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Genetic analysis of the ATG7 gene promoter in sporadic Parkinson's disease
Neuroscience Letters 534 (2013) 193–198
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Genetic analysis of the ATG7 gene promoter in sporadic Parkinson’s disease Dongfeng Chen a,1 , Shuchao Pang a,1 , Xungang Feng b,1 , Wenhui Huang a , Robert G. Hawley c,d,∗ , Bo Yan a,c,d,∗∗ a Shandong Provincial Key Laboratory of Cardiac Disease Diagnosis and Treatment, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong 272029, China b Division of Neurology, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong 272029, China c Department of Anatomy and Regenerative Biology, The George Washington University, 2300 Eye Street, N.W., Washington, DC 20037, USA d Sino-US Joint Laboratory of Translational Medicine, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong 272029, China
h i g h l i g h t s
The ATG7 gene promoter was genetically and functionally analyzed in sporadic PD. Four novel variants were identified in five PD patients, but in none of the controls. These variants significantly decreased transcriptional activities of the ATG7 gene promoter. These variants identified in PD may contribute to PD onset as a risk factor.
a r t i c l e
i n f o
Article history: Received 12 September 2012 Received in revised form 25 November 2012 Accepted 20 December 2012 Keywords: Parkinson’s disease ATG7 Promoter Sequence variants Autophagy
a b s t r a c t Parkinson’s disease (PD) is the second most common neurodegenerative disease. The majority of PD cases are sporadic, for which genetic causes and underlying molecular mechanisms remain largely unclear. Autophagy, a highly conserved cellular process that governs the breakdown of long-lived proteins and organelles, has been involved in the degradation of ␣-synuclein (␣-Syn), the main component of Lewy bodies. Accumulating evidence implicates deregulation of autophagy in the development and progression of sporadic PD. Altered autophagic gene expression has been observed in the brain tissues from PD patients and animal models. We hypothesized that changes in expression levels of autophagy-related genes (ATGs), rather than mutations associated with amino acid changes, may contribute to PD onset. In this study, the ATG7 gene promoter was sequenced bi-directionally in groups of sporadic PD patients and ethnic-matched healthy controls. As predicted, four novel heterozygous variants, 11313449G>A, 11313811T>C, 11313913G>A and 11314041G>A, were identified in five PD patients, but in none of the controls, which significantly decreased transcriptional activities of the ATG7 gene promoter. Two novel heterozygous variants, 11312947G>A and 11313006C>G, were only found in controls, which did not affect transcriptional activities of the ATG7 gene promoter. The other five novel variants were found in PD patients and controls with similar frequencies. Taken together, the sequence variants within the ATG7 gene promoter identified in PD patients may change ATG7 protein levels, which in turn would influence autophagic activity, contributing to PD onset as a risk factor. © 2013 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: AP2, Activator protein-2; ␣-Syn, ␣-syncuclein; ATGs, autophagy-related genes; ATG3, autophagy-related gene 3; ATG7, autophagy-related gene 7; ATG12, autophagy-related gene 12; CREB, cAMP-responsive element binding protein; HNF-1, hepatocyte nuclear factor-1 alpha; LC3, microtubule-associated protein 1 light chain 3; NF1, nuclear factor 1; PD, Parkinson’s disease; SIRT1, surtuin 1; SP1, specificity protein 1; TFII-I, an E-box-binding transcription factor; YY1, ying-yang 1. ∗ Corresponding author at: Department of Anatomy and Regenerative Biology, The George Washington University, 2300 Eye Street, N.W., Washington, DC 20037, USA. Tel.: +1 202 994 3511; fax: +1 202 994 8885. ∗∗ Corresponding author at: Shandong Provincial Key Laboratory of Cardiac Disease Diagnosis and Treatment, Jining Medical University Affiliated Hospital, Jining Medical University, 79 Guhuai Road, Jining, Shandong 272029, China. Tel.: +86 0537 2903579; fax: +86 0537 2213030. E-mail addresses: [email protected] (R.G. Hawley), [email protected], [email protected] (B. Yan). 1 These authors contributed equally to the work. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.12.039
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1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease, the majority of which is sporadic. Lewy body formation and dopaminergic neuronal loss in the substantia nigra are the pathological characteristics of PD. Although a number of genes and genetic loci have been associated with familial PD, the genetic causes and underlying molecular mechanism for sporadic PD remain largely unclear [26,36]. Alpha-synculein (␣-Syn), the main component of Lewy bodies, has been shown to play a central role in PD pathogenesis [8,43]. Autophagy, a highly conserved cellular process that delivers long-lived proteins and organelles to lysosomes for digestion, is composed of three subtypes, macroautophagy, microautophagy and chaperone-mediated autophagy. Autophagy is involved in development, differentiation, starvation adaptation, intracellular quality control, tumor suppression, aging, innate immunity and other processes [28,32]. Dysfunctional autophagy has been implicated in inflammation, cancer, cardiovascular diseases and neurodegenerative diseases [24,46]. Accumulating evidence indicates that deregulation of autophagy is involved in ␣-Syn degradation, contributing to the development and progression of sporadic PD [7,24,30,44–46]. Macroautophagy (hereafter referred to as autophagy) has been extensively studied. More than 30 autophagy related genes (ATGs) have been identified in yeast, most of which are conserved in mammals. ATG7, a ubiquitin-like modifier-activating enzyme, forms a complex with ubiquitin-like-conjugating enzyme ATG3. ATG7 is essential for microtubule-associated protein 1 light chain 3 (LC3) – phosphatidylethanolamine conjugation and the ATG12 conjugation systems, two critical systems in autophagosome formation [15,16,29,39–41]. Many studies in experimental animals with systemic and tissue-specific deletion of the ATG7 gene have demonstrated that the gene, and by inference autophagy, plays an essential role in neural development, neuronal survival and neurodegeneration [5,18–20]. In addition, recent studies involving tissue-specific deletion of ATG7 gene have revealed its roles in gluconeogenesis, adipogenesis and lipid metabolism in mice [9,37,38,52]. Alterations of autophagy have been observed in the brain tissues of PD patients, and in cellular and animal models of PD [1,4,48–50]. In the brain tissues of patients with dementia with Lewy bodies and in ␣-Syn transgenic mice, ATG7 gene expression is reduced [6]. Cell-specific deletion of the ATG7 gene in dopamine neurons leads to delayed neurodegeneration and ubiquitinated inclusions in a mouse model [10]. Therefore, we hypothesized that changes in the expression level of ATG7 gene may contribute to PD pathogenesis by altering autophagic activity. We have previously shown that expression of autophagic marker genes is significantly changed in sporadic PD patients [31,47]. Herein, as part of a systemic analysis of the critical ATG genes in PD, we genetically analyzed the ATG7 gene promoter in groups of sporadic PD patients and ethnic-matched healthy controls.
2. Materials and methods 2.1. Study subjects Sporadic PD patients (n = 101, mean age 66.99 years, male 54, female 47) were recruited from the Division of Neurology, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong, China. Ethnic-matched healthy controls (n = 148, mean age 60.16 years, male 71, female 77) were recruited from the Health and Physical Examination Center in the same hospital. All PD patients were diagnosed by two neurologists. PD patients
and controls with a family history of PD were excluded. This study was approved by the Human Ethics Committee of Jining Medical University Affiliated Hospital and informed consent was obtained in all cases. 2.2. DNA sequencing Peripheral leukocytes were isolated from venous blood by conventional density gradient centrifugation. Genomic DNAs were extracted with QIAGEN DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). The ATG7 gene promoter from −1145 bp to +112 bp was analyzed. Two overlapping DNA fragments, −1145 bp to −446 bp (700 bp) and −510 bp to +112 bp (623 bp), were generated by PCR and directly sequenced. PCR primers were designed based on genomic sequence of the human ATG7 gene (GenBank accession number, NC 000003.11) (Table 1). The DNA fragments were sequenced on a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned and compared with the wild-type human ATG7 gene promoter. 2.3. Functional analysis Wild-type and variant ATG7 gene promoters (1194 bp, from −1082 bp to +112 bp to the transcription start site, GenBank accession number, NC 000003.11) were generated by PCR (Table 1), and subcloned into KpnI and HindIII sites of a luciferase reporter vector (pGL3-basic) to construct expression vectors. Designated expression vectors were transiently transfected into human embryonic kidney cells (HEK-293) with lipofectamine-2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activities were measured 48 h posttransfection using dual-luciferase reporter assay system. All experiments were repeated at least three times, in triplicates. 2.4. Statistical analysis Quantitative data are represented as mean ± SE and analyzed by Student’s t-test. The distributions of sequence variants were compared between PD patients and controls by 2 test using SPSS v13.0. P < 0.05 was considered as statistically significant. 3. Results The sequence variants within the ATG7 gene promoter identified in PD patients and controls are summarized in Table 2. The locations of the variants are depicted in Fig. 1A. The chromatograms of the sequence variants identified only in PD patients or controls are shown in Fig. 1B. A total of eleven sequence variants were identified, all of which are novel. Among these variants, two are both heterozygous and homozygous, while the remaining nine are heterozygous. Four heterozygous variants, 11313449G>A, 11313811T>C, 11313913G>A and 11314041G>A, were identified in five PD patients, but in none of the controls. Two heterozygous variants, 11312947G>A and 11313006C>G, were only found in two controls. The other two heterozygous and homozygous variants, 11313215A>G and 11313910G>A, and three heterozygous variants, 11313127C>G, 11313666G>A and 11313988T>G, were found in both PD patients and controls with similar frequencies (P > 0.05). It is worth noting that the variant 11313913G>A was linked with a heterozygous variant 11313910G>A, which was identified in two PD patients. In addition, the allele at position of 11314041 within the human ATG7 gene genomic sequence (NC 000003.11) is A in the NCBI database. However, the same allele was G in all the PD patients and controls in this study, except one PD patient. The allele A was found in this PD patient. Thus, the allele A was considered as a heterozygous variant and defined as 11314041G>A.
D. Chen et al. / Neuroscience Letters 534 (2013) 193–198
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Table 1 PCR primers for the human ATG7 gene promoter.a PCR primers Sequencing ATG7-F1 ATG7-R1 ATG7-F2 ATG7-R2 Functional analysis ATG7-F ATG7-R a
Sequences
Location (bp)
5 -GGTTCCTTCTCTCCCACCTC-3 5 -GCACTGGACAGGTGTTGAAG-3 5 -GCCTTACAGGCCAGACAGAG-3 5 -CTTACCGCCGCTCAACTT-3
−1145 −446 −510 +112
5 -(KpnI)-ACGGAGTCTCGCTCTGTCGC-3 5 -(HindIII)-CTTACCGCCGCTCAACTTCC-3
−1082 +112
PCR products (bp) 700
623 1194
PCR primers were designed based on the genomic DNA sequence of the human ATG7 gene (NC 000003.11). The transcription start site (+1) is at position of 11314010.
Table 2 Sequence variants within the human ATG7 gene promoter. Variants
Genotype
Locationsa (bp)
Controls (n = 148)
PD (n = 101)
P-value
11312947G>A 11313006C>G 11313127C>G 11313215A>G
GA CG CG AA AG GG GA GA TC GG GA AA GA TG GA
−1064 −1005 −884 −796
1 1 6 69 68 11 0 2 0 20 72 56 0 4 0
0 0 7 45 42 14 1 1 1 19 49 33 2 1 1
– – 0.316 0.248
11313449G>A 11313666G>A 11313811T>C 11313910G>A
11313913G>A 11313988T>G 11314041G>A a
−561 −345 −199 −100
−97 −22 +32
– 1.000 – 0.467
– 0.651 –
The transcription start site of the ATG7 gene was set as +1.
Analysis of the ATG7 gene promoter with the TESS (transcription element search system) web tool (University of Pennsylvania) suggested that putative transcription factor binding sites may be altered by the identified variants. For example, the variant 11313449G>A abolished a GATA binding site and created a binding site for transcription factor TFII-I. The variant 11313449G>A abolished the binding site for HNF1. The variant 11313913G>A abolished an SP1 binding site and modified an AP2 binding site. The variant 11314041G>A created a binding site for NF1. In addition, the variant 11312947G>A abolished a YY1 binding site and the variant 11313006C>G created a CREB binding site. To examine the effects of these variants on the ATG7 gene promoter, luciferase reporter gene vectors containing wild type or variant promoters, pGL3-WT, pGL3-11312947A, pGL3-11313006G, pGL3-11313215G, pGL3-11313449A, pGL3-11313811C, pGL311313913A and pGL3-11314041A, were constructed and transfected into HEK-293 cells and dual-luciferase activities were determined. Compared to wild type ATG7 gene promoter, transcriptional activities were significantly decreased for the variant promoters identified in PD patients, pGL3-11313449A (P < 0.001), pGL3-11313811C (P < 0.01), pGL3-11313913A (P < 0.01) and pGL3-11314041A (P < 0.001). In contrast, transcriptional activities of pGL3-11312947A and pGL3-11313006G were not significantly different from that of pGL3-WT (P > 0.05). In addition, no significant difference was observed between pGL3-WT and pGL3-11313215G, which contained the variant that was found in PD patients and controls and was used as an internal control (Fig. 2). 4. Discussion In this study, we genetically analyzed the ATG7 gene promoter in sporadic PD patients and ethnic-matched healthy controls. Four novel heterozygous variants were identified in five PD patients,
but in none of the controls. All of the four variants significantly decreased transcriptional activities of the ATG7 gene promoter, probably by modifying, abolishing and creating binding sites for putative transcription factors. Although two variants only found in controls were suggested to change transcription factor binding sites, these variants did not affect transcriptional activities of the ATG7 gene promoter. To date, few mutations and variants in the ATG7 gene have been associated with human diseases. A polymorphism in the coding region of the ATG7 gene that substitutes alanine for valine has been associated with an earlier onset of Huntington’s disease [27]. In this study, we linked genetic variants within the ATG7 gene promoter to sporadic PD, the frequency of which was ∼5% (5/101). These variants may thus contribute to PD onset as a risk factor by altering autophagic activity.The human ATG7 gene has been localized to chromosome 3p25.3, encoding a protein of 703 amino acids [51]. The promoter region of the human ATG gene has not been characterized. At the epigenetic level, ATG7 gene expression has been shown to be modified by SIRT1, an NAD-dependent deacetylase, and by the acetyltransferase p300 protein [22,23]. In diverse human cancer cells, ATG7 gene expression is increased by proteasome inhibitors and epidermal growth factor receptor tyrosine kinase inhibitors [11,53], suggesting that ATG7 gene expression can be regulated. In this study, the sequence variants within the ATG7 gene promoter identified in PD patients affected its transcriptional activities, which may change ATG7 levels and lead to impaired autophagic activity. Several molecular pathways have been suggested for PD pathogenesis, including ␣-Syn accumulation, oxidative damage, mitochondrial dysfunction and inflammation [13,33,35,42]. Changes in ATG7 levels may contribute to PD onset via different pathways. First, as in vitro and in vivo studies indicate an important role of autophagy in ␣-Syn protein degradation, impaired autophagy may directly result in accumulation of ␣-Syn protein [30,44,45]. Second, impaired autophagy may lead to abnormal
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Fig. 1. Sequence variants within the ATG7 gene promoter identified in PD patients and controls. (A) Schematic representation of the sequence variants. The numbers correspond to those of the ATG7 gene genomic sequence (GenBank accession number, NC 000003.11). Transcription starts at position 11314010 in the first exon. (B) Chromatograms of the sequence variants only identified in either PD or controls. Sequence orientations are indicated. The top panels show wild-type sequences and the bottom panels homozygous variants. All the variants are marked with arrows. The variant 11313913G>A was linked with a heterozygous variant 11313910G>A, which is marked with a dashed arrow.
Fig. 2. Transcriptional activities of the ATG7 gene promoters. Wild type and variant ATG7 gene promoters were inserted into reporter gene vector pGL3-basic to generate expression vectors. These vectors were transfected into HEK-293 cells and dual-luciferase activities were measured. The transcriptional activity of wild type ATG7 gene promoter was designated as 100%. pGL3-basic was used as a negative control. The data were represented as mean ± SE from three independent transfection experiments, each in triplicate. Lanes 1, pGL3-basic; 2, pGL3-WT; 3, pGL3-11312947A; 4, pGL3-11313006G; 5, pGL3-11313215G, which was used as an internal negative control; 6, pGL3-11313449A; 7, pGL3-11313811C; 8, pGL311313913A; 9, pGL3-11314041A. *, P < 0.01, compared to pGL3-WT. **, P < 0.001, compared to pGL3-WT.
lipid metabolism. Elevated oxidized cholesterol metabolites have been shown to accelerate ␣-Syn accumulation by interacting with ␣-Syn [3,25]. In cellular and transgenic mouse models of PD, cholesterol-reducing agents reduce the accumulation of ␣-Syn and ameliorate the associated neuronal deficits [2,14,21]. Third, impaired autophagy may contribute to PD through inflammatory pathways. Autophagy has been shown to be required for monocyte differentiation and macrophage functions [17]. Fourth, ATG7 is involved in the phagocytosis of macrophages independent of autophagosome formation [34], which may be involved in ␣Syn clearance and inflammatory processes. The precise molecular mechanisms by which the ATG7 gene is linked to PD need further investigation. In conclusion, the ATG7 gene promoter was genetically and functionally analyzed in sporadic PD patients and ethnic-matched controls. The four novel heterozygous variants identified in PD patients significantly decreased the transcriptional activity of the ATG7 gene promoter, which would result in changed ATG7 protein levels and impaired autophagic activity, contributing to PD onset as a risk factor. As control of ATG7 gene expression and autophagic activity has been suggested as a therapy for neurodegenerative diseases [12], manipulation of ATG7 gene expression with different approaches may provide a novel potential therapy for PD patients.
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Genetic analysis of the ATG7 gene promoter in sporadic Parkinson’s disease Dongfeng Chen a,1 , Shuchao Pang a,1 , Xungang Feng b,1 , Wenhui Huang a , Robert G. Hawley c,d,∗ , Bo Yan a,c,d,∗∗ a Shandong Provincial Key Laboratory of Cardiac Disease Diagnosis and Treatment, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong 272029, China b Division of Neurology, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong 272029, China c Department of Anatomy and Regenerative Biology, The George Washington University, 2300 Eye Street, N.W., Washington, DC 20037, USA d Sino-US Joint Laboratory of Translational Medicine, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong 272029, China
h i g h l i g h t s
The ATG7 gene promoter was genetically and functionally analyzed in sporadic PD. Four novel variants were identified in five PD patients, but in none of the controls. These variants significantly decreased transcriptional activities of the ATG7 gene promoter. These variants identified in PD may contribute to PD onset as a risk factor.
a r t i c l e
i n f o
Article history: Received 12 September 2012 Received in revised form 25 November 2012 Accepted 20 December 2012 Keywords: Parkinson’s disease ATG7 Promoter Sequence variants Autophagy
a b s t r a c t Parkinson’s disease (PD) is the second most common neurodegenerative disease. The majority of PD cases are sporadic, for which genetic causes and underlying molecular mechanisms remain largely unclear. Autophagy, a highly conserved cellular process that governs the breakdown of long-lived proteins and organelles, has been involved in the degradation of ␣-synuclein (␣-Syn), the main component of Lewy bodies. Accumulating evidence implicates deregulation of autophagy in the development and progression of sporadic PD. Altered autophagic gene expression has been observed in the brain tissues from PD patients and animal models. We hypothesized that changes in expression levels of autophagy-related genes (ATGs), rather than mutations associated with amino acid changes, may contribute to PD onset. In this study, the ATG7 gene promoter was sequenced bi-directionally in groups of sporadic PD patients and ethnic-matched healthy controls. As predicted, four novel heterozygous variants, 11313449G>A, 11313811T>C, 11313913G>A and 11314041G>A, were identified in five PD patients, but in none of the controls, which significantly decreased transcriptional activities of the ATG7 gene promoter. Two novel heterozygous variants, 11312947G>A and 11313006C>G, were only found in controls, which did not affect transcriptional activities of the ATG7 gene promoter. The other five novel variants were found in PD patients and controls with similar frequencies. Taken together, the sequence variants within the ATG7 gene promoter identified in PD patients may change ATG7 protein levels, which in turn would influence autophagic activity, contributing to PD onset as a risk factor. © 2013 Elsevier Ireland Ltd. All rights reserved.
Abbreviations: AP2, Activator protein-2; ␣-Syn, ␣-syncuclein; ATGs, autophagy-related genes; ATG3, autophagy-related gene 3; ATG7, autophagy-related gene 7; ATG12, autophagy-related gene 12; CREB, cAMP-responsive element binding protein; HNF-1, hepatocyte nuclear factor-1 alpha; LC3, microtubule-associated protein 1 light chain 3; NF1, nuclear factor 1; PD, Parkinson’s disease; SIRT1, surtuin 1; SP1, specificity protein 1; TFII-I, an E-box-binding transcription factor; YY1, ying-yang 1. ∗ Corresponding author at: Department of Anatomy and Regenerative Biology, The George Washington University, 2300 Eye Street, N.W., Washington, DC 20037, USA. Tel.: +1 202 994 3511; fax: +1 202 994 8885. ∗∗ Corresponding author at: Shandong Provincial Key Laboratory of Cardiac Disease Diagnosis and Treatment, Jining Medical University Affiliated Hospital, Jining Medical University, 79 Guhuai Road, Jining, Shandong 272029, China. Tel.: +86 0537 2903579; fax: +86 0537 2213030. E-mail addresses: [email protected] (R.G. Hawley), [email protected], [email protected] (B. Yan). 1 These authors contributed equally to the work. 0304-3940/$ – see front matter © 2013 Elsevier Ireland Ltd. All rights reserved. http://dx.doi.org/10.1016/j.neulet.2012.12.039
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1. Introduction Parkinson’s disease (PD) is the second most common neurodegenerative disease, the majority of which is sporadic. Lewy body formation and dopaminergic neuronal loss in the substantia nigra are the pathological characteristics of PD. Although a number of genes and genetic loci have been associated with familial PD, the genetic causes and underlying molecular mechanism for sporadic PD remain largely unclear [26,36]. Alpha-synculein (␣-Syn), the main component of Lewy bodies, has been shown to play a central role in PD pathogenesis [8,43]. Autophagy, a highly conserved cellular process that delivers long-lived proteins and organelles to lysosomes for digestion, is composed of three subtypes, macroautophagy, microautophagy and chaperone-mediated autophagy. Autophagy is involved in development, differentiation, starvation adaptation, intracellular quality control, tumor suppression, aging, innate immunity and other processes [28,32]. Dysfunctional autophagy has been implicated in inflammation, cancer, cardiovascular diseases and neurodegenerative diseases [24,46]. Accumulating evidence indicates that deregulation of autophagy is involved in ␣-Syn degradation, contributing to the development and progression of sporadic PD [7,24,30,44–46]. Macroautophagy (hereafter referred to as autophagy) has been extensively studied. More than 30 autophagy related genes (ATGs) have been identified in yeast, most of which are conserved in mammals. ATG7, a ubiquitin-like modifier-activating enzyme, forms a complex with ubiquitin-like-conjugating enzyme ATG3. ATG7 is essential for microtubule-associated protein 1 light chain 3 (LC3) – phosphatidylethanolamine conjugation and the ATG12 conjugation systems, two critical systems in autophagosome formation [15,16,29,39–41]. Many studies in experimental animals with systemic and tissue-specific deletion of the ATG7 gene have demonstrated that the gene, and by inference autophagy, plays an essential role in neural development, neuronal survival and neurodegeneration [5,18–20]. In addition, recent studies involving tissue-specific deletion of ATG7 gene have revealed its roles in gluconeogenesis, adipogenesis and lipid metabolism in mice [9,37,38,52]. Alterations of autophagy have been observed in the brain tissues of PD patients, and in cellular and animal models of PD [1,4,48–50]. In the brain tissues of patients with dementia with Lewy bodies and in ␣-Syn transgenic mice, ATG7 gene expression is reduced [6]. Cell-specific deletion of the ATG7 gene in dopamine neurons leads to delayed neurodegeneration and ubiquitinated inclusions in a mouse model [10]. Therefore, we hypothesized that changes in the expression level of ATG7 gene may contribute to PD pathogenesis by altering autophagic activity. We have previously shown that expression of autophagic marker genes is significantly changed in sporadic PD patients [31,47]. Herein, as part of a systemic analysis of the critical ATG genes in PD, we genetically analyzed the ATG7 gene promoter in groups of sporadic PD patients and ethnic-matched healthy controls.
2. Materials and methods 2.1. Study subjects Sporadic PD patients (n = 101, mean age 66.99 years, male 54, female 47) were recruited from the Division of Neurology, Jining Medical University Affiliated Hospital, Jining Medical University, Jining, Shandong, China. Ethnic-matched healthy controls (n = 148, mean age 60.16 years, male 71, female 77) were recruited from the Health and Physical Examination Center in the same hospital. All PD patients were diagnosed by two neurologists. PD patients
and controls with a family history of PD were excluded. This study was approved by the Human Ethics Committee of Jining Medical University Affiliated Hospital and informed consent was obtained in all cases. 2.2. DNA sequencing Peripheral leukocytes were isolated from venous blood by conventional density gradient centrifugation. Genomic DNAs were extracted with QIAGEN DNeasy Blood and Tissue Kit (Qiagen, Valencia, CA, USA). The ATG7 gene promoter from −1145 bp to +112 bp was analyzed. Two overlapping DNA fragments, −1145 bp to −446 bp (700 bp) and −510 bp to +112 bp (623 bp), were generated by PCR and directly sequenced. PCR primers were designed based on genomic sequence of the human ATG7 gene (GenBank accession number, NC 000003.11) (Table 1). The DNA fragments were sequenced on a 3730 DNA Analyzer (Applied Biosystems, Foster City, CA, USA). The sequences were aligned and compared with the wild-type human ATG7 gene promoter. 2.3. Functional analysis Wild-type and variant ATG7 gene promoters (1194 bp, from −1082 bp to +112 bp to the transcription start site, GenBank accession number, NC 000003.11) were generated by PCR (Table 1), and subcloned into KpnI and HindIII sites of a luciferase reporter vector (pGL3-basic) to construct expression vectors. Designated expression vectors were transiently transfected into human embryonic kidney cells (HEK-293) with lipofectamine-2000 (Invitrogen, Carlsbad, CA, USA). Luciferase activities were measured 48 h posttransfection using dual-luciferase reporter assay system. All experiments were repeated at least three times, in triplicates. 2.4. Statistical analysis Quantitative data are represented as mean ± SE and analyzed by Student’s t-test. The distributions of sequence variants were compared between PD patients and controls by 2 test using SPSS v13.0. P < 0.05 was considered as statistically significant. 3. Results The sequence variants within the ATG7 gene promoter identified in PD patients and controls are summarized in Table 2. The locations of the variants are depicted in Fig. 1A. The chromatograms of the sequence variants identified only in PD patients or controls are shown in Fig. 1B. A total of eleven sequence variants were identified, all of which are novel. Among these variants, two are both heterozygous and homozygous, while the remaining nine are heterozygous. Four heterozygous variants, 11313449G>A, 11313811T>C, 11313913G>A and 11314041G>A, were identified in five PD patients, but in none of the controls. Two heterozygous variants, 11312947G>A and 11313006C>G, were only found in two controls. The other two heterozygous and homozygous variants, 11313215A>G and 11313910G>A, and three heterozygous variants, 11313127C>G, 11313666G>A and 11313988T>G, were found in both PD patients and controls with similar frequencies (P > 0.05). It is worth noting that the variant 11313913G>A was linked with a heterozygous variant 11313910G>A, which was identified in two PD patients. In addition, the allele at position of 11314041 within the human ATG7 gene genomic sequence (NC 000003.11) is A in the NCBI database. However, the same allele was G in all the PD patients and controls in this study, except one PD patient. The allele A was found in this PD patient. Thus, the allele A was considered as a heterozygous variant and defined as 11314041G>A.
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Table 1 PCR primers for the human ATG7 gene promoter.a PCR primers Sequencing ATG7-F1 ATG7-R1 ATG7-F2 ATG7-R2 Functional analysis ATG7-F ATG7-R a
Sequences
Location (bp)
5 -GGTTCCTTCTCTCCCACCTC-3 5 -GCACTGGACAGGTGTTGAAG-3 5 -GCCTTACAGGCCAGACAGAG-3 5 -CTTACCGCCGCTCAACTT-3
−1145 −446 −510 +112
5 -(KpnI)-ACGGAGTCTCGCTCTGTCGC-3 5 -(HindIII)-CTTACCGCCGCTCAACTTCC-3
−1082 +112
PCR products (bp) 700
623 1194
PCR primers were designed based on the genomic DNA sequence of the human ATG7 gene (NC 000003.11). The transcription start site (+1) is at position of 11314010.
Table 2 Sequence variants within the human ATG7 gene promoter. Variants
Genotype
Locationsa (bp)
Controls (n = 148)
PD (n = 101)
P-value
11312947G>A 11313006C>G 11313127C>G 11313215A>G
GA CG CG AA AG GG GA GA TC GG GA AA GA TG GA
−1064 −1005 −884 −796
1 1 6 69 68 11 0 2 0 20 72 56 0 4 0
0 0 7 45 42 14 1 1 1 19 49 33 2 1 1
– – 0.316 0.248
11313449G>A 11313666G>A 11313811T>C 11313910G>A
11313913G>A 11313988T>G 11314041G>A a
−561 −345 −199 −100
−97 −22 +32
– 1.000 – 0.467
– 0.651 –
The transcription start site of the ATG7 gene was set as +1.
Analysis of the ATG7 gene promoter with the TESS (transcription element search system) web tool (University of Pennsylvania) suggested that putative transcription factor binding sites may be altered by the identified variants. For example, the variant 11313449G>A abolished a GATA binding site and created a binding site for transcription factor TFII-I. The variant 11313449G>A abolished the binding site for HNF1. The variant 11313913G>A abolished an SP1 binding site and modified an AP2 binding site. The variant 11314041G>A created a binding site for NF1. In addition, the variant 11312947G>A abolished a YY1 binding site and the variant 11313006C>G created a CREB binding site. To examine the effects of these variants on the ATG7 gene promoter, luciferase reporter gene vectors containing wild type or variant promoters, pGL3-WT, pGL3-11312947A, pGL3-11313006G, pGL3-11313215G, pGL3-11313449A, pGL3-11313811C, pGL311313913A and pGL3-11314041A, were constructed and transfected into HEK-293 cells and dual-luciferase activities were determined. Compared to wild type ATG7 gene promoter, transcriptional activities were significantly decreased for the variant promoters identified in PD patients, pGL3-11313449A (P < 0.001), pGL3-11313811C (P < 0.01), pGL3-11313913A (P < 0.01) and pGL3-11314041A (P < 0.001). In contrast, transcriptional activities of pGL3-11312947A and pGL3-11313006G were not significantly different from that of pGL3-WT (P > 0.05). In addition, no significant difference was observed between pGL3-WT and pGL3-11313215G, which contained the variant that was found in PD patients and controls and was used as an internal control (Fig. 2). 4. Discussion In this study, we genetically analyzed the ATG7 gene promoter in sporadic PD patients and ethnic-matched healthy controls. Four novel heterozygous variants were identified in five PD patients,
but in none of the controls. All of the four variants significantly decreased transcriptional activities of the ATG7 gene promoter, probably by modifying, abolishing and creating binding sites for putative transcription factors. Although two variants only found in controls were suggested to change transcription factor binding sites, these variants did not affect transcriptional activities of the ATG7 gene promoter. To date, few mutations and variants in the ATG7 gene have been associated with human diseases. A polymorphism in the coding region of the ATG7 gene that substitutes alanine for valine has been associated with an earlier onset of Huntington’s disease [27]. In this study, we linked genetic variants within the ATG7 gene promoter to sporadic PD, the frequency of which was ∼5% (5/101). These variants may thus contribute to PD onset as a risk factor by altering autophagic activity.The human ATG7 gene has been localized to chromosome 3p25.3, encoding a protein of 703 amino acids [51]. The promoter region of the human ATG gene has not been characterized. At the epigenetic level, ATG7 gene expression has been shown to be modified by SIRT1, an NAD-dependent deacetylase, and by the acetyltransferase p300 protein [22,23]. In diverse human cancer cells, ATG7 gene expression is increased by proteasome inhibitors and epidermal growth factor receptor tyrosine kinase inhibitors [11,53], suggesting that ATG7 gene expression can be regulated. In this study, the sequence variants within the ATG7 gene promoter identified in PD patients affected its transcriptional activities, which may change ATG7 levels and lead to impaired autophagic activity. Several molecular pathways have been suggested for PD pathogenesis, including ␣-Syn accumulation, oxidative damage, mitochondrial dysfunction and inflammation [13,33,35,42]. Changes in ATG7 levels may contribute to PD onset via different pathways. First, as in vitro and in vivo studies indicate an important role of autophagy in ␣-Syn protein degradation, impaired autophagy may directly result in accumulation of ␣-Syn protein [30,44,45]. Second, impaired autophagy may lead to abnormal
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Fig. 1. Sequence variants within the ATG7 gene promoter identified in PD patients and controls. (A) Schematic representation of the sequence variants. The numbers correspond to those of the ATG7 gene genomic sequence (GenBank accession number, NC 000003.11). Transcription starts at position 11314010 in the first exon. (B) Chromatograms of the sequence variants only identified in either PD or controls. Sequence orientations are indicated. The top panels show wild-type sequences and the bottom panels homozygous variants. All the variants are marked with arrows. The variant 11313913G>A was linked with a heterozygous variant 11313910G>A, which is marked with a dashed arrow.
Fig. 2. Transcriptional activities of the ATG7 gene promoters. Wild type and variant ATG7 gene promoters were inserted into reporter gene vector pGL3-basic to generate expression vectors. These vectors were transfected into HEK-293 cells and dual-luciferase activities were measured. The transcriptional activity of wild type ATG7 gene promoter was designated as 100%. pGL3-basic was used as a negative control. The data were represented as mean ± SE from three independent transfection experiments, each in triplicate. Lanes 1, pGL3-basic; 2, pGL3-WT; 3, pGL3-11312947A; 4, pGL3-11313006G; 5, pGL3-11313215G, which was used as an internal negative control; 6, pGL3-11313449A; 7, pGL3-11313811C; 8, pGL311313913A; 9, pGL3-11314041A. *, P < 0.01, compared to pGL3-WT. **, P < 0.001, compared to pGL3-WT.
lipid metabolism. Elevated oxidized cholesterol metabolites have been shown to accelerate ␣-Syn accumulation by interacting with ␣-Syn [3,25]. In cellular and transgenic mouse models of PD, cholesterol-reducing agents reduce the accumulation of ␣-Syn and ameliorate the associated neuronal deficits [2,14,21]. Third, impaired autophagy may contribute to PD through inflammatory pathways. Autophagy has been shown to be required for monocyte differentiation and macrophage functions [17]. Fourth, ATG7 is involved in the phagocytosis of macrophages independent of autophagosome formation [34], which may be involved in ␣Syn clearance and inflammatory processes. The precise molecular mechanisms by which the ATG7 gene is linked to PD need further investigation. In conclusion, the ATG7 gene promoter was genetically and functionally analyzed in sporadic PD patients and ethnic-matched controls. The four novel heterozygous variants identified in PD patients significantly decreased the transcriptional activity of the ATG7 gene promoter, which would result in changed ATG7 protein levels and impaired autophagic activity, contributing to PD onset as a risk factor. As control of ATG7 gene expression and autophagic activity has been suggested as a therapy for neurodegenerative diseases [12], manipulation of ATG7 gene expression with different approaches may provide a novel potential therapy for PD patients.
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