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. 2015 Jan 30;290(5):2784-97.
doi: 10.1074/jbc.M114.593384. Epub 2014 Dec 17.

HTRA1 (high temperature requirement A serine peptidase 1) gene is transcriptionally regulated by insertion/deletion nucleotides located at the 3' end of the ARMS2 (age-related maculopathy susceptibility 2) gene in patients with age-related macular degeneration

Daisuke Iejima  1 , Takeshi Itabashi  1 , Yuich Kawamura  1 , Toru Noda  2 , Shinsuke Yuasa  3 , Keiichi Fukuda  3 , Chio Oka  4 , Takeshi Iwata  5
Affiliations

HTRA1 (high temperature requirement A serine peptidase 1) gene is transcriptionally regulated by insertion/deletion nucleotides located at the 3' end of the ARMS2 (age-related maculopathy susceptibility 2) gene in patients with age-related macular degeneration

Daisuke Iejima et al. J Biol Chem. .

Abstract

Dry age-related macular degeneration (AMD) accounts for over 85% of AMD cases in the United States, whereas Japanese AMD patients predominantly progress to wet AMD or polypoidal choroidal vasculopathy. Recent genome-wide association studies have revealed a strong association between AMD and an insertion/deletion sequence between the ARMS2 (age-related maculopathy susceptibility 2) and HTRA1 (high temperature requirement A serine peptidase 1) genes. Transcription regulator activity was localized in mouse retinas using heterozygous HtrA1 knock-out mice in which HtrA1 exon 1 was replaced with β-galactosidase cDNA, thereby resulting in dominant expression of the photoreceptors. The insertion/deletion sequence significantly induced HTRA1 transcription regulator activity in photoreceptor cell lines but not in retinal pigmented epithelium or other cell types. A deletion construct of the HTRA1 regulatory region indicated that potential transcriptional suppressors and activators surround the insertion/deletion sequence. Ten double-stranded DNA probes for this region were designed, three of which interacted with nuclear extracts from 661W cells in EMSA. Liquid chromatography-mass spectrometry (LC-MS/MS) of these EMSA bands subsequently identified a protein that bound the insertion/deletion sequence, LYRIC (lysine-rich CEACAM1 co-isolated) protein. In addition, induced pluripotent stem cells from wet AMD patients carrying the insertion/deletion sequence showed significant up-regulation of the HTRA1 transcript compared with controls. These data suggest that the insertion/deletion sequence alters the suppressor and activator cis-elements of HTRA1 and triggers sustained up-regulation of HTRA1. These results are consistent with a transgenic mouse model that ubiquitously overexpresses HtrA1 and exhibits characteristics similar to those of wet AMD patients.

Keywords: ARMS2; Age-related Macular Degeneration; DNA-binding Protein; EMSA; HTRA1; Photoreceptor; Retina; Retinal Degeneration; Transcription Regulation.

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Figures

FIGURE 1.
FIGURE 1.
Transcription regulatory region of ARMS2 and HTRA1. A, a schematic diagram of the transcription regulatory region of ARMS2 and HTRA1. Sequencing of the indicated transcription regulatory region spanned 10.5 kbp and was performed for both AMD and non-AMD controls. Two unique sequences, a C/A repeat variant in the ARMS2 transcription regulator (indicated with a blue box) and an insertion/deletion variant downstream of exon 2 of ARMS2 (indicated with a red box), were identified in a patient with AMD that carried the SNP, rs10490924. B, characteristic C/A mutations present in the ARMS2 transcription regulatory region (blue, C/A variant; red, C to A substitution). C, the insertion/deletion mutations present in the HTRA1 transcription regulatory region (blue, ARMS2 exon 2 region; red, point mutations). D–G, analysis of ARMS2 transcription regulator activity using a luciferase assay system. D, four luciferase vectors were generated to analyze ARMS2 transcription regulator activity: normal (1,000 bp), normal (600 bp), risk (1,000 bp), and risk (600 bp). The 1,000-bp sequence contained a C/T SNP in the upper 730-bp region from ARMS2 exon 1. ARMS2 transcription regulator activity detected in ARPE19 cells (E), RGC-5 cells (F), and 661W cells (G). Error bars, S.D. H–K, HTRA1 transcription regulator activity detected in various retinal cell lines. H, schematic diagram of the ARMS2-HTRA1 constructs used in the luciferase assays performed (black line, common regulator sequence; blue line, non-insertion/deletion regulator unique sequence; red line, insertion/deletion regulator unique sequence). Full-length HTRA1 transcription regulator activity detected in ARPE19 cells (I), RGC5 cells (*, p = 7.5 × 10−6) (J), and 661W cells (**, p = 1.07 × 10−6) (K). Error bars, S.D. EV, empty vector; NI, non-insertion/deletion regulator; I, insertion/deletion regulator.
FIGURE 2.
FIGURE 2.
The effect of the ARMS2 C/A variant on the HTRA1 transcription regulator. A, schematic diagram of the C/A variant plus HTRA1 regulatory element in the constructs used for luciferase assays (blue region, 600-bp AMRS2 transcription regulatory region (contains C/A variant); orange region, region that contains the HTRA1 regulatory element; yellow region, the luciferase reporter gene). B, C/A variant plus non-insertion/deletion type HTRA1 regulatory element activity in 661W cells. C, C/A variant plus insertion/deletion type HTRA1 regulatory element activity in 661W cells. Error bars, S.D. EV, empty vector; Normal, normal type C/A variant; Risk, risk type C/A variant; NI, non-insertion/deletion type HTRA1 regulatory element; I, insertion/deletion type HTRA1 regulatory element.
FIGURE 3.
FIGURE 3.
Htra1 transcription regulator activity and Htra1 protein expression in the mouse retina. A, immunohistochemistry to detect HtrA1 expression in HtrA1+/+, HtrA1+/−, HtrA1−/−, and HtrA1-Tg mouse retinas (blue, DAPI; green, HtrA1). NC (mouse IgG), staining control; scale bars, 100 μm. B, immunohistochemistry to detect HtrA1 expression in HtrA1+/− mouse retinas (red, β-galactosidase; green: HtrA1; blue, DAPI; yellow, merge). Scale bars, 100 μm. C, in situ hybridization to detect HtrA1 expression in HtrA1+/− mouse retina tissues (red arrowheads, HtrA1; blue arrowheads, β-galactosidase). Scale bars, 100 μm. OS, outer segment; IS, inner segment; ONL, outer nuclear layer; OPL, outer plexiform layer; INL, inner nuclear layer; IPL, inner plexiform layer; GCL, ganglion cell layer.
FIGURE 4.
FIGURE 4.
Enhanced HTRA1 transcription regulator activity is associated with the insertion/deletion variant in the photoreceptor cell line, 661W. A, constructs used for luciferase assay analysis (black line, common sequence between both regulators; blue line, non-insertion/deletion regulator (443 bp); red line, insertion/deletion regulator (54 bp); red boxes 1–5, non-insertion/deletion regulator constructs; green boxes 6–9, insertion/deletion regulator constructs; gray line 9, insertion/deletion region). B and C, transcription regulator activity detected for the non-insertion/deletion and insertion/deletion HTRA1 transcription regulator assayed (in B, *, p = 1.05 × 10−6; **, p = 3.46 × 10−5; in C, *, p = 2.2 × 10−6; **, p = 0.0086; ***, p = 0.0062). EV, empty vector; error bars, S.D.
FIGURE 5.
FIGURE 5.
HTRA1 transcription in human iPSCs derived from wet AMD patients. A, genotyping of non-insertion/deletion versus insertion/deletion versions of the human HTRA1 gene transcription regulatory region. B, detection of human iPSC marker genes by RT-PCR. Detection of GAPDH was used as an internal control. C, relative transcriptional level of HTRA1 determined by quantitative RT-PCR for iPSCs derived from individuals with non-insertion/deletion (control) versus insertion/deletion (wet AMD) transcription regulators. Sample 1, 30-year-old non-AMD male; sample 2, 72-year-old wet AMD female; sample 3, 38-year-old non-AMD male; sample 4, 71-year-old non-AMD female; sample 5, 71-year-old wet AMD female. Error bars, S.D.
FIGURE 6.
FIGURE 6.
Identification of the transcription factors binding to non-insertion/deletion and insertion/deletion regions of the HTRA1 transcription regulators. A, location of the double-stranded DNA probes, 1–10, in the region upstream of the HTRA1 coding region that were designed for EMSAs. Both non-insertion/deletion and insertion/deletion HTRA1 transcription regulatory regions were targeted. B, EMSAs of probes 1–10 using nuclear extracts from 661W cells cultured in 2 and 10% FBS. Red arrowheads, detected signals. C, increased expression of HtrA1 was detected in Western blot assays performed following treatment of 661W cells with LPS (1 μg/ml, 0–60 min) using anti-mouse HtrA1 antibodies. Detection of α-tubulin was used as an internal control. D, a Venn diagram shows the number of proteins found to bind the non-insertion/deletion versus insertion/deletion regions of the HTRA1 transcription regulator based on LC-MS/MS data. E, gene ontology term of non-insertion/deletion- and insertion/deletion-binding protein (categorized by molecular function).
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