Curr Protoc. 2023 Aug;3(8):e855. doi: 10.1002/cpz1.855.

ABSTRACT

Here we describe a Drosophila genome engineering technique that can scarlessly modify genomic sequences near any mapped attP attachment site previously integrated by transposon mobilization or gene targeting. This technique combines two highly efficient and robust procedures: phiC31 integrase-mediated site-specific integration and homing endonuclease-mediated resolution of local duplications. In this technique, a donor fragment containing the desired mutation(s) is first integrated into a selected attP site near the target locus by phiC31 integrase-mediated site-specific integration, which creates local duplications consisting of the mutant-containing donor fragment and the wild-type target locus. Next, homing endonuclease-induced double-stranded DNA breaks trigger recombination between the duplications and resolve the target locus to generate scarless mutant alleles. In every step, the desired flies can be easily identified by patterns of dominant markers, so no large-scale screens are needed. This technique is highly efficient and can be used to generate scarless point mutations, insertions, and deletions. The availability of large libraries of mapped attP site-containing transposon/CRISPR insertions in Drosophila allows the modification of more than half of the euchromatic Drosophila genome at a high efficiency. As more and more attP-containing insertions are generated and mapped, this technique will be able to modify larger portions of the Drosophila genome. The principles of this technique are applicable to other organisms where modifications to the genome are feasible. © 2023 Wiley Periodicals LLC. Basic Protocol 1: Verifying attP-containing insertions Support Protocol: Extracting genomic DNA Basic Protocol 2: Generating the donor plasmid Basic Protocol 3: Injecting the donor plasmid and establishing transformant stocks Basic Protocol 4: Verifying the transformants Basic Protocol 5: Generating the final scarless alleles Basic Protocol 6: Verifying the final alleles.

PMID:37540775 | DOI:10.1002/cpz1.855

Sci Adv. 2023 Aug 4;9(31):eadf3984. doi: 10.1126/sciadv.adf3984. Epub 2023 Aug 4.

ABSTRACT

The glioblastoma (GBM) stem cell-like cells (GSCs) are critical for tumorigenesis/therapeutic resistance of GBM. Mounting evidence supports tumor-promoting function of long noncoding RNAs (lncRNAs), but their role in GSCs remains poorly understood. By combining CRISPRi screen with orthogonal multiomics approaches, we identified a lncRNA DARS1-AS1-controlled posttranscriptional circuitry that promoted the malignant properties of GBM cells/GSCs. Depleting DARS1-AS1 inhibited the proliferation of GBM cells/GSCs and self-renewal of GSCs, prolonging survival in orthotopic GBM models. DARS1-AS1 depletion also impaired the homologous recombination (HR)-mediated double-strand break (DSB) repair and enhanced the radiosensitivity of GBM cells/GSCs. Mechanistically, DARS1-AS1 interacted with YBX1 to promote target mRNA binding and stabilization, forming a mixed transcriptional/posttranscriptional feed-forward loop to up-regulate expression of the key regulators of G1-S transition, including E2F1 and CCND1. DARS1-AS1/YBX1 also stabilized the mRNA of FOXM1, a master transcription factor regulating GSC self-renewal and DSB repair. Our findings suggest DARS1-AS1/YBX1 axis as a potential therapeutic target for sensitizing GBM to radiation/HR deficiency-targeted therapy.

PMID:37540752 | DOI:10.1126/sciadv.adf3984

PLoS Biol. 2023 Aug 4;21(8):e3002212. doi: 10.1371/journal.pbio.3002212. Online ahead of print.

ABSTRACT

The mature mammalian cortex is composed of 6 architecturally and functionally distinct layers. Two key steps in the assembly of this layered structure are the initial establishment of the glial scaffold and the subsequent migration of postmitotic neurons to their final position. These processes involve the precise and timely regulation of adhesion and detachment of neural cells from their substrates. Although much is known about the roles of adhesive substrates during neuronal migration and the formation of the glial scaffold, less is understood about how these signals are interpreted and integrated within these neural cells. Here, we provide in vivo evidence that Cas proteins, a family of cytoplasmic adaptors, serve a functional and redundant role during cortical lamination. Cas triple conditional knock-out (Cas TcKO) mice display severe cortical phenotypes that feature cobblestone malformations. Molecular epistasis and genetic experiments suggest that Cas proteins act downstream of transmembrane Dystroglycan and β1-Integrin in a radial glial cell-autonomous manner. Overall, these data establish a new and essential role for Cas adaptor proteins during the formation of cortical circuits and reveal a signaling axis controlling cortical scaffold formation.

PMID:37540708 | DOI:10.1371/journal.pbio.3002212

Cell Rep. 2023 Aug 3;42(8):112924. doi: 10.1016/j.celrep.2023.112924. Online ahead of print.

ABSTRACT

Lymphoid tissue inducer (LTi) cells, a subset of innate lymphoid cells (ILCs), play an essential role in the formation of secondary lymphoid tissues. However, the regulation of the development and functions of this ILC subset is still elusive. In this study, we report that the transcription factor T cell factor 1 (TCF-1), just as GATA3, is indispensable for the development of non-LTi ILC subsets. While LTi cells are still present in TCF-1-deficient mice, the organogenesis of Peyer's patches (PPs), but not of lymph nodes, is impaired in these mice. LTi cells from different tissues have distinct gene expression patterns, and TCF-1 regulates the expression of lymphotoxin specifically in PP LTi cells. Mechanistically, TCF-1 may directly and/or indirectly regulate Lta, including through promoting the expression of GATA3. Thus, the TCF-1-GATA3 axis, which plays an important role during T cell development, also critically regulates the development of non-LTi cells and tissue-specific functions of LTi cells.

PMID:37540600 | DOI:10.1016/j.celrep.2023.112924

Methods Mol Biol. 2023;2693:39-60. doi: 10.1007/978-1-0716-3342-7_4.

ABSTRACT

RNA sequencing (RNA-seq) is a powerful method of transcriptional analysis that allows for the sequence identification and quantification of cellular transcripts. RNA-seq can be used for differential gene expression (DGE) analysis, gene fusion detection, allele-specific expression, isoform and splice variant quantification, and identification of novel genes. These applications can be used for downstream systems biology analyses such as gene ontology or pathway analysis to provide insight into processes altered between biological conditions. Given the wide range of signaling pathways subject to chaperone activity as well as numerous chaperone functions in RNA metabolism, RNA-seq may provide a valuable tool for the study of chaperone proteins in biology and disease. This chapter outlines an example RNA-seq workflow to determine differentially expressed (DE) genes between two or more sample conditions and provides some considerations for RNA-seq experimental design.

PMID:37540425 | DOI:10.1007/978-1-0716-3342-7_4

Methods Mol Biol. 2023;2686:509-536. doi: 10.1007/978-1-0716-3299-4_24.

ABSTRACT

Understanding the global and dynamic nature of plant developmental processes requires not only the study of the transcriptome, but also of the proteome, including its largely uncharacterized peptidome fraction. Recent advances in proteomics and high-throughput analyses of translating RNAs (ribosome profiling) have begun to address this issue, evidencing the existence of novel, uncharacterized, and possibly functional peptides. To validate the accumulation in tissues of sORF-encoded polypeptides (SEPs), the basic setup of proteomic analyses (i.e., LC-MS/MS) can be followed. However, the detection of peptides that are small (up to ~100 aa, 6-7 kDa) and novel (i.e., not annotated in reference databases) presents specific challenges that need to be addressed both experimentally and with computational biology resources. Several methods have been developed in recent years to isolate and identify peptides from plant tissues. In this chapter, we outline two different peptide extraction protocols and the subsequent peptide identification by mass spectrometry using the database search or the de novo identification methods.

PMID:37540375 | DOI:10.1007/978-1-0716-3299-4_24

Methods Mol Biol. 2023;2686:495-508. doi: 10.1007/978-1-0716-3299-4_23.

ABSTRACT

Developmental processes in multicellular organisms depend on the proficiency of cells to orchestrate different gene expression programs. Over the past years, several studies of reproductive organ development have considered genomic analyses of transcription factors and global gene expression changes, modeling complex gene regulatory networks. Nevertheless, the dynamic view of developmental processes requires, as well, the study of the proteome in its expression, complexity, and relationship with the transcriptome. In this chapter, we describe a dual extraction method-for protein and RNA-for the characterization of genome expression at proteome level and its correlation to transcript expression data. We also present a shotgun proteomic procedure (LC-MS/MS) followed by a pipeline for the imputation of missing values in mass spectrometry results.

PMID:37540374 | DOI:10.1007/978-1-0716-3299-4_23

Hum Mol Genet. 2023 Aug 4:ddad127. doi: 10.1093/hmg/ddad127. Online ahead of print.

NO ABSTRACT

PMID:37540221 | DOI:10.1093/hmg/ddad127

OMICS. 2023 Aug 4. doi: 10.1089/omi.2023.0072. Online ahead of print.

ABSTRACT

Idiopathic pulmonary fibrosis (IPF) is a chronic progressive fibrotic disease of the lung with poor prognosis. Fibrosis results from remodeling of the interstitial tissue. A wide range of gene expression changes are observed, but the role of micro RNAs (miRNAs) and circular RNAs (circRNA) is still unclear. Therefore, this study aimed to establish an messenger RNA (mRNA)-miRNA-circRNA competing endogenous RNA (ceRNA) regulatory network to uncover novel molecular signatures using systems biology tools. Six datasets were used to determine differentially expressed genes (DEGs) and miRNAs (DEmiRNA). Accordingly, protein-protein, mRNA-miRNA, and miRNA-circRNA interactions were constructed. Modules were determined and further analyzed in the Drug Gene Budger platform to identify potential therapeutic compounds. We uncovered common 724 DEGs and 278 DEmiRNAs. In the protein-protein interaction network, TMPRSS4, ESR2, TP73, CLEC4E, and TP63 were identified as hub protein coding genes. The mRNA-miRNA interaction network revealed two modules composed of ADRA1A, ADRA1B, hsa-miR-484 and CDH2, TMPRSS4, and hsa-miR-543. The DEmiRNAs in the modules further analyzed to propose potential circRNA regulators in the ceRNA network. These results help deepen the understanding of the mechanisms of IPF. In addition, the molecular leads reported herein might inform future innovations in diagnostics and therapeutics research and development for IPF.

PMID:37540140 | DOI:10.1089/omi.2023.0072

Plant Physiol. 2023 Aug 4:kiad439. doi: 10.1093/plphys/kiad439. Online ahead of print.

ABSTRACT

Autophagy serves as an important recycling route for the growth and survival of eukaryotic organisms in nutrient-deficient conditions. Since starvation induces massive changes in metabolic flux that are coordinated by key metabolic enzymes, specific processing steps of autophagy may be linked with metabolic flux-monitoring enzymes. We attempted to identify carbon metabolic genes that modulate autophagy using VIGS screening of 45 glycolysis- and Calvin-Benson cycle-related genes in Arabidopsis (Arabidopsis thaliana). Here, we report that three consecutive triose-phosphate-processing enzymes involved in cytosolic glycolysis, TPI (triose-phosphate-isomerase), GAPC (glyceraldehyde-3-phosphate dehydrogenase), and PGK (phosphoglycerate kinase), designated TGP, negatively regulate autophagy. Depletion of TGP enzymes causes spontaneous autophagy induction and increases AUTOPHAGY-RELATED 1 (ATG1) kinase activity. TGP enzymes interact with ATG101, a regulatory component of the ATG1 kinase complex. Spontaneous autophagy induction and abnormal growth under insufficient sugar in the TGP mutants are suppressed by crossing with the atg101 mutant. Considering that triose-phosphates are photosynthates transported to the cytosol from active chloroplasts, the TGP enzymes would be strategically positioned to monitor the flow of photosynthetic sugars and modulate autophagy accordingly. Collectively, these results suggest that TGP enzymes negatively control autophagy acting upstream of the ATG1 complex, which is critical for seedling development.

PMID:37539947 | DOI:10.1093/plphys/kiad439

iScience. 2023 Jul 13;26(8):107370. doi: 10.1016/j.isci.2023.107370. eCollection 2023 Aug 18.

ABSTRACT

Mitochondria play important roles in angiogenesis. However, the mechanisms remain elusive. In this study, we found that mitochondrial ubiquinol-cytochrome c reductase complex assembly factor 3 (UQCC3) is a key regulator of angiogenesis. TALEN-mediated knockout of Uqcc3 in mice caused embryonic lethality at 9.5-10.5 days postcoitum, and vessel density was dramatically reduced. Similarly, knockout of uqcc3 in zebrafish induced lethality post-fertilization and impaired vascular development. Knockout of UQCC3 resulted in slower tumor growth and angiogenesis. Mechanistically, UQCC3 was upregulated under hypoxia, promoted reactive oxygen species (ROS) generation, enhanced HIF-1α stability and increased VEGF expression. Finally, higher expression of UQCC3 was associated with poor prognosis in multiple types tumors, implying a role for UQCC3 in tumor progression. In conclusion, our findings highlight the important contribution of UQCC3 to angiogenesis under both physiological and pathological conditions, indicating the potential of UQCC3 as a therapeutic target for cancer.

PMID:37539028 | PMC:PMC10393800 | DOI:10.1016/j.isci.2023.107370

iScience. 2023 Jul 13;26(8):107369. doi: 10.1016/j.isci.2023.107369. eCollection 2023 Aug 18.

ABSTRACT

Extranodal natural killer/T cell lymphoma, nasal type (ENKTL) is an aggressive lymphoid malignancy with a poor prognosis and lacks standard treatment. Targeted therapies are urgently needed. Here we systematically investigated the druggable mechanisms through chemogenomic screening and identified that Bcl-xL-specific BH3 mimetics effectively induced ENKTL cell apoptosis. Notably, the specific accumulation of Bcl-xL, but not other Bcl-2 family members, was verified in ENKTL cell lines and patient tissues. Furthermore, Bcl-xL high expression was shown to be closely associated with worse patient survival. The critical role of Bcl-xL in ENKTL cell survival was demonstrated utilizing selective inhibitors, genetic silencing, and a specific degrader. Additionally, the IL2-JAK1/3-STAT5 signaling was implicated in Bcl-xL dysregulation. In vivo, Bcl-xL inhibition reduced tumor burden, increased apoptosis, and prolonged survival in ENKTL cell line xenograft and patient-derived xenograft models. Our study indicates Bcl-xL as a promising therapeutic target for ENKTL, warranting monitoring in ongoing clinical trials by targeting Bcl-xL.

PMID:37539026 | PMC:PMC10393801 | DOI:10.1016/j.isci.2023.107369

R Soc Open Sci. 2023 Aug 2;10(8):221469. doi: 10.1098/rsos.221469. eCollection 2023 Aug.

ABSTRACT

Transcription is a complex phenomenon that permits the conversion of genetic information into phenotype by means of an enzyme called RNA polymerase, which erratically moves along and scans the DNA template. We perform Bayesian inference over a paradigmatic mechanistic model of non-equilibrium statistical physics, i.e. the asymmetric exclusion processes in the hydrodynamic limit, assuming a Gaussian process prior for the polymerase progression rate as a latent variable. Our framework allows us to infer the speed of polymerases during transcription given their spatial distribution, while avoiding the explicit inversion of the system's dynamics. The results, which show processing rates strongly varying with genomic position and minor role of traffic-like congestion, may have strong implications for the understanding of gene expression.

PMID:37538742 | PMC:PMC10394410 | DOI:10.1098/rsos.221469

Front Bioinform. 2023 Jul 19;3:1254668. doi: 10.3389/fbinf.2023.1254668. eCollection 2023.

NO ABSTRACT

PMID:37538347 | PMC:PMC10395825 | DOI:10.3389/fbinf.2023.1254668

Cell Rep. 2023 Aug 1;42(8):112879. doi: 10.1016/j.celrep.2023.112879. Online ahead of print.

ABSTRACT

Neuroblastoma is a lethal childhood solid tumor of developing peripheral nerves. Two percent of children with neuroblastoma develop opsoclonus myoclonus ataxia syndrome (OMAS), a paraneoplastic disease characterized by cerebellar and brainstem-directed autoimmunity but typically with outstanding cancer-related outcomes. We compared tumor transcriptomes and tumor-infiltrating T and B cell repertoires from 38 OMAS subjects with neuroblastoma to 26 non-OMAS-associated neuroblastomas. We found greater B and T cell infiltration in OMAS-associated tumors compared to controls and showed that both were polyclonal expansions. Tertiary lymphoid structures (TLSs) were enriched in OMAS-associated tumors. We identified significant enrichment of the major histocompatibility complex (MHC) class II allele HLA-DOB∗01:01 in OMAS patients. OMAS severity scores were associated with the expression of several candidate autoimmune genes. We propose a model in which polyclonal auto-reactive B lymphocytes act as antigen-presenting cells and drive TLS formation, thereby supporting both sustained polyclonal T cell-mediated anti-tumor immunity and paraneoplastic OMAS neuropathology.

PMID:37537844 | DOI:10.1016/j.celrep.2023.112879

J Proteomics. 2023 Aug 1:104983. doi: 10.1016/j.jprot.2023.104983. Online ahead of print.

ABSTRACT

BRCA2 and RAD51 are two proteins that play a central role in homologous recombination (HR) and DNA double strand break (DSB) repair. BRCA2 assists RAD51 fibrillation and defibrillation through binding with its eight BRC repeats, with BRC4 being one of the most efficient and best characterized. RAD51 inactivation by small molecules has been proposed as a strategy to impair BRCA2/RAD51 binding and, ultimately, the HR pathway, with the aim of making cancer cells more sensitive to PARP inhibitors (PARPi). This strategy, which mimics a synthetic lethality (SL) approach, has been successfully performed in vitro by using the myristoylated derivative of BRC4 (myr-BRC4), designed for a more efficient cell entry. The present study applies a method to obtain a proteomic fingerprint after cellular treatment with the myr-BRC4 peptide using a mass spectroscopy (MS) proteomic approach. (Data are available via ProteomeXchange with identifier PXD042696.) We performed a comparative proteomic profiling of the myr-BRC4 treated vs. untreated BxPC-3 pancreatic cancer cells and evaluated the differential expression of proteins. Among the identified proteins, we focused our attention on proteins shared by both the RAD51 and the BRCA2 interactomes, and on those whose reduction showed high statistical significance. Three downregulated proteins were identified (FANCI, FANCD2, and RPA3), and protein downregulation was confirmed through immunoblotting analysis, validating the MS approach. Our results suggest that, being a direct consequence of myr-BRC4 treatment, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring HR impairment. SIGNIFICANCE: RAD51's inhibition has gained increasing attention because of its possible implications in personalized medicine through the SL approach. Chemical disruption of protein-protein interactions (PPIs) between RAD51 and BRCA2, or some of its partner proteins, could potentiate PARPi DNA damage-induced cell death. This could have application for difficult to treat cancers, such as BRCA-competent and olaparib (PARPi) resistant pancreatic adenocarcinoma. Despite RAD51 being a widely studied target, researchers still lack detailed mechanistic information. This has stifled progress in the field with only a few RAD51 inhibitors having been identified, none of which have gained regulatory approval. Nevertheless, the peptide BRC4 is one of the most specific and best characterized RAD51 binder and inhibitor reported to date. Our study is the first to report the proteomic fingerprint consequent to cellular treatment of myr-BRC4, to offer a reference for the discovery of specific protein/pathway alterations within DNA damage repair. Our results suggest that, being a direct consequence of myr-BRC4 treatment, and ultimately ofBRCA2/RAD51 disruption, the detection of FANCD2, FANCI, and RPA3 downregulation could be used as an indicator for monitoring DNA damage repair impairment and therefore be used to potentiate the development of new effective therapeutic strategies.

PMID:37536521 | DOI:10.1016/j.jprot.2023.104983

Nat Biotechnol. 2023 Aug 3. doi: 10.1038/s41587-023-01881-x. Online ahead of print.

ABSTRACT

Single-cell assay for transposase-accessible chromatin by sequencing (scATAC-seq) has emerged as a powerful tool for dissecting regulatory landscapes and cellular heterogeneity. However, an exploration of systemic biases among scATAC-seq technologies has remained absent. In this study, we benchmark the performance of eight scATAC-seq methods across 47 experiments using human peripheral blood mononuclear cells (PBMCs) as a reference sample and develop PUMATAC, a universal preprocessing pipeline, to handle the various sequencing data formats. Our analyses reveal significant differences in sequencing library complexity and tagmentation specificity, which impact cell-type annotation, genotype demultiplexing, peak calling, differential region accessibility and transcription factor motif enrichment. Our findings underscore the importance of sample extraction, method selection, data processing and total cost of experiments, offering valuable guidance for future research. Finally, our data and analysis pipeline encompasses 169,000 PBMC scATAC-seq profiles and a best practices code repository for scATAC-seq data analysis, which are freely available to extend this benchmarking effort to future protocols.

PMID:37537502 | DOI:10.1038/s41587-023-01881-x

Nat Commun. 2023 Aug 3;14(1):4654. doi: 10.1038/s41467-023-40365-z.

ABSTRACT

Molecular biology aims to understand cellular responses and regulatory dynamics in complex biological systems. However, these studies remain challenging in non-model species due to poor functional annotation of regulatory proteins. To overcome this limitation, we develop a multi-layer neural network that determines protein functionality directly from the protein sequence. We annotate kinases and phosphatases in Glycine max. We use the functional annotations from our neural network, Bayesian inference principles, and high resolution phosphoproteomics to infer phosphorylation signaling cascades in soybean exposed to cold, and identify Glyma.10G173000 (TOI5) and Glyma.19G007300 (TOT3) as key temperature regulators. Importantly, the signaling cascade inference does not rely upon known kinase motifs or interaction data, enabling de novo identification of kinase-substrate interactions. Conclusively, our neural network shows generalization and scalability, as such we extend our predictions to Oryza sativa, Zea mays, Sorghum bicolor, and Triticum aestivum. Taken together, we develop a signaling inference approach for non-model species leveraging our predicted kinases and phosphatases.

PMID:37537196 | DOI:10.1038/s41467-023-40365-z

Nat Commun. 2023 Aug 3;14(1):4669. doi: 10.1038/s41467-023-40380-0.

ABSTRACT

Constraint-based metabolic models have been used for decades to predict the phenotype of microorganisms in different environments. However, quantitative predictions are limited unless labor-intensive measurements of media uptake fluxes are performed. We show how hybrid neural-mechanistic models can serve as an architecture for machine learning providing a way to improve phenotype predictions. We illustrate our hybrid models with growth rate predictions of Escherichia coli and Pseudomonas putida grown in different media and with phenotype predictions of gene knocked-out Escherichia coli mutants. Our neural-mechanistic models systematically outperform constraint-based models and require training set sizes orders of magnitude smaller than classical machine learning methods. Our hybrid approach opens a doorway to enhancing constraint-based modeling: instead of constraining mechanistic models with additional experimental measurements, our hybrid models grasp the power of machine learning while fulfilling mechanistic constrains, thus saving time and resources in typical systems biology or biological engineering projects.

PMID:37537192 | DOI:10.1038/s41467-023-40380-0

Nat Commun. 2023 Aug 3;14(1):4667. doi: 10.1038/s41467-023-40248-3.

ABSTRACT

Warming shifts the thermal optimum of net photosynthesis (ToptA) to higher temperatures. However, our knowledge of this shift is mainly derived from seedlings grown in greenhouses under ambient atmospheric carbon dioxide (CO2) conditions. It is unclear whether shifts in ToptA of field-grown trees will keep pace with the temperatures predicted for the 21st century under elevated atmospheric CO2 concentrations. Here, using a whole-ecosystem warming controlled experiment under either ambient or elevated CO2 levels, we show that ToptA of mature boreal conifers increased with warming. However, shifts in ToptA did not keep pace with warming as ToptA only increased by 0.26-0.35 °C per 1 °C of warming. Net photosynthetic rates estimated at the mean growth temperature increased with warming in elevated CO2 spruce, while remaining constant in ambient CO2 spruce and in both ambient CO2 and elevated CO2 tamarack with warming. Although shifts in ToptA of these two species are insufficient to keep pace with warming, these boreal conifers can thermally acclimate photosynthesis to maintain carbon uptake in future air temperatures.

PMID:37537190 | DOI:10.1038/s41467-023-40248-3