Biology subjects

Xie, J.

Publications and source records attributed to Xie, J..

At least 109 records · Page 6Linked to original sources

bioRxiv2024

Positive impact of Type-A and -C response regulator genes on growth and panicle architecture in rice

Cytokinin signal transduction occurs through a "two-component system." Type-A and -C response regulators (RRs) are groups of proteins of similar structures constituting significant components of cytokinin signal transduction. In rice, 13 (Type-A) and two (Type-C) RRs have been identified to date; however, their functions remain partially known. In this study, we examined the expression patterns of Type-A and Type-C RRs in rice using RNA-Seq and confirmed their functions by constructing mutants of the 15 genes with CRISPR/Cas9. Almost all Type-A RRs played positive roles in the development of secondary branches and secondary spikelets, particularly RR2 and RR4. Notably, rr1 rr2 and rr8 rr12 rr13 higher-order mutants displayed small panicle sizes and decreased plant height. Additionally, both Type-C RRs played positive roles in regulating heading date. RNA-seq revealed several genes with significantly altered expression in the rr2 and rr4 mutants, with almost half of the differentially expressed genes (DEGs) overlapping between the two mutants. Many of the DEGs were associated with the cytokinin and abscisic acid pathways. Our findings provide new insights into the functions of Type-A and -C RRs in rice growth and may serve as a foundation for future studies focusing on cytokinin signaling.

plant biology↗

bioRxiv2024

PNPLA3-I148M is a Neomorph that Interferes with Two Primary Hepatic Triglyceride Clearance Pathways

A common variant of PNPLA3, encoding PNPLA3-I148M, is the most significant genetic determinant of fatty liver disease worldwide. However, it is unclear precisely how PNPLA3-I148M drives disease risk. Here, we show that endogenous human PNPLA3-I148M impairs secretion of Apolipoprotein B (ApoB), the scaffolding protein of very low-density lipoproteins (VLDLs), from hepatocytes. This is not due to a generalized secretory pathway defect, nor is it equivalent to loss of function of PNPLA3. The VLDL secretory defect is conserved in mice expressing human I148M. Untargeted lipidomics reveal that I148M human cells are enriched in polyunsaturated fatty acid (PUFA)-containing triglycerides at the expense of PUFA-containing phosphatidylcholine, causing reduced membrane dynamics, concomitantly hindering biogenesis of secreted VLDLs. ApoB secretion is substantially rescued in I148M cells that overexpress ABHD5/CGI-58, an I148M binding partner that activates lipolysis by ATGL/PNPLA2 when not bound to I148M. Conversely, knocking down CGI-58 or PNPLA2 mimics I148M. We propose that neomorphic PNPLA3-I148M exacerbates fatty liver risk by simultaneously impeding two major CGI-58-dependent pathways for liver triglyceride clearance: lipolysis and secretion.

cell biology↗

bioRxiv2024

Low RT-based Genome Editing Fidelity in Mouse Hepatocytes: Challenges and Solutions

Abstract/SummaryIntegrase-mediated Programmable Genomic Integration (I-PGI) uses a Cas9 nickase (nCas9) with a reverse transcriptase (RT), to write a large serine integrase (LSI) target site (attB/P, here called "beacon") in a programmed location. Co-delivery of the LSI and a DNA template containing the cognate recognition site results in precise integration of the template in a specific genomic location. While we were able to achieve high-fidelity beacon placement in a range of primate cycling and non-dividing cells, when translating our technology into an in vivo rodent model (liver) we surprisingly observed very low beacon fidelity, with the vast majority of beacons being unsuitable for integration. This phenomenon was independent of mouse strain, but was specific to non-dividing cells, as a cycling mouse hepatocyte cell line (Hepa1-6) demonstrated very high levels of fidelity. To address this issue we utilized neonatal mice, which have a much higher proportion of proliferating hepatocytes than adult mice. This resulted in a significant increase in the placement of high-fidelity beacons, and achieved functional gene expression after I-PGI in a therapeutically relevant target site. In an alternate approach, we engineered transgenic mice with intact beacons placed in specific genomic locations, allowing us to optimize integrase and DNA template dosing and kinetics. In summary, we have identified a previously undescribed challenge when using RT-based editing to write long sequences (~40 bp) in non-dividing rodent hepatocytes. This phenomenon was specific to rodents and was not observed in primate dividing or non-dividing cells. This previously unidentified challenge using RTs will limit the use of I-PGI in mouse models, however here we describe two methods that address this issue.

bioengineering↗

bioRxiv2024

CIGB-300 reverses chemo-resistance in MDR1-transfected lung squamous cancer cells

BackgroundInhibition of ABC transporter protein activity is considered to be the most effective method to reverse multidrug resistance (MDR). In this study, we evaluated the MDR reversal potential of CIGB-300, a potent CK2 kinase inhibitor. MethodsABCB1 overexpressing lung adenocarcinoma NCI-H226 cells were constructed using lentivirus, and the expression of ABCB1 gene and protein was detected by real-time fluorescence quantitative PCR and Western blotting. MTT assay was used to assess the cytotoxicity and MDR reversal effect of CIGB-300.The effect of CIGB-300 on ABCB1 expression was determined by Western Blotting. Cell surface expression and subcellular localization of ABCB1 were examined by Flow Cytometry and Immunofluorescence Staining. Rh123 efflux and accumulation were measured by Fluorescent Enzyme Labeler and Flow Cytometry. ResultsCIGB-300 significantly increased the sensitivity of drug-resistant cells overexpressing the ABCB1 drug efflux pump (NCI-H226-ABCB1), while it had no effect on their parental cell lines. At the same time, its mechanism of action may be related to the inhibition of ABCB1 expression, which was dose-dependent, Moreover, in addition, we demonstrated that CIGB-300 reduced the expression of NFKB and CDC37 proteins. ConclusionsOur study elucidated that CIGB-300 reverses ABCB1-mediated MDR by inhibiting ABCB1 protein expression or intracellular signaling and provides a potential therapeutic strategy to improve tumor chemosensitivity.

cancer biology↗

bioRxiv2024

Breaking Free: Development of Circular AAV Cargos for Targeted Seamless Integration in the Liver

Recent advancements in gene insertion have shifted from DNA repair-dependent mechanisms to more precise approaches, enhancing safety and predictability for editing outcomes. Integrase-mediated programmable genomic integration (I-PGI) utilizes a DNA cargo to insert transgenes in a targeted, unidirectional manner. In vivo, where nuclear delivery of DNA is challenging, adeno-associated virus (AAV) can act as the cargo vector. While I-PGI does not require DNA double-stranded breaks (DSBs) for activity, linear DNA cargo, like AAV, stimulates DNA end joining activity after integration. To mitigate potential risks from DSBs with linear viral cargo, we developed two circular genome types capable of seamless gene insertion in non-dividing cells. We first harnessed the orthogonal property of large serine integrases to produce circle-AAV (cAAV) from linear viral genomes in cells. cAAV demonstrated faithful seamless cargo integration in primary human hepatocytes (PHH) and robust DSB-free insertion structures in vivo. We then investigated the delivery of packaged circular AAV cargo (AAV.AD), which eliminates the need for enzymatic manipulation in the cell. AAV.AD proved to be a viable cargo for I-PGI, exhibiting functional integration in PHH and in vivo, that resulted in seamless insertion structures. Together, these findings provide the first reported evidence of DSB-free programmable genomic integration using integrase and AAV cargo, addressing a previously unrecognized challenge in the field.

bioengineering↗

bioRxiv2024

Biocontrol efficiency and mechanism of novel Streptomyces luomodiensis SCA4-21 against banana Fusarium wilt

The soil-borne fungi Fusarium oxysporum f. sp. cubense tropical race 4 (Foc TR4) causes banana Fusarium wilt, which seriously threats global banana production. Biocontrol has been considered viable alternative method to manage banana Fusarium wilt. In previous study, we found that novel S. luomodiensis SCA4-21 exhibited antifungal activity. Here, we revealed that the extracts of strain SCA4-21 significantly inhibited the growth of Foc TR4 hyphae and spore germination, severely disrupting the ultrastructure of Foc TR4 hyphal cells and spore morphology. These extracts also exhibited broad-spectrum antifungal activity against eight other phytopathogenic fungi. Furthermore, we demonstrated that strain SCA4-21 produce 32 volatile organic compounds, including five antifungal compounds. In a pot experiment, we discovered that the inoculation of strain SCA4-21 significantly inhibited the infection of Foc TR4 in banana seedling corms, achieving a biocontrol efficiency of 59.3%, and promoted the growth of banana seedlings. Additionally, this inoculation significantly enhanced the abundances of beneficial bacterial genera Streptomyces, Bacillus, Sphingomonas, and Massilia, as well as fungal genera Mortierella, Purpureocillium, Gibellulopsis, and Xenomyrothecium, while significantly reduced the abundances of pathogenic bacteria genus Pantoea and fungal genus Fusarium, in the banana rhizosphere soil. Moreover, the inoculation of strain SCA4-21 significantly increased the enrichment of pathways such as carbohydrate metabolism, amino acid metabolism, and metabolism of terpenoids and polyketides. Therefore, we postulated that strain SCA4-21 may synergistically combat banana Fusarium wilt by producing antifungal compounds and enriching beneficial bacteria and fungi. Our findings present a promising biocontrol agent for the management of banana Fusarium wilt. IMPORTANCEBanana (Musa spp.) is one of the most popular fruit crops and the fourth largest food crop in developing countries within tropical and subtropical regions. However, the emergence and rapid spread of strain Foc TR4 seriously hinder the development of banana industry. Currently, there is no effective control measure available. Biological control holds potential due to its safety and effectiveness. Here, we found that the extracts of S. luomodiensis SCA4-21 exhibited significant inhibitory effects on the hyphal growth and spore germination of Foc TR4, as well as severe destructive effects on its cell morphology and ultrastructure, and broad-spectrum antifungal activity. We also discovered that the inoculation of strain SCA4-21 significantly inhibited the infection of Foc TR4 in banana seedling corms, reduced the incidence index of banana Fusarium wilt, promoted the growth of banana seedlings, and enhances beneficial microbes and metabolic pathways, suggesting that strain SCA4-21 is a promising biocontrol agent.

physiology↗

bioRxiv2024

LP-Micro Offers Interpretable Disease Outcome Prediction by Leveraging Microbial Biomarkers and Their Time-Varying Effects

Information generated from longitudinally-sampled microbial data has the potential to illuminate important aspects of development and progression for many human conditions and diseases. Identifying microbial biomarkers and their time-varying effects can not only advance our understanding of pathogenetic mechanisms, but also facilitate early diagnosis and guide optimal timing of interventions. However, longitudinal predictive modeling of highly noisy and dynamic microbial data (e.g., metagenomics) poses analytical challenges. To overcome these challenges, we introduce a robust and interpretable machine-learning-based longitudinal microbiome analysis framework, LP-Micro, that encompasses: (i) longitudinal microbial feature screening via a polynomial group lasso, (ii) disease outcome prediction implemented via machine learning methods (e.g., XGBoost, deep neural networks), and (iii) interpretable association testing between time points, microbial features, and disease outcomes via permutation feature importance. We demonstrate in simulations that LP-Micro can not only identify incident disease-related microbiome taxa but also offers improved prediction accuracy compared to existing approaches. Applications of LP-Micro in two longitudinal microbiome studies with clinical outcomes of childhood dental disease and weight loss following bariatric surgery yield consistently high prediction accuracy. The identified critical early predictive time points are informative and aligned with clinical expectations.

bioinformatics↗

bioRxiv2024

Curative levels of endogenous gene replacement achieved in non-human primate liver using programmable genomic integration

The ability to efficiently place a large piece of DNA in a specific genomic location has been a goal for the gene therapy field since its inception; however, despite significant advances in gene editing technology, this had yet to be achieved. Here we describe two methods of programmable genomic integration (PGI) that overcome some of the limitations of current approaches. Using a combination of clinically validated delivery technologies (LNP, AAV), we demonstrate the ability to specifically integrate large (>2 kb) DNA sequences into endogenous introns in the liver of non-human primates (NHP). PGI was effective across multiple genomic locations and transgenes, and insertion led to expression from the endogenous promoter. PGI was highly efficient, achieving expression in >50% of liver cells after a single course of treatment, which would be curative for most monogenic recessive liver diseases. This is the first report of clinically curative level of gene insertion at endogenous loci in NHP.

bioengineering↗

bioRxiv2024

Deciphering gene regulatory programs in mouse embryonic skin through single-cell multiomics analysis

BackgroundCell type-specific transcriptional heterogeneity in embryonic mouse skin is well-documented, but few studies have investigated the regulatory mechanisms. ResultsHere, we present high throughput single-cell chromatin accessibility and transcriptome sequencing (HT-scCAT-seq), a method that simultaneously profiles transcriptome and chromatin accessibility. We utilized HT-scCAT-seq to dissect the gene regulatory mechanism governing epidermal stratification, periderm terminal differentiation, and fibroblast specification. ConclusionsBy linking chromatin accessibility to gene expression, we identified candidate cis-regulatory elements (cCREs) and target genes crucial for dermal and epidermal development. We described cells with similar gene expression profiles that exhibit distinct chromatin accessibility statuses during periderm terminal differentiation. Finally, we characterized the underlying lineage-determining transcription factors (TFs), and demonstrated that ALX4 and RUNX2 were candidate TF regulators of the dermal papilla lineage development through in silico perturbation analysis.

developmental biology↗

bioRxiv2024

Inhibition of Androgen Receptor Exposes Replication Stress Vulnerability in Prostate Cancer

Standard initial systemic treatment for patients with metastatic prostate cancer includes agents that target androgen receptor (AR) signaling. Despite an initial positive response to these AR pathway inhibitors (ARPIs), acquired resistance remains a significant challenge. We show that treatment of AR-positive prostate cancer cells with the frontline ARPI enzalutamide induces DNA replication stress. Such stress is exacerbated by suppression of translesion DNA synthesis (TLS), leading to aberrant accumulation of single-stranded DNA (ssDNA) gaps and persistent DNA damage biomarkers. We further demonstrate that the TLS inhibitor, JH-RE-06, markedly sensitizes AR-positive prostate cancer cells, but not AR-negative benign cells, to enzalutamide in vitro. Combination therapy with enzalutamide and JH-RE-06 significantly suppresses cancer growth in a syngeneic murine tumor model over vehicle control or individual treatment groups. These findings suggest that AR inhibition broadly triggers DNA replication stress in hormone-sensitive prostate cancer, thereby exposing a unique vulnerability that can be exploited by a TLS-disrupting adjuvant for targeted therapy.

cancer biology↗

bioRxiv2024

Ligase-mediated programmable genomic integration (L-PGI): an efficient site-specific gene editing system that overcomes the limitations of reverse transcriptase-based editing systems

Since their discovery, CRISPR/Cas9 systems have been repurposed for programmable targeted genomic editing. This has led to unprecedented advancement of gene editing for therapeutic benefit. Initial uses of CRISPR/Cas9 were focused on gene disruption via DNA cleavage, but significant engineering led to systems for single base editing as well as insertion, deletion and manipulation of short stretches of genomic sequences using nicking Cas9 and RT-based methods. These technologies allowed safer and more precise editing but were limited to small corrections and showed significantly reduced efficiencies in nondividing cells, presenting difficulty for translation to in vivo therapies. To find an alternate editing strategy that could address these shortcomings, we revisited the mechanism of DNA nicking by nCas9. nCas9 nicking creates a free 5 phosphate group and a 3 hydroxyl group on the complementary strand of the target sequence. Under ordinary conditions in the cell these ends are re-joined by endogenously expressed ligases to repair DNA back to wild-type. If, however, a DNA fragment containing the desired edit were present, ligation of the nicked genomic DNA with the delivered fragment could result in gene editing. We demonstrate that optimization of each component and introduction of a chemically modified high affinity splinting DNA allows a variety of ligase-based edits, including longer edits not efficient with RT-based systems, at high efficiencies and fidelities that minimize genomic byproducts in both dividing and nondividing cells as well as in vivo in adult mice. Here we present the first therapeutically relevant ligation-based programmable gene editing technology, L-PGI.

bioengineering↗

bioRxiv2024

GPR30 in spinal cholecystokinin-positive neurons modulates neuropathic pain via mediating descending facilitation

Neuropathic pain, a major health problem affecting 7-10% of the global population, lacks effective treatment due to its elusive mechanisms. Cholecystokinin-positive (CCK+) neurons in the spinal dorsal horn (SDH) are critical for neuropathic pain, yet the underlying molecular mechanisms remain unclear. Here, we show that the membrane estrogen receptor G-protein coupled estrogen receptor (GPER/GPR30) in spinal neurons was significantly upregulated in chronic constriction injury (CCI) mice and that inhibition of GPR30 in CCK+ neurons reversed CCI-induced neuropathic pain. Furthermore, GPR30 in spinal CCK+ neurons was essential for the enhancement of AMPA-mediated excitatory synaptic transmission in CCI mice. Moreover, GPR30 was expressed in spinal CCK+ neurons that received direct projection from the primary sensory cortex (S1-SDH). Chemogenetic inhibition of S1-SDH post-synaptic neurons alleviated CCI-induced neuropathic pain. Conversely, chemogenetic activation of these neurons mimicked neuropathic pain symptoms, which were attenuated by spinal inhibition of GPR30. Finally, we confirmed that GPR30 in S1-SDH post-synaptic neurons was required for CCI-induced neuropathic pain. Taken together, our findings suggest that GPR30 in spinal CCK+ neurons and S1-SDH postsynaptic neurons is pivotal for neuropathic pain, thereby representing a promising therapeutic target for neuropathic pain.

neuroscience↗

bioRxiv2024

spaDesign: A Statistical Framework to Improve the Design of Sequencing-based Spatial Transcriptomics Experiments

MotivationRigorous experimental design is essential for obtaining biologically meaningful findings and high-throughput spatial transcriptomics studies are not an exception. While some efforts have been made to improve the design of these studies, it is still significantly understudied yet how to optimize key experimental factors of these experiments. In the case of sequencing-based spatial transcriptomics studies, determining the minimum sequencing depth is an important experimental factor to decide. ResultsTo address this critical limitation, here we propose spaDesign, a statistical framework to improve the design of sequencing-based spatial transcriptomics experiments. spaDesign is a statistically rigorously designed framework that employs Poisson Gaussian process and Fisher-Gaussian kernel mixture. It can easily simulate a range of spatial transcriptomics data with various sequencing depths, effect sizes, and spatial patterns, which allows rigorous estimation of needed total sequencing depths to detect spatial domains based on spatial transcriptomics experiments. We demonstrated the utility and power of spaDesign using 10X Visium data of the human brain and the chicken heart. AvailabilityThe R package and associated tutorial are freely available at https://github.com/JuanXie19/spaDesign.

bioinformatics↗

bioRxiv2024

IL-34 empowers regulatory T cells with novel non-canonical function to safeguard brain barrier integrity during neuro-inflammation.

In efforts to find reparative strategies for brain damage, brain-associated regulatory T cells (Tregs) have gained increasing attention in recent years. Beyond their textbook immunoregulatory function, Tregs have emerged as key players in the response to brain trauma and the restoration of damaged brain tissue. Here, we are the first to describe a novel, non-canonical function of Tregs in maintaining the sealing capacity of both the blood-brain barrier (BBB) and the blood-cerebrospinal fluid (CSF) barrier. Moreover, we identified the cytokine IL-34 as a critical determinant in this newly unveiled Treg function. Mechanistically, IL-34 exerts its influence by modulating the expression and localization of the tight junction protein ZO-1 in both BBB endothelial cells and choroid plexus epithelial cells, thereby reinforcing the strength of the brain barriers. Given the well-established notion of leaky brain barriers and the involvement of immunological components in neurological diseases such as Alzheimers disease (AD) and multiple sclerosis (MS), we further demonstrate diminished IL-34 expression in Tregs derived from patients with relapsing-remitting MS (RR-MS) and patients with AD and even mild cognitive impairment (MCI). Remarkably, our study reveals the potential of IL-34 treatment in reinstating the integrity of brain barriers within murine models mimicking these neurological disorders. These ground-breaking findings shed light on the intricate relationship between Tregs, IL-34, and the integrity of brain barriers. They offer novel avenues for therapeutic approaches to ameliorate brain barrier dysfunction in the context of neurological disorders.

immunology↗

bioRxiv2024

LYMTACs: Chimeric Small Molecules Repurpose Lysosomal Membrane Proteins for Target Protein Relocalization and Degradation

Proximity-inducing modalities that co-opt cellular pathways offer new opportunities to regulate oncogenic drivers. Inspired by the success of proximity-based chimeras in both intracellular and extracellular target space, here we describe the development of LYsosome Membrane TArgeting Chimeras (LYMTACs) as a novel small molecule-based platform that functions intracellularly to modulate the membrane proteome. Conceptually, LYMTACs are heterobifunctional small molecules that co-opt short-lived lysosomal membrane proteins (LMPs) as effectors to deliver targets for lysosomal degradation. We demonstrate that a promiscuous kinase inhibitor-based LYMTAC selectively targets membrane proteins for lysosomal degradation via RNF152, a short-lived LMP. To extend these findings, we show that oncogenic, membrane-associated KRASG12D protein can be tethered to RNF152, inducing KRAS relocalization to the lysosomal membrane, inhibiting downstream phospho-ERK signaling, and leading to lysosomal degradation of KRASG12D in a LYMTAC-dependent manner. Notably, potent cell killing could be attributed to the multi-pharmacology displayed by LYMTACs, which differentiates the LYMTAC technology from existing modalities. Thus, LYMTACs represent a proximity-based therapeutic approach that promises to expand the target space for challenging membrane proteins through targeted protein relocalization and degradation.

cancer biology↗

bioRxiv2024

Osterix Facilitates Osteocytic Communication by Targeting Connexin43

Osteocytes, terminal-differentiated cells in bone, are now considered as more pivotal regulators of mature bone homeostasis than other bone cells, since they constitute 90- 95% of the bone cell population. Given their non-migratory nature within the mineralized matrix, their unique dendrites are crucial for cell-to-cell communication in response to both intracellular and extracellular stimuli, such as bone fracture or mechanical load. Here, we showed that Osterix (Osx), usually recognized as a specific doorkeeper for osteoblast differentiation during new bone formation marked by collagen type I 1 (Col11), was unexpectedly co-expressed with Col11 in osteocytes within the cortical bone of mice. Deleting Osx in Col11-positive osteocytes disrupted cortical bone structure and osteocytic dendrites in mice, thus impairing transcellular fluid flow and intercellular communication. Conversely, overexpression of Osx in osteocytes enhanced these processes. Furthermore, we identified Connexin43, a critical protein of gap junction channel, was a direct transcriptional target of Osx in regulating dendrites of osteocytes. Pharmacological restoration of Connexin43 levels rescued the dysfunction in Osx-deficient osteocytes both in vitro and in vivo. Taken together, this work demonstrated Osxs distinct role in osteocyte function through maintaining intercellular signaling, which broadened the current understanding of its role in Col11-positive bone cells, extending beyond osteoblasts and bone mineralization, offering new insights into bone diseases such as fracture nonunion or disuse osteoporosis.

cell biology↗

bioRxiv2024

Large Serine Integrase Off-target Discovery and Validation for Therapeutic Genome Editing

While numerous technologies for the characterization of potential off-target editing by CRISPR/Cas9 have been described, the development of new technologies and analytical methods for off-target recombination by Large Serine Integrases (LSIs) are required to advance the application of LSIs for therapeutic gene integration. Here we describe a suite of off-target recombination discovery technologies and a hybrid capture validation approach as a comprehensive framework for off-target characterization of LSIs. HIDE- Seq (High-throughput Integrase-mediated DNA Event Sequencing) is a PCR-free unbiased genome-wide biochemical assay capable of discovering sites with LSI- mediated free DNA ends (FDEs) and off-target recombination events. Cryptic-Seq is a PCR-based unbiased genome-wide biochemical or cellular-based assay that is more sensitive than HIDE-Seq but is limited to the discovery of sites with off-target recombination. HIDE-Seq and Cryptic-Seq discovered 38 and 44,311 potential off-target sites respectively. 2,455 sites were prioritized for validation by hybrid capture NGS in LSI- edited K562 cells and off-target integration was detected at 52 of the sites. We benchmarked the sensitivity of our LSI off-target characterization framework against unbiased whole genome sequencing (WGS) on LSI-edited samples, and off-target integration was detected at 5 sites with an average genome coverage of 40x. This reflects a greater than 10-fold increase in sensitivity for off-target detection compared to WGS, however only 4 of the 5 sites detected by WGS were also validated by hybrid capture NGS. The dissemination of these technologies will help advance the application of LSIs in therapeutic genome editing by establishing methods and benchmarks for the sensitivity of off-target detection.

genomics↗

bioRxiv2024

Impacts of nucleosome positioning elements and pre-assembled chromatin states on expression and retention of transgenes

Transgene applications ranging from gene therapy to development of stable cell lines and organisms rely on maintaining expression of transgenes. To-date, the use of plasmid-based transgenes has been limited by loss of their expression shortly after delivery into target cells. This short-lived expression of plasmid-based transgenes has been largely attributed to host cell-mediated degradation and/or silencing of transgenes. To assess the impact of "priming" plasmid-based transgenes to adopt accessible chromatin states to promote gene expression, nucleosome positioning elements were introduced at promoters of transgenes, or vectors were pre-assembled into nucleosomes containing unmodified histones or histone mutants mimicking constitutively acetylated states at residues 9 and 14 of histone H3 or residue 16 of histone H4 prior to their introduction into cells, then transgene expression was monitored over time. DNA sequences capable of positioning nucleosomes could positively impact expression of adjacent transgenes in a distance-dependent manner in the absence of pre-assembly into chromatin. Intriguingly, pre-assembly of plasmids into chromatin facilitated prolonged expression of transgenes relative to plasmids that were not pre-packaged into chromatin. Interactions between pre-assembled chromatin states and nucleosome positioning-derived effects on expression of reporter genes were also assessed and, generally, nucleosome positioning played the predominant role in influencing gene expression relative to priming with hyperacetylated chromatin states.

molecular biology↗