Browsing by Subject "RNAseq"
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Item Open Access Genomic characterization of the evolutionary potential of the sea urchin Strongylocentrotus droebachiensis facing ocean acidification.(Genome Biol Evol, 2017-01-12) Runcie, Daniel E; Dorey, Narimane; Garfield, David A; Stumpp, Meike; Dupont, Sam; Wray, Gregory AOcean acidification (OA) is increasing due to anthropogenic CO2 emissions, and poses a threat to marine species and communities worldwide. To better project the effects of acidification on organisms' health and persistence an understanding is needed of (1) the mechanisms underlying developmental and physiological tolerance, and (2) the potential populations have for rapid evolutionary adaptation. This is especially challenging in non-model species where targeted assays of metabolism and stress physiology may not be available or economical for large-scale assessments of genetic constraints. We used mRNA sequencing and a quantitative genetics breeding design to study mechanisms underlying genetic variability and tolerance to decreased seawater pH (-0.4 pH units) in larvae of the sea urchin Strongylocentrotus droebachiensis We used a gene ontology-based approach to integrate expression profiles into indirect measures of cellular and biochemical traits underlying variation in larval performance (i.e., growth rates). Molecular responses to OA were complex, involving changes to several functions such as growth rates, cell division, metabolism, and immune activities. Surprisingly, the magnitude of pH effects on molecular traits tended to be small relative to variation attributable to segregating functional genetic variation in this species. We discuss how the application of transcriptomics and quantitative genetics approaches across diverse species can enrich our understanding of the biological impacts of climate change.Item Open Access The RNA-binding protein DND1 acts Sequentially as a negative regulator of pluripotency and a positive regulator of epigenetic modifiers required for germ cell reprogramming.(Development (Cambridge, England), 2019-06-28) Ruthig, Victor A; Friedersdorf, Matthew B; Garness, Jason A; Munger, Steve C; Bunce, Corey; Keene, Jack D; Capel, BlancheThe adult spermatogonial stem cell population arises from pluripotent primordial germ cells (PGCs) that enter the fetal testis around embryonic day (E)10.5. PGCs undergo rapid mitotic proliferation, then enter prolonged cell cycle arrest (G1/G0) during which they transition to pro-spermatogonia. In mice homozygous for the Ter mutation in the RNA-binding protein Dnd1 (Dnd1 Ter/Ter ), many male germ cells (MGCs) fail to enter G1/G0, and form teratomas, tumors containing many embryonic cell types. To investigate the origin of these tumors, we sequenced the MGC transcriptome in Dnd1 Ter/Ter mutants at E12.5, E13.5, and E14.5, just prior to teratoma formation, and correlated this information with DO-RIP-Seq identified DND1 direct targets. Consistent with previous results, we found DND1 controls down-regulation of many genes associated with pluripotency and active cell cycle, including mTor, Hippo and Bmp/Nodal signaling pathway elements. However, DND1 targets also include genes associated with male differentiation including a large group of chromatin regulators activated in wild type but not mutant MGCs during the E13.5 and E14.5 transition. Results suggest multiple DND1 functions, and link DND1 to initiation of epigenetic modifications in MGCs.Item Open Access Transcriptional Profiling of CD8+ CMV-Specific T Cell Functional Subsets Obtained Using a Modified Method for Isolating High-Quality RNA From Fixed and Permeabilized Cells.(Frontiers in immunology, 2020-01) Healy, Zachary R; Weinhold, Kent J; Murdoch, David MPrevious studies suggest that the presence of antigen-specific polyfunctional T cells is correlated with improved pathogen clearance, disease control, and clinical outcomes; however, the molecular mechanisms responsible for the generation, function, and survival of polyfunctional T cells remain unknown. The study of polyfunctional T cells has been, in part, limited by the need for intracellular cytokine staining (ICS), necessitating fixation and cell membrane permeabilization that leads to unacceptable degradation of RNA. Adopting elements from prior research efforts, we developed and optimized a modified protocol for the isolation of high-quality RNA (i.e., RIN > 7) from primary human T cells following aldehyde-fixation, detergent-based permeabilization, intracellular cytokines staining, and sorting. Additionally, this method also demonstrated utility preserving RNA when staining for transcription factors. This modified protocol utilizes an optimized combination of an RNase inhibitor and high-salt buffer that is cost-effective while maintaining the ability to identify and resolve cell populations for sorting. Overall, this protocol resulted in minimal loss of RNA integrity, quality, and quantity during cytoplasmic staining of cytokines and subsequent flourescence-activated cell sorting. Using this technique, we obtained the transcriptional profiles of functional subsets (i.e., non-functional, monofunctional, bifunctional, polyfunctional) of CMV-specific CD8+T cells. Our analyses demonstrated that these functional subsets are molecularly distinct, and that polyfunctional T cells are uniquely enriched for transcripts involved in viral response, inflammation, cell survival, proliferation, and metabolism when compared to monofunctional cells. Polyfunctional T cells demonstrate reduced activation-induced cell death and increased proliferation after antigen re-challenge. Further in silico analysis of transcriptional data suggested a critical role for STAT5 transcriptional activity in polyfunctional cell activation. Pharmacologic inhibition of STAT5 was associated with a significant reduction in polyfunctional cell cytokine expression and proliferation, demonstrating the requirement of STAT5 activity not only for proliferation and cell survival, but also cytokine expression. Finally, we confirmed this association between CMV-specific CD8+ polyfunctionality with STAT5 signaling also exists in immunosuppressed transplant recipients using single cell transcriptomics, indicating that results from this study may translate to this vulnerable patient population. Collectively, these results shed light on the mechanisms governing polyfunctional T cell function and survival and may ultimately inform multiple areas of immunology, including but not limited to the development of new vaccines, CAR-T cell therapies, and adoptive T cell transfer.