PDYN, a gene implicated in brain/mental disorders, is targeted by REST in the adult human brain

https://doi.org/10.1016/j.bbagrm.2014.09.001Get rights and content

Highlights

  • We study transcriptional control of PDYN in SH-SY5Y cells and the human brain.

  • We determine the REST-PDYN binding profile in SH-SY5Y cells and the human brain.

  • Interfering with REST activity results in increased PDYN expression in SH-SY5Y cells.

  • Reduced REST binding coincides with higher PDYN expression in the human brain.

  • REST represses PDYN expression in both SH-SY5Y cells and the human brain.

Abstract

The dynorphin κ-opioid receptor system is implicated in mental health and brain/mental disorders. However, despite accumulating evidence that PDYN and/or dynorphin peptide expression is altered in the brain of individuals with brain/mental disorders, little is known about transcriptional control of PDYN in humans. In the present study, we show that PDYN is targeted by the transcription factor REST in human neuroblastoma SH-SY5Y cells and that that interfering with REST activity increases PDYN expression in these cells. We also show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain and may have implications for mental health and brain/mental disorders.

Introduction

The dynorphin κ-opioid receptor system modulates neurotransmission in the brain and is involved in a variety of processes that influence mental health, for example the stress response and reward processing [1]. Altered PDYN and/or dynorphin peptide expression is observed in a number of brain/mental disorders including but not limited to drug addiction, Alzheimer's disease and epilepsy [1]. Moreover, genetic variants in PDYN are associated with a growing number of psychopathologies, for example drug addiction and schizophrenia [1]. However, little is known about transcriptional control of PDYN in humans.

In silico and in vitro studies have implicated a number of transcription factors in regulation of Pdyn, most notably AP-1, CREB, DREAM, NF-κB and YY1 [1]. However, in vivo evidence is available only for AP-1, CREB and DREAM. Moreover, all but one of the target sites for AP-1 and CREB identified in these studies are poorly conserved and there are no reports that they are bound by AP-1 or CREB in humans [1], [2], [3]. On the contrary, the target site for DREAM is conserved and bound by DREAM in human neuroblastoma NB69 cells [4]. This makes DREAM the only transcription factor for which evidence from living cells is available that it is involved in transcriptional control of PDYN in humans.

To identify transcription factors that may regulate PDYN expression in humans, we screened publicly available chromatin immunoprecipitation-sequencing (ChIP-Seq) data on 161 transcription factors from 91 cell lines generated by ENCODE [2], [3]. In this way, we identified REST, a transcriptional repressor that regulates a large number of neuronal genes [5], [6]. REST both regulates and is regulated by the microRNA MIR-9 and together they mediate a switch in chromatin remodeling complexes that is essential for neural development [7], [8]. REST and MIR-9 are also implicated in brain/mental disorders, for example drug addiction, Alzheimer's disease, epilepsy and Huntington's disease [9].

In the present study, we show that PDYN is targeted by REST in human neuroblastoma SH-SY5Y cells and that interfering with REST activity by means of ectopic expression of dominant negative REST or MIR-9 increases PDYN expression in these cells. We further show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Our results suggest that REST represses PDYN expression in SH-SY5Y cells and the adult human brain and may have implications for mental health and brain/mental disorders.

Section snippets

Human subjects

This study was approved by Stockholm's ethic vetting board. Postmortem samples from the prefrontal cortex, Bordmann's area 9 of 30 adult subjects were obtained from the New South Wales Tissue Resource Center. Samples were collected by qualified pathologists under full ethical clearance and with informed, written consent from the next of kin. A demographic and clinical data table is given in Table S1.

Plasmids

REEX1 contains the entire REST coding sequence (aa 1-1097) [5]. DN REST p73 contains a partial

PDYN is targeted by REST in a number of human cells including neurons derived from embryonic stem cells

Screening of ChIP-Seq data on 161 transcription factors from 91 cell lines generated by ENCODE revealed that PDYN (gene plus 100 kilo base pairs upstream of the transcription start site) is targeted by at least one of 48 transcription factors/chromatin modifiers per cell line [2], [3]. The most compelling binding evidence is for REST as determined by signal intensity, motif score and number of cell lines.

PDYN contains two REST target sites/RE1s; both of which were identified prior to ENCODE [21]

Discussion

In the present study, we show that PDYN is targeted by REST in SH-SY5Y cells and that interfering with REST activity increases PDYN expression in these cells. We also show that REST binding to PDYN is reduced in the adult human brain compared to SH-SY5Y cells, which coincides with higher PDYN expression. This may be related to MIR-9 mediated down-regulation of REST as suggested by a strong inverse correlation between REST and MIR-9 expression. Combined, our results suggest that REST represses

Conflict of interest

The authors declare no conflict of interest.

Acknowledgements

This study is dedicated to Toni S. Shippenberg, a devoted mentor and outstanding scientist. Thanks to: 1) doctors Noel Buckley, Elena Cattaneo, Johan Franck, Therese Garrick, Clive Harper, Donna Sheedy, Ranjan Sen, Tatiana Yakovleva and Chiara Zuccato for sharing their expertise; 2) doctors David J. Anderson, Elisa Caffarelli, William J. Freed, Gail Mandel, Raja Jothi, Michael J. Pazin, Igor Ponomarev, Romano Regazzi, Peisu Zhang and Keji Zhao for sharing their material and/or data; and 3) our

References (37)

  • H.A. Tejeda et al.

    The dynorphin/kappa-opioid receptor system and its role in psychiatric disorders

    Cell. Mol. Life Sci.

    (2012)
  • M.B. Gerstein et al.

    Architecture of the human regulatory network derived from ENCODE data

    Nature

    (2012)
  • J. Wang et al.

    Sequence features and chromatin structure around the genomic regions bound by 119 human transcription factors

    Genome Res.

    (2012)
  • C.J. Schoenherr et al.

    The neuron-restrictive silencer factor (NRSF): a coordinate repressor of multiple neuron-specific genes

    Science

    (1995)
  • A.N. Packer et al.

    The bifunctional microRNA miR-9/miR-9* regulates REST and CoREST and is downregulated in Huntington's disease

    J. Neurosci.

    (2008)
  • A.S. Yoo et al.

    MicroRNA-mediated switching of chromatin-remodelling complexes in neural development

    Nature

    (2009)
  • L. Ooi et al.

    Chromatin crosstalk in development and disease: lessons from REST

    Nat. Rev. Genet.

    (2007)
  • J. Tapia-Ramirez et al.

    A single zinc finger motif in the silencing factor REST represses the neural-specific type II sodium channel promoter

    Proc. Natl. Acad. Sci. U. S. A.

    (1997)
  • Cited by (15)

    • Endogenous opiates and behavior: 2014

      2016, Peptides
      Citation Excerpt :

      DYN A binding was directly detected in the second extracellular loop of the KOR using a soluble protein scaffold [56]. PDYN is targeted by REST in adult human brain neuroblastoma cells [291]. DYN up-regulation in the dentate granule cell mossy fiber pathway following chronic inhibition of GluN2B-containing NMDAR was associated with increased CREB (Ser 133) phosphorylation, but was independent of BDNF/TrkB signaling pathways [836].

    View all citing articles on Scopus
    1

    These authors contributed equally to this work.

    View full text