Biochem Genet DOI 10.1007/s10528-014-9653-x

Identification of Functional Haplotypes in the Promoter Region of the LST1 Gene Jeyoung Woo • Chaeyoung Lee

Received: 23 July 2013 / Accepted: 20 March 2014 Ó Springer Science+Business Media New York 2014

Abstract Psoriasis, an inflammatory skin disease, was associated with a promoter variant (rs9267502; P = 3.786 9 10-19) of LST1 that may regulate immune response and cellular morphogenesis. Promoter activity was examined to identify functional variants in the proximity of the associated variant. Five natural haplotypes (H1–H5) of four variants (rs7758790, rs9267502, rs41268884, rs17200775) in strong linkage disequilibrium were investigated. The most common haplotype (H1) was TGAG (frequency 0.78), and the second most frequent haplotype (H2) was CGGG (0.09). Luciferase assay was conducted using reporter constructs including each haplotype and firefly luciferase gene. As a result, promoter activity of H1 was smaller than the construct without the promoter region (P \ 0.05). The promoter activities of H3, H4, and H5 corresponded to that of H1 (P [ 0.05), and H2 promoter activity was larger than that of H1, H3, H4, and H5 (P \ 0.05). This might result from changes in binding affinity to transcription factors by nucleotide substitutions of the promoter variants of the LST1 gene. Keywords

LST1  Promoter  Psoriasis  Single nucleotide variant

Introduction Psoriasis is a common inflammatory skin disease with a prevalence of 1–3% (Chandran and Raychaudhuri 2010). Although psoriasis has been considered a complex disease caused by many genetic and environmental factors (Lebwohl 2003), its genetic associations were tentatively reported from candidate gene analyses (e.g., PSORS1; Trembath et al. 1997). Recent genome-wide association J. Woo  C. Lee (&) School of Systems Biomedical Science, Soongsil University, 511 Sangdo-dong, Dongjak-gu, Seoul 156-743, Korea e-mail: [email protected]

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studies (GWAS) have identified signals of genetic association of susceptibility to psoriasis (Cargill et al. 2007; Manolio et al. 2007; Zhang et al. 2009; Ellinghaus et al. 2010). They were located largely in noncoding (intergenic and intronic) regions, yet their functions have not been examined. Three of them were suspected as potential promoter variants, located in a 50 upstream (-2 to 0 kb) region of the genes encoding proteins. Among them, rs9267502 in the promoter of the leukocytespecific transcript 1 (LST1) gene on chromosome 6 showed the most significant association with psoriasis (P = 3.786 9 10-19; Manolio et al. 2007). This study aimed to identify functional sequence variants in the proximity of rs9267502 and their promoter activity as a post-GWAS functional study.

Materials and Methods Linkage Disequilibrium and Haplotyping In order to search for putative functional single nucleotide polymorphisms (SNPs) in proximity to rs9267502, a linkage disequilibrium (LD) map was drawn with SNPs located in the 50 upstream region (-2 to 0 kb) of the gene encoding LST1. We utilized genotypes of 379 Europeans from the 1000 Genomes Project (http:// browser.1000genomes.org/index.html). The LD blocks were estimated using the Haploview freeware, version 4.2 (Barrett et al. 2005). Plasmid Construction and Site-directed Mutagenesis A pair of primers was designed to amplify a fragment of 1257 bp (–1164 to ?84) of the LST1 promoter region by PCR. The forward primer was 50 -ctcgagCAC TTTGGAAGGCTAAGGTG-30 (restriction site XhoI for cloning is underlined), and the reverse primer was 50 -aagcttACTGGCCAGTGCTGTCTGGC-30 (restriction site HindIII underlined). The amplicon included the selected SNPs linked to rs9267502 and was cloned into a T-blunt vector (Solgent, Deajeon, Korea). After digestion with XhoI and HindIII (NEB, Herts, UK), the amplicon was ligated into the pGL3Basic vector (Promega, Madison, WI, USA). Primers were designed to make appropriate mutations for each haplotype. They were 50 -GACCAGGAGTTCAAAACCAGCCTGGGCAACATA-30 (forward) and 50 -TAT GTTGCCCAGGCTGGTTTTGAACTCCTGGTC-30 (reverse) for rs7758790 T?C, 50 -CTGAATATTATTCAGCATAGGGAACATGGAGGATGGG-30 (forward) and 50 -CCCATCCTCCATGTTCCCTATGCTGAATAATATTCAG-30 (reverse) for rs9267502 G?A, 50 -ACTTAGTTCTGTGTTCAGTGCCAGAATGCTGCCTC-30 (forward) and 50 -GAGGCAGCATTCTGGCACTGAACACAGAACTAAGT-30 (reverse) for rs41268884 A?G, and 50 -CCTAACCCTGGAGCCAAACTTCC TCTCCTAAC-30 (forward) and 50 -GTTAGGAGAGGAAGTTTGGCTCCAGGGT TAGG-30 (reverse) for rs17200775 G?A. We used a QuickChange XL Site-Directed mutagenesis kit (Stratagene, La Jolla, CA, USA) for mutagenesis. Every construct was verified by direct sequencing (Solgent).

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Luciferase Reporter Assay Human embryonic kidney (HEK) 293 cells were cultured in Dulbecco’s modified Eagle’s medium (Wellgene, Daegu, Korea) with 10% fetal bovine serum (Wellgene) and 1% penicillin streptomycin (Wellgene) at 37°C under 5% CO2. The HEK293 cells (2.4 9 105 cells/well) were seeded into a six-well plate 24 h prior to transfection. The cells in one well were transfected with 1.2 lg of each haplotype construct and 4 lg of Lipofectamine 2000 (Invitrogen, USA). In the corresponding well, 10 ng of pRL-CMV vector (Promega) with the gene encoding Renilla luciferase was cotransfected as a negative control. Transfected cells were incubated for 24 h at 37°C under 5% CO2. The cells were washed with phosphatebuffered saline 1 day after transfection and lysed by 19 passive lysis buffer. Luciferase activity was measured using the Dual-Luciferase Reporter Assay system (Promega) and luminescence microplate reader (CentroPRO LB 962; Berthold, USA). All the experiments were conducted at least three times. Statistical Analysis Relative luciferase activity was obtained based on luciferase activity of pGL3 without the promoter construct of the LST1 gene. Their mean was estimated for each construct. Differences in the mean luciferase activity among constructs were tested first by analysis of variance (ANOVA), followed by a post hoc analysis employing Tukey’s multiple comparison test. All the statistical analyses were conducted using SPSS 20.0 software (SPSS, Chicago, IL, USA).

Results The linkage analysis revealed an LD block including rs9267502, previously identified for association with psoriasis. The block included four SNPs and was 1087 bp long, ranging from –1106 to –20 bp distant from the gene (Fig. 1). The major haplotype estimated from this block had a frequency of 0.78, and the other haplotypes were all less than 0.1. Dual-luciferase assay showed differences in expression among the haplotypes (Fig. 2). The reporter construct with the major haplotype (H1) had a smaller (P \ 0.05) luciferase activity than the null promoter sequence (pGL3-Basic). The promoter activities of the other haplotypes corresponded to that of H1 (P [ 0.05). However, the promoter activity of H2 was greater than that of H1 and the other haplotypes (P \ 0.05), corresponding to that of the null promoter sequence (P [ 0.05).

Discussion LST1 modulates immune responses and cellular morphogenesis (Yu and Weissman 2000). In addition, its expression increased with autoimmune-induced inflammation

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Fig. 1 Linkage disequilibrium and haplotypes of nucleotide variants in the upstream (–2 to 0 kb) region of the LST1 gene. a LD map with pairwise r2 estimates. Asterisk (*) indicates the single nucleotide variant associated with psoriasis. Nucleotide variants that consist of an LD block are in bold. b Haplotypes (frequency [ 0.01) of the nucleotide variants within the LD block

Fig. 2 Promoter activity of reporter constructs with haplotypes in the promoter of the LST1 gene. Luciferase activity is presented as the value relative to that of pGL3 without the LST1 promoter. Bars with different lowercase letters (a and b) indicate significantly different promoter activities based on post hoc Tukey’s honest significant difference test (P \ 0.05)

and inflammatory mediators (Mulcahy et al. 2006). It was largely active in dendritic cells, which are antigen-presenting cells associated with the immune system, and led to the production of long and thin filopodia (Raghunathan et al. 2001). The dendritic cells transfer external environment information to other immune-related cells (Banchereau et al. 2000). In this process, filopodia play an important role in antigen presentation (Al-Alwan et al. 2001). Therefore, we hypothesized that LST1 might increase filopodia, induce antigen presentation of dendritic cells, overactivate T cells, produce autoimmune responses, and increase susceptibility to psoriasis.

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Fig. 3 Prediction of transcription factor binding sites by haplotype. Each cell shows transcription factors predicted by the haplotype in that column but not by the haplotype in that row

The current study identified functional nucleotide variants on promoter activity of the gene encoding LST1. The most frequent haplotype (H1), which consists of four nucleotide variants from –1106 to –20 bp, reduced promoter activity in comparison with the construct without the promoter region (pGL3). Decreased promoter activity was also found with the other haplotypes, except for the second most frequent haplotype (H2). This suggests that the nucleotide variants as components of the haplotype could individually or interactively change binding affinity to transcription factors. Unfortunately, the mechanisms underlying the transcriptional expression of the LST1 gene are unknown. The current study predicted transcription factor binding sites of the haplotypes by an in silico approach using Promo (Messeguer et al. 2002). As a result, a variety of transcription factor binding sites were altered by the natural haplotypes (Fig. 3). In particular, four transcription factor binding sites were predicted to be common to H1, H3, H4, and H5 (P \ 0.05) but not to H2 (P [ 0.05). We suggest a scenario that they reduced transcription activity by binding to the promoter. The transcription factors with binding sites not predicted in H2 were closely associated with a variety of diseases; e.g., CCAAT/enhancer binding protein a (C/EBPa) is critical for developing granulocytes, and its mutations could obstruct granulocyte maturation and cause acute myeloid leukemia (Park et al. 2013). The genes encoding hepatocyte nuclear factor 3a (HNF3a) and X-box binding protein 1 (XBP1) are coexpressed with the estrogen receptor-a gene (ESR1) in breast cancer (Lacroix and Leclercq 2004). Furthermore, the spliced form of XBP-1 is associated with chronic inflammatory diseases such as rheumatoid arthritis (Savic et al. 2013) and tumor necrosis factor receptor associated periodic syndrome (Bulua et al. 2011; Dickie et al. 2012).

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Functional studies are needed to understand transcription factors associated with LST1 expression and the corresponding underlying mechanisms. Further examination of the processes following the transcription initiation might uncover how the promoter sequence variants cause psoriasis. Acknowledgments This study was supported by the Basic Science Research Program through the National Research Foundation of Korea, funded by the Ministry of Education, Science, and Technology (Grant No. 2012002096).

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Identification of functional haplotypes in the promoter region of the LST1 gene.

Psoriasis, an inflammatory skin disease, was associated with a promoter variant (rs9267502; P = 3.786 × 10(-19)) of LST1 that may regulate immune resp...
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