Yeast lacking the H3 or H4 amino termini, and corresponding wild type strains, were grown in synthetic media. These conditions induce Gcn4-activated transcription.
Contribution of the histone H3 and H4 amino termini to Gcn4p- and Gcn5p-mediated transcription in yeast.
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Purpose: Avian photoreceptors are a diverse class of neurons, comprised of four single cones, the two members of the double cone, and rods. Many distinctive features of photoreceptor subtypes, including spectral tuning, oil droplet size and pigmentation, synaptic targets and spatial patterning, have been well characterized, but the molecular mechanisms underlying these attributes have not been explored. Furthermore, the signaling events and transcriptional regulators driving the differentiation of these diverse photoreceptors are currently unknown. Methods: To identify genes specifically expressed in distinct chicken (Gallus gallus) photoreceptor subtypes, we developed fluorescent reporters that label photoreceptor subpopulations, isolated these subpopulations using fluorescence-activated cell sorting, subjected them to next-generation sequencing, and conducted differential expression analysis. Results: We identified hundreds of differentially expressed genes from photoreceptor subpopulations labeled with rhodopsin, red opsin, green opsin, and violet opsin reporters. These genes are involved in a variety of processes, including phototransduction, transcriptional regulation, cell adhesion, maintenance of intra- and extra-cellular structure, and metabolism. Of particular note are a variety of differentially expressed transcription factors, which may drive and maintain photoreceptor diversity, and cell adhesion molecules that may mediate spatial patterning of photoreceptors and act to establish retinal circuitry. Conclusions: These analyses provide a framework for future studies that will dissect the role of these various factors in the differentiation of avian photoreceptor subtypes. Overall design: mRNA expression profiling of 5 pairs of photoreceptor subtypes isolated from chicken retinal explants, 3 replicates per sample
Transcriptome profiling of developing photoreceptor subtypes reveals candidate genes involved in avian photoreceptor diversification.
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Plastids emit signals that broadly affect cellular processes. Based on previous genetic analyses, we propose that plastid signaling regulates the downstream components of a light signaling network and that these interactions coordinate chloroplast biogenesis with both the light environment and development by regulating gene expression. We tested these ideas by analyzing light-regulated and plastid-regulated transcriptomes. We found that the plastid is a major regulator of light signaling, attenuating the expression of more than half of all light-regulated genes in our dataset and changing the nature of light regulation for a smaller fraction of these light-regulated genes.
Plastids are major regulators of light signaling in Arabidopsis.
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Analysis of gene expression in isolated mouse lung dendritic cells isolated during influenza A virus infection, with and without activaiton of the aryl hydrocarbon receptor (AHR). Overall design: To determine genome wide changes in dendritic cells mediated by aryl hydrocarbon receptor activation
Genome-Wide Transcriptional Analysis Reveals Novel AhR Targets That Regulate Dendritic Cell Function during Influenza A Virus Infection.
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The ability to generate defined null mutations in mice revolutionized the analysis of gene function in mammals. However, gene-deficient mice generated by using 129-derived embryonic stem (ES) cells may carry large segments of 129 DNA, even when extensively backcrossed to reference strains, such as C57BL/6J, and this may confound interpretation of experiments performed in these mice. Tissue plasminogen activator (tPA), encoded by the PLAT gene, is a fibrinolytic serine protease that is widely expressed in the brain. A large number of neurological abnormalities have been reported in tPA-deficient mice. The studies here compare genes differentially expressed in the brains of Plat-/- mice from two independent Plat-/- mouse derivations to wild-type C57BL/6J mice. One strain denoted “Old” was constructed in ES cells from a 129 mouse and backcrossed extensively to C57BL/6J, and one denoted “New” Plat-/- mouse was constructed using zinc finger nucleases directly in the C57BL/6J-Plat-/- mouse strain. We identify a significant set of genes that are differentially expressed in the brains of Old Plat-/- mice that preferentially cluster in the vicinity of Plat on chromosome 8, apparently linked to more than 20 Mbp of DNA flanking Plat being of 129 origin. No such clustering is seen in the New Plat-/- mice. Overall design: Whole-transcriptome profiling of the cerebral cortex of wild-type control C57BL/6J mice and two independent Plat-/- mice strains on the C57BL/6J background.
Passenger mutations and aberrant gene expression in congenic tissue plasminogen activator-deficient mouse strains.
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This present study is the first to investigate the global changes in host gene expression during the interaction of human bronchial epithelial cells and live Alternaria spores. Human bronchial epithelial cells (BEAS2-B) were exposed to spores or media alone for 24 hours. RNA was collected from three biological replicates/treatment and used to assess changes in gene expression patterns using Affymetrix Human Genome U133 Plus 2.0 Arrays. Interestingly, many cytokine/chemokine immune response genes were upregulated. Genes involved in cell death, retinoic acid signaling, TLR3, and interferon response pathways were also significantly upregulated.
Analysis of global gene expression changes in human bronchial epithelial cells exposed to spores of the allergenic fungus, Alternaria alternata.
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As part of a study of the role of the aryl hydrocarbon receptor (Ahr) in maintenance and senescence of hematopoietic stem cells (HSC), global gene expression profiling was done with HSC isolated from 18-month-old Ahr-knockout and wild-type mice. HSC from aged AhR-KO mice had changes in expression of many genes related to HSC maintenance, consistent with the phenotype observed in aging Ahr-KO mice: decreased survival rate, splenomegaly, increased circulating white blood cells, hematopoietic cell accumulation in tissues, anemia, increased numbers of stem/progenitor and lineage-committed cells in bone marrow, decreased erythroid progenitor cells in bone marrow, and decreased self-renewal capacity of HSC.
Conditional deletion of Ahr alters gene expression profiles in hematopoietic stem cells.
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Background: Degenerative disc disease (DDD) is a primary contributor to low back pain, a leading cause of disability. Progression of DDD is aided by inflammatory cytokines in the intervertebral disc (IVD), particularly TNF-a and IL-1ß, but current treatments fail to effectively target this mechanism. The objective of this study was to explore the feasibility of CRISPR epigenome editing based therapy for DDD, by modulation of TNFR1/IL1R1 signaling in pathological human IVD cells. Methods: Human IVD cells from the nucleus pulposus of patients receiving surgery for back pain were obtained and the regulation of TNFR1/IL1R1 signaling by a lentiviral CRISPR epigenome editing system was tested. These cells were tested for successful lentiviral transduction/expression of dCas9-KRAB system and regulation of TNFR1/IL1R1 expression. TNFR1/IL1R1 signaling disruption was investigated via measurement of NF-?B activity, apoptosis, and anabolic/catabolic changes in gene expression post inflammatory challenge. Results: CRISPR epigenome editing systems were effectively introduced into pathological human IVD cells and significantly downregulated TNFR1 and IL1R1. This downregulation significantly attenuated deleterious TNFR1 signaling but not IL1R1 signaling. This is attributed to less robust IL1R1 expression downregulation, and IL-1ß driven reversal of IL1R1 expression downregulation in a portion of patient IVD cells. Additionally, RNAseq data indicated a novel transcription factor targets, IRF1 and TFAP2C, as being a primary regulators of inflammatory signaling in IVD cells. Discussion: These results demonstrate the feasibility of CRISPR epigenome editing of inflammatory receptors in pathological IVD cells, but highlight a limitation in epigenome targeting of IL1R1. This method has potential application as a novel gene therapy for DDD, to attenuate the deleterious effect of inflammatory cytokines present in the degenerative IVD. Overall design: Patient nucleus pulposus cells (TNFR1kd and nontargeting control) were analyzed by RNA-seq with and without TNF-a treatment.
Lentiviral CRISPR Epigenome Editing of Inflammatory Receptors as a Gene Therapy Strategy for Disc Degeneration.
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RNASeq data from WT, Zmym2 knockout- and Zmym2 overexpressing- E14tg2a mouse embryonic stem cells Overall design: RNASeq of ESCs in medium containing Serum and Leukaemia Inhibitory Factor (LIF)
ZMYM2 inhibits NANOG-mediated reprogramming.
Successfully fighting infection requires a properly tuned immune system. Recent epidemiological studies link exposure to pollutants that bind the aryl hydrocarbon receptor (AHR) during development with poorer immune responses later in life. Yet, how developmental triggering of AHR durably alters immune cell function remains unknown. Using a mouse model, we show that developmental activation of AHR leads to long-lasting reduction in the response of CD8+ T cells during influenza virus infection, cells critical for resolving primary infection. Combining genome-wide approaches, we demonstrate that developmental activation alters DNA methylation and gene expression patterns in isolated CD8+ T cells prior to and during infection. Altered transcriptional profiles in CD8+ T cells from developmentally exposed mice reflect changes in pathways involved in proliferation and immunoregulation, with an overall pattern that bears hallmarks of T cell exhaustion. Developmental exposure also changed DNA methylation across the genome, but differences were most pronounced following infection, where we observed inverse correlation between promoter methylation and gene expression. This points to altered regulation of DNA methylation as one mechanism by which AHR causes durable changes in T cell function. Discovering that distinct gene sets and pathways were differentially changed in developmentally exposed mice prior to and after infection further reveals that the process of CD8+ T cell activation is rendered fundamentally different by early life AHR signaling. These findings reveal a novel role for AHR in the developing immune system: regulating DNA methylation and gene expression as immune cells respond to viral infection later in life. Overall design: In this study, two biological replicates were collected for each of four treatment groups: developmental exposure to vehicle control (Veh) and naïve, developmental exposure to 2,3,7,8-tetrachlorodibenzo-p-dioxin (TCDD) and naïve, developmental exposure to Veh and infected, developmental exposure to TCDD and infected. For each sample, both RNA-seq and methylated DNA immunoprecipitation (MeDIP)-seq were performed.
Linking the aryl hydrocarbon receptor with altered DNA methylation patterns and developmentally induced aberrant antiviral CD8+ T cell responses.
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