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• W2047916240 abstract "Loss-of-function mutations in DJ-1 cause a subset of familial Parkinson disease (PD). However, the mechanism underlying the selective vulnerability in dopaminergic pathway due to the inactivation of DJ-1 is unclear. Previously, we have reported that DJ-1 is a neuroprotective transcriptional co-activator interacting with the transcriptional co-repressor pyrimidine tract-binding protein-associated splicing factor (PSF). Here we show that DJ-1 and PSF bind and regulate the human tyrosine hydroxylase (TH) promoter. Inactivation of DJ-1 by small interference RNA (siRNA) results in decreased TH expression and l-DOPA production in human dopaminergic cell lines. Consistent with its role as a transcriptional regulator, DJ-1 specifically suppresses the global SUMO-1 modification. High molecular weight sumoylated protein species, including PSF, accumulate in the lymphoblast cells from the patients carrying pathogenic DJ-1 mutations. DJ-1 elevates the TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Furthermore, siRNA silencing of DJ-1 decreases the acetylation of TH promoter-bound histones, and histone deacetylase inhibitors restore the DJ-1 siRNA-induced repression of TH. Therefore, our results suggest DJ-1 as a regulator of protein sumoylation and directly link the loss of DJ-1 expression and transcriptional dysfunction to impaired dopamine synthesis. Loss-of-function mutations in DJ-1 cause a subset of familial Parkinson disease (PD). However, the mechanism underlying the selective vulnerability in dopaminergic pathway due to the inactivation of DJ-1 is unclear. Previously, we have reported that DJ-1 is a neuroprotective transcriptional co-activator interacting with the transcriptional co-repressor pyrimidine tract-binding protein-associated splicing factor (PSF). Here we show that DJ-1 and PSF bind and regulate the human tyrosine hydroxylase (TH) promoter. Inactivation of DJ-1 by small interference RNA (siRNA) results in decreased TH expression and l-DOPA production in human dopaminergic cell lines. Consistent with its role as a transcriptional regulator, DJ-1 specifically suppresses the global SUMO-1 modification. High molecular weight sumoylated protein species, including PSF, accumulate in the lymphoblast cells from the patients carrying pathogenic DJ-1 mutations. DJ-1 elevates the TH expression by inhibiting the sumoylation of PSF and preventing its sumoylation-dependent recruitment of histone deacetylase 1. Furthermore, siRNA silencing of DJ-1 decreases the acetylation of TH promoter-bound histones, and histone deacetylase inhibitors restore the DJ-1 siRNA-induced repression of TH. Therefore, our results suggest DJ-1 as a regulator of protein sumoylation and directly link the loss of DJ-1 expression and transcriptional dysfunction to impaired dopamine synthesis. Parkinson disease (PD) 4The abbreviations used are: PD, Parkinson disease; TH, tyrosine hydroxylase; PSF, pyrimidine tract-binding protein-associated splicing factor; SUMO, small ubiquitin-like modifier; PIAS, protein inhibitors of activated STAT; STAT, signal transducers and activators of transcription; HDAC, histone deacetylase; ChIP, chromatin immunoprecipitation; siRNA, small interference RNA; DJBP, DJ-1 binding protein; E1, ubiquitin-activating enzyme; E2, ubiquitin carrier protein; E3, ubiquitin-protein isopeptide ligase; CMV, cytomegalovirus; WT, wild type; IP, immunoprecipitation; Q-PCR, quantitative PCR; DEL, exon 1–5 deletion; HA, hemagglutinin; RNAi, RNA interference. is a common progressive movement disease characterized by the selective loss of dopaminergic neurons and the decrease of striatal dopamine levels (1Lang A.E. Lozano A.M. N. Engl. J. Med. 1998; 339: 1044-1053Crossref PubMed Scopus (1795) Google Scholar). Dopamine deficiency in PD patients contributes to the typical clinical features, which include tremor, bradykinesia, rigidity, and postural instability. These symptoms can be temporally controlled by administering medications targeting dopamine metabolism and function, such as the dopamine precursor l-DOPA and dopamine agonists. The rate-limiting enzyme for dopamine synthesis is tyrosine hydroxylase (TH). Loss-of-function mutations in the DJ-1 gene cause early-on-set Parkinsonism (2Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2307) Google Scholar), although the disease-causing mechanism remains to be fully resolved. The evolutionarily conserved DJ-1 has been shown to regulate oxidative stress, apoptosis, protein aggregation, and transcription in various subcellular compartments (3Taira T. Saito Y. Niki T. Iguchi-Ariga S.M. Takahashi K. Ariga H. EMBO Rep. 2004; 5: 213-218Crossref PubMed Scopus (760) Google Scholar, 4Yokota T. Sugawara K. Ito K. Takahashi R. Ariga H. Mizusawa H. Biochem. Biophys. Res. Commun. 2003; 312: 1342-1348Crossref PubMed Scopus (331) Google Scholar, 5Martinat C. Shendelman S. Jonason A. Leete T. Beal M.F. Yang L. Floss T. Abeliovich A. PLoS Biol. 2004; 2: e327Crossref PubMed Scopus (250) Google Scholar, 6Shendelman S. Jonason A. Martinat C. Leete T. Abeliovich A. PLoS Biol. 2004; 2: e362Crossref PubMed Scopus (510) Google Scholar, 7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar, 8Junn E. Taniguchi H. Jeong B.S. Zhao X. Ichijo H. Mouradian M.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9691-9696Crossref PubMed Scopus (291) Google Scholar). In vitro experiments from several laboratories have clearly demonstrated the neuroprotective activity of DJ-1, although different molecular mechanisms have been proposed (4Yokota T. Sugawara K. Ito K. Takahashi R. Ariga H. Mizusawa H. Biochem. Biophys. Res. Commun. 2003; 312: 1342-1348Crossref PubMed Scopus (331) Google Scholar, 7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar, 8Junn E. Taniguchi H. Jeong B.S. Zhao X. Ichijo H. Mouradian M.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9691-9696Crossref PubMed Scopus (291) Google Scholar, 9Canet-Aviles R.M. Wilson M.A. Miller D.W. Ahmad R. McLendon C. Bandyopadhyay S. Baptista M.J. Ringe D. Petsko G.A. Cookson M.R. Proc. Natl. Acad. Sci. U. S. A. 2004; 101: 9103-9108Crossref PubMed Scopus (929) Google Scholar). The role of DJ-1 in neuronal survival is strengthened by in vivo studies using Drosophila lacking DJ-1, which exhibit increased sensitivity to oxidative stress or environmental mitochondrial toxins (10Yang Y. Gehrke S. Haque M.E. Imai Y. Kosek J. Yang L. Beal M.F. Nishimura I. Wakamatsu K. Ito S. Takahashi R. Lu B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13670-13675Crossref PubMed Scopus (309) Google Scholar, 11Meulener M. Whitworth A.J. Armstrong-Gold C.E. Rizzu P. Heutink P. Wes P.D. Pallanck L.J. Bonini N.M. Curr. Biol. 2005; 15: 1572-1577Abstract Full Text Full Text PDF PubMed Scopus (315) Google Scholar). In one study, inactivation of a Drosophila DJ-1 homolog by siRNA leads to the degeneration of the TH-positive dopaminergic neurons as in PD patients (10Yang Y. Gehrke S. Haque M.E. Imai Y. Kosek J. Yang L. Beal M.F. Nishimura I. Wakamatsu K. Ito S. Takahashi R. Lu B. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 13670-13675Crossref PubMed Scopus (309) Google Scholar). On the other hand, DJ-1-deficient mice do not reproduce the typical neuropathology of PD patients, such as the loss of the dopaminergic neurons and the formation of intracellular inclusion Lewy bodies, although they exhibit subtle defects in the nigral-striatal pathway and motor functions (12Goldberg M.S. Pisani A. Haburcak M. Vortherms T.A. Kitada T. Costa C. Tong Y. Martella G. Tscherter A. Martins A. Bernardi G. Roth B.L. Pothos E.N. Calabresi P. Shen J. Neuron. 2005; 45: 489-496Abstract Full Text Full Text PDF PubMed Scopus (449) Google Scholar, 13Kim R.H. Smith P.D. Aleyasin H. Hayley S. Mount M.P. Pownall S. Wakeham A. You-Ten A.J. Kalia S.K. Horne P. Westaway D. Lozano A.M. Anisman H. Park D.S. Mak T.W. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 5215-5220Crossref PubMed Scopus (598) Google Scholar, 14Chen L. Cagniard B. Mathews T. Jones S. Koh H.C. Ding Y. Carvey P.M. Ling Z. Kang U.J. Zhuang X. J. Biol. Chem. 2005; 280: 21418-21426Abstract Full Text Full Text PDF PubMed Scopus (211) Google Scholar). Patients carrying DJ-1 mutations demonstrate reduced dopamine uptake indistinguishable to patients with sporadic PD (15Dekker M.C. Eshuis S.A. Maguire R.P. Veenma-van der Duijn L. Pruim J. Snijders P.J. Oostra B.A. van Duijn C.M. Leenders K.L. J. Neural Transm. 2004; 111: 1575-1581Crossref PubMed Scopus (33) Google Scholar). Due to the lack of pathological evidence from patients with DJ-1 mutations, it remains unclear whether the loss of DJ-1 function affects both dopaminergic neuronal survival and nigral-striatal pathways in humans. Before mutations in the DJ-1 gene were linked to familial PD, DJ-1 had been suggested to regulate transcription by modulating androgen receptor function in the testis (16Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar, 17Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). DJ-1 restores the activities of androgen receptor by interacting with and antagonizing the transcriptional repressors PIASxa and DJBP (16Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar, 17Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Nevertheless, PIASxa and DJBP are mainly expressed in the testis (16Niki T. Takahashi-Niki K. Taira T. Iguchi-Ariga S.M. Ariga H. Mol. Cancer Res. 2003; 1: 247-261PubMed Google Scholar, 17Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), and whether transcriptional regulation by DJ-1 is related to PD pathogenesis is yet to be fully characterized. We have recently demonstrated that DJ-1 forms complexes with nuclear proteins pyrimidine tract-binding protein-associated splicing factor (PSF) and p54nrb (7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar), two proteins that are highly expressed in the brain and affect RNA metabolism and transcription. DJ-1 serves as a transcriptional co-activator and prevents co-repressor PSF-mediated gene silencing and apoptosis. Consistent with the role of DJ-1 in transcriptional regulation, several protein interaction studies suggest a potential functional link between DJ-1 and small ubiquitin-like modifiers (SUMO). Sumoylation is a reversible ATP-dependent process to covalently attach SUMO to target proteins, usually nuclear proteins (18Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1399) Google Scholar). Similar to ubiquitination, this process requires the participation of E1 activating enzymes, E2 conjugating enzymes, and E3 ligases (18Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1399) Google Scholar, 19Melchior F. Schergaut M. Pichler A. Trends Biochem. Sci. 2003; 28: 612-618Abstract Full Text Full Text PDF PubMed Scopus (337) Google Scholar, 20Gill G. Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (628) Google Scholar). Sumoylation has been shown to modulate the subcellular localization and transcriptional activity of the substrates (18Johnson E.S. Annu. Rev. Biochem. 2004; 73: 355-382Crossref PubMed Scopus (1399) Google Scholar, 20Gill G. Genes Dev. 2004; 18: 2046-2059Crossref PubMed Scopus (628) Google Scholar, 21Girdwood D.W. Tatham M.H. Hay R.T. Semin. Cell. Dev. Biol. 2004; 15: 201-210Crossref PubMed Scopus (152) Google Scholar). DJ-1 interacts with PIASxa and PIASy (17Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar), which are SUMO E3 ligases belonging to the protein inhibitor of the activated STAT (PIAS) family. In addition, DJ-1 interacts with SUMO-1, SUMO-activating enzyme Uba2, and conjugating enzyme ubc-9 in the yeast two-hybrid systems (8Junn E. Taniguchi H. Jeong B.S. Zhao X. Ichijo H. Mouradian M.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9691-9696Crossref PubMed Scopus (291) Google Scholar, 22Shinbo Y. Niki T. Taira T. Ooe H. Takahashi-Niki K. Maita C. Seino C. Iguchi-Ariga S.M. Ariga H. Cell Death Differ. 2006; 13: 96-108Crossref PubMed Scopus (148) Google Scholar). However, the functional significance of the link between sumoylation and DJ-1 and the potential relevance to PD pathogenesis are unclear. In the current study, we extend our prior findings and identify the human TH as a cellular target of transcriptional regulation by DJ-1 and PSF. DJ-1 promotes the expression of TH by inhibiting the SUMO-1 modification of PSF and preventing the recruitment of histone deacetylase (HDAC) 1 by PSF. Therefore, our results indicate that the transcriptional dysregulation caused by DJ-1 inactivation and the subsequent impairment of dopamine synthesis contribute to the development of PD. Cell Culture, Plasmids, and Chemicals—Human CHP-212 cells were purchased from ATCC (Manassas, VA) and maintained in Eagle's minimum essential medium/F-12 (50%/50%) containing 10% fetal bovine serum and antibiotics. Human SH-SY5Y, HeLa, and HEK293 cells were maintained in Dulbecco's modified Eagle's medium supplemented with 10% fetal bovine serum and antibiotics. For immunofluorescence, cells were grown on coverslips. Wild-type and mutant DJ-1 constructs and SH-SY5Y cells stably expressing these constructs were described previously (7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar). The lymphoblasts from patients carrying pathogenic DJ-1 mutations (2Bonifati V. Rizzu P. van Baren M.J. Schaap O. Breedveld G.J. Krieger E. Dekker M.C. Squitieri F. Ibanez P. Joosse M. van Dongen J.W. Vanacore N. van Swieten J.C. Brice A. Meco G. van Duijn C.M. Oostra B.A. Heutink P. Science. 2003; 299: 256-259Crossref PubMed Scopus (2307) Google Scholar) were described previously (23Macedo M.G. Anar B. Bronner I.F. Cannella M. Squitieri F. Bonifati V. Hoogeveen A. Heutink P. Rizzu P. Hum. Mol. Genet. 2003; 12: 2807-2816Crossref PubMed Scopus (127) Google Scholar). Human HDAC1 construct (pCMV-FLAG-HDAC1) was kindly provided by Dr. Fang Liu at Rutgers University. Human SUMO-1 construct (pCMV-HA-SUMO-1) was a gift from Dr. Kim Orth at the University of Texas Southwestern Medical Center. Human PIASy (pCMV-PIASy) was purchased from ATCC. Rat TH-luc reporter plasmid was kindly provided by Dr. Kwang Soo Kim at McLean Hospital, Harvard Medical School, and the pTK-Renilla luciferase plasmid for transfection control was obtained from Promega (Madison, WI). A FLAG tag was fused in-frame to the N terminus of human wild-type PSF (7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar) to generate a tagged PSF construct (pCMV-FLAG-PSF). To introduce point mutations at the PSF sumoylation site (I337A and K338A), we used a two-step PCR mutagenesis approach to mutate and amplify the PSF fragments flanked by the EcoRI and BamHI sites. The resulting PCR products (979 bp) were then subcloned into an expression vector using pCMV-FLAG-PSF as backbone. The PSF plasmids were confirmed by sequencing. The flanking primers were: forward, 5′-gatgtcggttgtttgttg-3′; reverse, 5′-atctcccatgttcattgct-3′. Mutagenesis primers for PSF-I337A were: forward, 5′-ggattcggatttgctaagcttgaatctagagc-3′; reverse, 5′-gctctagattcaagcttagcaaatccgaatcc-3′. Mutagenesis primers for PSF-K338A were: forward, 5′-ggattcggatttattgcgcttgaatctagagc-3′; reverse, 5′-gctctagattcaagcgcaataaatccgaatcc-3′. Sodium butyrate and trichostatin A were from Sigma. Transfection of siRNA and Plasmids—CHP-212 or SH-SY5Y cells plated in 6-well culture dishes were transfected with 100 nm siRNA constructs against the human DJ-1 (SMARTpool reagent, Dharmacon, Lafayette, CO) or nonspecific control siRNA constructs (siControl non-targeting pool, Dharmacon) using the Transfectin reagent (Bio-Rad, Hercules, CA) following the manufacturer's suggested protocol. Cells were harvested at 48 h post-transfection for RNA extraction or at days 1, 2, and 4 for Western blotting. To analyze the effects of HDAC inhibitors on the TH expression after DJ-1 inactivation, indicated sodium butyrate or trichostatin A was added to fresh medium 48 h after siRNA transfection, and the mixture was cultured for an additional 2 days. Lipofectamine 2000 (Invitrogen) was used to transfect various plasmids. To study the transcriptional repression of TH by PSF, CHP-212 cells plated in 10-cm dishes were co-transfected with 20 μg of control vector, WT, or mutant PSF plasmids and 2 μg of green fluorescent protein. Transfected cells were harvested 48 h later and enriched using a Cytomation Mo-Flo cell sorter (Core facility, Dana Farber Cancer Institute, Boston, MA) for subsequent total RNA extraction and mRNA analysis using quantitative PCR. Western Blotting, Immunoprecipitation, and Antibodies—The procedures for Western blotting and immunoprecipitation were described previously (7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar). For SUMO-1-conjugated PSF IP, cells were lysed in denaturing radioimmune precipitation assay-deoxycholate buffer; for various co-immunoprecipitation experiments, cells were lysed in non-denaturing lysis buffer containing 1% Triton X-100. Antibodies used for IP included: a mouse monoclonal anti-PSF (Sigma), a rabbit polyclonal anti-DJ-1 (7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar), and a mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology, Santa Cruz, CA). Antibodies for Western blotting included: a mouse monoclonal anti-TH (Sigma); monoclonal (Stressgen, San Diego, CA) and a polyclonal anti-DJ-1; a goat anti-β-actin and a mouse monoclonal anti-HDAC1 (Santa Cruz Biotechnology); a mouse monoclonal anti-SUMO-1 and a rabbit polyclonal anti-SUMO-2/3 (Invitrogen); and a rabbit polyclonal anti-FLAG tag and a rabbit polyclonal anti-acetylated histones (Histone sampler kit, Cell Signaling, Beverly, MA). RNA Extraction and Real-time Quantitative PCR—RNA was extracted using TRIzol reagent (Invitrogen) and purified with an RNeasy Kit or an RNeasy Micro Kit (Qiagen). The quality of extracted RNA was determined by agarose gel electrophoresis. Reference RNA used for the calibration curve was made by pooling equal amounts of RNA from all samples. Q-PCRs were performed using a LightCycler (Roche Applied Science) and a One-Step QuantiTect™ SYBR Green RT-PCR kit (Qiagen). Experimental conditions and primer design parameter were set according to the Q-PCR kit instruction manual. Results were normalized to an internal control PCR amplified with glyceraldehyde-3-phosphate dehydrogenase or β-actin primers included in the same run of Q-PCR. The specificity of PCR amplifications was further confirmed by agarose gel electrophoresis. Primers for the human TH were: forward, 5′-cctcgcccatgcactc-3′; reverse, 5′-cctcgcccatgcactc-3′. Primers for PSF were: forward, 5′-accaccagcagcatcacc-3′; reverse, 5′-tcccaacaaacaaccgaca-3′. Chromatin Immunoprecipitation Assays—ChIP assays using cultured cells were performed following the instructions of the EZ ChIP Kit (Upstate) with the following modifications. After protein-DNA cross-linking and harvesting, the cell pellets were resuspended in lysis buffer and sonicated on ice using a Branson Digital Sonifier (Branson Ultrasonics Corp., Danbury, CT) with 16 sets of 4-s pulse at 17% maximum power. The genomic DNA was sheared to 300–1200 bp of length. Aliquots of chromatin solution (each equivalent to 1 × 106 cells) were pre-cleared with Protein G-agarose and incubated with species-matched IgG or specific antibodies overnight at 4 °C with rotation. ChIP assays using brain tissues from normal patients were performed as described before (24Lu T. Pan Y. Kao S.Y. Li C. Kohane I. Chan J. Yankner B.A. Nature. 2004; 429: 883-891Crossref PubMed Scopus (1481) Google Scholar). The substantia nigra tissues from a male (postmortem interval, 4.5 h) and a female (postmortem interval, 13 h) patient with no neurological or psychiatric conditions were acquired according to institutional regulations. Tissues surrounding the substantia nigra pars compacta were trimmed off to yield relatively pure nigral tissue. Samples were snapfrozen in liquid nitrogen after dissection and stored under –80 °C until usage. The specific antibodies applied in ChIP assays for DJ-1, PSF, HDAC1, and acetylated histones are described above. The final immunoprecipitated DNA fragments were used as templates for PCR with hot start Platinum Taq (Invitrogen) using the following conditions: 3 min at 94 °C; 32 cycles of 30 s denaturation at 95 °C, 30 s annealing at 57 °C, and 30 s elongation at 72 °C; with one final incubation for 2 min at 72 °C. 27–33 cycles were used for semiquantitative PCR in the TH-promoter-associated acetylated histones IP. Primer 3 software was used to design the PCR primers for amplifying the human TH promoter. The forward primer was 5′-gagccttcctggtgtttgtg-3′; the reverse primer was 5′-ctctccgattccagatggtg-3′. The PCR products correspond to the promoter sequences –2909 to –2707 upstream to the transcriptional initiation site of the human TH gene, and were analyzed by electrophoresis on 2% Tris-acetate-EDTA agarose gels. The primers used to amplify the control human glyceraldehyde-3-phosphate dehydrogenase promoter were: forward, 5′-tactagcggttttacgggcg-3′; reverse, 5′-tcgaacaggaggagcagagagcga-3′. TH Activity Assay—The TH activity in CHP-212 cells transfected with control or DJ-1-specific siRNA was determined by measuring l-DOPA formation using methods as described (25Hayashi M. Yamaji Y. Kitajima W. Saruta T. Am. J. Physiol. 1990; 258: F28-F33PubMed Google Scholar). In brief, 4 days after the siRNA transfection, CHP-212 cells plated in 6-well dishes were rinsed twice with 1 ml of the physiological medium and then incubated for 12 h at 37 °C with 100 mm l-tyrosine and 500 μm n-hydroxybenzylhydrozine (Sigma), a selective DOPA decarboxylase inhibitor. After the incubation, medium was discarded and the cells were collected, resuspended with 0.1 ml of 0.1 n perchloric acid, and sonicated. An aliquot (10 μl) was used to determine protein concentration, and the remainder was centrifuged at 15,000 × g at 4 °C for 10 min. A 20-μl aliquot of the supernatant was injected onto an ODS Hypersil column (150 × 4.6 mm, 5-μm particle size, Keystone Scientific Inc., Bellefonte, PA). Mobile phase consisted of (in mm): 50 potassium phosphate, 0.1 EDTA, 0.2 heptane sulfonic acid, and 10% methanol, pH 2.6. l-DOPA peaks were detected with an Antec Leyden Intro amperometric high-performance liquid chromatography system (GBC Analytical Systems). Peaks from samples were compared against a daily 200-pictrogram l-DOPA external standard. Results were normalized per microgram of protein and analyzed through one-way analysis of variance. DJ-1 and PSF Transcriptionally Regulate the Human TH Promoter—Since we have shown that DJ-1 is a transcriptional co-activator, we examined whether DJ-1 regulated the expression of genes involved in dopaminergic neurotransmission, such as tyrosine hydroxylase, the rate-limiting enzyme that converts tyrosine to the dopamine precursor l-DOPA. To mimic the loss-of-function effects as seen in PD patients with DJ-1 mutations, we used DJ-1-specific siRNA constructs to inhibit the synthesis of endogenous DJ-1 in two human dopaminergic neuroblastoma cell lines, CHP-212 and SH-SY5Y cells. The protein levels of TH and DJ-1 showed time-dependent decreases in CHP-212 cells transfected with DJ-1-specific siRNA (Fig. 1A). 4 days after DJ-1 siRNA transfection, the TH protein expression was reduced by 90% (Fig. 1A). Quantitative real-time PCR results indicated that DJ-1 inactivation by siRNA significantly decreased the TH mRNA levels in both CHP-212 and SH-SY5Y cells as determined by quantitative real-time PCR (Fig. 1B). In addition, the reduction in the TH expression following siRNA knockdown of DJ-1 decreased the TH activity by almost 40% in CHP-212 cells, as determined by the production of l-DOPA using high-performance liquid chromatography (Fig. 1C). Consistent with these observations, the TH mRNA expression was increased by >100% in SH-SY5Y cells stably expressing the human wild-type DJ-1 (Fig. 1D). Previously, we have shown that DJ-1 interacts with and blocks the functions of a transcriptional repressor PSF in human dopaminergic cells (7Xu J. Zhong N. Wang H. Elias J.E. Kim C.K. Woldman I. Pifl C. Gygi S.P. Geula C. Yankner B.A. Hum. Mol. Genet. 2005; 14: 1231-1241Crossref PubMed Scopus (227) Google Scholar). As in SH-SY5Y cells, PSF specifically interacted with DJ-1 in untransfected CHP-212 cells (data not shown). Therefore, we tested whether PSF repressed TH transcription. Transient expression of the wild-type PSF inhibited the human TH mRNA expression in CHP-212 cells (Fig. 1E). To further confirm the transcriptional regulation of the human TH promoter by both DJ-1 and PSF, we performed ChIP assays to assess the physical interactions between these two transcriptional regulators and the TH promoter in vitro and in vivo. The DNA co-immunoprecipitated with either a monoclonal anti-PSF or a polyclonal anti-DJ-1 antibody using the lysates from CHP-212 cells or human substantia nigra pars compacta tissues were amplified by primers specifically recognizing the human TH promoter but not by primers recognizing the human glyceraldehyde-3-phosphate dehydrogenase promoter (Fig. 1F). Taken together, our results strongly demonstrate that DJ-1 activates the human TH expression and regulates dopamine synthesis. DJ-1 Inhibits the Sumoylation of PSF and Its Repression of the TH Promoter—To elucidate the molecular mechanism of this transcriptional regulation, we assessed the possibility that DJ-1 might regulate the sumoylation of PSF. DJ-1 has been shown to interact with SUMO-1, SUMO-activating enzyme Uba2, and conjugating enzyme ubc-9 (8Junn E. Taniguchi H. Jeong B.S. Zhao X. Ichijo H. Mouradian M.M. Proc. Natl. Acad. Sci. U. S. A. 2005; 102: 9691-9696Crossref PubMed Scopus (291) Google Scholar, 22Shinbo Y. Niki T. Taira T. Ooe H. Takahashi-Niki K. Maita C. Seino C. Iguchi-Ariga S.M. Ariga H. Cell Death Differ. 2006; 13: 96-108Crossref PubMed Scopus (148) Google Scholar) using the yeast two-hybrid system. In addition, DJ-1 interacts with SUMO ligases PIASxa and PIASy and potentially regulates their functions (17Takahashi K. Taira T. Niki T. Seino C. Iguchi-Ariga S.M. Ariga H. J. Biol. Chem. 2001; 276: 37556-37563Abstract Full Text Full Text PDF PubMed Scopus (289) Google Scholar). Meanwhile, PSF harbors a potential sumoylation site (IKLE) that completely matches the consensus sumoylation motif ΨKXE, where the conserved lysine (K) and glutamic acid (E) are preceded by a hydrophobic amino acid (Ψ) and any amino acid (X), respectively (Fig. 2A). Recently, a proteomic study suggested that PSF is sumoylated, although the site of modification has not been mapped (26Rosas-Acosta G. Russell W.K. Deyrieux A. Russell D.H. Wilson V.G. Mol. Cell. Proteomics. 2005; 4: 56-72Abstract Full Text Full Text PDF PubMed Scopus (194) Google Scholar). We first examined the effect of DJ-1 on global sumoylation in SH-SY5Y cells stably expressing the human wild-type DJ-1. Overexpression of the wild-type DJ-1 resulted in an overall decrease in the amount of high molecular weight proteins modified by SUMO-1 but not by SUMO-2 or SUMO-3 (Fig. 2B). To examine whether DJ-1 similarly regulates sumoylation in vivo, we analyzed the lymphoblast cells harvested from normal or PD patients carrying pathogenic mutations in the DJ-1 gene. Consistent with the results from Fig. 2B, DJ-1 inactivation caused by the homozygous deletion of exons 1 to 5 (DEL) or the L166P point mutation resulted in a slight but reproducible increase in the amount of SUMO-1-modified high molecular weight proteins (Fig. 2C, -fold change relative to WT, mean ± S.E., DEL: 1.47 ± 0.09; L166P: 3.07 ± 0.61; p < 0.05, n = 3 for both conditions). Next, we examined whether PSF was sumoylated and whether DJ-1 affected this process. Immunoprecipitation of PSF and Western blotting of SUMO species and PSF using lysates from SH-SY5Y cells stably expressing various DJ-1 constructs indicated that PSF was modified by SUMO-1 (Fig. 2D) but not by SUMO-2 or SUMO-3 (data not shown). Further, the wild-type and the non-pathogenic R98Q DJ-1, but not the pathogenic DJ-1 mutants expressed at similar levels, specifically reduced the abundance of SUMO-1-conjugated PSF (Fig. 2, D and E). The inability of the pathogenic DJ-1 mutants to suppress PSF sumoylation suggests a functional link between the reg" @default.
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• W2047916240 title "DJ-1 Transcriptionally Up-regulates the Human Tyrosine Hydroxylase by Inhibiting the Sumoylation of Pyrimidine Tract-binding Protein-associated Splicing Factor" @default.
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• W2047916240 doi "https://doi.org/10.1074/jbc.m601935200" @default.
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