scholarly journals A dopamine-induced gene expression signature regulates neuronal function and cocaine response

2019 ◽  
Author(s):  
Katherine E. Savell ◽  
Morgan E. Zipperly ◽  
Jennifer J. Tuscher ◽  
Corey G. Duke ◽  
Robert A. Phillips ◽  
...  
Keyword(s):  
Gene Expression ◽  
Drugs Of Abuse ◽  
Gene Expression Signature ◽  
Receptor Activation ◽  
Early Gene ◽  
Expression Signature ◽  
Behavioral Adaptations ◽  
Drug Actions

Drug addiction is a worldwide health problem, with overdose rates of both psychostimulants and opioids currently on the rise in many developed countries. Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. However, even with well-studied drugs such as cocaine, drug-induced transcriptional responses remain poorly understood due to the cellular heterogeneity of the NAc and complex drug actions via multiple neurotransmitter systems. Here, we leveraged high-throughput single-nucleus RNA-sequencing to create a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type-specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified specific neuronal subpopulations that are activated by cocaine, and defined an immediate early gene expression program that is upregulated following cocaine experience in vivo and dopamine (DA) receptor activation in vitro. To characterize the neuronal response to this DA-mediated gene expression signature, we engineered a large-scale CRISPR/dCas9 activation strategy to recreate this program. Multiplexed induction of this gene program initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Taken together, these results define the genome-wide transcriptional response to cocaine with cellular precision, and demonstrate that drug-responsive gene programs are sufficient to initiate both physiological and behavioral adaptations to drugs of abuse.

scholarly journals A dopamine-induced gene expression signature regulates neuronal function and cocaine response

2020 ◽  
Vol 6 (26) ◽  
pp. eaba4221 ◽  
Author(s):  
Katherine E. Savell ◽  
Jennifer J. Tuscher ◽  
Morgan E. Zipperly ◽  
Corey G. Duke ◽  
Robert A. Phillips ◽  
...  
Keyword(s):  
Gene Expression ◽  
Large Scale ◽  
Drugs Of Abuse ◽  
Transcriptional Response ◽  
Gene Expression Signature ◽  
Receptor Activation ◽  
Early Gene ◽  
Behavioral Adaptations

Drugs of abuse elevate dopamine levels in the nucleus accumbens (NAc) and alter transcriptional programs believed to promote long-lasting synaptic and behavioral adaptations. Here, we leveraged single-nucleus RNA-sequencing to generate a comprehensive molecular atlas of cell subtypes in the NAc, defining both sex-specific and cell type–specific responses to acute cocaine experience in a rat model system. Using this transcriptional map, we identified an immediate early gene expression program that is up-regulated following cocaine experience in vivo and dopamine receptor activation in vitro. Multiplexed induction of this gene program with a large-scale CRISPR-dCas9 activation strategy initiated a secondary synapse-centric transcriptional profile, altered striatal physiology in vitro, and enhanced cocaine sensitization in vivo. Together, these results define the transcriptional response to cocaine with cellular precision and demonstrate that drug-responsive gene programs can potentiate both physiological and behavioral adaptations to drugs of abuse.

scholarly journals Location of sequences in polyomavirus DNA that are required for early gene expression in vivo and in vitro.

1984 ◽  
Vol 4 (12) ◽  
pp. 2594-2609 ◽  
Author(s):  
C R Mueller ◽  
A M Mes-Masson ◽  
M Bouvier ◽  
J A Hassell
Keyword(s):  
Gene Expression ◽  
Thymidine Kinase ◽  
Dna Sequences ◽  
Viral Genome ◽  
Simian Virus 40 ◽  
Early Gene ◽  
Wild Type ◽  
Transcription In Vitro

To define the DNA sequences required for the expression of the polyomavirus early transcription unit, we cloned part of the viral genome in a plasmid vector, isolated mutants bearing lesions introduced in vitro within DNA sequences upstream of the transcriptional start site, and measured the capacity of these various mutant genomes to transform cells and to function as templates for transcription in vitro by comparison with wild-type DNA. One set of mutants bore 5' unidirectional deletions beginning at position -810 and extending downstream to position +4. Another set of mutants bore 3' undirectional deletions starting at position +4 and progressing upstream to position -311. The last set of mutants bore internal deletions between positions -810 and +4. Analyses of the properties of these mutant DNAs led us to conclude that the region between positions -403 and -311 includes an enhancer of gene expression. Deletion of this area from the viral genome reduced gene expression in vivo to 1 to 2% of wild-type levels, as measured by transformation assays. Moreover, this region increased the frequency of transformation of thymidine kinase-negative Rat-2 cells by the herpes simplex virus thymidine kinase (tk) gene from 5- to 20-fold. This occurred only if the polyomavirus sequences were covalently linked to the tk gene and then occurred independently of their orientation or position relative to the tk gene. A second transcriptional element is located downstream of the enhancer between positions -311 and -213. This element together with the enhancer was sufficient to bring about transformation of Rat-1 cells at nearly wild-type frequencies, and together these elements constitute the minimal sequences required for gene expression in vivo. The sequences making up the second element may be functionally duplicated downstream of position -165 (between positions -165 and -60). This was revealed by the characterization of mutant genomes with deletions between positions -349 and -60. The role of these redundant elements is not known; however, they may be analogous to the 21-base-pair repeats of simian virus 40. Finally, sequences between positions -57 and -1 were required for accurate and efficient transcription in vitro. However, this DNA stretch, which includes the TATA box and major transcriptional start sites, was not absolutely required for gene expression in vivo. We conclude that the polyomavirus promoter comprises multiple functional elements which are distributed across a DNA stretch of about 400 base pairs.

scholarly journals Graded regulation of cellular quiescence depth between proliferation and senescence by a lysosomal dimmer switch

2019 ◽  
Vol 116 (45) ◽  
pp. 22624-22634 ◽  
Author(s):  
Kotaro Fujimaki ◽  
Ruoyan Li ◽  
Hengyu Chen ◽  
Kimiko Della Croce ◽  
Hao Helen Zhang ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Expression Signature ◽  
Cell Types ◽  
The Body ◽  
Cellular Mechanisms ◽  
Lysosomal Function ◽  
Quiescent Cells ◽  
Common Transition

The reactivation of quiescent cells to proliferate is fundamental to tissue repair and homeostasis in the body. Often referred to as the G0 state, quiescence is, however, not a uniform state but with graded depth. Shallow quiescent cells exhibit a higher tendency to revert to proliferation than deep quiescent cells, while deep quiescent cells are still fully reversible under physiological conditions, distinct from senescent cells. Cellular mechanisms underlying the control of quiescence depth and the connection between quiescence and senescence are poorly characterized, representing a missing link in our understanding of tissue homeostasis and regeneration. Here we measured transcriptome changes as rat embryonic fibroblasts moved from shallow to deep quiescence over time in the absence of growth signals. We found that lysosomal gene expression was significantly up-regulated in deep quiescence, and partially compensated for gradually reduced autophagy flux. Reducing lysosomal function drove cells progressively deeper into quiescence and eventually into a senescence-like irreversibly arrested state; increasing lysosomal function, by lowering oxidative stress, progressively pushed cells into shallower quiescence. That is, lysosomal function modulates graded quiescence depth between proliferation and senescence as a dimmer switch. Finally, we found that a gene-expression signature developed by comparing deep and shallow quiescence in fibroblasts can correctly classify a wide array of senescent and aging cell types in vitro and in vivo, suggesting that while quiescence is generally considered to protect cells from irreversible arrest of senescence, quiescence deepening likely represents a common transition path from cell proliferation to senescence, related to aging.

Estrogen abrogates zoledronic acid induced gene expression signature in endocrine sensitive tumor cell lines in vitro.

2018 ◽  
Vol 36 (15_suppl) ◽  
pp. e12570-e12570
Author(s):  
Sigrid Weingartshofer ◽  
Martin Bilban ◽  
Marie Theres Kastner ◽  
Juraj Hlavaty ◽  
Ingrid Walter ◽  
...  
Keyword(s):  
Gene Expression ◽  
Zoledronic Acid ◽  
Tumor Cell ◽  
Cell Lines ◽  
Gene Expression Signature ◽  
Tumor Cell Lines ◽  
Expression Signature

Molecular Effects of Rituximab on CLL Cells In Vivo: Rapid Increase in Serum Interferon gamma, Induction of a Distinct Tumor Cell Gene Expression Signature and Loss of CD20 Expression.

Blood ◽  
2007 ◽  
Vol 110 (11) ◽  
pp. 2048-2048
Author(s):  
Adrian Wiestner ◽  
Elinor Lee ◽  
Berengere Vire ◽  
Federica Gibellini ◽  
Ndegwa Njuguna ◽  
...  
Keyword(s):  
Gene Expression ◽  
Tumor Cells ◽  
Dominant Role ◽  
Tumor Biology ◽  
Gene Expression Signature ◽  
Leukemic Cells ◽  
Mrna Levels ◽  
Expression Signature ◽  
Protein Levels

Abstract Proposed mechanisms on how the monoclonal anti-CD20 antibody rituximab (R) depletes B-cells include antibody-dependent cellular cytotoxicity and complement-dependent cytotoxicity. In vitro studies have suggested that R induced pro-apoptotic signals contribute to clinical efficacy and may sensitize cells to chemotherapy. To investigate the effect of R on tumor biology in vivo, we analyzed the molecular changes in leukemic cells of 12 previously untreated CLL patients during the first R (375mg/m2) infusion. The median reduction of circulating tumor cells within 24h was 50% (range 0–67%). We first determined whether R affects gene expression in CLL cells obtained before and at 6h and 24h after the start of R. Cells were purified by CD19+ selection and gene expression was measured on Affymetrix HU133A 2.0 arrays. A one-way ANOVA test with a stringent cutoff (false discovery rate of <20%) identified 69 genes whose expression increased >50% at 6h compared to pre treatment, and 31 genes whose expression decreased by >30%. Most of the up-regulated genes are known to be regulated by interferon (IFN) and include the pro-apoptotic genes IRF1, STAT1, FAS and OAS2. Of 12 cytokines assayed in the serum, we found that only IFNy, IL–6, IL–8, IL–10, and TNFa were induced by R with a peak at 2h. Consistent with a dominant role of IFNy on gene expression in the CLL cells, STAT1, a direct and essential mediator of IFNy signaling, was activated in circulating leukemic cells in vivo. In addition, when comparing the response between patients, IFNy serum protein levels correlated strongly with the intensity of the gene expression changes in the tumor cells (r=0.83, p=0.008). We could not detect any IFNy mRNA in CLL cells and conclude that the IFNy is most likely released by NK cells activated through FcyRIII signaling. Considering the long half-life of R, we were surprised to see that both cytokine serum levels and gene expression changes almost completely subsided by 24h. Intriguingly, among the few genes that were down-regulated by treatment, the gene encoding CD20 was the most strongly and consistently affected showing a 50% decrease in expression at 24h. We also assessed CD20 protein levels by Western blotting. Total CD20 levels were markedly decreased already at 6h and by 24h almost all CD20 had been lost. The more rapid and more pronounced decrease of CD20 protein as opposed to mRNA levels is consistent with a process previously described as shaving, during which R bound CD20 is pulled of the cell surface (Kennedy AD, J Immunol. 2004). Despite the absence of a clinical cytokine release syndrome, we observed basically identical changes in serum cytokines and gene expression with subsequent infusions in 2 patients analyzed. In summary, R induced a characteristic gene expression signature in CLL cells that is dominated by IFN response genes, many of which have well characterized pro-apoptotic functions. Thus, our data suggest that signaling for apoptosis is not so much a direct effect of R, but due to a complex immune response to the R coated CLL cells. Modified treatment schedules capable of delivering sustained pro-apoptotic signals hold promise for improved efficacy of R and should be explored.

scholarly journals A Minimal Subset of Seven Genes Associated with Tumor Hepatocyte Differentiation Predicts a Poor Prognosis in Human Hepatocellular Carcinoma

Cancers ◽  
2021 ◽  
Vol 13 (22) ◽  
pp. 5624
Author(s):  
Matthis Desoteux ◽  
Corentin Louis ◽  
Kevin Bévant ◽  
Denise Glaise ◽  
Cédric Coulouarn
Keyword(s):  
Gene Expression ◽  
Hepatocellular Carcinoma ◽  
Poor Prognosis ◽  
Gene Expression Signature ◽  
Hepatocyte Differentiation ◽  
Expression Signature ◽  
Heparg Cell Line ◽  
Minimal Subset ◽  
Differentiation Status

Hepatocellular carcinoma (HCC) is a deadly cancer worldwide as a result of a frequent late diagnosis which limits the therapeutic options. Tumor progression in HCC is closely correlated with the dedifferentiation of hepatocytes, the main parenchymal cells in the liver. Here, we hypothesized that the expression level of genes reflecting the differentiation status of tumor hepatocytes could be clinically relevant in defining subsets of patients with different clinical outcomes. To test this hypothesis, an integrative transcriptomics approach was used to stratify a cohort of 139 HCC patients based on a gene expression signature established in vitro in the HepaRG cell line using well-controlled culture conditions recapitulating tumor hepatocyte differentiation. The HepaRG model was first validated by identifying a robust gene expression signature associated with hepatocyte differentiation and liver metabolism. In addition, the signature was able to distinguish specific developmental stages in mice. More importantly, the signature identified a subset of human HCC associated with a poor prognosis and cancer stem cell features. By using an independent HCC dataset (TCGA consortium), a minimal subset of seven differentiation-related genes was shown to predict a reduced overall survival, not only in patients with HCC but also in other types of cancers (e.g., kidney, pancreas, skin). In conclusion, the study identified a minimal subset of seven genes reflecting the differentiation status of tumor hepatocytes and clinically relevant for predicting the prognosis of HCC patients.

Rituximab Induces an Interferon Gene Expression Signature in Patients with CLL.

Blood ◽  
2006 ◽  
Vol 108 (11) ◽  
pp. 4988-4988
Author(s):  
Elinor Lee ◽  
Xiuli Xu ◽  
Peter Munson ◽  
Ronald Cooper ◽  
Nalini Raghavachari ◽  
...  
Keyword(s):  
Gene Expression ◽  
B Cells ◽  
Gene Expression Signature ◽  
Specific Gene ◽  
Cytokine Release ◽  
Cytokine Release Syndrome ◽  
Ifn Gamma ◽  
Expression Signature ◽  
Specific Gene Expression

Abstract Rituximab, a monoclonal anti-CD20 antibody, is used to treat Chronic Lymphocytic Leukemia (CLL) in combination with fludarabine. Rituximab is thought to deplete B-cells through antibody-dependent cellular cytotoxicity, complement-dependent cytotoxicity, and possibly signaling for apoptosis. Whether or not signaling by rituximab contributes to its clinical efficacy and can sensitize the malignant cells to chemotherapy is controversial. To investigate if rituximab can induce a specific gene expression signature, we used genomic-scale gene expression profiling (Affymetrix HU133A 2.0 arrays) of B-cells from CLL patients receiving their first rituximab infusion. During the infusion, patients experienced a cytokine release syndrome (fever, chills, and hypotension) that led to interruption and symptomatic treatment in most; however, all patients were able to finish the treatment. Absolute lymphocyte counts decreased on average by 50% over this initial 24h period. We analyzed CD19+ selected CLL cells from eight patients obtained pre and 6 and 24 hours after the start of rituximab. A one-way ANOVA test was used to identify genes up- or down-regulated with a false discovery rate (FDR = number of expected chance findings / number of observations) of <10%. We identified 80 genes with at least 1.5× higher expression at 6h versus 0h including many interferon (IFN)-regulated genes like IRF1, IFITM1, STAT1, JAK3 and several apoptosis related genes such as FAS and Caspase 8. The majority of these genes were at least 2-fold up-regulated at 6 hours, but most returned to pre-treatment levels by 24 hours. Thus, rituximab induced a transient gene expression signature that correlated with the cytokine release syndrome during the infusion. To determine whether or not this IFN signature was caused directly by rituximab signaling or indirectly by cytokines released during the infusion, we compared rituximab and IFN gamma effects on CLL cells in-vitro. Both rituximab (10ug/ml with cross-linking) and IFN-gamma (1000U/ml) induced FAS (CD95) expression in CLL cells measured by flow cytometry. CD95 expression was low on untreated CLL cells, at 6 hours, up-regulation of CD95 expression with rituximab was stronger than with IFN, while at 24 hours, IFN treated cells showed slightly higher CD95 expression. Next, we investigated whether rituximab is able to activate STAT1, the main transcription factor regulating IFN target genes. IFN gamma induced rapid phosphorylation of STAT1 in CLL cells, but rituximab did not. However, we observed phosphorylation of ERK in response to rituximab as has been reported by others. After in-vitro stimulation with rituximab and IFN-gamma, IRF-1 and STAT-1 were up-regulated at 2 and 6 hours as measured by real time PCR, albeit with a stronger response after IFN. We conclude that rituximab is associated with a specific gene expression signature in CLL patients that is characterized by IFN response genes. At this point, we cannot rule out that this signature is contributed in part by cytokines released during the rituximab infusion. However, the rapid up-regulation of CD95, STAT1, and IRF1 under controlled in-vitro conditions is consistent with a direct effect of rituximab. Ongoing studies aim to better characterize rituximab signaling in CLL and to determine whether this can contribute to apoptosis or sensitize the leukemic cells to chemotherapy.

ΔNp63α is a super enhancer-enriched master factor controlling the basal-to-luminal differentiation transcriptional program and gene regulatory networks in nasopharyngeal carcinoma

2019 ◽  
Vol 41 (9) ◽  
pp. 1282-1293 ◽  
Author(s):  
Jing Cai ◽  
Shengnan Chen ◽  
Mei Yi ◽  
Yixin Tan ◽  
Qian Peng ◽  
...  
Keyword(s):  
Gene Expression ◽  
Nasopharyngeal Carcinoma ◽  
Basal Cell ◽  
Regulatory Networks ◽  
Regulation Of Gene Expression ◽  
Gene Expression Signature ◽  
Molecular Signatures ◽  
Chip Sequencing

Abstract Nasopharyngeal carcinoma (NPC) originates via malignant transformation of the pseudostratified nasopharyngeal epithelium, composed of basal and luminal cells. Super enhancers (SEs) are large clusters of cis-elements involved in the regulation of gene expression through epigenetic regulatory mechanisms. In this study, we demonstrated that basal cell-specific proteins are highly expressed, whereas luminal cell proteins are downregulated in NPC, implying a perturbation of basal-to-luminal differentiation during NPC development. We characterized NPC cell models according to different molecular signatures associated with their differentiation status and found that distinct SE landscapes are tightly associated with basal or luminal-like molecular signatures in NPC cells. Furthermore, the transcription of ΔNP63α, a prominent isoform of TP63, was found to be driven by SEs in NPC cells. Data from chromatin immunoprecipitation (ChIP)-sequencing showed that ΔNP63α largely occupied regions of SEs associated with basal cell-specific genes. Silencing of ΔNP63α led to a loss of H3K27ac occupancy at basal-type SEs and triggered a basal-to-luminal gene expression signature switch, suggesting that ΔNP63α is a master factor contributing to the perturbation of luminal differentiation. Integrative transcriptomics analysis also revealed that ΔNP63α acts as a core factor involved in the dysregulation of gene expression in NPC. Furthermore, ΔNP63α enhanced EGF-stimulated NF-κB activation in NPC cells by activating SE-mediated EGFR transcription. Finally, depletion of ΔNP63α in NPC cells induced robust growth inhibition of NPC cells in vitro and in vivo. Our data revealed that ΔNP63α-dependent SE reprogramming contributes to the blockade of luminal differentiation and uncontrolled proliferation in NPC.

scholarly journals Protein Kinase Cδ Blocks Immediate-Early Gene Expression in Senescent Cells by Inactivating Serum Response Factor

2004 ◽  
Vol 24 (16) ◽  
pp. 7298-7311 ◽  
Author(s):  
Keith Wheaton ◽  
Karl Riabowol
Keyword(s):  
Gene Expression ◽  
Protein Kinase ◽  
Serum Response Factor ◽  
Immediate Early Gene ◽  
Early Gene ◽  
Immediate Early ◽  
Response Factor ◽  
Serum Response

ABSTRACT Fibroblasts lose the ability to replicate in response to growth factors and become unable to express growth-associated immediate-early genes, including c-fos and egr-1, as they become senescent. The serum response factor (SRF), a major transcriptional activator of immediate-early gene promoters, loses the ability to bind to the serum response element (SRE) and becomes hyperphosphorylated in senescent cells. We identify protein kinase C delta (PKCδ) as the kinase responsible for inactivation of SRF both in vitro and endogenously in senescent cells. This is due to a higher level of PKCδ activity as cells age, production of the PKCδ catalytic fragment, and its nuclear localization in senescent but not in low-passage-number cells. The phosphorylation of T160 of SRF by PKCδ in vitro and in vivo led to loss of SRF DNA binding activity. Both the PKCδ inhibitor rottlerin and ectopic expression of a dominant negative form of PKCδ independently restored SRE-dependent transcription and immediate-early gene expression in senescent cells. Modulation of PKCδ activity in vivo with rottlerin or bistratene A altered senescent- and young-cell morphology, respectively. These observations support the idea that the coordinate transcriptional inhibition of several growth-associated genes by PKCδ contributes to the senescent phenotype.

Early Gene Expression and Morphogenesis of the Murine Chorioallantoic Placenta In vivo and In vitro

Placenta ◽  
2009 ◽  
Vol 30 (1) ◽  
pp. 96-104 ◽  
Author(s):  
L.K. Proctor ◽  
C. Dunk ◽  
D. Baczyk ◽  
J.C.P. Kingdom ◽  
S. Lee Adamson
Keyword(s):  
Gene Expression ◽  
Early Gene ◽  
Early Gene Expression ◽  
Chorioallantoic Placenta
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