ABSTRACT
Goosecoid (GSC), translated from a homeobox gene, is a protein that participates in metastasis of various cancers. Pancreatic adenocarcinoma (PAAD) is one of the deadliest malignancies associated with a poor diagnosis and prognosis. To develop new treatment target or biomarker for PAAD, this study intended to assess the effects and the molecular mechanism of GSC on PAAD metastasis. The expressive discrepancy of GSC in PAAD and normal tissues/cells was compared by both the quantitative PCR and western blot. The effects of GSC silencing and GSC over-expression on PAAD cells and TGF-ß signaling were proved by wound-healing assay, cell counting kit-8, Transwell assay and western blot. From the results, GSC mRNA and protein levels were enriched in PAAD cancer tissues and cells. GSC silencing prohibited metastasis of PAAD cells including the ability to invade, migrate and epithelial-mesenchymal transition (EMT), whereas GSC upregulation stimulated these cells behaviors above. GSC silencing reversed the effects on cellular processes induced by activation of the TGF-ß pathway. Furthermore, silencing of GSC postponed tumor growth in xenograft model. In summary, GSC was abundantly expressed in PAAD, which activated the TGF-ß pathway to enhance cell metastasis and tumor development.
Subject(s)
Adenocarcinoma , Epithelial-Mesenchymal Transition , Neoplasm Metastasis , Pancreatic Neoplasms , Signal Transduction , Transforming Growth Factor beta , Animals , Humans , Mice , Adenocarcinoma/metabolism , Adenocarcinoma/pathology , Adenocarcinoma/genetics , Cell Line, Tumor , Cell Movement/genetics , Cell Proliferation , Epithelial-Mesenchymal Transition/genetics , Gene Expression Regulation, Neoplastic , Gene Silencing , Intercellular Signaling Peptides and Proteins , Mice, Inbred BALB C , Mice, Nude , Pancreatic Neoplasms/pathology , Pancreatic Neoplasms/metabolism , Pancreatic Neoplasms/genetics , Transforming Growth Factor beta/metabolismABSTRACT
Limited effective targets have challenged the treatment of oral squamous cell carcinoma (OSCC). Casein kinase 2 interacting protein 1 (CKIP-1) is a scaffold protein involved in various diseases. However, the role of CKIP-1 in OSCC remains unclear. The aim of this study was to explore the regulatory role of CKIP-1 in OSCC, as well as the involved mechanism. First, higher expression of CKIP-1 in OSCC tissues and cell lines were found. Series of gain- and loss-of-function experiments demonstrated suppressed malignant behaviours and enhanced apoptosis of OSCC cells when CKIP-1 was silenced. Also, inhibited tumour growth in CKIP-1-silenced group were proved. Further, mitochondrial transcription factor A (TFAM) downregulation, increased ROS production, decreased mitochondrial membrane potential and cGAS-STING activation in CKIP-1-silenced group were observed. The involvement of mitochondrial homeostasis-related TFAM/cGAS-STING axis in CKIP-1-silenced OSCC cells was finally demonstrated by tetramethylpyrazine (TMP) that inhibits TFAM degradation. Taken together, our study demonstrated that CKIP-1 silencing could significantly antagonize OSCC via TFAM/cGAS-STING axis, which may provide a candidate target for OSCC treatment.
Subject(s)
Apoptosis , Carcinoma, Squamous Cell , DNA-Binding Proteins , Membrane Proteins , Mitochondria , Mouth Neoplasms , Signal Transduction , Transcription Factors , Humans , Mitochondria/metabolism , Mitochondria/drug effects , Mouth Neoplasms/metabolism , Mouth Neoplasms/genetics , Mouth Neoplasms/pathology , Cell Line, Tumor , Membrane Proteins/metabolism , Membrane Proteins/genetics , Transcription Factors/metabolism , Transcription Factors/genetics , Carcinoma, Squamous Cell/metabolism , Carcinoma, Squamous Cell/genetics , Carcinoma, Squamous Cell/pathology , DNA-Binding Proteins/metabolism , DNA-Binding Proteins/genetics , Animals , Homeostasis , Mitochondrial Proteins/metabolism , Mitochondrial Proteins/genetics , Membrane Potential, Mitochondrial/drug effects , Gene Expression Regulation, Neoplastic , Cell Proliferation , Carrier Proteins/metabolism , Carrier Proteins/genetics , Mice , Gene Silencing , Reactive Oxygen Species/metabolism , Male , Intracellular Signaling Peptides and ProteinsABSTRACT
In animals, evolutionarily conserved Polycomb repressive complex 2 (PRC2) catalyzes histone H3 lysine 27 trimethylation (H3K27me3) and PRC1 functions in recruitment and transcriptional repression. However, the mechanisms underlying H3K27me3-mediated stable transcriptional silencing are largely unknown, as PRC1 subunits are poorly characterized in fungi. Here, we report that in the filamentous fungus Magnaporthe oryzae, the N-terminal chromodomain and C-terminal MRG domain of Eaf3 play key roles in facultative heterochromatin formation and transcriptional silencing. Eaf3 physically interacts with Ash1, Eed, and Sin3, encoding an H3K36 methyltransferase, the core subunit of PRC2, and a histone deacetylation co-suppressor, respectively. Eaf3 co-localizes with a set of repressive Ash1-H3K36me2 and H3K27me3 loci and mediates their transcriptional silencing. Furthermore, Eaf3 acts as a histone reader for the repressive H3K36me2 and H3K27me3 marks. Eaf3-occupied regions are associated with increased nucleosome occupancy, contributing to transcriptional silencing in M. oryzae. Together, these findings reveal that Eaf3 is a repressive H3K36me2 reader and plays a vital role in Polycomb gene silencing and the formation of facultative heterochromatin in fungi.
Subject(s)
Fungal Proteins , Gene Silencing , Heterochromatin , Histones , Histones/metabolism , Histones/genetics , Heterochromatin/metabolism , Heterochromatin/genetics , Fungal Proteins/metabolism , Fungal Proteins/genetics , Methylation , Gene Expression Regulation, Fungal , Polycomb Repressive Complex 2/metabolism , Polycomb Repressive Complex 2/genetics , Nucleosomes/metabolism , Polycomb-Group Proteins/metabolism , Polycomb-Group Proteins/genetics , Lysine/metabolismABSTRACT
RNA interference (RNAi) technology has emerged as a pivotal strategy in sustainable pest management, offering a targeted approach that significantly mitigates the environmental and health risks associated with traditional insecticides. Originally implemented through genetically modified organisms (GMOs) to produce specific RNAi constructs, the technology has evolved in response to public and regulatory concerns over GMOs. This evolution has spurred the development of non-transgenic RNAi applications such as spray-induced gene silencing (SIGS), which employs double-stranded RNA (dsRNA) to silence pest genes directly without altering the plant's genetic makeup. Despite its advantages in specificity and reduced ecological footprint, SIGS faces significant obstacles, particularly the instability of dsRNA in field conditions, which limits its practical efficacy. To overcome these limitations, innovative delivery mechanisms have been developed. These include nanotechnology-based systems, minicells, and nanovesicles, which are designed to protect dsRNA from degradation and enhance its delivery to target organisms. While these advancements have improved the stability and application efficiency of dsRNA, comprehensive assessments of their environmental safety and the potential for increased exposure risks to non-target organisms remain incomplete. This comprehensive review aims to elucidate the environmental fate of dsRNA and evaluate the potential risks associated with its widespread application on non-target organisms, encompassing soil microorganisms, beneficial insects, host plants, and mammals. The objective is to establish a more refined framework for RNAi risk assessment within environmental and ecotoxicological contexts, thereby fostering the development of safer, non-transgenic RNAi-based pest control strategies.
Subject(s)
RNA Interference , RNA, Double-Stranded , Ecotoxicology , Animals , Plants, Genetically Modified , Gene SilencingABSTRACT
Strigolactones (SLs) are plant hormones that regulate diverse developmental processes and environmental responses in plants. It has been discovered that SLs play an important role in regulating plant immune resistance to pathogens but there are currently no reports on their role in the interaction between Nicotiana benthamiana and the tobacco mosaic virus (TMV). In this study, the exogenous application of SLs weakened the resistance of N. benthamiana to TMV, promoting TMV infection, whereas the exogenous application of Tis108, a SL inhibitor, resulted in the opposite effect. Virus-induced gene silencing (VIGS) inhibition of two key SL synthesis enzyme genes, NtCCD7 and NtCCD8, enhanced the resistance of N. benthamiana to TMV. Additionally, we conducted a screening of N. benthamiana related to TMV infection. TMV-infected plants treated with SLs were compared to the control by using RNA-seq. The KEGG enrichment analysis and weighted gene co-expression network analysis (WGCNA) of differentially expressed genes (DEGs) suggested that plant hormone signaling transduction may play a significant role in the SL-TMV-N. benthamiana interactions. This study reveals new functions of SLs in regulating plant immunity and provides a reference for controlling TMV diseases in production.
Subject(s)
Disease Resistance , Gene Expression Regulation, Plant , Lactones , Nicotiana , Plant Diseases , Tobacco Mosaic Virus , Nicotiana/virology , Nicotiana/genetics , Nicotiana/immunology , Tobacco Mosaic Virus/physiology , Lactones/pharmacology , Disease Resistance/genetics , Plant Diseases/virology , Plant Diseases/genetics , Plant Diseases/immunology , Plant Proteins/genetics , Plant Proteins/metabolism , Plant Growth Regulators/metabolism , Plant Growth Regulators/pharmacology , Plant Immunity/genetics , Plant Immunity/drug effects , Gene SilencingABSTRACT
piRNAs are crucial for transposon silencing, germ cell maturation, and fertility in male mice. Here, we report on the genetic landscape of piRNA dysfunction in humans and present 39 infertile men carrying biallelic variants in 14 different piRNA pathway genes, including PIWIL1, GTSF1, GPAT2, MAEL, TDRD1, and DDX4. In some affected men, the testicular phenotypes differ from those of the respective knockout mice and range from complete germ cell loss to the production of a few morphologically abnormal sperm. A reduced number of pachytene piRNAs was detected in the testicular tissue of variant carriers, demonstrating impaired piRNA biogenesis. Furthermore, LINE1 expression in spermatogonia links impaired piRNA biogenesis to transposon de-silencing and serves to classify variants as functionally relevant. These results establish the disrupted piRNA pathway as a major cause of human spermatogenic failure and provide insights into transposon silencing in human male germ cells.
Subject(s)
DNA Transposable Elements , Infertility, Male , RNA, Small Interfering , Spermatogenesis , Testis , Male , Humans , Spermatogenesis/genetics , Infertility, Male/genetics , Infertility, Male/metabolism , Infertility, Male/pathology , RNA, Small Interfering/metabolism , RNA, Small Interfering/genetics , DNA Transposable Elements/genetics , Animals , Testis/metabolism , Mice , Adult , Gene Silencing , Mice, Knockout , Argonaute Proteins/metabolism , Argonaute Proteins/genetics , Long Interspersed Nucleotide Elements/genetics , Spermatogonia/metabolism , DEAD-box RNA Helicases/genetics , DEAD-box RNA Helicases/metabolism , Piwi-Interacting RNAABSTRACT
Pulmonary arterial hypertension (PAH) is a chronic disease characterized by a progressive increase in mean pulmonary arterial pressure. Mutations in the BMPR2 and AQP1 genes have been described in familial PAH. The bone morphogenetic proteins BMP9 and BMP10 bind with high affinity to BMPR2. Administration of BMP9 has been proposed as a potential therapeutic strategy against PAH, although recent conflicting evidence dispute the effect of such a practice. Considering the involvement of the above molecules in PAH onset, progression, and therapeutic value, we examined the effects of modulation of BMP9, BMPR2, and AQP1 on BMP9, BMP10, BMPR2, AQP1, and TGFB1 expression in human pulmonary microvascular endothelial cells in vitro. Our results demonstrated that silencing the BMPR2 gene resulted in increased expression of its two main ligands, namely BMP9 and BMP10. Exogenous administration of BMP9 caused the return of BMP10 to basal levels, while it restored the decreased AQP1 protein levels and the decreased TGFB1 mRNA and protein expression levels caused by BMPR2 silencing. Moreover, AQP1 gene silencing also resulted in increased expression of BMP9 and BMP10. Our results might possibly imply that the effect of exogenously administered BMP9 on molecules participating in the BMP signaling pathway could depend on the expression levels of BMPR2. Taken together, these results may provide insight into the highly complex interactions of the BMP signaling pathway.
Subject(s)
Aquaporin 1 , Bone Morphogenetic Protein Receptors, Type II , Endothelial Cells , Growth Differentiation Factor 2 , Signal Transduction , Transforming Growth Factor beta1 , Humans , Aquaporin 1/metabolism , Aquaporin 1/genetics , Growth Differentiation Factor 2/metabolism , Growth Differentiation Factor 2/genetics , Endothelial Cells/metabolism , Bone Morphogenetic Protein Receptors, Type II/metabolism , Bone Morphogenetic Protein Receptors, Type II/genetics , Transforming Growth Factor beta1/metabolism , Lung/metabolism , Lung/blood supply , Microvessels/metabolism , Microvessels/cytology , Cells, Cultured , Gene Silencing , Pulmonary Arterial Hypertension/metabolism , Pulmonary Arterial Hypertension/genetics , Bone Morphogenetic ProteinsABSTRACT
Instability of transgene expression is a major challenge for the biopharmaceutical industry, which can impact yields and regulatory approval. Some tRNA genes (tDNAs) can resist epigenetic silencing, the principal mechanism of expression instability, and protect adjacent genes against the spread of repressive heterochromatin. We have taken two naturally occurring clusters of human tDNAs and tested their ability to reduce epigenetic silencing of transgenes integrated into the genome of Chinese hamster ovary (CHO) cells. We find sustained improvements in productivity both in adherent CHO-K1 cells and in an industrially relevant CHO-DG44 expression system (Apollo X, FUJIFILM Diosynth Biotechnologies). We conclude that specific tDNA clusters offer potential to mitigate the widespread problem of production instability.
Subject(s)
Cricetulus , RNA, Transfer , Transgenes , CHO Cells , Animals , RNA, Transfer/genetics , Humans , Cricetinae , Epigenesis, Genetic/genetics , Gene Silencing , Recombinant Proteins/genetics , Recombinant Proteins/metabolismABSTRACT
Colorectal cancer (CRC) is one of the most common malignant carcinoma worldwide. DNA polymerase epsilon 2, accessory subunit (POLE2) participates in DNA replication, repair, and cell cycle control, but its association with CRC development remains unclear. In the present study, the differentially expressed genes (DEGs) in CRC were screened from bioinformatics analysis based on GEO database. RT-qPCR was used to assess mRNA expression. CCK-8 and colony formation assays were applied for the evaluation of cell proliferation. Wound healing and transwell assays were used to detect cell migration and invasion. Protein levels were determined by Western blotting assay. We found that POLE2 was highly expressed in CRC tissues and cell lines. Inhibition of POLE2 suppressed the proliferation, migration and invasion of CRC cells. Mechanistically, Wnt/ß-catenin signaling pathway was inactivated by inhibition of POLE2. Activation of Wnt/ß-catenin pathway can reverse the function of POLE2 knockdown on CRC cells. In vivo studies demonstrated that POLE2 silencing could notably inhibit the growth of tumors, which was consistent with the results in vitro. In conclusion, we found POLE2 as a novel oncogene in CRC, providing a potential therapeutic or diagnostic target in CRC.
Subject(s)
Cell Proliferation , Colorectal Neoplasms , Wnt Signaling Pathway , Humans , Colorectal Neoplasms/genetics , Colorectal Neoplasms/pathology , Colorectal Neoplasms/metabolism , Mice , Animals , Cell Movement , Cell Line, Tumor , Disease Progression , Gene Expression Regulation, Neoplastic , Mice, Nude , Gene Silencing , DNA Polymerase II/genetics , DNA Polymerase II/metabolismSubject(s)
Brain , Gene Silencing , Prion Proteins , Animals , Humans , Mice , Brain/metabolism , Prion Diseases/genetics , Prion Diseases/metabolism , Prion Proteins/genetics , Prion Proteins/metabolismABSTRACT
The malaria-causing parasites have to complete a complex infection cycle in the mosquito vector that also involves attack by the insect's innate immune system, especially at the early stages of midgut infection. However, Anopheles immunity to the late Plasmodium sporogonic stages, such as oocysts, has received little attention as they are considered to be concealed from immune factors due to their location under the midgut basal lamina and for harboring an elaborate cell wall comprising an external layer derived from the basal lamina that confers self-properties to an otherwise foreign structure. Here, we investigated whether Plasmodium berghei oocysts and sporozoites are susceptible to melanization-based immunity in Anopheles gambiae. Silencing of the negative regulator of melanization response, CLIPA14, increased melanization prevalence without significantly increasing the numbers of melanized oocysts, while co-silencing CLIPA14 with CLIPA2, a second negative regulator of melanization, resulted in a significant increase in melanized oocysts and melanization prevalence. Only late-stage oocysts were found to be melanized, suggesting that oocyst rupture was a prerequisite for melanization-based immune attack, presumably due to the loss of the immune-evasive features of their wall. We also found melanized sporozoites inside oocysts and in the hemocoel, suggesting that sporozoites at different maturation stages are susceptible to melanization. Silencing the melanization promoting factors TEP1 and CLIPA28 rescued oocyst melanization in CLIPA2/CLIPA14 co-silenced mosquitoes. Interestingly, silencing of CTL4, that protects early stage ookinetes from melanization, had no effect on oocysts and sporozoites, indicating differential regulation of immunity to early and late sporogonic stages. Similar to previous studies addressing ookinete stage melanization, the melanization of Plasmodium falciparum oocysts was significantly lower than that observed for P. berghei. In summary, our results provide conclusive evidence that late sporogonic malaria parasite stages are susceptible to melanization, and we reveal distinct regulatory mechanisms for ookinete and oocyst melanization.
Subject(s)
Anopheles , Melanins , Oocysts , Plasmodium berghei , Sporozoites , Animals , Anopheles/parasitology , Anopheles/immunology , Plasmodium berghei/immunology , Oocysts/metabolism , Melanins/metabolism , Sporozoites/immunology , Sporozoites/metabolism , Mosquito Vectors/parasitology , Mosquito Vectors/immunology , Insect Proteins/metabolism , Insect Proteins/genetics , Insect Proteins/immunology , Malaria/immunology , Malaria/parasitology , Gene Silencing , Immunity, Innate , FemaleABSTRACT
BACKGROUND: The SET domain group (SDG) genes encode histone lysine methyltransferases, which regulate gene transcription by altering chromatin structure and play pivotal roles in plant flowering determination. However, few studies have investigated their role in the regulation of flowering in upland cotton. RESULTS: A total of 86 SDG genes were identified through genome-wide analysis in upland cotton (Gossypium hirsutum). These genes were unevenly distributed across 25 chromosomes. Cluster analysis revealed that the 86 GhSDGs were divided into seven main branches. RNA-seq data and qRTâPCR analysis revealed that lysine methyltransferase 3 (KMT3) genes were expressed at high levels in stamens, pistils and other floral organs. Using virus-induced gene silencing (VIGS), functional characterization of GhKMT3;1a and GhKMT3;2a revealed that, compared with those of the controls, the GhKMT3;1a- and GhKMT3;2a-silenced plants exhibited later budding and flowering and lower plant heightwere shorter. In addition, the expression of flowering-related genes (GhAP1, GhSOC1 and GhFT) significantly decreased and the expression level of GhSVP significantly increased in the GhKMT3;1a- and GhKMT3;2a-silenced plants compared with the control plants. CONCLUSION: A total of 86 SDG genes were identified in upland cotton, among which GhKMT3;1a and GhKMT3;2a might regulate flowering by affecting the expression of GhAP1, GhSOC1, GhFT and GhSVP. These findings will provide genetic resources for advanced molecular breeding in the future.
Subject(s)
Flowers , Gene Expression Regulation, Plant , Gossypium , Histone-Lysine N-Methyltransferase , Plant Proteins , Gossypium/genetics , Gossypium/enzymology , Gossypium/physiology , Flowers/genetics , Flowers/growth & development , Plant Proteins/genetics , Plant Proteins/metabolism , Histone-Lysine N-Methyltransferase/genetics , Histone-Lysine N-Methyltransferase/metabolism , Genes, Plant , Gene SilencingABSTRACT
Retrotransposition is one of the main factors responsible for gene duplication and thus genome evolution. However, the sequences that undergo this process are not only an excellent source of biological diversity, but in certain cases also pose a threat to the integrity of the DNA. One of the mechanisms that protects against the incorporation of mobile elements is the HUSH complex, which is responsible for silencing long, intronless, transcriptionally active transposed sequences that are rich in adenine on the sense strand. In this study, broad sets of human and porcine retrocopies were analysed with respect to the above factors, taking into account evolution of these molecules. Analysis of expression pattern, genomic structure, transcript length, and nucleotide substitution frequency showed the strong relationship between the expression level and exon length as well as the protective nature of introns. The results of the studies also showed that there is no direct correlation between the expression level and adenine content. However, protein-coding retrocopies, which have a lower adenine content, have a significantly higher expression level than the adenine-rich non-coding but expressed retrocopies. Therefore, although the mechanism of HUSH silencing may be an important part of the regulation of retrocopy expression, it is one component of a more complex molecular network that remains to be elucidated.
Subject(s)
Evolution, Molecular , Gene Silencing , Retroelements , Retroelements/genetics , Animals , Humans , Swine/genetics , Introns , ExonsABSTRACT
BACKGROUND: The piRNA pathway in animal gonads functions as an 'RNA-based immune system', serving to silence transposable elements and prevent inheritance of novel invaders. In Drosophila, this pathway relies on three gonad-specific Argonaute proteins (Argonaute-3, Aubergine and Piwi) that associate with 23-28 nucleotide piRNAs, directing the silencing of transposon-derived transcripts. Transposons constitute a primary driver of genome evolution, yet the evolution of piRNA pathway factors has not received in-depth exploration. Specifically, channel nuclear pore proteins, which impact piRNA processing, exhibit regions of rapid evolution in their promoters. Consequently, the question arises whether such a mode of evolution is a general feature of transposon silencing pathways. RESULTS: By employing genomic analysis of coding and promoter regions within genes that function in transposon silencing in Drosophila, we demonstrate that the promoters of germ cell-specific piRNA factors are undergoing rapid evolution. Our findings indicate that rapid promoter evolution is a common trait among piRNA factors engaged in germline silencing across insect species, potentially contributing to gene expression divergence in closely related taxa. Furthermore, we observe that the promoters of genes exclusively expressed in germ cells generally exhibit rapid evolution, with some divergence in gene expression. CONCLUSION: Our results suggest that increased germline promoter evolution, in partnership with other factors, could contribute to transposon silencing and evolution of species through differential expression of genes driven by invading transposons.
Subject(s)
DNA Transposable Elements , Evolution, Molecular , Gene Silencing , Germ Cells , Promoter Regions, Genetic , RNA, Small Interfering , Animals , DNA Transposable Elements/genetics , Germ Cells/metabolism , RNA, Small Interfering/genetics , RNA, Small Interfering/metabolism , Drosophila Proteins/genetics , Drosophila/genetics , Argonaute Proteins/geneticsABSTRACT
Multiplex gene regulation enables the controlled and simultaneous alteration of the expression levels of multiple genes and is generally pursued to precisely alter complex cellular pathways with a single intervention. Thus far, this has been typically exploited in combination with genome editing tools (i.e., base-/prime-editing, designer nucleases) to enable simultaneous genetic alterations and modulate complex physiologic cellular pathways. In the field of cancer immunotherapy, multiplex genome editing has been used to simultaneously inactivate three genes (i.e., TRAC, B2M, and PDCD1) and generate universal chimeric antigen receptor (CAR) T cells resistant to the inhibitory activity of the PD-1 ligand. However, the intrinsic risk of genomic aberrations driven by such tools poses concerns because of the generation of multiple single-strand or double-strand DNA breaks followed by DNA repair. Modulating gene expression without DNA damage using epigenome editing promises a safer and efficient approach to alter gene expression. This method enables for simultaneous activation and/or repression of target genes, offering superior fine-tuning capabilities with reduced off-targeting effects and potential reversibility as compared to genome editing. Here we describe a detailed protocol for achieving multiplexed and sustainable gene silencing in CAR T cells. In an exemplary approach, we use designer epigenome modifiers (DEMs) for the simultaneous inactivation of two T cell inhibitory genes, PDCD1 and LAG3 to generate CAR T cells with increased resistance to tumor-induced exhaustion.
Subject(s)
Gene Editing , Gene Silencing , Receptors, Chimeric Antigen , Gene Editing/methods , Humans , Receptors, Chimeric Antigen/genetics , Receptors, Chimeric Antigen/immunology , Receptors, Chimeric Antigen/metabolism , T-Lymphocytes/immunology , T-Lymphocytes/metabolism , CRISPR-Cas Systems , Immunotherapy, Adoptive/methods , Programmed Cell Death 1 Receptor/genetics , EpigenomeABSTRACT
Expression of Protocadherin (Pcdh) genes is critical to the generation of neuron identity and wiring of the nervous system. Pcdhα genes are arranged in clusters and exhibit a range of expression profiles, from stochastic to deterministic. Because Pcdhα promoters have high sequence identity and share distal enhancers, how distinct neurons choose which gene to express remains unclear. We show that the interplay between multiple enhancers, epigenetics, and genome folding orchestrates differential readouts of the locus across neurons. The probability of Pcdhα promoter choice depends on enhancer/promoter encounters catalyzed by cohesin, whose extrusion trajectories determine the likelihood that an individual promoter can "escape" heterochromatin-mediated silencing. We propose that tunable locus-specific regulatory elements and cell type-specific cohesin activity underlie the generation of cellular diversity by Pcdh genes.
Subject(s)
Cadherins , Cohesins , Gene Silencing , Heterochromatin , Neurons , Animals , Humans , Mice , Cadherins/genetics , Cadherins/metabolism , Cohesins/metabolism , Enhancer Elements, Genetic , Heterochromatin/metabolism , Heterochromatin/genetics , Mice, Inbred C57BL , Multigene Family , Neurons/metabolism , Neurons/physiology , Promoter Regions, Genetic , Male , FemaleABSTRACT
BACKGROUND: PD-1/PD-L1 blockade has become a powerful method to treat malignant tumors. However, a large proportion of patients still do not benefit from this treatment, due to low tumor immunogenicity and low tumor penetration of the agents. Recently, neutrophil elastase has been shown to induce robust tumor immunogenicity, while the insufficient enzyme activity at the tumor site restricted its anti-tumor application. Here, we designed polyethyleneimine-modified neutrophil elastase (PEI-elastase) loaded with PD-L1small interfering RNA (PD-L1 siRNA) for improving enzymatic activity and delivering siRNA to tumor, which was expected to solve the above-mentioned problems. RESULTS: We first demonstrated that PEI-elastase possessed high enzymatic activity, which was also identified as an excellent gene-delivery material. Then, we synthesized anti-tumor lipopolymer (P-E/S Lip) by encapsulating PEI-elastase and PD-L1siRNA with pH-responsive anionic liposomes. The P-E/S Lip could be rapidly cleaved in tumor acidic environment, leading to exposure of the PEI-elastase/PD-L1 siRNA. Consequently, PEI-elastase induced powerful tumor immunogenicity upon direct tumor killing with minimal toxicity to normal cells. In parallel, PEI-elastase delivered PD-L1siRNA into the tumor and reduced PD-L1 expression. Orthotopic tumor administration of P-E/S Lip not only attenuated primary tumor growth, but also produced systemic anti-tumor immune response to inhibit growth of distant tumors and metastasis. Moreover, intravenous administration of P-E/S Lip into mice bearing subcutaneous tumors leaded to an effective inhibition of established B16-F10 tumor and 4T1 tumor, with histological analyses indicating an absence of detectable toxicity. CONCLUSIONS: In our study, a protease-based nanoplatform was used to cooperatively provoke robust tumor immunogenicity and down-regulate PD-L1 expression, which exhibited great potential as a combination therapy for precisely treating solid tumors.
Subject(s)
B7-H1 Antigen , Immunotherapy , Polyethyleneimine , RNA, Small Interfering , Animals , Polyethyleneimine/chemistry , RNA, Small Interfering/chemistry , B7-H1 Antigen/metabolism , Mice , Immunotherapy/methods , Cell Line, Tumor , Female , Humans , Mice, Inbred BALB C , Liposomes/chemistry , Nanoparticles/chemistry , Neoplasms/drug therapy , Neoplasms/therapy , Neoplasms/immunology , Mice, Inbred C57BL , Gene SilencingABSTRACT
Despite the widely accepted involvement of DNA methylation in the regulation of rDNA transcription, the relative participation of different cytosine methylation pathways is currently described only for a few model plants. Using PacBio, Bisulfite, and RNA sequencing; PCR; Southern hybridizations; and FISH, the epigenetic consequences of rDNA copy number variation were estimated in two T. porrifolius lineages, por1 and por2, the latter with more than twice the rDNA copy numbers distributed approximately equally between NORs on chromosomes A and D. The lower rDNA content in por1 correlated with significantly reduced (>90%) sizes of both D-NORs. Moreover, two (L and S) prominent rDNA variants, differing in the repetitive organization of intergenic spacers, were detected in por2, while only the S-rDNA variant was detected in por1. Transcriptional activity of S-rDNA in por1 was associated with secondary constriction of both A-NORs. In contrast, silencing of S-rDNA in por2 was accompanied by condensation of A-NORs, secondary constriction on D-NORs, and L-rDNA transcriptional activity, suggesting (i) bidirectional nucleolar dominance and (ii) association of S-rDNAs with A-NORs and L-rDNAs with D-NORs in T. porrifolius. Each S- and L-rDNA array was formed of several sub-variants differentiating both genetically (specific SNPs) and epigenetically (transcriptional efficiency and cytosine methylation). The most significant correlations between rDNA silencing and methylation were detected for symmetric CWG motifs followed by CG motifs. No correlations were detected for external cytosine in CCGs or asymmetric CHHs, where methylation was rather position-dependent, particularly for AT-rich variants. We conclude that variations in rDNA copy numbers in plant diploids can be accompanied by prompt epigenetic responses to maintain an appropriate number of active rDNAs. The methylation dynamics of CWGs are likely to be the most responsible for regulating silent and active rDNA states.
Subject(s)
Cytosine , DNA Methylation , DNA, Ribosomal , Gene Silencing , Cytosine/metabolism , DNA, Ribosomal/genetics , DNA Copy Number Variations , Transcription, Genetic , Epigenesis, Genetic , Gene Expression Regulation, Plant , Chromosomes, Plant/geneticsABSTRACT
BACKGROUND: Neuropathic pain (NP) is a challenging health condition owing to its complex nature and associated multiple etiologies. The occurrence of NP involves the abnormal activity of neurons mediated by oxidative stress (OS). Previous research has demonstrated that m6A methylation plays a role in the regulatory pathway of NP. This study aimed to investigate the specific molecular pathways through which m6A methylation modifiers alleviate NP. METHODS: For this purpose, an NO rat model was developed via spared nerve injury (SNI), followed by quantifying the animal's pain assessment via paw withdrawal threshold (PWT) and paw withdrawal latency (PWL). The OS in SNI rats was evaluated by measuring reactive oxygen species, superoxide dismutase, and catalase (CAT) in spinal cord tissues. Moreover, quantitative-real-time polymerase chain reaction and western blot analysis were employed for detecting fat mass and obesity-associated (FTO) and GPR177 levels, while m6A levels of GPR117 were analyzed via MeRIP. RESULTS: The results indicated an enhanced OS with highly expressed FTO in spinal cord tissue samples, where knocking down Fto effectively relieved NP and OS in SNI rats. Mechanistic investigations revealed that Fto-mediated reduction of Grp177 m6A modification was involved in the WNT5a/TRPV1 axis-mediated OS remission of NP. Moreover, in vitro experiment results indicated that YTHDF2 was an important m6A methylated reading protein for this process. CONCLUSIONS: Fto silencing leads to increased m6A methylation of Grp177 through a YTHDF2-dependent mechanism, resulting in decreased Grp177 stability and ultimately reducing NP in rats by OS suppression.
Subject(s)
Alpha-Ketoglutarate-Dependent Dioxygenase FTO , Neuralgia , Oxidative Stress , Receptors, G-Protein-Coupled , Animals , Neuralgia/metabolism , Neuralgia/genetics , Neuralgia/etiology , Rats , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/metabolism , Alpha-Ketoglutarate-Dependent Dioxygenase FTO/genetics , Receptors, G-Protein-Coupled/metabolism , Receptors, G-Protein-Coupled/genetics , Male , Disease Models, Animal , Rats, Sprague-Dawley , Gene Silencing , Methylation , Adenosine/metabolism , Adenosine/analogs & derivatives , Spinal Cord/metabolism , Spinal Cord/pathologyABSTRACT
In eukaryotes, repetitive DNA can become silenced de novo, either transcriptionally or post-transcriptionally, by processes independent of strong sequence-specific cues. The mechanistic nature of such processes remains poorly understood. We found that in the fungus Neurospora crassa, de novo initiation of both transcriptional and post-transcriptional silencing was linked to perturbed chromatin, which was produced experimentally by the aberrant activity of transcription factors at the tetO operator array. Transcriptional silencing was mediated by canonical constitutive heterochromatin. On the other hand, post-transcriptional silencing resembled repeat-induced quelling but occurred normally when homologous recombination was inactivated. All silencing of the tetO array was dependent on SAD-6, fungal ortholog of the SWI/SNF chromatin remodeler ATRX (Alpha Thalassemia/Mental Retardation Syndrome X-Linked), which was required to maintain nucleosome occupancy at the perturbed locus. In addition, we found that two other types of sequences (the lacO array and native AT-rich DNA) could also undergo recombination-independent quelling associated with perturbed chromatin. These results suggested a model in which the de novo initiation of transcriptional and post-transcriptional silencing is coupled to the remodeling of perturbed chromatin.