Rest Regulates DYRK1A Transcription In A Negative Feedback Loop

Backgrounds and objectiveAlzheimer's disease (AD) is the most common reason for dementia, with5%incidence among seniors older than65years. The incidence doubles every5years. The characteristic pathology of AD include neurofibrillary tangles, the major component of which is insoluble hyperphosphorylated tau protein, and senile plaque, the major component of which is insoluble Aβ deposits. It is found that dual-specificity tyrosine-(Y)-phosphorylation regulated kinase1A (DYRK1A) is highly expressed in the hippocampus of AD patients. DYRK1A is the homolog of Drosophila Minibrain kinase (MNB) in human, and plays important roles in growth, development, and neurogenesis. DYRK1A overexpression is thought to be necessary for the pathogenesis of AD as it participates the hyperphosphorylation of APP, the formation of tau protein, and the catalyzation of APP to enhance the production of intracellular Aβ. In addition, by phosphorylating the substrates containing RPX(S/T)P consensus amino acid, DYRK1A involves in various pathologic conditions. As DYRK1A has gene-dosage effects, it is meaningful to study the regulation of DYRK1A transcription.RE-1silencer of transcription factor (REST; also known as neuron-restrictive silencing factor, NRSF) is mainly expressed in neuron precursor cells and non-neuronal cells, and can regulate the transcription of target genes. But recent researches reveal that REST is also highly expressed in central nervous system, especially in hippocampus. The co-localization of DYRK1A and REST in hippocampus indicates the potential interaction between the two proteins.The role of DYRKIA in the pathogenesis of AD is extensively explored, and the most concerned is the effects of DYRKIA phophorylation to the production of tau protein and Aβ. It has been found that REST mRNA can be reduced by DYRKIA dosage imbalance, while the influence of DYRKIA dosage imbalance to DYRKIA protein and the underlying mechanisms have not been revealed. There are only two papers concerning the regulation of DYRKIA gene, but there is no research on the regulation of REST to DYRKIA gene. Both overexpression and knockdown of DYRKIA are hazardous to the human body as to the dosage effects of DYRKIA.By studying the transcriptional regulation of DYRKIA by REST and the feedback effects of DYRKIA dosage imbalance to REST mRNA and protein, we try to further understand the how DYRKIA and REST are involved in the pathogenesis of AD, and thus may provide evidence for new therapeutic approaches.Materials and methods1. Cell culture:HEK293, C6, PC12and N2a were cultured and used for subsequent experiments.2. Detect the regulation of REST to human DYRKIA gene transcription. The promoter region of the human DYRKIA gene obtained by PCR was cloned into the promoter-less plasmid pGL3-Basic with proper restriction enzyme sites; the promoter construct was transfected into HEK293cells with or without REST expression vector and the promoter activity was determined; RANi was used to construct siREST to knockdown REST expression in HEK293cells, total RNA was extracted and DYRKIA mRNA levels were determined by semi-quantitative RT-PCR analysis, BDNF mRNA was also measured to verify REST activity; the results were further confirmed in murine-derived PC12and C6cell lines; after REST overexpression in HEK293cells for48hours, DYRKIA mRNA levels were determined by Taqman real time quantitative RT-PCR analysis; the promoter construct was transfected into N2a cells with pDYRK1A or psi-DY and the luciferase activity was measured to reflect the response of DYRKIA to its dosage imbalance. 3. The determination of the functional NRSE site(s) in DYRKIA promoter region. In silico analysis of DYRKIA promoter region reveals3putative NRSE sites. Various deletion plasmids of DYRKIA promoter region and pBDNFluc, which indicates REST transcriptional activity, were transfected into REST-competent C6or REST-incompetent PC12cells alone, and the difference between luciferase activities of the deletion constructs in C6and PC12cells were measured to reflect the REST disparity and to indicate the possible positions of NRSE site(s); DYRKIA promoter deletion plasmids were transfected into PC12cells with REST expression vector to verify activity differences among the promoters in C6and PC12cells; EMS A and ChIP were used to confirm in vitro and in vivo the functional NRSE site(s) found in the above experiments.4. Detect the expression pattern of DYRKIA and REST in normal adult C57BL/6mouse brain, during neurodevelopment and in different cell lines. The expression pattern of DYRKIA and REST proteins in neurons of hippocampus and cerebral cortex of adult mouse brain was detected by immunofluoresecnce; the mRNA levels of Dyrkla and Rest from normal mouse brain aging at embryonic day13(E13), E17, postnatal day1(P1), P7, P14, and P30were determined by real time quantification RT-PCR (qRT-PCR); the mRNA levels of Dyrk1a and Rest in PC12,C6,N2cells were measured by semi-quantitative RT-PCR.5. Detect the influence of DYRKIA dosage imbalance to REST expression and to reveal the possible underlying mechanisms. SiRNA against mouse and human DYRKIA (psi-DY) was constructed and verified in N2a and HEK293cells; DYRKIA expression vector (pDYRK1A) and psi-DY were transfected into N2a cells, total RNA was extracted at48hours and endogenous Rest mRNA levels were determined using semi-quantitative RT-PCR; REST expression vector together with pDYRKIA or psi-DY were co-transfected into HEK293cells and then REST protein levels were determined by western blotting; after chased with the protein synthesis inhibitor cycloheximide for1or2hours, the degradation rate of proteins in HEK293cells transfected with both pDYRK1A and psi-DY were determined to find the relationship between REST protein degradation and DYRKIA dosage imbalance. Because the degradation of REST protein is associated with the ubiquitination of its C-terminus, we transfected ubiquitin expression vector, REST expression vector and pDYRKIA or psi-DY into HEK293cells; anti-Ubi antibody was used as the immunoprecipitation antibody and anti-REST antibody was used for subsequent western blotting analysis to detect the influence of DYRK1A dosage imbalance to the ubiquitination of REST protein. REST truncated mutant, REST-FS, which lacks the C terminus, together with pDYRK1A or psi-DY were transfected into HEK293cells to verify the above results.6. Detect how the transcriptional activity of REST was influenced by DYRK1A dosage imbalance. BDNF, the well-known target gene of REST, was used to reflect the transcriptional activity of REST. When pDYRK1A or psi-DY was transfected into mouse N2a cells, the mRNA level and luciferase activity of BDNF were measured; EMSA was performed on N2a cell nuclear extract transfected with pDYRK1A, psi-DY, or empty vector alone or with REST expression plasmid to detect binding ability of REST protein to its target sequence in nuclear extracts disposed with pDYRK1A or psi-DY.Results1. REST can activate human DYRK1A gene transcription. The promoter region of the human DYRK1A gene obtained by PCR was cloned into the promoter-less plasmid pGL3-Basic and was transfected into HEK293cells. The activity of the promoter was significantly higher than pGL3-Basic and can be remarkably activated by REST; contrary to the reduction of BDNF mRNA, REST overexpression increased DYRK1A mRNA, while REST knockdown significantly reduced DYRK1A mRNA as confirmed by semi-quantative RT-PCR analysis; the results were further confirmed in murine-derived PC12and C6cell lines. Real time quantification RT-PCR (qRT-PCR) also showed the significant increase of DYRK1A mRNA by REST overexpression; the promoter activity of DYRK1A gene reduced both DYRK1A overexpression and knockdown.2.-833to-815bp in human DYRK1A promoter region may be a functional NRSE site that is responsive to REST overexpression. The positions of three putative NRSE sites are:-833to-815bp corresponds to the first putative NRSE sites (NRSE-DY1),-644to-626bp corresponds to the second putative NRSE sites (NRSE-DY2), and-550to-534bp corresponds to the third putative NRSE sites (NRSE-DY3). Luciferase assay showed that pBDNFluc, containing a repressing NRSE site in the BDNF promoter upstream of luciferase gene, had higher promoter activity in PC12cells compared to C6cells; DYRK1A promoter constructs containing-833to-815bp had higher promoter activity in PC12cells than in C6cells, while further deletion abolished the differential effect of C6and PC12cell lines, indicating the NRSE site was located in the region of-833to-815bp; PC12cells co-transfected with DYRK1A deletion constructs and REST expression vector also showed that REST expression can decrease the promoter activity of BDNFluc and increase the promoter activity of DYRK1A deletion constructs containing the NRSE site from-833to-815bp; the results from electrophoretic mobility shift assay (EMSA) and chromatin immunoprecipitation (ChIP) also revealed that it is the first NRSE site, corresponding to DYRK1A promoter-833to-815bp, that actually responsible for the up-regulation of DYRK1A gene by REST.3. DYRK1A and REST coordinately expressed in mouse brain, during neurodevelopment and in cell lines. Immunofluoresecnce revealed that DYRK1A and REST proteins coordinately expressed in neurons of hippocampus and cerebral cortex of adult mouse brain; Real time quantification RT-PCR (qRT-PCR) performed on cDNA templates prepared from normal mouse brain aging at embryonic day13(E13), E17, postnatal day1(P1), P7, P14, and P30revealed that mRNA levels of Dyrk1a and Rest were expressed with significant correlation during neurodevelopment, with both showed increased expression at P7to P14and then decreased at P30but still detectable; strong Dyrkla signals were detected in C6and N2a cell lines which have relatively much more Rest mRNA, while Dyrkla expression is nearly undetectable in PC12cell line which shows very low levels of Rest expression.4. REST expression is down-regulated by DYRK1A protein imbalance. SiRNA against mouse Dyrkl a (psi-DY) was constructed and verified in N2a cells. Both the transfection of DYRKIA expression vector and psi-DY into N2a cells significantly reduced Rest mRNA expression; the co-transfection of REST expression vector with pDYRK1A and psi-DY showed that both DYRKIA overexpression and knockdown significantly reduced REST protein levels; after chased with the protein synthesis inhibitor cycloheximide for1or2hours, the proteins in HEK293cells transfected with both pDYRK1A and psi-DY degraded much more quickly than control; using anti-Ubi antibody as the immunoprecipitation antibody, we found that both DYRKIA overexpression and knockdown significantly increased the ubiquitination of REST when REST expression vector was cotransfected with ubiquitin expression construct and pDYRK1A or psi-DY into HEK293cells; while the co-transfection of REST truncated mutant, REST-FS, which lacks the C terminus, together with pDYRK1A or psi-DY did not caused the reduction of REST protein.5. Transcriptional activity of REST was reduced by DYRKIA imbalance. The mRNA level of Bdnf was elevated in mouse N2a cells transfected with pDYRK1A or psi-DY compared with controls; the promoter activity of BDNF was also significantly elevated by overexpression or knockdown of DYRKIA; EMSA performed on N2a cell nuclear extract transfected with pDYRK1A, psi-DY, or empty vector alone or with REST expression plasmid showed REST protein had significantly lower binding ability to nuclear extracts disposed with pDYRK1A or psi-DY.Conclusions1. The transcription of human DYRKIA gene can be activated by REST.2. The first NRSE site, corresponding to DYRKIA promoter-833to-815bp, is responsible for the up-regulation of DYRKIA gene by REST.3. Dyrk1a and Rest coordinately expressed in adult mouse brain, during neurodevelopment and in PC12, C6and N2a cell lines.4. REST expression is down-regulated by DYRKIA protein imbalance.5. Transcriptional activity of REST was reduced by DYRKIA imbalance.