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

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.4.12.2594 ◽

1984 ◽

Vol 4 (12) ◽

pp. 2594-2609 ◽

Cited By ~ 18

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.

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

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1915905116 ◽

2019 ◽

Vol 116 (45) ◽

pp. 22624-22634 ◽

Cited By ~ 9

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.

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

Carcinogenesis ◽

10.1093/carcin/bgz203 ◽

2019 ◽

Vol 41 (9) ◽

pp. 1282-1293 ◽

Cited By ~ 3

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.

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

Molecular and Cellular Biology ◽

10.1128/mcb.24.16.7298-7311.2004 ◽

2004 ◽

Vol 24 (16) ◽

pp. 7298-7311 ◽

Cited By ~ 25

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.

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

Placenta ◽

10.1016/j.placenta.2008.09.014 ◽

2009 ◽

Vol 30 (1) ◽

pp. 96-104 ◽

Cited By ~ 6

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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Regulation of SpeB in Streptococcus pyogenes by pH and NaCl: a Model for In Vivo Gene Expression

Journal of Bacteriology ◽

10.1128/jb.188.2.399-408.2006 ◽

2006 ◽

Vol 188 (2) ◽

pp. 399-408 ◽

Cited By ~ 70

Author(s):

Jennifer A. Loughman ◽

Michael Caparon

Keyword(s):

Gene Expression ◽

Streptococcus Pyogenes ◽

Growth Phase ◽

Soft Tissues ◽

Expression Profiles ◽

Expression Patterns ◽

Transcriptional Response ◽

Culture Conditions

ABSTRACT For a pathogen such as Streptococcus pyogenes, ecological success is determined by its ability to sense the environment and mount an appropriate adaptive transcriptional response. Thus, determining conditions for analyses of gene expression in vitro that are representative of the in vivo environment is critical for understanding the contributions of transcriptional response pathways to pathogenesis. In this study, we determined that the gene encoding the SpeB cysteine protease is up-regulated over the course of infection in a murine soft-tissue model. Conditions were identified, including growth phase, acidic pH, and an NaCl concentration of <0.1 M, that were required for expression of speB in vitro. Analysis of global expression profiles in response to these conditions in vitro identified a set of coregulated genes whose expression patterns showed a significant correlation with that of speB when examined during infection of murine soft tissues. This analysis revealed that a culture medium that promotes high levels of SpeB expression in vitro produced an expression profile that showed significant correlation to the profile observed in vivo. Taken together, these studies establish culture conditions that mimic in vivo expression patterns; that growth phase, pH, and NaCl may mimic relevant cues sensed by S. pyogenes during infection; and that identification of other environmental cues that alter expression of speB in vitro may provide insight into the signals that direct global patterns of gene expression in vivo.

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Computational drug repositioning of bortezomib to reverse metastatic effect ofGALNT14in lung cancer

10.1101/394163 ◽

2018 ◽

Cited By ~ 1

Author(s):

Ok-Seon Kwon ◽

Haeseung Lee ◽

Hyeon-Joon Kong ◽

Ji Eun Park ◽

Wooin Lee ◽

...

Keyword(s):

Gene Expression ◽

Lung Cancer ◽

In Silico ◽

Large Scale ◽

Cancer Metastasis ◽

Drug Repositioning ◽

Expression Patterns ◽

Lung Cancer Metastasis

AbstractAlthough many molecular targets for cancer therapy have been discovered, they often show poor druggability, which is a major obstacle to develop targeted drugs. As an alternative route to drug discovery, we adopted anin silicodrug repositioning (in silicoDR) approach based on large-scale gene expression signatures, with the goal of identifying inhibitors of lung cancer metastasis. Our analysis of clinicogenomic data identified GALNT14, an enzyme involved in O-linked N-acetyl galactosamine glycosylation, as a putative driver of lung cancer metastasis leading to poor survival. To overcome the poor druggability of GALNT14, we leveraged Connectivity Map approach, anin silicoscreening for drugs that are likely to revert the metastatic expression patterns. It leads to identification of bortezomib (BTZ) as a potent metastatic inhibitor, bypassing direct inhibition of poorly druggable target, GALNT14. The anti-metastatic effect of BTZ was verifiedin vitroandin vivo. Notably, both BTZ treatment andGALNT14knockdown attenuated TGFβ-mediated gene expression and suppressed TGFβ-dependent metastatic genes, suggesting that BTZ acts by modulating TGFβ signalingTaken together, these results demonstrate that ourin silicoDR approach is a viable strategy to identify a candidate drug for undruggable targets, and to uncover its underlying mechanisms.

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A lysosomal dimmer switch regulates cellular quiescence depth

10.1101/413989 ◽

2018 ◽

Cited By ~ 1

Author(s):

Kotaro Fujimaki ◽

Ruoyan Li ◽

Hengyu Chen ◽

Kimiko Della Croce ◽

Hao Helen Zhang ◽

...

Keyword(s):

Gene Expression ◽

Gene Expression Signature ◽

Serum Starvation ◽

Quiescent State ◽

Cellular Mechanisms ◽

Lysosomal Function ◽

Quiescent Cells ◽

Cellular Quiescence

ABSTRACTNumerous physiological and pathological phenomena are associated with the quiescent state of a cell. Cellular quiescence is a heterogeneous resting state; cells in deep than shallow quiescence require stronger growth stimulation to exit quiescence and reenter the cell cycle. Despite the importance of quiescent cells such as stem and progenitor cells to tissue homeostasis and repair, cellular mechanisms controlling the depth of cellular quiescence are poorly understood. Here we began by analyzing transcriptome changes as rat embryonic fibroblasts moved progressively deeper into quiescence under increasingly longer periods of serum starvation. We found that lysosomal gene expression was significantly upregulated in deep than shallow quiescence, which compensated for gradually reduced autophagy flux observed during quiescence deepening. Consistently, we show that inhibiting lysosomal function drove cells deeper into quiescence and eventually into a senescence-like irreversibly arrested state. By contrast, increasing lysosomal function progressively pushed cells into shallower quiescence. That is, lysosomal function modulates quiescence depth continuously like a dimmer switch. Mechanistically, we show that lysosomal function prevents quiescence deepening by reducing oxidative stress in the cell. Lastly, we show that a gene expression signature developed by comparing deep and shallow quiescent cells can correctly classify senescent and aging cells in a wide array of cell lines in vitro and tissues in vivo, suggesting that quiescence deepening, senescence, and aging may share common regulatory mechanisms.

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

Molecular and Cellular Biology ◽

10.1128/mcb.4.12.2594-2609.1984 ◽

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.

Download Full-text