Single mammalian cells compensate for differences in cellular volume and DNA copy number through independent global transcriptional mechanisms
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ABSTRACT:
ORGANISM(S): Homo sapiens
PROVIDER: GSE66053 | GEO | 2015/03/01
SECONDARY ACCESSION(S): PRJNA275801
REPOSITORIES: GEO
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Project description:The number of tRNA isodecoder genes has increased dramatically in mammals, but the specific molecular and physiological reasons for this expansion remain elusive. To address this fundamental question we used CRISPR editing to knockout the seven-membered phenylalanine tRNA gene family in mice, both individually and combinatorially. Using ATAC-seq, RNA-seq and proteomics we observed distinct molecular consequences of individual tRNA deletions. We show that tRNA-Phe-1-1 is required for neuronal function and its loss is partially compensated by increased expression of other tRNAs but results in mistranslation. In contrast, the other tRNA-Phe isodecoders compensate for the loss of each of the remaining six tRNA-Phe genes. In the tRNA-Phe gene family, the expression of at least four tRNA-Phe isodecoders is required for embryonic development and survival. The loss of tRNA-Phe-1-1 and any other three tRNA-Phe genes causes embryonic lethality indicating that tRNA-Phe-1-1 is most important for development and survival. Our results reveal that the multi-copy configuration of tRNA isodecoder genes is required to buffer translation and viability in mammals.
2023-04-19 | PXD029684 | Pride
Project description:There is only a limited understanding of the relation between copy number and expression for mammalian genes. We fine mapped cis and trans regulatory loci due to copy number change for essentially all genes using a human-hamster radiation hybrid (RH) panel. These loci are called copy number expression quantitative trait loci (ceQTLs).Unexpected findings from a previous study of a mouse-hamster RH panel were replicated. These findings included decreased expression as a result of increased copy number for 30% of genes and an attenuated relationship between expression and copy number on the X chromosome suggesting an Xist independent form of dosage compensation. In a separate glioblastoma dataset, we found conservation of genes in which dosage was negatively correlated with gene expression. These genes were enriched in signaling and receptor activities. The observation of attenuated X-linked gene expression in response to increased gene number was also replicated in the glioblastoma dataset. Of 523 gene deserts of size > 600 kb in the human RH panel, 325 contained trans ceQTLs with -log10 P > 4.1. Recently discovered genes, ultra conserved regions, noncoding RNAs and microRNAs explained only a small fraction of the results, suggesting a substantial portion of gene deserts harbor as yet unidentified functional elements.Radiation hybrids are a useful tool for high resolution mapping of cis and trans loci capable of affecting gene expression due to copy number change. Analysis of two independent radiation hybrid panels show agreement in their findings and may serve as a discovery source for novel regulatory loci in noncoding regions of the genome.
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Project description:MotivationSomatic DNA copy number alterations (CNAs) arise in tumor tissue because of underlying genomic instability. Recurrent CNAs that occur in the same genomic region across multiple independent samples are of interest to researchers because they may contain genes that contribute to the cancer phenotype. However, differences in copy number states between cancers are also commonly of interest, for example when comparing tumors with distinct morphologies in the same anatomic location. Current methodologies are limited by their inability to perform direct comparisons of CNAs between tumor cohorts, and thus they cannot formally assess the statistical significance of observed copy number differences or identify regions of the genome where these differences occur.ResultsWe introduce the DiNAMIC.Duo R package that can be used to identify recurrent CNAs in a single cohort or recurrent copy number differences between two cohorts, including when neither cohort is copy neutral. The package utilizes Python scripts for computational efficiency and provides functionality for producing figures and summary output files.Availability and implementationThe DiNAMIC.Duo R package is available from CRAN at https://cran.r-project.org/web/packages/DiNAMIC.Duo/index.html. This article uses publicly available data from the Broad Institute TCGA Genome Data Analysis Center, https://doi.org/10.7908/C11G0KM9.Supplementary informationSupplementary data are available at Bioinformatics online.
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Project description:Electrotransfection is a non-viral method for delivery of nucleic acids into cells. In our previous study, we have determined the minimal copy number of plasmid DNA (pDNA) per cell required for transgene expression post electrotransfection, and developed a statistical framework to predict the pDNA copy number in the nucleus. To experimentally verify the prediction, the current study was designed to quantify the average copy number of pDNA per nucleus post electrotransfection. To achieve it, we developed a novel approach to effectively obtain isolated nuclei with minimal contamination by extranuclear pDNA. This sample preparation method enabled us to accurately measure intranuclear pDNA using quantitative real-time PCR. The data showed that the copy number of pDNA per nucleus was dependent on the period of cell culture post pulsing and the pDNA dose for electrotransfection. Additionally, the data were used to improve the statistical framework for understanding kinetics of pDNA transport in cells, and predicting how the kinetics depended on different factors. It is expected that the framework and the methodology developed in the current study will be useful for evaluating factors that may affect kinetics and mechanisms of pDNA transport in cells.
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Project description:The number of tRNA isodecoders has increased dramatically in mammals, but the specific molecular and physiological reasons for this expansion remain elusive. To address this fundamental question we used CRISPR editing to knockout the seven-membered phenylalanine tRNA gene family in mice, both individually and combinatorially. Using ATAC-Seq, RNA-seq, ribo-profiling and proteomics we observed distinct molecular consequences of single tRNA deletions. We show that tRNA-Phe-1-1 is required for neuronal function and its loss is partially compensated by increased expression of other tRNAs but results in mistranslation. In contrast, the other tRNA-Phe isodecoder genes buffer the loss of each of the remaining six tRNA-Phe genes. In the tRNA-Phe gene family, the expression of at least six tRNA-Phe alleles is required for embryonic viability and tRNA-Phe-1-1 is most important for development and survival. Our results reveal that the multi-copy configuration of tRNA genes is required to buffer translation and viability in mammals.
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Project description:The amylase gene (AMY), which codes for a starch-digesting enzyme in animals, underwent several gene copy number gains in humans (Perry et al., 2007), dogs (Axelsson et al., 2013), and mice (Schibler et al., 1982), possibly along with increased starch consumption during the evolution of these species. Here, we present comprehensive evidence for AMY copy number expansions that independently occurred in several mammalian species which consume diets rich in starch. We also provide correlative evidence that AMY gene duplications may be an essential first step for amylase to be expressed in saliva. Our findings underscore the overall importance of gene copy number amplification as a flexible and fast evolutionary mechanism that can independently occur in different branches of the phylogeny.
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Project description:ContextTSPY1 is a tandemly-repeated gene on the human Y chromosome forming an array of approximately 21-35 copies. The testicular expression pattern and the inferred function of the TSPY1 protein suggest possible involvement in spermatogenesis. However, data are scarce on TSPY1 copy number variation in different Y lineages and its role in spermatogenesis.ObjectivesWe sought to define: 1) the extent of TSPY1 copy number variation within and among Y chromosome haplogroups; and 2) the role of TSPY1 dosage in spermatogenic efficiency.Materials and methodsA total of 154 idiopathic infertile men and 130 normozoospermic controls from Central Italy were analyzed. We used a quantitative PCR assay to measure TSPY1 copy number and also defined Y haplogroups in all subjects.ResultsWe provide evidence that TSPY1 copy number shows substantial variation among Y haplogroups and thus that population stratification does represent a potential bias in case-control association studies. We also found: 1) a significant positive correlation between TSPY1 copy number and sperm count (P < 0.001); 2) a significant difference in mean TSPY1 copy number between patients and controls (28.4 +/- 8.3 vs. 33.9 +/- 10.7; P < 0.001); and 3) a 1.5-fold increased risk of abnormal sperm parameters in men with less than 33 copies (P < 0.001).ConclusionsTSPY copy number variation significantly influences spermatogenic efficiency. Low TSPY1 copy number is a new risk factor for male infertility with potential clinical consequences.
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