TY - JOUR
AU1 - Padhy,, Biswajit
AU2 - Hayat,, Bushra
AU3 - Nanda, Gargi, Gouranga
AU4 - Mohanty, Pranjya, Paramita
AU5 - Alone, Debasmita, Pankaj
AB - Abstract Genetic variants at PTK2B–CLU locus pose as high-risk factors for many age-related disorders. However, the role of these variants in disease progression is less characterized. In this study, we aimed to investigate the functional significance of a clusterin intronic SNP, rs2279590, that has been associated with pseudoexfoliation, Alzheimer’s disease (AD) and diabetes. We have previously shown that the alleles at rs2279590 differentially regulate clusterin (CLU) gene expression in lens capsule tissues. This polymorphism resides in an active regulatory region marked by H3K27Ac and DNase I hypersensitive site and is an eQTL for CLU expression. Here, we report the presence of an enhancer element in surrounding region of rs2279590. Deletion of a 115 bp intronic region flanking the rs2279590 variant through CRISPR-Cas9 genome editing in HEK293 cells demonstrated a decreased clusterin gene expression. Electrophoretic mobility shift and chromatin immunoprecipitation assays show that rs2279590 with allele ‘A’ constitutes a transcription factor binding site for heat shock factor-1 (HSF1) but not with allele ‘G’. By binding to allele ‘A’, HSF1 abrogates the enhancer effect of the locus as validated by reporter assays. Interestingly, rs2279590 locus has a widespread enhancer effect on two nearby genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); both of which have been previously associated with AD as risk factors. To summarize, our study unveils a mechanistic role of the common variant rs2279590 that can affect a variety of aging disorders by regulating the expression of a specific set of genes. Introduction Clusterin or Apolipoprotein-J (8p21.1) is a multifunctional protein with diverse roles. Stabilization of cell–cell and cell–matrix interactions, lipid transport, apoptosis, inhibition of complement activation and clearance of extracellular deposits are some of the crucial functions attributed to clusterin (CLU) (1,2). CLU has been associated at both genomic and proteomic levels with age-related disorders, such as, pseudoexfoliation (PEX), Alzheimer’s disease (AD) and Type 2 Diabetes mellitus (3–7). The former two conditions, PEX (OMIM: 177650) and AD (OMIM: 104300), share similar pathological alterations like characteristic deposition of fibrillar protein aggregates and gradual deterioration of optic and brain nerves, respectively. Studies have shown that Clusterin accumulation differentiates the severe advanced stage of PEX called pseudoexfoliation glaucoma (PEXG) from the less severe syndrome stage, pseudoexfoliation syndrome (PEXS) (3). Additionally, brain function in AD-affected individuals with over accumulated Clusterin has been reported to deteriorate faster than normal individuals (8–10). It is however unclear, whether CLU accumulation in such disorders is the cause or a consequence. Although studies suggest that CLU plays a crucial role in preventing extracellular deposits; it also has been shown that knockout of clusterin in PDAPP mice (AD mice model) have reduced fibrillar plaque and neuritic dystrophy (11). Furthermore, an alternative truncated form of Clusterin called nuclear Clusterin (nCLU), is known to induce apoptosis by acting as a pro-death protein (12). Although, nCLU level is in low abundance in a healthy cell, stress induced increase in its expression can lead to apoptosis of affected neurons; a pathogenic feature for both PEX and AD. At the genomic level, common variants within CLU locus also have been associated with PEX and AD (3,4,6). A variant, rs2279590 within the clusterin gene is of particular importance as it has been picked up as a genetic risk factor for PEX, AD and diabetes (3,4,6). Earlier, we have shown that individuals homozygous for the risk allele ‘G’ at rs2279590 have a 2-fold higher clusterin expression in lens capsule tissues than those with allele ‘A’ (3); suggesting a regulatory role of rs2279590 over CLU expression. Association of rs2279590 with such diverse disorders and its location within a high-risk locus demands further investigation into its functional implications in disease progression. The current study focuses on finding the functional role, if any, of rs2279590 in clusterin gene regulation. We observed that the genomic region surrounding rs2279590 is located in an active locus marked by H3K27Ac and DNase I hypersensitivity site (ENCODE project) and is a quantitative trait loci (eQTL) for CLU expression as evident through GTEx project. Luciferase reporter assays validated the rs2279590 region as a regulatory element. Deletion of a 115 bp genomic region flanking rs2279590 polymorphic site by CRISPR/Cas9 system, leads to downregulation of clusterin gene expression in HEK293 cells; suggesting that the variant resides within an enhancer element. Transition from risk allele ‘G’ to ‘A’ at rs2279590 was found to create a transcription factor binding site (TFBS) for heat shock factor 1 (HSF1) that abrogated the enhancer activity of the region. The locus surrounding this variant was also found to regulate two neighbouring AD associated candidate genes, protein tyrosine kinase 2 beta (PTK2B) and epoxide hydrolase-2 (EPHX2); suggesting a distal enhancer effect contributed by this locus harbouring rs2279590. In conclusion, our study suggests that a common risk variant at the CLU locus has a widespread enhancer effect in regulating multiple candidate genes associated with PEX/AD and assists in the pathogenesis of such aging disorders. Results Locus containing rs2279590 acts as an enhancer and regulates clusterin gene expression Using the data from ENCODE (Encyclopedia of DNA Elements) project in UCSC genome browser, we observed that the region surrounding the polymorphic site, rs2279590 located in 7th intron of clusterin gene, is within a DNase I hypersensitive site and has modified H3K27Ac mark, suggesting the presence of an active regulatory region (Fig. 1). In addition, eQTL data from GTEx project also indicate a regulatory role of rs2279590 over CLU expression in both muscle-skeletal and skin-sun exposed tissue samples examined (Supplementary Material, Fig. S1). We, therefore, cloned a 201 bp region surrounding rs2279590 into a luciferase reporter vector to check its regulatory effect in HEK293 cells. As shown in Figure 2A, the luciferase activity was significantly higher (∼2-fold, P = 0.03) in constructs containing the rs2279590 locus as compared with the cells with empty luciferase vector; implying a regulatory effect of this locus. Figure 1. Open in new tabDownload slide rs2279590 is located within an enhancer element as indicated by active regulatory marks. Region around rs2279590 viewed in UCSC genome browser using ENCODE data. Three selected tracks: DNase hypersensitivity site, H3K27Ac (Histone H3 acetylated at lysine 27) and TFBS (Transcription factor binding site) are shown. Location of rs2279590 is indicated by a central vertical grey line. Figure 1. Open in new tabDownload slide rs2279590 is located within an enhancer element as indicated by active regulatory marks. Region around rs2279590 viewed in UCSC genome browser using ENCODE data. Three selected tracks: DNase hypersensitivity site, H3K27Ac (Histone H3 acetylated at lysine 27) and TFBS (Transcription factor binding site) are shown. Location of rs2279590 is indicated by a central vertical grey line. Figure 2. Open in new tabDownload slide Enhancer effect of rs2279590 locus on clusterin gene expression. (A) Normalized luciferase activity (214.4 ± 28.2) is shown for reporter construct containing the region surrounding rs2279590 (201 bp with major allele ‘G’) compared with that of empty vector (100 ± 21.9) (P = 0.03). (B) A representative figure showing deletion of 115 bp region around rs2279590 by using a pair of sgRNA through CRISPR/Cas9 genome editing method in HEK293 cells which are heterozygote (AG) for rs2279590. (C) Gel picture depicting confirmed deletion of a 115 bp region around rs2279590 generating homozygous knockout HEK293 cells. (D) qRT-PCR assay showing significant downregulation with a P value of 0.01 for secretory form of clusterin (sCLU) in cells with deletion (0.36 ± 0.1) compared with that of non deleted cells (1 ± 0.02). (E) Western blotting also showed a significant downregulation of CLU protein in cells with deletion than that of control non deleted cells. (F) An alternate transcript form of clusterin called as nuclear clusterin (nCLU) is also found to be significantly downregulated (P = 0.02) in cells with deletion (0.47 ± 0.12) compared with that of non deleted cells (1 ± 0.05) is shown through qRT-PCR assay. Het and Homo KO corresponds to homozyogus and heterozygous knockout deletions, respectively. All experiments were performed at least three times and values are represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05. Figure 2. Open in new tabDownload slide Enhancer effect of rs2279590 locus on clusterin gene expression. (A) Normalized luciferase activity (214.4 ± 28.2) is shown for reporter construct containing the region surrounding rs2279590 (201 bp with major allele ‘G’) compared with that of empty vector (100 ± 21.9) (P = 0.03). (B) A representative figure showing deletion of 115 bp region around rs2279590 by using a pair of sgRNA through CRISPR/Cas9 genome editing method in HEK293 cells which are heterozygote (AG) for rs2279590. (C) Gel picture depicting confirmed deletion of a 115 bp region around rs2279590 generating homozygous knockout HEK293 cells. (D) qRT-PCR assay showing significant downregulation with a P value of 0.01 for secretory form of clusterin (sCLU) in cells with deletion (0.36 ± 0.1) compared with that of non deleted cells (1 ± 0.02). (E) Western blotting also showed a significant downregulation of CLU protein in cells with deletion than that of control non deleted cells. (F) An alternate transcript form of clusterin called as nuclear clusterin (nCLU) is also found to be significantly downregulated (P = 0.02) in cells with deletion (0.47 ± 0.12) compared with that of non deleted cells (1 ± 0.05) is shown through qRT-PCR assay. Het and Homo KO corresponds to homozyogus and heterozygous knockout deletions, respectively. All experiments were performed at least three times and values are represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05. In addition, to check the in vivo effect of the locus on clusterin gene expression, we generated HEK293115−/− cells in which 115 bp genomic region around rs2279590 was deleted using CRISPR/Cas9 system (Fig. 2B and C). Two independent single-cell derived homozygous clones were subsequently used to check for CLU gene expression compared with that of control non-deleted cells. Quantitative real-time PCR (qRT-PCR) assays (Fig. 2D) confirmed a significant downregulation (P = 0.01) of transcripts for secretory form of clusterin (sCLU) in HEK293115−/− cells versus non-deleted cells and validated through western blot analysis (Fig. 2E). Furthermore, we have also checked the effect of this locus on another transcript variant of clusterin coding for a proapoptotic nuclear form of clusterin or nCLU which only expresses during high level of cytotoxic stress (e.g. ionic radiation) including lethal heat shock (13). nCLU expression was also found to be downregulated (Fig. 2F) in rs2279590 locus deleted cells (HEK293115−/−) similar to its secretory isoform, suggesting a regulatory effect of the said locus on both sCLU and nCLU expressions. Transition from G→A at rs2279590 creates a binding site for HSF1 Suspecting the presence of a transcription factor binding site (TFBS) at rs2279590, we analysed its surrounding region by online TFSEARCH program. In silico results showed that heat shock factors (HSFs) specifically bound to the flanking region of rs2279590 with allele ‘A’ but not with allele ‘G’. To validate the same, electrophoretic mobility shift assays (EMSAs) were performed using nuclear extracts from HEK293 cells and a 29 bp labelled oligo identical to surrounding genomic region at rs2279590 with allele ‘A’. It was observed that a specific protein complex bound to the labelled oligos (Lane 2, Fig. 3A) marked by a shift (arrowhead). With addition of unlabelled oligos comprising allele ‘A’ (Lane 3) the shift disappears but not with unlabelled oligos with allele ‘G’ (Lane 4); suggesting the binding complex is specific for allele ‘A’. Similar binding assays with labelled oligos comprising ‘G’ allele do not show a prominent shift as seen with allele ‘A’ (Supplementary Material, Fig. S2A). Figure 3. Open in new tabDownload slide HSF1 binds to allele ‘A’ but not to allele ‘G’ at rs2279590. EMSA was performed using heat shocked nuclear extract from HEK293 cells. (A) Unlike unlabelled oligo with allele ‘A’, addition of unlabelled oligo with allele ‘G’ at rs2279590 couldn’t abolish the shift suggesting the specificity of binding complex to allele ‘A’. (B) Two biotin labelled probes: a 28 bp reported heat shock element (HSE) (Lane 1-3) and one identical to 29 bp flanking region of rs2279590 with allele ‘A’ (Lane 4-6) were used. The presence of a shift in lane 2 and 5 (arrowhead) implies binding of a protein complex from HEK293 nuclear extract to both HSE and to the genomic region surrounding rs2279590. However, addition of their identical but unlabelled oligos in excess (lane 3 and 6) dissolves the shift suggesting specificity of the binding complex. (C) Specific competitive assays were done with increasing concentrations of unlabelled HSE (1× and 100× fold excess) or unlabelled rs2279590 probe with allele ‘A’ (1×, 10×, 50×, 100×, 200× and 400× fold excess). The shift abolished in the lanes 3-10 indicates that the same protein complex binds to HSE and rs2279590 region. (D) Supershift assay with EMSA validated HSF1 antibody (Lane 4) shows elimination of the shift which signifies the binding of HSF1 to allele ‘A’ at rs2279590. All experiments were replicated at least three times. Arrowhead and Starmark represent the specific and nonspecific shift, respectively. Figure 3. Open in new tabDownload slide HSF1 binds to allele ‘A’ but not to allele ‘G’ at rs2279590. EMSA was performed using heat shocked nuclear extract from HEK293 cells. (A) Unlike unlabelled oligo with allele ‘A’, addition of unlabelled oligo with allele ‘G’ at rs2279590 couldn’t abolish the shift suggesting the specificity of binding complex to allele ‘A’. (B) Two biotin labelled probes: a 28 bp reported heat shock element (HSE) (Lane 1-3) and one identical to 29 bp flanking region of rs2279590 with allele ‘A’ (Lane 4-6) were used. The presence of a shift in lane 2 and 5 (arrowhead) implies binding of a protein complex from HEK293 nuclear extract to both HSE and to the genomic region surrounding rs2279590. However, addition of their identical but unlabelled oligos in excess (lane 3 and 6) dissolves the shift suggesting specificity of the binding complex. (C) Specific competitive assays were done with increasing concentrations of unlabelled HSE (1× and 100× fold excess) or unlabelled rs2279590 probe with allele ‘A’ (1×, 10×, 50×, 100×, 200× and 400× fold excess). The shift abolished in the lanes 3-10 indicates that the same protein complex binds to HSE and rs2279590 region. (D) Supershift assay with EMSA validated HSF1 antibody (Lane 4) shows elimination of the shift which signifies the binding of HSF1 to allele ‘A’ at rs2279590. All experiments were replicated at least three times. Arrowhead and Starmark represent the specific and nonspecific shift, respectively. A three-tier experimental validation was carried out to validate the binding of HSFs at rs2279590. Figure 3B shows a comparative shift in lanes 2 and 5 when a labelled consensus heat shock element (HSE) and labelled rs2279590 oligo with allele ‘A’ were used, respectively. A competitive EMSA on labelled oligo containing rs2279590/A allele, when challenged with increased concentrations (1- and 100-fold excess) of consensus unlabelled HSE oligo (lanes 3 and 4, Fig. 3C) or unlabelled rs2279590/A oligo (1-, 10-, 50-, 100-, 200- and 400-fold excess) (lanes 5–10, Fig. 3C) the shift disappears; indicating that the 29 bp rs2279590/A sequence binds to protein complexes similar to those binding to a heat shock element. As shown in Figure 3C, 1-fold addition of either unlabelled HSE or rs2279590/A (lane 3 and 5, respectively) oligos was sufficient to compete for the binding complex, thereby drastically reducing the intensity of the shift. Later, when the binding complex was challenged with HSF1-specific antibody, disappearance of the shift was noted (lane 4, Fig. 3D) indicating that the protein binding complex bound to allele ‘A’ at rs2279590 comprises of HSF1 protein. However, absence of any super-shift could be because the antibodies are bound to the epitope of proteins necessary for DNA-protein complex formation. Super-shift assays with other HSFs (HSF2 and HSF4) did not dissolve the shift completely, suggesting the binding complex consists of HSF1 but not HSF2 and HSF4 (Supplementary Material, Fig. S2B). Additionally, to validate the binding of HSF1 to rs2279590 in vivo, chromatin immunoprecipitation (ChIP) assays were performed in HEK293 cells. As shown in Figure 4A and B the genomic region surrounding rs2279590 was enriched by qRT-PCR after immunoprecipitation with anti-HSF1 antibody but not by rabbit IgG (negative control). This confirms in vivo binding of HSF1 to the sequences comprising of rs2279590. Figure 4. Open in new tabDownload slide Enhancer effect of rs2279590 locus was lost after binding of HSF1 to allele ‘A’ at rs2279590. (A) Chromatin immunoprecipitated samples were used in qRT-PCR assay which shows more enrichment of rs2279590 flanking region with HSF1 antibody than with that of rabbit IgG in HEK293 cells. (B) Fold enrichment of genomic region around rs2279590 is significantly higher (P = 0.004) in IP samples with HSF1 (3.19 ± 0.55) antibody than that of rabbit IgG (1.04 ± 0.08). (C) Normalized luciferase activity is shown in heat shocked, MG132 (5 µM) treated and siRNA (HSF1) treated HEK293 cells to differentiate the allelic effect on reporter activity. Cells transfected with constructs containing allele ‘A’ show reduced reporter activity in both heat shocked (41.23 ± 13.61, P = 0.01) and MG132 (76.72 ± 34.73, P = 0.03) treated cells than cells with allele ‘G’ (226.11 ± 27.43 and 201.39 ± 12.84, respectively); while it was found to be similar with P value of 0.31 between constructs with allele ‘G’ (191.6 ± 7.43) and ‘A’ (144.46 ± 16.05) after knockdown of HSF1 with a pool of HSF1 specific siRNA. All experiments were performed at least three times and values are represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05, **P < 0.005. Figure 4. Open in new tabDownload slide Enhancer effect of rs2279590 locus was lost after binding of HSF1 to allele ‘A’ at rs2279590. (A) Chromatin immunoprecipitated samples were used in qRT-PCR assay which shows more enrichment of rs2279590 flanking region with HSF1 antibody than with that of rabbit IgG in HEK293 cells. (B) Fold enrichment of genomic region around rs2279590 is significantly higher (P = 0.004) in IP samples with HSF1 (3.19 ± 0.55) antibody than that of rabbit IgG (1.04 ± 0.08). (C) Normalized luciferase activity is shown in heat shocked, MG132 (5 µM) treated and siRNA (HSF1) treated HEK293 cells to differentiate the allelic effect on reporter activity. Cells transfected with constructs containing allele ‘A’ show reduced reporter activity in both heat shocked (41.23 ± 13.61, P = 0.01) and MG132 (76.72 ± 34.73, P = 0.03) treated cells than cells with allele ‘G’ (226.11 ± 27.43 and 201.39 ± 12.84, respectively); while it was found to be similar with P value of 0.31 between constructs with allele ‘G’ (191.6 ± 7.43) and ‘A’ (144.46 ± 16.05) after knockdown of HSF1 with a pool of HSF1 specific siRNA. All experiments were performed at least three times and values are represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05, **P < 0.005. Binding of HSF1 to allele ‘A’ at rs2279590 abrogates the enhancer effect of the locus In order to check the allele specific effect on the reporter activity, a 201 bp intronic region harbouring rs2279590 (with either ‘A’ or ‘G’ allele) was cloned into pGL4.23 luciferase vector and transfected into HEK293 cells and the reporter activity was checked. As represented in Figure 4C, changing the allele from ‘G’ to ‘A’ in HEK293 transfected cells, under heat shock or MG-132 treatment drastically reduced the reporter activity; suggesting a loss of enhancer effect with rs2279590/A allele. Although a similar effect was also found in non-treated cells growing at normal conditions but the difference was not found to be significant (Supplementary Material, Fig. S3). To understand allele specific enhancer activity in absence of HSF1, luciferase activity in HEK293 transfected cells was checked after knocking down HSF1 expression by a pool of HSF1 specific siRNA (Supplementary Material, Fig. S4). As expected, no significant difference (P = 0.31) was observed in the reporter activity between constructs containing either allele ‘G’ or ‘A’ in HSF1 knocked down cells (Fig. 4C). This suggests binding of HSF1 to allele ‘A’ abrogates the enhancer effect of the locus. Deletion of rs2279590 locus alters the expression of PTK2B and EPHX2, known modulators in AD pathogenesis GWAS studies have previously shown that the region containing CLU and PTK2B is situated within a high-risk locus for AD pathogenesis (14). As rs2279590 is also within the same high-risk locus, we wanted to investigate the effect of this variant in regulating the neighbouring genes around CLU. For this, expression of nine crucial genes clustered around 5’ and 3’ ends of CLU gene, within a stretch of around 558 kb was analysed in HEK293115−/− cells lacking the regulatory locus (Fig. 5A). One of the genes, CHRNA2 did not show any expression in control HEK293 cells and is therefore omitted from analysis. Interestingly, a significant downregulation in the expression of two upstream genes, PTK2B and EPHX2 was observed in HEK293115−/− cells as compared with control HEK293 cells (Fig. 5B). This indicates that the genomic region around rs2279590 has an extended regulatory role with a widespread enhancer effect on PTK2B and EPHX2. Figure 5. Open in new tabDownload slide Locus containing rs2279590 also regulates PTK2B and EPHX2 gene expression. (A) A stretched genomic region of ∼700 kb around clusterin gene in chromosome 8 is shown. Position of crucial genes relative to that of clusterin is depicted. (B) qRT-PCR assays were done for genes located in the PTK2B-CLU locus in rs2279590 region deleted cells and control non-deleted cells. Expression of PTK2B and EPHX2 is significantly downregulated with a P value of 0.006 and 0.008 respectively in deleted cells (0.58 ± 0.08 and 0.34 ± 0.04, respectively) compared with control non-deleted cells (1 ± 0.05 and 1 ± 0.04, respectively). qRT-PCR assay did not show any expression of CHRNA2 in control HEK293 cells, hence omitted from analysis. Experiments were performed at least three times and values are represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05. Figure 5. Open in new tabDownload slide Locus containing rs2279590 also regulates PTK2B and EPHX2 gene expression. (A) A stretched genomic region of ∼700 kb around clusterin gene in chromosome 8 is shown. Position of crucial genes relative to that of clusterin is depicted. (B) qRT-PCR assays were done for genes located in the PTK2B-CLU locus in rs2279590 region deleted cells and control non-deleted cells. Expression of PTK2B and EPHX2 is significantly downregulated with a P value of 0.006 and 0.008 respectively in deleted cells (0.58 ± 0.08 and 0.34 ± 0.04, respectively) compared with control non-deleted cells (1 ± 0.05 and 1 ± 0.04, respectively). qRT-PCR assay did not show any expression of CHRNA2 in control HEK293 cells, hence omitted from analysis. Experiments were performed at least three times and values are represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05. Upregulation of HSF1 suggests proteotoxic stress in anterior eye tissues of PEX-affected subjects In response to proteotoxic stress, cell activates HSF1, which in turn upregulates the heat shock proteins (HSPs) to prevent protein misfolding. Earlier studies have reported that HSF1 overexpression has a protective role in a variety of neurodegenerative disorders caused due to accumulation of misfolded proteins (15–17). Further, aberrant expression of HSF1 has also been implicated in neurodegeneration (18). Assuming an involvement of proteotoxic stress in anterior eye tissues of PEX-affected subjects, we checked the expression of HSF1. Interestingly, HSF1 level was found to be significantly upregulated in both lens capsule and conjunctiva of PEXS-affected individuals (Fig. 6). However, we didn’t find any upregulation of HSF1 in PEXG-affected individuals; a later stage of PEX with degenerated optic nerve head (ONH) cells. Figure 6. Open in new tabDownload slide HSF1 upregulation indicates a proteotoxic stress in anterior eye tissues of PEXS-affected study subjects. qRT-PCR assays were done to check the expression of HSF1 in anterior eye tissues of PEX (including both PEXS and PEXG) and control subjects. HSF1 is found to be upregulated in both lens capsule (1.39 ± 0.15, P = 0.02) and conjunctiva (2.68 ± 0.29, P = 0.0003) in PEXS individuals compared with that of control (1 ± 0.03 and 1.06 ± 0.12, respectively). However, no difference was observed between control and PEXG in lens capsule (1.01 ± 0.05 versus 1.11 ± 0.08) and conjunctiva (1.01 ± 0.06 versus 1.15 ± 0.08), respectively). This implicates a proteotoxic stress in the anterior eye tissues of PEXS subjects and a failed stimulation to upregulate HSF1 in PEXG individuals might be responsible for the death of ONH cells. LC and Conj correspond to lens capsule and conjunctiva, respectively. Sample size is denoted by ‘n’ and expression change in fold is represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05, ***P < 0.0005. Figure 6. Open in new tabDownload slide HSF1 upregulation indicates a proteotoxic stress in anterior eye tissues of PEXS-affected study subjects. qRT-PCR assays were done to check the expression of HSF1 in anterior eye tissues of PEX (including both PEXS and PEXG) and control subjects. HSF1 is found to be upregulated in both lens capsule (1.39 ± 0.15, P = 0.02) and conjunctiva (2.68 ± 0.29, P = 0.0003) in PEXS individuals compared with that of control (1 ± 0.03 and 1.06 ± 0.12, respectively). However, no difference was observed between control and PEXG in lens capsule (1.01 ± 0.05 versus 1.11 ± 0.08) and conjunctiva (1.01 ± 0.06 versus 1.15 ± 0.08), respectively). This implicates a proteotoxic stress in the anterior eye tissues of PEXS subjects and a failed stimulation to upregulate HSF1 in PEXG individuals might be responsible for the death of ONH cells. LC and Conj correspond to lens capsule and conjunctiva, respectively. Sample size is denoted by ‘n’ and expression change in fold is represented as mean ± SEM. Student’s t-test was used to calculate statistical significance between groups, *P < 0.05, ***P < 0.0005. Discussion Clusterin, a multifunctional protein has divergent roles from being cytoprotective to cytotoxic. Extensive studies have reported the association of CLU variants with the risk of developing various diseases like PEX, diabetes and AD (3,4,6,7). This indicates that a complex mechanism employed by CLU is responsible for the progression of these diseases. Understanding the role of risk variants within CLU can help in better diagnosis and treatment of such disorders. In this study, we aimed to characterize the functional significance of a risk variant, rs2279590, housed in 7th intron of CLU gene. This particular variant has been reported to be a risk factor for both AD and PEX (3,4,6). We, therefore, intended to understand the functional role of this SNP. rs2279590 is a functional intronic variant and regulates clusterin gene expression Recent studies have reported the involvement of genetic variants in CLU gene with the risk of developing PEX and AD. Since then, attempts have been made to identify the significance of these risk variants in regulating CLU expression. One such study reported that the AD-risk allele at CLU, rs9331888, increased the clusterin expression as compared with its counter allele (19). Similarly, two other GWAS-associated risk variants; rs9331896 and rs11136000 which are located in the second and third intron of CLU gene (6,14), respectively have regulatory role over CLU expression as evident through eQTL data from GTEx project. Accordingly, examined tissue samples with risk allele of both these SNPs, rs9331896 (T) and rs11136000 (C) reportedly have elevated CLU expression than their respective reference alleles (GTEx Portal on 07/11/17). This suggests a cumulative effect of these risk variants in CLU upregulation during pathogenesis of AD. However, same wasn’t true for another AD-risk variant, rs7982; suggesting that not all associated variants in clusterin gene have functional implications in disease causation (10). Similar to the above studies, we previously reported that individuals homozygous for risk allele ‘G’ at rs2279590 (another risk variant for AD/PEX) within CLU showed 2-fold increased clusterin gene expression compared with ‘AA’ carriers in lens capsules (3). In the present study, we found an enhancer effect of the genomic region surrounding rs2279590 with the risk allele ‘G’ on CLU expression. This is also supported by the eQTL data from GTEx that indicates tissue samples with genotype ‘GG’ (alternative allele- G) at rs2279590 have elevated CLU expression than samples with that of ‘AA’ (reference allele- A) with an effect size of 0.2 (Supplementary Material, Fig. S1). However, reverse allelic association (allele ‘A’) in German cohort suggests a profound effect of other nearby SNPs compared with moderate effect shown by rs2279590 (4). Analysis of eQTL data for entire CLU gene (GTEx Portal on 07/11/17) also indicates that there are dozens of SNPs acting as eQTL for CLU expression each with an effect size around 0.2. Altogether, the combined effect of nearby SNPs including rs9331896 and rs11136000 may supersede the moderate effect shown by rs2279590 in different ethnic background. Further ongoing functional studies of these eQTL SNPs in relation to rs2279590 will define the role of CLU in AD and PEXG progression. This substantiates the fact that certain risk alleles in CLU gene enhance clusterin expression and thereby contribute towards AD/PEX pathogenesis. Studies have shown that elevated level of CLU is linked to disease severity in both PEXG- and AD-affected patients (3,8,20). Both in the anterior eye tissues of PEXG and in the brain of AD-affected individuals CLU was found to be upregulated compared with their respective controls (3,20,21). Although CLU was upregulated, whether it’s the cause or an effect is still speculative. Clusterin knockout in PDAPP mice has shown to be beneficial due to reduced fibrillar plaque formation and neuritic dystrophy (11). Nonetheless, being an extracellular chaperone, the protective role of CLU cannot be undermined. A study suggests that it is the extracellular CLU: substrate ratio that decides the fate of CLU to be pathogenic or protective. When substrate is in large molar excess, CLU coincorporates itself within the complex in a failed attempt to prevent aggregation; leading to large insoluble aggregates (22). Further, in chronic stress a nuclear form of Clusterin (nCLU) tends to increase which initiates caspase-3 dependent apoptosis (12). Enhancer effect associated with risk allele ‘G’ at rs2279590 locus, on both secretory and nuclear forms of Clusterin suggests a cytotoxic effect of both sCLU and nCLU can augment neurodegeneration. HSF1 is a critical regulator in clusterin gene expression Through bioinformatic and molecular analysis, we identified HSF1 to bind preferentially to protective allele ‘A’ at rs2279590 and consequently regulate clusterin gene expression. HSF1 is a prominent member of a family of transcription factors called heat shock factors known to be activated upon heat shock, stress or inflammatory triggering agents. Upon activation, it differentially regulates either by upregulating or downregulating a cascade of genes. It is a complex regulator and affect gene expression differently under similar conditions in different tissues (23). Here, binding of HSF1 to the protective allele ‘A’ negatively regulates CLU gene expression; suggesting a suppressor effect. By negatively regulating, HSF1 might help in reducing the cytotoxicity associated with CLU overaccumulation. Earlier studies have related HSF1 to various types of neurodegenerative proteinopathies. Downregulation of HSF1 accelerated the formation of protein aggregates in Huntington’s disease (24); whereas, activated HSF1 in R6/2 Huntington disease mice prevents polyglutamine aggregate formation (16). Thus, HSF1 could be regarded as a key controller of endogenous protein aggregation (25). Similarly, in AD animal models, HSF1 expression was remarkably decreased in the cerebellum; whereas, overexpression of HSF1 reduces brain β-amyloid levels and improves memory function (26,27). These studies suggest a protective role of HSF1 in such aging disorders. This was in-sync with the recent reports where the Alzheimer’s patients showed significantly lower levels of HSF1 than control individuals (28). In this study, we identified an upregulated HSF1 mRNA levels in the anterior eye tissues of PEXS subjects but not in the later more severe form of disease, i.e. PEXG; suggesting the protective role of HSF1 is diminished in the later stages of the disease condition and may augment severity. rs2279590 enhancer element also regulates two distal genes: PTK2B and EPHX2 GWAS studies have previously shown an association of PTK2B-CLU loci with AD (14). Protein tyrosine kinase 2 beta or PTK2B, belongs to a non-receptor protein kinase family involved in calcium induced regulation of ion channels and MAPK pathway activation. It plays a role in inducing phosphorylation of GSK3 (Glycogen synthase kinase 3) which then promotes Tau fibrillar pathology. In hippocampus of AD-affected individuals, heightened phosphotyrosine immunoreactivity was found in the neuritic plaques, tangle-bearing neurons and microglia, which are characteristic features of increased PTK2B activity (29). Accumulation of PTK2B was also reported in the early event of AD pathogenesis with progressive Tau pathology. However, it is unclear whether accumulation of PTK2B in aiding the Tau pathology, is a consequence or a cause (30). In this study, we found decreased levels of PTK2B mRNA in cells with the deleted enhancer element containing rs2279590; suggesting a distal enhancer effect of the locus over PTK2B expression. Although, regulatory effect of rs2279590 on PTK2B was not established in eQTL data from GTEx project, it may depend on various confounding factors including the type of tissue analysed. Further, earlier reports have shown an increased PTK2B expression with AD associated risk alleles at rs28834970 and rs2718058 (a cis-pQTL within PTK2B locus and a trans-pQTL within NME8 locus) (31). Cumulatively, these results suggest that AD risk variants are capable of regulating PTK2B expression to promote AD pathogenesis. Deletion of rs2279590 locus also leads to downregulation of EPHX2, another candidate gene for AD pathogenesis. EPHX2 or epoxide hydrolase-2 metabolises epoxyeicosatrienoic acids which are proven to be neuroprotective. Upregulation of hydrolase activity of soluble EPHX2 leads to an increased OGD-induced (oxygen-glucose deprived) neuronal cell death (32). Recently, EPHX2 too has been associated with AD as a risk factor through GWAS (33). According to all the cumulative data known till date, a model is being proposed as shown in Figure 7 that rs2279590 resides within a regulatory genomic region and with risk allele ‘G’ shows a widespread enhancer effect on three PEX/AD risk associated candidate genes; CLU, PTK2B and EPHX2. However, binding of HSF1 to the protective allele ‘A’ abolishes the enhancer effect of the locus, which consequent to decreased expression of the target genes; thereby implying a lowered risk of developing PEX or AD. Figure 7. Open in new tabDownload slide Proposed model explaining the effect of SNP-dependent HSF1 binding at rs2279590, target gene expression and AD/PEX risk. Genomic region containing rs2279590/G resides within an active enhancer element and increases gene expression of CLU, PTK2B and EPHX2. However, binding of HSF1 to protective allele ‘A’ at rs2279590 diminishes the enhancer effect of the locus; which leads to decrease in target gene expression with a lowered risk of developing PEX/AD. Figure 7. Open in new tabDownload slide Proposed model explaining the effect of SNP-dependent HSF1 binding at rs2279590, target gene expression and AD/PEX risk. Genomic region containing rs2279590/G resides within an active enhancer element and increases gene expression of CLU, PTK2B and EPHX2. However, binding of HSF1 to protective allele ‘A’ at rs2279590 diminishes the enhancer effect of the locus; which leads to decrease in target gene expression with a lowered risk of developing PEX/AD. Summary and Conclusion With this study, we confirm that a PEX/AD associated risk variant, rs2279590, resides within an enhancer element and regulates the expression of three candidate genes CLU, PTK2B and EPHX2, which were previously known to be modulators in the progression of AD. Increase in CLU expression by the risk allele at rs2279590 provides a mechanistic insight into the cytotoxic role of CLU in PEXG individuals. Future studies are needed to explore the role of PTK2B and EPHX2 genes in PEX pathogenesis. Materials and Methods Study subjects recruitment This study was approved by the ethics review boards of the National Institute of Science Education and Research and JPM Rotary Club of Cuttack Eye Hospital and Research Institute, India and adhered to the tenets of the Declaration of Helsinki. All participants underwent a detailed ocular examination, including slit lamp, ocular biometry, Goldman applanation tonometry, +90 D biomicroscopic fundus evaluation and 4 mirror gonioscopy. Inclusion and exclusion criteria for the grouping of control and PEX-affected individuals were followed as reported previously (3). Anterior eye tissues (lens capsules and conjunctiva) from PEX-affected study subjects and age matched controls were collected in RNAlater stabilisation solution (Invitrogen, USA) during cataract surgery and stored at −80°C until further use. Cell culture The human cell line, HEK293 was cultured in HiGlutaXL Dulbecco’s Modified Eagle Medium, High Glucose (AL007G) with 10% fetal bovine serum (RM9952) and 1% penicillin (100 U/ml) and streptomycin (0.1 mg/ml) (A001), maintained at 37°C and 5% CO2. All cell culture chemicals were procured from HiMedia, Mumbai, India. Luciferase reporter assays For reporter assays, pGL4.23 luciferase reporter vector with minimal promoter and pGL4.74 renilla vector were used (Promega, Madison, Wisconsin). Genomic DNA was extracted from peripheral blood leucocytes of the study subjects by phenol-chloroform extraction method. An intronic region of 201 bp surrounding rs2279590 variant (harbouring either ‘AA’ or ‘GG’ genotype at the polymorphic site) was PCR-amplified using a specific primer pair (Supplementary Material, Table S1) from the extracted genomic DNA. The amplified products were then cloned into pGL4.23 vector by double digestion at KpnI-XhoI site (KpnI-HF and XhoI-HF, New England Biolabs, Ipswich, MA, USA). For transfection, HEK293 cells were seeded in a 12-well plate. At 80% confluency, the cells were transiently co-transfected (Lipofectamine 2000, Invitrogen) with luciferase constructs (1 µg) and renilla vector (pGL4.74, 10 ng). Transfection efficiency was normalized by renilla reporter activity. After 24 h of post-transfection, cell lysates were prepared following Dual-Luciferase® Reporter Assay System (Promega). Reporter activities were measured with Varioskan® Flash Multimode reader (Thermo Fisher Scientific, Waltham, Massachusetts) following manufacturer’s instructions. Values for luciferase activity, post normalisation with Renilla reporter activity, were used for further analysis. Each of the experiments was repeated independently with at least three replicates. CRISPR/Cas9 construct preparation and genome editing Genome editing of HEK293 was done by CRISPR/Cas9 system as described previously (34). A pair of sgRNA was designed (http://crispr.mit.edu/; date last accessed September 7, 2016) with a little off-target specificity to delete a 115 bp region around the SNP, rs2279590 (Supplementary Material, Table S1). Annealed oligonucleotides were phosphorylated and ligated into BbsI digested PX459 (Plasmid #62988; Addgene, Cambridge, Massachusetts). At 50% confluency, the HEK293 cells were transfected with 2.5 µg of each CRISPR construct (with sgRNA1 and sgRNA2) simultaneously using lipofectamine 2000 (Invitrogen, Carlsbad, California). Transfected cells were selected after 24-h post-transfection in complete media supplemented with 2.5 µg/µl puromycin. Single cell clones were then isolated and cultured by dilution cloning and subsequently genomic DNA was isolated using DNeasy Blood & Tissue Kit (Qiagen, Hilden, Germany) following manufacturer’s instructions. Screening was done to find out homozygous deletion of genomic region containing rs2279590 by using a specific set of primers (Supplementary Material, Table S1) outside the targeted sites and subsequently confirmed by sequencing. Positive clones were used for subsequent experiments. Electrophoretic mobility shift assay Online program, TFSEARCH (http://www.cbrc.jp/research/db/TFSEARCH.html; date last accessed December 27, 2014) was used for candidate search with a default threshold score set to 85. Two pairs of complementary 29-mer oligonucleotides, centred around rs2279590 with allele ‘A’ or ‘G’ and a previously reported heat shock element (HSE) were ordered (35); both with and without biotin 5’-end labelling [Integrated DNA Technologies (IDT), Iowa, USA]. Sequences of the oligomers are listed in Supplementary Material, Table S1. Annealing of complementary oligos was done by incubating them at 95°C for 5 min, followed by step-cooling to room temperature. Nuclear extract from heat shocked (1 h at 42 °C) HEK293 was prepared by using NE-PER kit (Thermo Fisher Scientific) and protein concentrations were estimated by Bradford’s assay. EMSA was performed using LightShift Chemiluminescent EMSA Kit (Thermo Fisher Scientific) following the manufacturer’s instructions. DNA-protein binding assays were carried out using 3 µg of total nuclear extract and 100 fmol of biotinylated annealed oligonucleotides for each 20 µl total reaction volume. Competitive EMSA was done using 400-fold excess (40 pmol) of unlabelled double-stranded oligonucleotides. For supershift assays, 2 µg of EMSA validated HSF1, HSF2 and HSF4 antibody (sc-9144X, sc-13056 and sc-366983; respectively) were procured from Santa Cruz Biotechnology, Dallas, Texas and were incubated with the final reaction mixture for an additional 30 min on ice. Protein-DNA complexes were separated on 6% native polyacrylamide gels in 0.5X TBE, transferred to nylon membranes (Thermo Fisher Scientific) and observed by chemiluminescent detection methods post-UV crosslinking. Chromatin immunoprecipitation Pierce Agarose ChIP kit (Thermo Fisher Scientific) was used for in vivo ChIP assays and the protocol suggested by the manufacturer was followed. Briefly, 2x106 HEK293 cells were seeded and used for each experiment. Micrococcal nuclease (20 U) was used for efficient digestion of formaldehyde fixed chromatin. Digested chromatin with an average length of 500 bp was subsequently used for immunoprecipitation (IP). For each IP, 25 µg of digested chromatin was incubated overnight at 4°C with 5 µg of ChIP validated HSF1 antibody (Santa Cruz Biotechnology, sc-9144X). As negative control, normal Rabbit IgG (1 µl) provided in the ChIP kit was used. Later, 20 µl of ChIP grade protein A/G plus agarose beads was added to each IP and incubated for 2 h at 4°C with continuous rocking. IP complexes were then washed twice with each wash buffer supplied in the kit. Elution of the IP complex was done by incubating in IP elution buffer at 65°C for 1 hr. Reverse crosslinking and protein digestion were done by NaCl and Proteinase K, respectively. Subsequently, DNA was purified by using DNA Clean-Up column supplied in the kit. Fold enrichment of the target region in IP compared with that of input was assayed by a specific primer set (Supplementary Material, Table S1) through quantitative real time PCR (qRT-PCR). Knockdown assays siRNA pool for targeting HSF1 was procured from Santa Cruz Biotechnology (sc-35611) and transfection was done as per manufacturer’s protocol. For luciferase assays, reporter vector and siRNA pool were transfected together, cell lysates were prepared after 36 h and subsequently checked for reporter activity. Quantitative real-time PCR Total RNA was isolated from individual lens capsules or HEK293 cells by using an RNA extraction kit (RNeasy Mini Kit, QIAGEN). cDNA was synthesized with 1 µg of total RNA, using a Reverse Transcription Kit (Verso cDNA Synthesis Kit - AB1453A; Thermo Fisher Scientific). Gene specific primers (Supplementary Material, Table S1) overlapping exon-exon junction were designed by using PrimerQuest Tool (IDT). qRT-PCR was performed using 7500 Real time PCR Systems (Applied Biosystems, Foster city, California). Total 5 ng of cDNA and 0.8 µM each of forward and reverse primers were used per 20 µl reaction volume in triplicate for each sample. Amplification specificity of the PCR product was checked via melt curve analysis and sequencing. ΔΔCt method was used to calculate expression change in fold for each target gene. For normalization, GAPDH expression was taken as an endogenous control. Western blotting Cytosolic extract from HEK293 cells was prepared using NE-PER kit (Thermo Fisher Scientific). Denatured cytosolic extract (10 µg) was then loaded on a 12% SDS-PAGE and subsequently transferred onto a PVDF membrane (Immobilion-P PVDF from Merck Millipore, Billerica, Massachusetts). Subsequent steps were followed as previously described (3). A polyclonal antibody for Clusterin (sc-6419; Santa Cruz Biotechnology) was used as primary antibody and HRP-conjugated rabbit anti-goat IgG (480011730; Imgenex, India) was used as secondary antibody. GAPDH antibody (6665 A; Imgenex, India) was used for endogenous control experiments. Detection was done using chemiluminescence kit (Super Signal Femto Maximum Sensitivity Substrate, Thermo Fisher Scientific) in a Chemi-Doc (Bio-Rad, Hercules, California). DNA sequencing Bidirectional Sanger’s sequencing of all the constructs was done using BigDye Terminator v.3.1 cycle sequencing kit (Applied Biosystem), with their respective primers (Supplementary Material, Table S1) on 3130xl Genetic Analyser (Applied Biosystem) platform. For sequencing analysis, BioEdit v7.1 (http://www.mbio.ncsu.edu/bioedit/bioedit.html; date last accessed March 4, 2016) and sequence analysis software v5.3 (Applied Biosystem) were used. Statistical analysis All experiments were repeated at least three times. Descriptive data were presented as mean ± SEM. Group wise results for statistical significance was compared by Student’s t-test and P < 0.05 was considered as statistically significant. Supplementary Material Supplementary Material is available at HMG online. Acknowledgement The authors thank the study participants for their contribution and consent for this study. Conflict of Interest statement. None declared. Funding National Institute of Science Education and Research, Department of Atomic Energy (India); Council of Scientific and Industrial Research (India) Grant no. 27(0317)/16/EMR-II. References 1 Rosenberg M.E. , Silkensen J. ( 1995 ) Clusterin: physiologic and pathophysiologic considerations . Int. J. Biochem. Cell Biol ., 27 , 633 – 645 . Google Scholar Crossref Search ADS PubMed WorldCat 2 Jones S.E. , Jomary C. ( 2002 ) Clusterin . Int. J. Biochem. Cell. Biol ., 34 , 427 – 431 . Google Scholar Crossref Search ADS PubMed WorldCat 3 Padhy B. , Nanda G.G. , Chowdhury M. , Padhi D. , Rao A. , Alone D.P. ( 2014 ) Role of an extracellular chaperone, clusterin in the pathogenesis of pseudoexfoliation syndrome and pseudoexfoliation glaucoma . Exp. Eye Res ., 127 , 69 – 76 . Google Scholar Crossref Search ADS PubMed WorldCat 4 Krumbiegel M. , Pasutto F. , Mardin C.Y. , Weisschuh N. , Paoli D. , Gramer E. , Zenkel M. , Weber B.H. , Kruse F.E. , Schlotzer-Schrehardt U. et al. ( 2009 ) Exploring functional candidate genes for genetic association in german patients with pseudoexfoliation syndrome and pseudoexfoliation glaucoma . Invest. Ophthalmol. Vis. Sci ., 50 , 2796 – 2801 . Google Scholar Crossref Search ADS PubMed WorldCat 5 Chen L.H. , Kao P.Y. , Fan Y.H. , Ho D.T. , Chan C.S. , Yik P.Y. , Ha J.C. , Chu L.W. , Song Y.Q. ( 2012 ) Polymorphisms of CR1, CLU and PICALM confer susceptibility of Alzheimer's disease in a southern Chinese population . Neurobiol. Aging , 33 , 210 e211 – 217 . WorldCat 6 Lambert J.C. , Heath S. , Even G. , Campion D. , Sleegers K. , Hiltunen M. , Combarros O. , Zelenika D. , Bullido M.J. , Tavernier B. et al. ( 2009 ) Genome-wide association study identifies variants at CLU and CR1 associated with Alzheimer's disease . Nat. Genet ., 41 , 1094 – 1099 . Google Scholar Crossref Search ADS PubMed WorldCat 7 Daimon M. , Oizumi T. , Karasawa S. , Kaino W. , Takase K. , Tada K. , Jimbu Y. , Wada K. , Kameda W. , Susa S. et al. ( 2011 ) Association of the clusterin gene polymorphisms with type 2 diabetes mellitus . Metabolism , 60 , 815 – 822 . Google Scholar Crossref Search ADS PubMed WorldCat 8 Giri M. , Zhang M. , Lu Y. ( 2016 ) Genes associated with Alzheimer's disease: an overview and current status . Clin. Interv. Aging , 11 , 665 – 681 . Google Scholar Crossref Search ADS PubMed WorldCat 9 Schrijvers E.M. , Koudstaal P.J. , Hofman A. , Breteler M.M. ( 2011 ) Plasma clusterin and the risk of Alzheimer disease . JAMA , 305 , 1322 – 1326 . Google Scholar Crossref Search ADS PubMed WorldCat 10 Karch C.M. , Jeng A.T. , Nowotny P. , Cady J. , Cruchaga C. , Goate A.M. ( 2012 ) Expression of novel Alzheimer's disease risk genes in control and Alzheimer's disease brains . PloS One , 7 , e50976 . Google Scholar Crossref Search ADS PubMed WorldCat 11 DeMattos R.B. , O'Dell M.A. , Parsadanian M. , Taylor J.W. , Harmony J.A. , Bales K.R. , Paul S.M. , Aronow B.J. , Holtzman D.M. ( 2002 ) Clusterin promotes amyloid plaque formation and is critical for neuritic toxicity in a mouse model of Alzheimer's disease . Proc. Natl. Acad. Sci. U.S.A , 99 , 10843 – 10848 . Google Scholar Crossref Search ADS PubMed WorldCat 12 Caccamo A.E. , Scaltriti M. , Caporali A. , D'Arca D. , Scorcioni F. , Astancolle S. , Mangiola M. , Bettuzzi S. ( 2004 ) Cell detachment and apoptosis induction of immortalized human prostate epithelial cells are associated with early accumulation of a 45 kDa nuclear isoform of clusterin . Biochem. J ., 382 , 157 – 168 . Google Scholar Crossref Search ADS PubMed WorldCat 13 Trougakos I.P. , Djeu J.Y. , Gonos E.S. , Boothman D.A. ( 2009 ) Advances and challenges in basic and translational research on clusterin . Cancer Res ., 69 , 403 – 406 . Google Scholar Crossref Search ADS PubMed WorldCat 14 Lambert J.C. , Ibrahim-Verbaas C.A. , Harold D. , Naj A.C. , Sims R. , Bellenguez C. , DeStafano A.L. , Bis J.C. , Beecham G.W. , Grenier-Boley B. et al. ( 2013 ) Meta-analysis of 74, 046 individuals identifies 11 new susceptibility loci for Alzheimer's disease . Nat. Genet ., 45 , 1452 – 1458 . Google Scholar Crossref Search ADS PubMed WorldCat 15 Verma P. , Pfister J.A. , Mallick S. , D'Mello S.R. ( 2014 ) HSF1 protects neurons through a novel trimerization- and HSP-independent mechanism . J. Neurosci ., 34 , 1599 – 1612 . Google Scholar Crossref Search ADS PubMed WorldCat 16 Fujimoto M. , Takaki E. , Hayashi T. , Kitaura Y. , Tanaka Y. , Inouye S. , Nakai A. ( 2005 ) Active HSF1 significantly suppresses polyglutamine aggregate formation in cellular and mouse models . J. Biol. Chem ., 280 , 34908 – 34916 . Google Scholar Crossref Search ADS PubMed WorldCat 17 Neef D.W. , Jaeger A.M. , Thiele D.J. ( 2011 ) Heat shock transcription factor 1 as a therapeutic target in neurodegenerative diseases . Nat. Rev. Drug Discov ., 10 , 930 – 944 . Google Scholar Crossref Search ADS PubMed WorldCat 18 Kim E. , Wang B. , Sastry N. , Masliah E. , Nelson P.T. , Cai H. , Liao F.F. ( 2016 ) NEDD4-mediated HSF1 degradation underlies alpha-synucleinopathy . Hum. Mol. Genet ., 25 , 211 – 222 . Google Scholar Crossref Search ADS PubMed WorldCat 19 Szymanski M. , Wang R. , Bassett S.S. , Avramopoulos D. ( 2011 ) Alzheimer's risk variants in the clusterin gene are associated with alternative splicing . Transl. Psychiatry , 1 , e18. WorldCat 20 Zhou Y. , Hayashi I. , Wong J. , Tugusheva K. , Renger J.J. , Zerbinatti C. , Götz J. ( 2014 ) Intracellular clusterin interacts with brain isoforms of the bridging integrator 1 and with the microtubule-associated protein Tau in Alzheimer's disease . PloS One , 9 , e103187 . Google Scholar Crossref Search ADS PubMed WorldCat 21 Zenkel M. , Kruse F.E. , Junemann A.G. , Naumann G.O. , Schlotzer-Schrehardt U. ( 2006 ) Clusterin deficiency in eyes with pseudoexfoliation syndrome may be implicated in the aggregation and deposition of pseudoexfoliative material . Invest. Ophthalmol. Vis. Sci ., 47 , 1982 – 1990 . Google Scholar Crossref Search ADS PubMed WorldCat 22 Yerbury J.J. , Poon S. , Meehan S. , Thompson B. , Kumita J.R. , Dobson C.M. , Wilson M.R. ( 2007 ) The extracellular chaperone clusterin influences amyloid formation and toxicity by interacting with prefibrillar structures . faseb J ., 21 , 2312 – 2322 . Google Scholar Crossref Search ADS PubMed WorldCat 23 Wang X. , Cattaneo F. , Ryno L. , Hulleman J. , Reixach N. , Buxbaum J.N. ( 2014 ) The systemic amyloid precursor transthyretin (TTR) behaves as a neuronal stress protein regulated by HSF1 in SH-SY5Y human neuroblastoma cells and APP23 Alzheimer's disease model mice . J. Neurosci ., 34 , 7253 – 7265 . Google Scholar Crossref Search ADS PubMed WorldCat 24 Anckar J. , Sistonen L. ( 2011 ) Regulation of HSF1 function in the heat stress response: implications in aging and disease . Annu. Rev. Biochem ., 80 , 1089 – 1115 . Google Scholar Crossref Search ADS PubMed WorldCat 25 Lechler M.C. , Crawford E.D. , Groh N. , Widmaier K. , Jung R. , Kirstein J. , Trinidad J.C. , Burlingame A.L. , David D.C. ( 2017 ) Reduced Insulin/IGF-1 Signaling Restores the Dynamic Properties of Key Stress Granule Proteins during Aging . Cell Rep ., 18 , 454 – 467 . Google Scholar Crossref Search ADS PubMed WorldCat 26 Jiang Y.Q. , Wang X.L. , Cao X.H. , Ye Z.Y. , Li L. , Cai W.Q. ( 2013 ) Increased heat shock transcription factor 1 in the cerebellum reverses the deficiency of Purkinje cells in Alzheimer's disease . Brain Res ., 1519 , 105 – 111 . Google Scholar Crossref Search ADS PubMed WorldCat 27 Gouras G.K. ( 2013 ) mTOR: at the crossroads of aging, chaperones, and Alzheimer's disease . J. Neurochem ., 124 , 747 – 748 . Google Scholar Crossref Search ADS PubMed WorldCat 28 Goetzl E.J. , Boxer A. , Schwartz J.B. , Abner E.L. , Petersen R.C. , Miller B.L. , Carlson O.D. , Mustapic M. , Kapogiannis D. ( 2015 ) Low neural exosomal levels of cellular survival factors in Alzheimer's disease . Ann. Clin. Transl. Neurol ., 2 , 769 – 773 . Google Scholar Crossref Search ADS PubMed WorldCat 29 Kohler C. , Dinekov M. , Gotz J. ( 2013 ) Active glycogen synthase kinase-3 and tau pathology-related tyrosine phosphorylation in pR5 human tau transgenic mice . Neurobiol. Aging , 34 , 1369 – 1379 . Google Scholar Crossref Search ADS PubMed WorldCat 30 Dourlen P. , Fernandez-Gomez F.J. , Dupont C. , Grenier-Boley B. , Bellenguez C. , Obriot H. , Caillierez R. , Sottejeau Y. , Chapuis J. , Bretteville A. et al. ( 2017 ) Functional screening of Alzheimer risk loci identifies PTK2B as an in vivo modulator and early marker of Tau pathology . Mol. Psychiatry , 22 , 874 – 883 . Google Scholar Crossref Search ADS PubMed WorldCat 31 Chan G. , White C.C. , Winn P.A. , Cimpean M. , Replogle J.M. , Glick L.R. , Cuerdon N.E. , Ryan K.J. , Johnson K.A. , Schneider J.A. et al. ( 2015 ) CD33 modulates TREM2: convergence of Alzheimer loci . Nat. Neurosci ., 18 , 1556 – 1558 . Google Scholar Crossref Search ADS PubMed WorldCat 32 Koerner I.P. , Jacks R. , DeBarber A.E. , Koop D. , Mao P. , Grant D.F. , Alkayed N.J. ( 2007 ) Polymorphisms in the human soluble epoxide hydrolase gene EPHX2 linked to neuronal survival after ischemic injury . J. Neurosci ., 27 , 4642 – 4649 . Google Scholar Crossref Search ADS PubMed WorldCat 33 Chapuis J. , Flaig A. , Grenier-Boley B. , Eysert F. , Pottiez V. , Deloison G. , Vandeputte A. , Ayral A.M. , Mendes T. , Desai S. et al. ( 2017 ) Genome-wide, high-content siRNA screening identifies the Alzheimer's genetic risk factor FERMT2 as a major modulator of APP metabolism . Acta Neuropathol ., 133 , 955 – 966 . Google Scholar Crossref Search ADS PubMed WorldCat 34 Ran F.A. , Hsu P.D. , Wright J. , Agarwala V. , Scott D.A. , Zhang F. ( 2013 ) Genome engineering using the CRISPR-Cas9 system . Nat. Protoc ., 8 , 2281 – 2308 . Google Scholar Crossref Search ADS PubMed WorldCat 35 Ortner V. , Ludwig A. , Riegel E. , Dunzinger S. , Czerny T. ( 2015 ) An artificial HSE promoter for efficient and selective detection of heat shock pathway activity . Cell Stress Chaperones , 20 , 277 – 288 . Google Scholar Crossref Search ADS PubMed WorldCat © The Author 2017. Published by Oxford University Press. All rights reserved. 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TI - Pseudoexfoliation and Alzheimer’s associated CLU risk variant, rs2279590, lies within an enhancer element and regulates CLU, EPHX2 and PTK2B gene expression
JF - Human Molecular Genetics
DO - 10.1093/hmg/ddx329
DA - 2017-11-15
UR - https://www.deepdyve.com/lp/oxford-university-press/pseudoexfoliation-and-alzheimer-s-associated-clu-risk-variant-8MBdBtvctl
SP - 4519
VL - 26
IS - 22
DP - DeepDyve
ER -