Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.

Xiayang Xie, Jonathan R. Mathias, Marie Ange Smith, Steven L. Walker, Yong Teng, Martin Distel, Reinhard W. Köster, Howard I. Sirotkin, Meera T. Saxena, Jeff S. Mumm

Research output: Contribution to journalArticle

13 Citations (Scopus)

Abstract

Background: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations.Results: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing.Conclusions: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.

Original languageEnglish (US)
Article number93
JournalBMC Biology
Volume10
DOIs
StatePublished - Nov 30 2012

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Transcriptional Silencer Elements
Gene Transfer Techniques
Transgenes
Neurons
transgenes
Transcription Factors
transcription factors
neurons
Neurology
Tissue
nervous system
Nervous System
enhancer elements
promoter regions
targeting
Connectome
transgenesis
silencer
Optogenetics
Morpholinos

Keywords

  • enhancer trap
  • Gal4/UAS
  • neuron
  • NRSE/RE1
  • NRSF/REST
  • transgenesis
  • zebrafish

ASJC Scopus subject areas

  • Physiology
  • Biotechnology
  • Structural Biology
  • Developmental Biology
  • Plant Science
  • Ecology, Evolution, Behavior and Systematics
  • Cell Biology
  • Biochemistry, Genetics and Molecular Biology(all)
  • Agricultural and Biological Sciences(all)

Cite this

Silencer-delimited transgenesis : NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner. / Xie, Xiayang; Mathias, Jonathan R.; Smith, Marie Ange; Walker, Steven L.; Teng, Yong; Distel, Martin; Köster, Reinhard W.; Sirotkin, Howard I.; Saxena, Meera T.; Mumm, Jeff S.

In: BMC Biology, Vol. 10, 93, 30.11.2012.

Research output: Contribution to journalArticle

Xie, X, Mathias, JR, Smith, MA, Walker, SL, Teng, Y, Distel, M, Köster, RW, Sirotkin, HI, Saxena, MT & Mumm, JS 2012, 'Silencer-delimited transgenesis: NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner.', BMC Biology, vol. 10, 93. https://doi.org/10.1186/1741-7007-10-93
Xie, Xiayang ; Mathias, Jonathan R. ; Smith, Marie Ange ; Walker, Steven L. ; Teng, Yong ; Distel, Martin ; Köster, Reinhard W. ; Sirotkin, Howard I. ; Saxena, Meera T. ; Mumm, Jeff S. / Silencer-delimited transgenesis : NRSE/RE1 sequences promote neural-specific transgene expression in a NRSF/REST-dependent manner. In: BMC Biology. 2012 ; Vol. 10.
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abstract = "Background: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations.Results: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing.Conclusions: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.",
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AU - Mathias, Jonathan R.

AU - Smith, Marie Ange

AU - Walker, Steven L.

AU - Teng, Yong

AU - Distel, Martin

AU - Köster, Reinhard W.

AU - Sirotkin, Howard I.

AU - Saxena, Meera T.

AU - Mumm, Jeff S.

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N2 - Background: We have investigated a simple strategy for enhancing transgene expression specificity by leveraging genetic silencer elements. The approach serves to restrict transgene expression to a tissue of interest - the nervous system in the example provided here - thereby promoting specific/exclusive targeting of discrete cellular subtypes. Recent innovations are bringing us closer to understanding how the brain is organized, how neural circuits function, and how neurons can be regenerated. Fluorescent proteins enable mapping of the 'connectome', optogenetic tools allow excitable cells to be short-circuited or hyperactivated, and targeted ablation of neuronal subtypes facilitates investigations of circuit function and neuronal regeneration. Optimally, such toolsets need to be expressed solely within the cell types of interest as off-site expression makes establishing causal relationships difficult. To address this, we have exploited a gene 'silencing' system that promotes neuronal specificity by repressing expression in non-neural tissues. This methodology solves non-specific background issues that plague large-scale enhancer trap efforts and may provide a means of leveraging promoters/enhancers that otherwise express too broadly to be of value for in vivo manipulations.Results: We show that a conserved neuron-restrictive silencer element (NRSE) can function to restrict transgene expression to the nervous system. The neuron-restrictive silencing factor/repressor element 1 silencing transcription factor (NRSF/REST) transcriptional repressor binds NRSE/repressor element 1 (RE1) sites and silences gene expression in non-neuronal cells. Inserting NRSE sites into transgenes strongly biased expression to neural tissues. NRSE sequences were effective in restricting expression of bipartite Gal4-based 'driver' transgenes within the context of an enhancer trap and when associated with a defined promoter and enhancer. However, NRSE sequences did not serve to restrict expression of an upstream activating sequence (UAS)-based reporter/effector transgene when associated solely with the UAS element. Morpholino knockdown assays showed that NRSF/REST expression is required for NRSE-based transgene silencing.Conclusions: Our findings demonstrate that the addition of NRSE sequences to transgenes can provide useful new tools for functional studies of the nervous system. However, the general approach may be more broadly applicable; tissue-specific silencer elements are operable in tissues other than the nervous system, suggesting this approach can be similarly applied to other paradigms. Thus, creating synthetic associations between endogenous regulatory elements and tissue-specific silencers may facilitate targeting of cellular subtypes for which defined promoters/enhancers are lacking.

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KW - enhancer trap

KW - Gal4/UAS

KW - neuron

KW - NRSE/RE1

KW - NRSF/REST

KW - transgenesis

KW - zebrafish

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