Identification Of Transcriptional Regulatory Elements Within ATP13A2(PARK9) Gene Promoter And Hypoxia Regulation

Background:Parkinson's disease (PD) is the second most common neurodeg-enerative disorders with a variable combination of motor and non-motor symptoms. Its characteristic motor symptoms include tremor at rest, rigidity, bradykinesia. Non-motor dysfunctions, such as depression, anxiety, dementia and sexual dysfunction may appear as the disease progresses. The etiology of PD is unclear, however, both genetic and environmental causes contribute to it. Since the first missense mutation in the a-synuclein gene reported in a large PD family of Italian descent in1997, up to date,13genes are reported to be associated with PD. a-synuclein, LRRK2, Parkin, DJ-1, PINK1and ATP13A2have been confirmed to be the pathogenic genes for PD.In2006Alfredo and colleagues identified ATP13A2mutations from a non-consanguineous Chilean family with PD and named it PARK9. Following this report, the mutation screening of ATP13A2was performed on PD patients from different ethnic groups, and several novel mutations in ATP13A2were identified. The human ATP13A2gene (OMIM*610513), locates in chromosome1p36encoding for a protein of1180amino acids with10transmembrane domains which belongs to the P-type ATPase superfamily. However, the protein function of ATP13A2and the pathogenic mechanism of PARK9is still not clear.Recently, abnormal gene expression has been implicated in neurodegenerative disorders. However, the transcriptional regulation of the ATP13A2gene is unknown.Objective:To define the molecular mechanism underlying its transcription and its transcriptional regulation.Methods:1. RNA Ligase Mediated Rapid Amplification of cDNA Ends (RLM-RACE) assay was performed to determine the transcription start site of the human ATP13A2gene.2. To examine the transcriptional regulation of the human ATP13A2, fragments of the5'flanking region of the ATP13A2gene were pulled out by PCR from the human genome DNA and cloned into pG13-basic vector. Luciferase assay was performed to detect the promoter activity of these vectors.3. To further identify the transcription factor binding sites, Computer-based transcription factor binding site analysis was employed and the putative binding sties were confirmed by EMSA and ChIP.4. To determine the effect of hypoxia and HIF-1α on the human ATP13A2promoter activity, HEK293cells were co-transfected by pPK9-A with pHIF-1α or exposed to hypoxic condition after trans-fection with pPK9-A.5. To further investigate the transcrptional regulation of ATP13A2by hypoxia on the mRNA level, HEK293cells and MN9D cells were exposed to hypoxic condition. The endogenous mRNA level of ATP13A2from both cell lines were measured and compared to its own control after hypoxia treatment.Results:1. PCR product from RLM-RACE was sequenced and the sequence result revealed the adenine locating at206bp upstream of the translation start site was the base after RNA adapter;2. A series of luciferase reporter plasmids with different upstream deletions of the ATP13A2gene promoter region were constructed and named according to the original location of insert fragment (TSS was designated as+1); pPK9-A (-1954to+84bp), pPK9-C (-1538to+84bp), pPK9-E (-846to+84bp), pPK9-F (-201to+84bp), pPK9-G(-78to+84bp), pPK9-H(-78to+50bp) present significant promoter activiey;3. Computer-based transcription factor binding site analysis revealed that there are nine putative hypoxia response elements (HRE) in the2.0kb promoter region (-1954to+84) of the human ATP13A2gene;4. EMSA showed that oligos of HRE-1, HRE-3, HRE-4, HRE-8and HRE-9can sharped induced the intensity of the DNA-protein band formed by HIF-1a probe and HIF-1a protein; ChIP assay showed PCR products can be amplified from chromatin samples immune-precipitated by HIF-1a antibody using specific primers of HRE-1, HRE-3+4and HRE-8+9respectively;5. pPK9-A was transfected into HEK293cells with pHIF-1a or empty vector as control, pPK9-A promoter activity was significantly increased compared to control (2.483+0.241, P<0.05); pPK9-A transfected HEK293cells were subjected to hypoxia (2%oxygen) or normoxia (21%oxygen) for24hours and the promoter activity of pPK9-A was significant increased by2.466+0.293(P<0.05) after24hours exposure to low oxygen;6. HEK293cells were incubated in hypoxia chamber with2%oxygen for0,12,24,48hours. Endogenous ATP13A2mRNA levels were measured by qRT-PCR and the results showed the exposure to hypoxic condition for24and48hours resulted in a significant increase of ATP13A2mRNA level,2.356+0.320fold and2.526+0.203fold, respectively; MN9D cells were also inbubated in hypoxia chamber with2%oxygen for0,12, and24hours, and the mRNA level of Atpl3a2after12and24hours hypoxic treatment were increased to1.668±0.148and1.606±0.130, respectively (P<0.05).Conclusions:1. We identified the adenine locating at206bp upstream of the translation start site is the major transcription start site of the ATP-13A2gene;2. We constructed a series of plasmids with different deletions of the5' flanking region of the ATP13A2gene; the5'flanking region from-78to+50bp contains the minimal promoter sequence necessary for the transcriptional activation of the human ATP13A2gene expression;3. EMS A and ChIP assay confirmed the HRE sites1,3,4,8and9are functional HIF-1a binding sites;4. Overexpression of transcription factor HIF-1a can increase the promoter activity of ATP13A2, and hypoxia can up-regulate the promoter activity of the ATP13A2via HIF-1α;5. Finally, we further confirmed hypoxia can up-regulate the expression of ATP13A2gene at transcription level.