Biochimica et Biophysica Acta (BBA) - Gene Regulatory Mechanisms
PDYN, a gene implicated in brain/mental disorders, is targeted by REST in the adult human brain
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
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These authors contributed equally to this work.