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

Keywords: MIR-9; NRSF; PDYN; Prefrontal cortex; REST; SH-SY5Y cell.

Figure 1.. PDYN/NM_024411 viewed in the University of California, Santa Cruz genome browser [34].

Shown are: 1) the single nucleotide polymorphism, rs910080 [35]; 3) annotated, experimentally validated cis-regulatory regions corresponding to the REST target sites/RE1s upstream of PDYN (OREG0004403) and in the 3’-untranslated region (3’-UTR) of this gene (OREG0007105) [36]; 4) single base pair resolution images of these sites and the corresponding regions in chimp, mouse and rat [37]; 5) genome-wide chromatin immunoprecipitation (ChIP)-sequencing data on REST in human embryonic stem cells (green) and neurons derived from these cells (black) and the corresponding control/reverse cross-linked chromatin data as tracks [2, 3]; and 6) the positions of the ChIP primers used in this study as inserts.

Figure 2.. REST and MIR-9 in transcriptional control of PDYN in human neuroblastoma SH-SY5Y cells.

a. Effects of ectopic expression of MIR-9 or dominant negative REST on REST binding to the RE1s in PDYN determined using ChIP. Results obtained with an intronic sequence in PDYN not containing an RE1 and an unspecific IgG are shown for comparison. b. Effects of ectopic expression of MIR-9 or dominant negative REST on PDYN expression determined using real-time polymerase chain reaction (PCR). c. Effects of ectopic expression of dominant negative REST or MIR-9 on REST and MIR-9 expression determined using real-time PCR and western blotting, respectively. Top: blot incubated with 17-641. Bottom left: MIR-9. Bottom right: REST, N = 6/group. Mock transfected cells = cells transfected with transfection agent alone; pSup-miR-9 = cells transfected with pSup-miR-9; DN REST p73 = cells transfected with DN REST p73; REEX1 = cells transfected with REEX1; REST Ab = 07-579; IgG = PP64B; and internal control = total extract from control cells. N = 3/group unless otherwise stated. Horizontal bars represent group means and standard error of the mean. * = P < 0.05; ** = P < 0.01; *** = P < 0.001.

Figure 3.. REST and MIR-9 in transcriptional control of PDYN in the adult human brain.

a. REST binding to the RE1s in PDYN in the prefrontal cortex determined using ChIP. N = 2 pooled samples from 3 men each. b. Correlation between REST and MIR-9 expression in the prefrontal cortex determined using real-time PCR and western blotting, respectively. Internal control = pooled extract from prefrontal cortex of all subjects; 0 = subject number 0; and U = unspecific. N = 29. Subject 26 was omitted from the statistical analysis as it was identified as an outlier (Grubb’s test, Z = 3.6, P < 0.05).