to abnormal neurodevelopment.

genomics↗