Knockdown of PGBD5 inhibits the malignant progression of glioma through upregulation of the PPAR pathway.

Glioma is the most common type of primary intracranial malignant tumor, and because of its high invasiveness and recurrence, its prognosis remains poor. The present study investigated the biological function of piggyBac transportable element derived 5 (PGBD5) in glioma. Glioma and para-cancerous tissues were obtained from five patients. Reverse transcription-quantitative PCR and western blotting were used to detect the expression levels of PGBD5. Transwell assay and flow cytometry were used to evaluate cell migration, invasion, apoptosis and cell cycle distribution. In addition, a nude mouse tumor transplantation model was established to study the downstream pathways of PGBD5 and the molecular mechanism was analyzed using transcriptome sequencing. The mRNA and protein expression levels of PGBD5 were increased in glioma tissues and cells. Notably, knockdown of PGBD5 in vitro could inhibit the migration and invasion of glioma cells. In addition, the knockdown of PGBD5 expression promoted apoptosis and caused cell cycle arrest in the G2/M phase, thus inhibiting cell proliferation. Furthermore, in vivo experiments revealed that knockdown of PGBD5 expression could inhibit Ki67 expression and slow tumor growth. Changes in PGBD5 expression were also shown to be closely related to the peroxisome proliferator-activated receptor (PPAR) signaling pathway. In conclusion, interference with PGBD5 could inhibit the malignant progression of glioma through the PPAR pathway, suggesting that PGBD5 may be a potential molecular target of glioma.

Flexible TAM requirement of TnpB enables efficient single-nucleotide editing with expanded targeting scope.

TnpBs encoded by the IS200/IS605 family transposon are among the most abundant prokaryotic proteins from which type V CRISPR-Cas nucleases may have evolved. Since bacterial TnpBs can be programmed for RNA-guided dsDNA cleavage in the presence of a transposon-adjacent motif (TAM), these nucleases hold immense promise for genome editing. However, the activity and targeting specificity of TnpB in homology-directed gene editing remain unknown. Here we report that a thermophilic archaeal TnpB enables efficient gene editing in the natural host. Interestingly, the TnpB has different TAM requirements for eliciting cell death and for facilitating gene editing. By systematically characterizing TAM variants, we reveal that the TnpB recognizes a broad range of TAM sequences for gene editing including those that do not elicit apparent cell death. Importantly, TnpB shows a very high targeting specificity on targets flanked by a weak TAM. Taking advantage of this feature, we successfully leverage TnpB for efficient single-nucleotide editing with templated repair. The use of different weak TAM sequences not only facilitates more flexible gene editing with increased cell survival, but also greatly expands targeting scopes, and this strategy is probably applicable to diverse CRISPR-Cas systems.

Deciphering cell types by integrating scATAC-seq data with genome sequences.

The single-cell assay for transposase-accessible chromatin using sequencing (scATAC-seq) technology provides insight into gene regulation and epigenetic heterogeneity at single-cell resolution, but cell annotation from scATAC-seq remains challenging due to high dimensionality and extreme sparsity within the data. Existing cell annotation methods mostly focus on the cell peak matrix without fully utilizing the underlying genomic sequence. Here we propose a method, SANGO, for accurate single-cell annotation by integrating genome sequences around the accessibility peaks within scATAC data. The genome sequences of peaks are encoded into low-dimensional embeddings, and then iteratively used to reconstruct the peak statistics of cells through a fully connected network. The learned weights are considered as regulatory modes to represent cells, and utilized to align the query cells and the annotated cells in the reference data through a graph transformer network for cell annotations. SANGO was demonstrated to consistently outperform competing methods on 55 paired scATAC-seq datasets across samples, platforms and tissues. SANGO was also shown to be able to detect unknown tumor cells through attention edge weights learned by the graph transformer. Moreover, from the annotated cells, we found cell-type-specific peaks that provide functional insights/biological signals through expression enrichment analysis, cis-regulatory chromatin interaction analysis and motif enrichment analysis.

Iterative assay for transposase-accessible chromatin by sequencing to isolate functionally relevant neuronal subtypes.

The Drosophila brain contains tens of thousands of distinct cell types. Thousands of different transgenic lines reproducibly target specific neuron subsets, yet most still express in several cell types. Furthermore, most lines were developed without a priori knowledge of where the transgenes would be expressed. To aid in the development of cell type-specific tools for neuronal identification and manipulation, we developed an iterative assay for transposase-accessible chromatin (ATAC) approach. Open chromatin regions (OCRs) enriched in neurons, compared to whole bodies, drove transgene expression preferentially in subsets of neurons. A second round of ATAC-seq from these specific neuron subsets revealed additional enriched OCR2s that further restricted transgene expression within the chosen neuron subset. This approach allows for continued refinement of transgene expression, and we used it to identify neurons relevant for sleep behavior. Furthermore, this approach is widely applicable to other cell types and to other organisms.

Childhood cancer mutagenesis caused by transposase-derived PGBD5.

Genomic rearrangements are a hallmark of most childhood tumors, including medulloblastoma, one of the most common brain tumors in children, but their causes remain largely unknown. Here, we show that PiggyBac transposable element derived 5 (Pgbd5) promotes tumor development in multiple developmentally accurate mouse models of Sonic Hedgehog (SHH) medulloblastoma. Most Pgbd5-deficient mice do not develop tumors, while maintaining normal cerebellar development. Ectopic activation of SHH signaling is sufficient to enforce cerebellar granule cell progenitor-like cell states, which exhibit Pgbd5-dependent expression of distinct DNA repair and neurodevelopmental factors. Mouse medulloblastomas expressing Pgbd5 have increased numbers of somatic structural DNA rearrangements, some of which carry PGBD5-specific sequences at their breakpoints. Similar sequence breakpoints recurrently affect somatic DNA rearrangements of known tumor suppressors and oncogenes in medulloblastomas in 329 children. This identifies PGBD5 as a medulloblastoma mutator and provides a genetic mechanism for the generation of oncogenic DNA rearrangements in childhood cancer.

Disease-associated astrocyte epigenetic memory promotes CNS pathology.

Disease-associated astrocyte subsets contribute to the pathology of neurologic diseases, including multiple sclerosis and experimental autoimmune encephalomyelitis1-8 (EAE), an experimental model for multiple sclerosis. However, little is known about the stability of these astrocyte subsets and their ability to integrate past stimulation events. Here we report the identification of an epigenetically controlled memory astrocyte subset that exhibits exacerbated pro-inflammatory responses upon rechallenge. Specifically, using a combination of single-cell RNA sequencing, assay for transposase-accessible chromatin with sequencing, chromatin immunoprecipitation with sequencing, focused interrogation of cells by nucleic acid detection and sequencing, and cell-specific in vivo CRISPR-Cas9-based genetic perturbation studies we established that astrocyte memory is controlled by the metabolic enzyme ATP-citrate lyase (ACLY), which produces acetyl coenzyme A (acetyl-CoA) that is used by histone acetyltransferase p300 to control chromatin accessibility. The number of ACLY+p300+ memory astrocytes is increased in acute and chronic EAE models, and their genetic inactivation ameliorated EAE. We also detected the pro-inflammatory memory phenotype in human astrocytes in vitro; single-cell RNA sequencing and immunohistochemistry studies detected increased numbers of ACLY+p300+ astrocytes in chronic multiple sclerosis lesions. In summary, these studies define an epigenetically controlled memory astrocyte subset that promotes CNS pathology in EAE and, potentially, multiple sclerosis. These findings may guide novel therapeutic approaches for multiple sclerosis and other neurologic diseases.

Mage transposon: a novel gene delivery system for mammalian cells.

Transposons, as non-viral integration vectors, provide a secure and efficient method for stable gene delivery. In this study, we have discovered Mage (MG), a novel member of the piggyBac(PB) family, which exhibits strong transposability in a variety of mammalian cells and primary T cells. The wild-type MG showed a weaker insertion preference for near genes, transcription start sites (TSS), CpG islands, and DNaseI hypersensitive sites in comparison to PB, approaching the random insertion pattern. Utilizing in silico virtual screening and feasible combinatorial mutagenesis in vitro, we effectively produced the hyperactive MG transposase (hyMagease). This variant boasts a transposition rate 60% greater than its native counterpart without significantly altering its insertion pattern. Furthermore, we applied the hyMagease to efficiently deliver chimeric antigen receptor (CAR) into T cells, leading to stable high-level expression and inducing significant anti-tumor effects both in vitro and in xenograft mice models. These findings provide a compelling tool for gene transfer research, emphasizing its potential and prospects in the domains of genetic engineering and gene therapy.

An optimized polymeric delivery system for piggyBac transposition.

The piggyBac transposon/transposase system has been explored for long-term, stable gene expression to execute genomic integration of therapeutic genes, thus emerging as a strong alternative to viral transduction. Most studies with piggyBac transposition have employed physical methods for successful delivery of the necessary components of the piggyBac system into the cells. Very few studies have explored polymeric gene delivery systems. In this short communication, we report an effective delivery system based on low molecular polyethylenimine polymer with lipid substitution (PEI-L) capable of delivering three components, (i) a piggyBac transposon plasmid DNA carrying a gene encoding green fluorescence protein (PB-GFP), (ii) a piggyBac transposase plasmid DNA or mRNA, and (iii) a 2 kDa polyacrylic acid as additive for transfection enhancement, all in a single complex. We demonstrate an optimized formulation for stable GFP expression in two model cell lines, MDA-MB-231 and SUM149 recorded till day 108 (3.5 months) and day 43 (1.4 months), respectively, following a single treatment with very low cell number as starting material. Moreover, the stability of the transgene (GFP) expression mediated by piggyBac/PEI-L transposition was retained following three consecutive cryopreservation cycles. The success of this study highlights the feasibility and potential of employing a polymeric delivery system to obtain piggyBac-based stable expression of therapeutic genes.

Application of RNA interference and protein localization to investigate housekeeping and developmentally regulated genes in the emerging model protozoan Paramecium caudatum.

Unicellular eukaryotes represent tremendous evolutionary diversity. However, the molecular mechanisms underlying this diversity remain largely unexplored, partly due to a limitation of genetic tools to only a few model species. Paramecium caudatum is a well-known unicellular eukaryote with an unexpectedly large germline genome, of which only two percent is retained in the somatic genome following sexual processes, revealing extensive DNA elimination. However, further progress in understanding the molecular mechanisms governing this process is hampered by a lack of suitable genetic tools. Here, we report the successful application of gene knockdown and protein localization methods to interrogate the function of both housekeeping and developmentally regulated genes in P. caudatum. Using these methods, we achieved the expected phenotypes upon RNAi by feeding, and determined the localization of these proteins by microinjection of fusion constructs containing fluorescent protein or antibody tags. Lastly, we used these methods to reveal that P. caudatum PiggyMac, a domesticated piggyBac transposase, is essential for sexual development, and is likely to be an active transposase directly involved in DNA cleavage. The application of these methods lays the groundwork for future studies of gene function in P. caudatum and can be used to answer important biological questions in the future.

Application of Single-Cell Assay for Transposase-Accessible Chromatin with High Throughput Sequencing in Plant Science: Advances, Technical Challenges, and Prospects.

The Single-cell Assay for Transposase-Accessible Chromatin with high throughput sequencing (scATAC-seq) has gained increasing popularity in recent years, allowing for chromatin accessibility to be deciphered and gene regulatory networks (GRNs) to be inferred at single-cell resolution. This cutting-edge technology now enables the genome-wide profiling of chromatin accessibility at the cellular level and the capturing of cell-type-specific cis-regulatory elements (CREs) that are masked by cellular heterogeneity in bulk assays. Additionally, it can also facilitate the identification of rare and new cell types based on differences in chromatin accessibility and the charting of cellular developmental trajectories within lineage-related cell clusters. Due to technical challenges and limitations, the data generated from scATAC-seq exhibit unique features, often characterized by high sparsity and noise, even within the same cell type. To address these challenges, various bioinformatic tools have been developed. Furthermore, the application of scATAC-seq in plant science is still in its infancy, with most research focusing on root tissues and model plant species. In this review, we provide an overview of recent progress in scATAC-seq and its application across various fields. We first conduct scATAC-seq in plant science. Next, we highlight the current challenges of scATAC-seq in plant science and major strategies for cell type annotation. Finally, we outline several future directions to exploit scATAC-seq technologies to address critical challenges in plant science, ranging from plant ENCODE(The Encyclopedia of DNA Elements) project construction to GRN inference, to deepen our understanding of the roles of CREs in plant biology.

Hi-Tag: a simple and efficient method for identifying protein-mediated long-range chromatin interactions with low cell numbers.

Protein-mediated chromatin interactions can be revealed by coupling proximity-based ligation with chromatin immunoprecipitation. However, these techniques require complex experimental procedures and millions of cells per experiment, which limits their widespread application in life science research. Here, we develop a novel method, Hi-Tag, that identifies high-resolution, long-range chromatin interactions through transposase tagmentation and chromatin proximity ligation (with a phosphorothioate-modified linker). Hi-Tag can be implemented using as few as 100,000 cells, involving simple experimental procedures that can be completed within 1.5 days. Meanwhile, Hi-Tag is capable of using its own data to identify the binding sites of specific proteins, based on which, it can acquire accurate interaction information. Our results suggest that Hi-Tag has great potential for advancing chromatin interaction studies, particularly in the context of limited cell availability.

Targeted design of synthetic enhancers for selected tissues in the Drosophila embryo.

Enhancers control gene expression and have crucial roles in development and homeostasis1-3. However, the targeted de novo design of enhancers with tissue-specific activities has remained challenging. Here we combine deep learning and transfer learning to design tissue-specific enhancers for five tissues in the Drosophila melanogaster embryo: the central nervous system, epidermis, gut, muscle and brain. We first train convolutional neural networks using genome-wide single-cell assay for transposase-accessible chromatin with sequencing (ATAC-seq) datasets and then fine-tune the convolutional neural networks with smaller-scale data from in vivo enhancer activity assays, yielding models with 13% to 76% positive predictive value according to cross-validation. We designed and experimentally assessed 40 synthetic enhancers (8 per tissue) in vivo, of which 31 (78%) were active and 27 (68%) functioned in the target tissue (100% for central nervous system and muscle). The strategy of combining genome-wide and small-scale functional datasets by transfer learning is generally applicable and should enable the design of tissue-, cell type- and cell state-specific enhancers in any system.

AAV-mediated delivery of a Sleeping Beauty transposon and an mRNA-encoded transposase for the engineering of therapeutic immune cells.

Engineering cells for adoptive therapy requires overcoming limitations in cell viability and, in the efficiency of transgene delivery, the duration of transgene expression and the stability of genomic integration. Here we report a gene-delivery system consisting of a Sleeping Beauty (SB) transposase encoded into a messenger RNA delivered by an adeno-associated virus (AAV) encoding an SB transposon that includes the desired transgene, for mediating the permanent integration of the transgene. Compared with lentiviral vectors and with the electroporation of plasmids of transposon DNA or minicircle DNA, the gene-delivery system, which we named MAJESTIC (for 'mRNA AAV-SB joint engineering of stable therapeutic immune cells'), offers prolonged transgene expression, as well as higher transgene expression, therapeutic-cell yield and cell viability. MAJESTIC can deliver chimeric antigen receptors (CARs) into T cells (which we show lead to strong anti-tumour activity in vivo) and also transduce natural killer cells, myeloid cells and induced pluripotent stem cells with bi-specific CARs, kill-switch CARs and synthetic T-cell receptors.

INO80-Dependent Remodeling of Transcriptional Regulatory Network Underlies the Progression of Heart Failure.

BACKGROUND: Progressive remodeling of cardiac gene expression underlies decline in cardiac function, eventually leading to heart failure. However, the major determinants of transcriptional network switching from normal to failed hearts remain to be determined. METHODS: In this study, we integrated human samples, genetic mouse models, and genomic approaches, including bulk RNA sequencing, single-cell RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, to identify the role of chromatin remodeling complex INO80 in heart homeostasis and dysfunction. RESULTS: The INO80 chromatin remodeling complex was abundantly expressed in mature cardiomyocytes, and its expression further increased in mouse and human heart failure. Cardiomyocyte-specific overexpression of Ino80, its core catalytic subunit, induced heart failure within 4 days. Combining RNA sequencing, chromatin immunoprecipitation followed by high-throughput sequencing, and assay for transposase-accessible chromatin with high-throughput sequencing, we revealed INO80 overexpression-dependent reshaping of the nucleosomal landscape that remodeled a core set of transcription factors, most notably the MEF2 (Myocyte Enhancer Factor 2) family, whose target genes were closely associated with cardiac function. Conditional cardiomyocyte-specific deletion of Ino80 in an established mouse model of heart failure demonstrated remarkable preservation of cardiac function. CONCLUSIONS: In summary, our findings shed light on the INO80-dependent remodeling of the chromatin landscape and transcriptional networks as a major mechanism underlying cardiac dysfunction in heart failure, and suggest INO80 as a potential preventative or interventional target.

Insertion sequence excision is enhanced by a protein that catalyzes branch migration and promotes microhomology-mediated end joining.

Insertion sequence (IS)-excision enhancer (IEE) promotes the excision of ISs in the genome of enterohemorrhagic Escherichia coli O157. Because IEE-dependent IS excision occurs in the presence of transposase, the process of IS transposition may be involved in IS excision; however, little is understood about the molecular mechanisms of IS excision. Our in vitro analysis revealed that IEE exhibits DNA-dependent ATPase activity, which is activated by branched DNA. IEE also catalyzes the branch migration of fork-structured DNA. These results suggest that IEE remodels branched structures of the IS transposition intermediate. Sequence analysis of recombination sites in IS-excision products suggested that microhomologous sequences near the ends of the IS are involved in IS excision. IEE promoted microhomology-mediated end joining (MMEJ), in which base pairing between 6-nucleotides complementary ends of two 3'-protruding DNAs and subsequent elongation of the paired DNA strand occurred. IS-excision frequencies were significantly decreased in cells producing IEE mutants that had lost either branch migration or MMEJ activity, which suggests that these activities of IEE are required for IS excision. Based on our results, we propose a model for IS excision triggered by IEE and transposase.

Dynamics of chromatin accessibility and genome wide control of desiccation tolerance in the resurrection plant Haberlea rhodopensis.

BACKGROUND: Drought is one of the main consequences of global climate change and this problem is expected to intensify in the future. Resurrection plants evolved the ability to withstand the negative impact of long periods of almost complete desiccation and to recover at rewatering. In this respect, many physiological, transcriptomic, proteomic and genomic investigations have been performed in recent years, however, few epigenetic control studies have been performed on these valuable desiccation-tolerant plants so far. RESULTS: In the present study, for the first time for resurrection plants we provide evidences about the differential chromatin accessibility of Haberlea rhodopensis during desiccation stress by ATAC-seq (Assay for Transposase Accessible Chromatin with high-throughput sequencing). Based on gene similarity between species, we used the available genome of the closely related resurrection plant Dorcoceras hygrometricum to identify approximately nine hundred transposase hypersensitive sites (THSs) in H. rhodopensis. The majority of them corresponds to proximal and distal regulatory elements of different genes involved in photosynthesis, carbon metabolism, synthesis of secondary metabolites, cell signalling and transcriptional regulation, cell growth, cell wall, stomata conditioning, chaperons, oxidative stress, autophagy and others. Various types of binding motifs recognized by several families of transcription factors have been enriched from the THSs found in different stages of drought. Further, we used the previously published RNA-seq data from H. rhodopensis to evaluate the expression of transcription factors putatively interacting with the enriched motifs, and the potential correlation between the identified THS and the expression of their corresponding genes. CONCLUSIONS: These results provide a blueprint for investigating the epigenetic regulation of desiccation tolerance in resurrection plant H. rhodopensis and comparative genomics between resurrection and non-resurrection species with available genome information.

Mechanism of target site selection by type V-K CRISPR-associated transposases.

CRISPR-associated transposases (CASTs) repurpose nuclease-deficient CRISPR effectors to catalyze RNA-guided transposition of large genetic payloads. Type V-K CASTs offer potential technology advantages but lack accuracy, and the molecular basis for this drawback has remained elusive. Here, we reveal that type V-K CASTs maintain an RNA-independent, "untargeted" transposition pathway alongside RNA-dependent integration, driven by the local availability of TnsC filaments. Using cryo-electron microscopy, single-molecule experiments, and high-throughput sequencing, we found that a minimal, CRISPR-less transpososome preferentially directs untargeted integration at AT-rich sites, with additional local specificity imparted by TnsB. By exploiting this knowledge, we suppressed untargeted transposition and increased type V-K CAST specificity up to 98.1% in cells without compromising on-target integration efficiency. These findings will inform further engineering of CAST systems for accurate, kilobase-scale genome engineering applications.

Eukaryotic RNA-guided endonucleases evolved from a unique clade of bacterial enzymes.

RNA-guided endonucleases form the crux of diverse biological processes and technologies, including adaptive immunity, transposition, and genome editing. Some of these enzymes are components of insertion sequences (IS) in the IS200/IS605 and IS607 transposon families. Both IS families encode a TnpA transposase and a TnpB nuclease, an RNA-guided enzyme ancestral to CRISPR-Cas12s. In eukaryotes, TnpB homologs occur as two distinct types, Fanzor1s and Fanzor2s. We analyzed the evolutionary relationships between prokaryotic TnpBs and eukaryotic Fanzors, which revealed that both Fanzor1s and Fanzor2s stem from a single lineage of IS607 TnpBs with unusual active site arrangement. The widespread nature of Fanzors implies that the properties of this particular lineage of IS607 TnpBs were particularly suited to adaptation in eukaryotes. Biochemical analysis of an IS607 TnpB and Fanzor1s revealed common strategies employed by TnpBs and Fanzors to co-evolve with their cognate transposases. Collectively, our results provide a new model of sequential evolution from IS607 TnpBs to Fanzor2s, and Fanzor2s to Fanzor1s that details how genes of prokaryotic origin evolve to give rise to new protein families in eukaryotes.

Sleeping Beauty Transposon Insertions into Nucleolar DNA by an Engineered Transposase Localized in the Nucleolus.

Transposons are nature's gene delivery vehicles that can be harnessed for experimental and therapeutic purposes. The Sleeping Beauty (SB) transposon shows efficient transposition and long-term transgene expression in human cells, and is currently under clinical development for gene therapy. SB transposition occurs into the human genome in a random manner, which carries a risk of potential genotoxic effects associated with transposon integration. Here, we evaluated an experimental strategy to manipulate SB's target site distribution by preferentially compartmentalizing the SB transposase to the nucleolus, which contains repetitive ribosomal RNA (rRNA) genes. We generated a fusion protein composed of the nucleolar protein nucleophosmin (B23) and the SB100X transposase, which was found to retain almost full transposition activity as compared to unfused transposase and to be predominantly localized to nucleoli in transfected human cells. Analysis of transposon integration sites generated by B23-SB100X revealed a significant enrichment into the p-arms of chromosomes containing nucleolus organizing regions (NORs), with preferential integration into the p13 and p11.2 cytobands directly neighboring the NORs. This bias in the integration pattern was accompanied by an enrichment of insertions into nucleolus-associated chromatin domains (NADs) at the periphery of nucleolar DNA and into lamina-associated domains (LADs). Finally, sub-nuclear targeting of the transposase resulted in preferential integration into chromosomal domains associated with the Upstream Binding Transcription Factor (UBTF) that plays a critical role in the transcription of 47S rDNA gene repeats of the NORs by RNA Pol I. Future modifications of this technology may allow the development of methods for specific gene insertion for precision genetic engineering.

Improved piggyBac Transformation with Capped Transposase mRNA in Pest Insects.

Creating transgenic insects is a key technology in insect genetics and molecular biology. A widely used instrument in insect transgenesis is the piggyBac transposase, resulting in essentially random genomic integrations. In contrast, site-specific recombinases allow the targeted integration of the transgene construct into a specific genomic target site. Both strategies, however, often face limitations due to low transgenesis efficiencies. We aimed to enhance transgenesis efficiencies by utilizing capped mRNA as a source of transposase or recombinase instead of a helper plasmid. A systematic comparison of transgenesis efficiencies in Aedes mosquitoes, as models for hard-to-transform insects, showed that suppling piggyBac transposase as mRNA increased the average transformation efficiency in Aedes aegypti from less than 5% with the plasmid source to about 50% with mRNA. Similar high activity was observed in Ae. albopictus with pBac mRNA. No efficiency differences between plasmid and mRNA were observed in recombination experiments. Furthermore, a hyperactive version of piggyBac transposase delivered as a plasmid did not improve the transformation efficiency in Ae. aegypti or the agricultural pest Drosophila suzukii. We believe that the use of mRNA has strong potential for enhancing piggyBac transformation efficiencies in other mosquitoes and important agricultural pests, such as tephritids.