Identification of a functional NRSE site in human DYRK1A promoter. A, schematic diagram of the human DYRK1A promoter deletion constructs consisting of a 5′-flanking region with serial deletions cloned into pGL3-Basic in front of the luciferase gene (Luc). Arrowheads indicate the directions of transcription. The numbers represent the end points of each construct. The positions of three putative NRSE sites are shown at the bottom. B, the deletion plasmids were confirmed by sequencing and restriction enzyme digestion checking on a 1.5% agarose gel. Vector size is 4.7 kb, and the DYRK1A gene 5′-flanking fragment inserts range from 0.5 to 1.5 kb. C, the deletion plasmids were transfected into REST-competent C6 or REST-incompetent PC12 cells. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 5). *, p < 0.001 by Student's t test. D, PC12 cells were co-transfected with REST expression vector and various DYRK1A promoter deletion constructs. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 5). *, p < 0.001 by Student's t test. E, pDYlucm was made to contain the mutant NRES site of DYRK1A promoter. The mutant plasmid, as well as the wild type pDYluc-A, was co-transfected with REST expression plasmid into HEK293 cells. Plasmid pRL-TK was used to normalize the transfection efficiency, and luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. F and G, EMSA was performed with IRDye 700-labeled consensus NRSE oligonucleotides (F) or NRSE site of DYRK1A promoter (G). First lane, labeled probe without nuclear extract. Second lane, incubation of infrared labeled NRSE probe with HeLa nuclear extracts retarded the migration rate of the labeled probe, which formed a new shifted DNA-protein complex band. The addition of anti-REST antibody further shifted the band to a larger mass shown by a longer running time (third lane in the lower panel of G). Competition assays were performed by further adding different concentrations of molar excess of unlabeled competitive oligonucleotides. H, anti-REST antibody was used to immunoprecipitate the cross-linked REST-DNA complex in ChIP assay in SH-SY5Y cells. A pair of primers targeting DYRK1A was used to amplify NRSE-DY1 and NRSE-DY3. Signals amplified from input were used as size markers. Mouse IgG was used as a negative control. Con, control.

REST expression is down-regulated by DYRK1A protein imbalance. A, psi-DY (siRNA against DYRK1A) was co-transfected with DYRK1A expression plasmid into N2a cells. DYRK1A was detected by anti-FLAG (M2) antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p < 0.0001 by Student's t test. B, N2a cells were transfected with DYRK1A expression vector pDYRK1A or knockdown vector psi-DY. Rest mRNA expression was detected by RT-PCR. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.0001 by Student's t test. C, N2a cells were co-transfected with REST expression vector and pDYRK1A or psi-DY. The cell lysates were separated with 12% SDS-PAGE. REST expression was detected by anti-REST antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. D, HEK293 cells co-transfected with REST expression vector, pUbi-His, and pDYRK1A or psi-DY were chased with 50 μg/ml cycloheximide (CHX) for 1 and 2 h. REST expression was detected by anti-REST antibody. β-Actin was used as loading control. E, HEK293 cells co-transfected with REST expression vector and pDYRK1A or psi-DY were lysed and immunoprecipitated with anti-ubiquitin antibody (1:1000) and detected with anti-REST antibody. The values represent the means ± S.E. (n = 3). *, p < 0.01 by Student's t test. F, N2a cells were co-transfected with C terminus REST truncated mutant, FLAG-tagged REST-FS, together with pDYRK1A or psi-DY. REST-FS protein was detected by anti-FLAG (M2) antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). p > 0.05 by Student's t test. Con, control; IP, immunoprecipitation.

Schematic diagram of the cross-interaction of REST and DYRK1A. The imbalance of DYRK1A expression in cells increases the phosphorylation and subsequent ubiquitination of REST protein. Ubiquitinated REST can be degraded by proteasome, which results in the weaker transcription of DYRK1A via a NRSE site located at DYRK1A gene promoter. Thus, a negative feedback loop is formed in DYRK1A gene regulation.

REST activates human DYRK1A gene transcription. A, pDYluc-A contains the functional promoter of DYRK1A gene. The promoter construct pDYluc-A, containing the 1127-bp fragment from the human DYRK1A gene 5′-UTR, was transfected into HEK293 cells. Luciferase activity was measured 24 h after transfection by a luminometer. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. B, DYRK1A gene promoter is activated by REST. REST was co-transfected with pDYluc-A into HEK293 cells. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. C, RT-PCR showed that REST knockdown construct psiREST can knock down Rest mRNA expression. β-Actin was used as the internal control. The values represent the means ± S.E. (n = 3). *, p = 0.0006 by Student's t test. D, the knockdown effect of psiREST was verified in protein level by Western blot. HEK293 cells were co-transfected with REST expression plasmid and psiREST. REST was detected with anti-REST antibody. β-Actin was used as loading control. The values represent the means ± S.E. (n = 3). *, p = 0.04 by Student's t test. E, RT-PCR showed that BDNF mRNA was repressed by REST overexpression in HEK293 cells. β-Actin was amplified as the internal control. *, p < 0.001 by Student's t test. F, RT-PCR showed that REST overexpression significantly increased DYRK1A mRNA, whereas REST knockdown significantly decreased DYRK1A mRNA levels. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. G, real time PCR showed that DYRK1A expression was elevated with REST overexpression. TaqMan gene expression assay of DYRK1A was performed with an ABI 7900HT real time PCR system. 18 S rRNA was chosen for the internal control. The values represent the means ± S.E. (n = 4). *, p = 0.0032 by Student's t test. Con, control.

DYRK1A and REST coordinately expressed in mouse brain during neurodevelopment and in cell lines. A and B, immunofluorescence staining was performed on coronal slices from adult mouse brain. A Cy3-conjugated anti-rabbit secondary antibody was used to visualize DYRK1A (A1 and B1, red) and REST (A4 and B5, red). MAP2 (microtubule-associated protein 2) was stained to serve as a neuronal marker. A fluorescein-conjugated anti-mouse secondary antibody was used to visualize MAP2 (B2 and B6, green). The nuclei were counterstained with blue DAPI (A2, A5, B3, and B7). Overlay showed that both DYRK1A and REST are enriched in neurons in hippocampus (A3 and A6) and cerebral cortex (B4 and B8). The images were captured with a Leica fluorescence microscope at ×400 (A) or ×600 (B). C, quantitative RT-PCR was performed on cDNA templates prepared from normal mouse brain aging at embryonic days 13 (E13) and 17 (E17); at P1, P7, P14, and P30; and in adult mice. One to three mice were used in each time point as indicated by the numbers after the hyphens. p = 0.0028 by correlation test. Rest (D) and Dyrk1a (E) mRNA levels were determined by RT-PCR in C6, N2A, and PC12 cells. β-Actin was amplified as the internal control. F, quantification of D and E showed that Rest and Dyrk1a were coordinately expressed at low levels in PC12 cells and at high levels in N2a and C6 cells. The values represent the means ± S.E. (n = 3). *, p = 0.0002 by correlation test.

Transcriptional activity of REST was reduced by DYRK1A protein imbalance. A and B, EMSA showed that REST activity was perturbed by DYRK1A imbalance. N2a cells were transfected with pDYRK1A and psi-DY (A) or co-transfected with REST expression vector (B). EMSA was performed with IRDye 700-labeled consensus NRSE. Nuclear extracts were obtained from N2a cells transfected with pDYRK1A (lane 2), psi-DY (lane 3), or empty vector (lane 4). The specificity of REST-NRSE binding was indicated by competition by consensus NRSE oligonucleotides (lane 5) or NRSE mutant oligonucleotides (lane 6). C and D, N2a cells were transfected with DYRK1A expression vector pDYRK1A or knockdown vector psi-DY. Bdnf mRNA levels were detected by RT-PCR. β-Actin was amplified as the internal control. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. E, the BDNF promoter construct pBDNFluc was co-transfected with pDYRK1A or psi-DY. Luciferase assay was performed 24 h after transfection. The values represent the means ± S.E. (n = 3). *, p < 0.001 by Student's t test. F, the DYRK1A promoter construct pDYluc-A was co-transfected with pDYRK1A or psi-DY into N2a cells. Luciferase activity was measured at 24 h by a luminometer. The values represent the means ± S.E. (n = 4). *, p < 0.001 by Student's t test. Con, control.