DYRK1A Gene Transcription Is Regulated By MEF2D

Background:Down syndrome(DS),also known as trisomy 21 syndrome,is a genetic disorder caused by the presence of all or part of a third copy of chromosome 21,It is typically associated with physical growth delays,characteristic facial features and mild to moderate intellectual disability.DS is one of the most common chromosome abnormalities in newborns,accounting about 10 per 10,000 live births and could not be cured.Dual-specificity tyrosine--phosphorylation regulated kinase 1A(DYRK1A),localized in the Down syndrome critical region of chromosome 21,encodes a member of the Dual-specificity tyrosine phosphorylation-regulated kinase(DYRK)family.DYRK1A contains 5 main regions:a bipartite nuclear localization signal(NLS)at N’-terminal,a protein kinase domain,a Pest region(rich in proline,glutamic acid,serine and threonine),and a highly conservative 13-consecutive-histidine repeat,a serine/threonine-rich sequence.DYRK1A catalyzes its autophosphorylation on serine/threonine and tyrosine residues and may play a significant role in a signaling pathway regulating cell proliferation and may be involved in brain development.DYRK1A is considered to be a strong candidate gene for intellectual disability related to DS.DYRK1A was first discovered as minibrain(mnb)gene and could cause an abnormal spacing of neuroblasts in the outer proliferation center of larval brain in Drosophila.This result suggested it may be involved in regulation of neurodevelopment.Several mice models also showed that DYRK1A knock-in could lead to neurodevelopmental delay,motor abnormalities,mental retardation,learning and memory deficit and reduced neuronal density.Overexpression of Dyrkla might inhibit neural cell proliferation and promote premature neuronal differentiation in the developing cerebral cortex without affecting cell fate and layer positioning,and cause the defects in synaptic vesicle endocytosis as well.Therefore,investigations for regulation of DYRK1A will contribute to elucidating mechanism of neurodegenerative disease.Myocyte-specific enhance factor 2(MEF2)family members are involved in control of not only muscle but also neuronal cell differentiation and neurodevelopment.Research has revealed that MEF2 was selectively expressed in newly generated postmitotic neurons and was required for the survival of these neurons.As a member of MEF2 family of transcription factors,MEF2D has been reported to be crucial for neurogenesis,neuronal differentiation and survival.Regulation of DYRK1A by MEF2D and interaction between them in glioblastoma cells were investigated for the first time.Hence,our research of regulatory mechanism between DYRK1A and MEF2D will be devoted to further elucidation of pathogenesis for neurodegenerative disease.Section IThe regulatory mechanisms of DYRK1A gene transcription by MEF2DObjectiveThe aim of this study was to investigate whether expression of DYRK1A different isoforms was regulated by MEF2D in glioblastoma cell lines.The underlying molecular mechanism of regulation of DYRK1A by MEF2D will be elucidated.Our original research will contribute to understanding function of both DYRK1A and MEF2D in neurodevelopment.Materials and methods1.MEF2D regulates DYRK1A gene transcription in glioblastoma cell line T98G.pCMV6-entry-MEF2D expression vector was transfected into T98G cells by LipofectamineTM2000.Empty vector was used as negative control.Cells were harvested in 48 hours.RNA was isolated by TRIzol reagent.Reverse transcription was performed with PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect Real Time).RT-PCR was used to quantify DYRK1A mRNA expression directly.The expression of DYRK1A mRNA was further quantified by TOYOBO SYBR Green gene Expression Analysis kit.2.MEF2D specifically activates DYRK1A Isoform 5 promoter.2.1 Constructs of truncations of DYRK1A promoter region:DYRK1A promoter region was amplified from human genome DNA by PCR.PCR product was cloned into pGL3-basic to generate pDYluc-long;2.2 Effect of MEF2D overexpression and knock-down.MEF2D expression construct or si-MEF2D was transfected into T98G cells by LipofectamineTM2000.Empty vector or si-CON was used as negative control.Cells were harvested and lysed in 48 hours.Western Blot was performed to examine MEF2D and DYRK1A expression at protein levels.β-ACTIN was used as internal control;2.3 pCMV6-entry-MEF2D expression vector and negative controls were transfected into HEK293 cells with pDYluc-long and pGL3-Basic vectors,respectively.Cells were harvested and lysed with Passive lysis buffer in 48 hours.Luciferase assay was performed to detect promoter activity.3.Identification and confirmation of the functional MEF2D response element on DYRK1A promoter region.3.1 Prediction of MEF2D response element on DYRK1A promoter region:the promoter sequence was analyzed at http://jaspar.genereg.net/.MEF2D specific binding sites were output in a list by score;3.2 Truncations of DYRK1A promoter were constructed into pGL3-Basic.Truncations and pGL3-Basic were transfected into HEK293 with pCMV6-entry-MEF2D and control vectors,respectively.Cells were harvested and lysed in 48 hours.Luciferase assay was performed to detect promoter activities of different truncations compared with pGL3-Basic.Functional MEF2D response element(DY-MRE)was identified in DYRK1A promoter region;3.3 pDYluc-MRE and pDYluc-MRE mutant containing putative MEF2D response element and mutant form were constructed into pGL3-Basic.Luciferase assay was achieved to confirm the influence to promoter activity by MEF2D;3.4 Probes containing putative DY-MRE were synthesized and labeled with IRDye700.Electrophoretic mobility shift assay(EMSA)was performed to investigate the specific binding of putative DY-MRE and MEF2D in vitro.Chromatin immunoprecipitation(ChIP)was achieved to further prove the binding specificity.Immunoprecipitation and western blotting were used to guarantee that anti-MEF2D antibody did pull-down MEF2D protein.4.To investigate the expression pattern of DYRK1A and MEF2D in normal adult C57BL/6 mice brains during neurodevelopment.RNA was isolated from normal mice brain aging at embryonic day 13.5(E13.5),18(E18),postnatal dayl(P1),7(P7),14(P14)and adult by TRIzol reagent.Reverse transcription was performed with PrimeScriptTM RT reagent Kit with gDNA Eraser(Perfect Real Time).The mRNA expressions of total DYRK1A and MEF2D in these samples were quantified by TOYOBO R SYBR Green gene Expression Analysis kit.5.To verify the effect of MEF2D to DYRK1A in glioblastoma T98 cells.pNFATc2 expression vector was transfected with pCMV6-entry-MEF2D and control vector into T98G cells,respectively.Cells were harvested in 48 hours.Western blot was performed to detect the influence of elevated DYRK1A to NFATc2 expression with MEF2D overexpression.As positive control,pNFATc2 expression vector was transfected with pCMV6-entry-DYRK1A and control vector,respectively.6.Statistical analysis:statistical analysis was processed by SPSS software(version 17.0).All of the experiments were repeated more than three times.Student’s t test and correlation analysis were used to determine significance between groups.Differences with P-values of less than 0.05 were considered statistically significant.Results1.MEF2D specifically upregulates total DYRK1A mRNA and DYRK1A isoform 5 expression,but have no influence on DYRK1A isoform 2 and 3 mRNA expression.Protein expression of total DYRK1A and DYRK1A isoform 5 could also be detected in both T98G cells and HEK293 cells as well.2.MEF2D increases DYRK1A mRNA expression by enhancing DYRK1A isoform 5 promoter activity.2.1 DYRK1A promoter activity was elevated by MEF2D overexpression;2.2 DYRK1A promoter activity was lowered by MEF2D knock-down.3.Identification of functional MEF2D binding site DY-MRE on DYRK1A promoter region.3.1 Sequence of DYRK1A promoter region was analyzed on Jaspar website(http://jaspar.genereg.net/)and putative MEF2D binding sites were searched out and listed by scores;3.2 Truncations of DYRK1A promoter were constructed into pGL3-basic vectors.Luciferase assay was performed to detect promoter activities of different truncations with MEF2D overexpression.Results showed that truncation vector covering-442～-254 bp was significantly upregulated in promoter activity by MEF2D.Jaspar sequence analysis indicated the existence of a funcational MEF2D binding site at-268～-254 bp;3.3 Vector containing-268～-254 bp was constructed.Meanwhile key bases in this sequence were mutated to generate the corresponding mutant form.Luciferase assay was achieved to further prove the elevation of promoter activity by MEF2D at this region.But promoter activity of mutant form could not be increased by MEF2D;3.4 EMSA and ChIP were performed to further confirm the functionel DY-MRE site located at-268～-254 bp.4.mRNA levels of MEF2D and total DYRK1A were coordinately expressed with significant positive correlation during neurodevelopment(p=0.0478,r=0.6182 by Spearman correlation).These results suggested a possible interaction between DYRK1A and MEF2D in neurodevelopment.5.NFATc2 protein expression was remarkably decreased by elevated DYRK1A expression through MEF2D overexpression.Conclusion1.MEF2D specifically upregulates DYRK1A isoform 5 expression.2.MEF2D specifically binds at-268～-254 bp at DYRK1A promoter region and activates DYRK1A transcriptional activity.3.MEF2D and total DYRK1A were coordinately expressed with significant positive correlation during neurodevelopment in mouse brain.4.MEF2D decreases NFATc2 protein expression by upregulation to DYRK1A expression in glioblastoma cell line.Section ⅡDYRK1A phosphorylates MEF2D and decreases its transcriptional activityObjectiveThe aim of this part was to investigate whether DYRK1A could phosphorylate MEF2D in HEK293 cell lines and influence its function.The underlying molecular mechanism of regulation of MEF2D by DYRK1A will be elucidated.Our original research will contribute to understanding interaction between DYRK1A and MEF2D in neurodevelopment.Materials and methods1.To detect the influence to MEF2D by DYRK1A in HEK293 cells.pCMV6-entry-MEF2D was transfected into HEK293 cells with pCMV6-entry-DYRK1A and control vector,respectively.Cells were harvested by centrifuging in 48 hours and lysed by sonication in western and IP lysis buffer at 4℃.Western Blotting was used to evaluate the expression of MEF2D by DYRK1A compared with control.2.Phosphorylation site of MEF2D by DYRK1A was determined by MS.2.1 MEF2D expression vector for in vitro purification was constructed.MEF2D coding sequence containing 521aa was inserted into pET32a(+)vector.pET32a-MEF2D vector was obtained and sent for sequencing;2.2 pET32a-MEF2D vector was transformed into BL21 competent cells for production.ITPG was used to induce protein expression over night.Cells were harvested for Ni-NTA filter purification;2.3 Purified protein was quantitated by RC DCTM Protein Assay(BIO-RAD)and testified by western blotting for validation;2.4 Purified MEF2D recombinant protein was applied for in vitro phosphorylation assay by DYRK1A recombinant protein(Thermofisher).Processed proteins were loaded on SDS-PAGE to be separated from each other.Protein gel was stained by coomassie brilliant blue R250 after electrophoresis.Specific protein band was carefully cut out after being properly destained and sent for MS analysis;2.5 MS analysis was completed by company and results were given back.3.MEF2D transcriptional activity was decreased by DYRK1A3.1 Multi-MEF2D specific responsive element was cloned into pGL3-Basic vector to generate p3xMRE construct;3.2 pCMV6-entry-DYRK1A、pCMV6-entry-DYRK1A(K188R)which is the kinase dead version of DYRK1A and control vector were transfected by LipofectamineTM2000 into HEK293 with pGL3-Basic or p3×MRE vector,respectively;3.3 Cells were harvested in 48 hours and lysed by Passive Lysis Buffer and applied for luciferase assay analysis.4.Degradation of MEF2D was influenced by DYRK1A4.1 DYRK1A expression vector and control vector were transfected into HEK293 with MEF2D expression vector by LipofectamineTM2000.Cells were devided into three 35 mm cell culture plates for each group 4 hours after transfection;4.2 NH4Cl was added into cells 24 hours after transfection with concentration of 10 mM and 20 mM.H2O was used as control.NH4Cl was maintained for 24 hours;4.3 Cells were harvested in 24 hours after NH4Cl was added.Western blotting was used to evaluate the degradation of MEF2D by DYRK1 A.5.Statistical analysis:statistical analysis was processed by SPSS software(version 17.0).All of the experiments were repeated more than three times.Student’s t test and correlation analysis were used to determine significance between groups.Differences with P-values of less than 0.05 were considered statistically significant.Results1.DYRK1A overexpression increased exogenous MEF2D protein expression and chaned its molecular weight by possible chemical modification;2.MEF2D recombinant protein was successfully purified and in vitro phosphorylated in presense of DYRK1A.MEF2D Ser-251 was determined by MS analysis;3.Luciferase Assay revealed that MEF2D could significantly elevate p3×MRE activity compared with control,which proved the effectiveness of p3×MRE.DYRK1A markedly decreased p3×MRE activity rather than DYRK1A(K188R).4.Degradation of MEF2D was alleviated by DYRK1 A.Conclusion1.DYRK1A phosphorylates MEF2D and elevated MEF2D expression;2.MS analysis revealed that MEF2D Ser-251 was phosphorylated by DYRK1 A;3.Transcriptional activity of MEF2D was decreased by DYRK1A.4.Degradation of MEF2D was suppressed by DYRK1A.