Báo cáo khoa học: Promoters of type I interferon genes from Atlantic salmon contain two main regulatory regions

Promoters Of Type I Interferon Genes From Atlantic Salmon Contain Two Main Regulatory Regions

Tác giả: Veronica Bergan, Silje Steinsvik, Hao Xu, Øyvind Kileng and Børre Robertsen

Lĩnh vực: Department of Marine Biotechnology, Norwegian College of Fishery Science, University of Tromsø, Norway

Nội dung tài liệu: Nghiên cứu này tập trung vào việc xác định và phân tích các vùng điều hòa chính trong các gen promoter loại I interferon (IFN) từ cá hồi Đại Tây Dương. Các nhà nghiên cứu đã xác định hai vùng promoter chính, PR-I và PR-II, có khả năng điều khiển sự phiên mã của các bản ghi IFN với vùng không dịch mã 5′ (5′-UTR) ngắn hoặc dài. Vùng PR-I, nằm ở vị trí gần điểm bắt đầu phiên mã, chứa các yếu tố điều hòa quan trọng như TATA-box, các motif gắn yếu tố điều hòa interferon (IRF) và motif gắn yếu tố hạt nhân kappa B (NFkB). Vùng PR-II, nằm xa hơn, cũng chứa các motif gắn IRF và một yếu tố ATF-2/c-Jun. Thử nghiệm hoạt tính promoter bằng cách sử dụng gen luciferase cho thấy PR-I là vùng điều hòa chính, chịu trách nhiệm cho việc cảm ứng mạnh mẽ sau khi tiếp xúc với poly(I:C) và hoạt động của nó phụ thuộc vào NFkB. Nghiên cứu cũng chỉ ra rằng chỉ một trong hai loại virus được thử nghiệm, virus bệnh thận hoại tử tuyến tụy truyền nhiễm, có khả năng kích thích hoạt động promoter. Khả năng thiết lập một thử nghiệm promoter IFN trên cá hồi Đại Tây Dương hứa hẹn hữu ích cho việc nghiên cứu tương tác giữa vật chủ và virus.

Mục lục chi tiết:

• Keywords
• Correspondence
• (Received 24 April 2006, revised 9 June 2006, accepted 15 June 2006)
• doi:10.1111/j.1742-4658.2006.05382.x
• Recognition of viral nucleic acids by vertebrate host cells results in the synthesis and secretion of type I interferons (IFN-α/β), which induce an antiviral state in neighboring cells. We have cloned the genes and promoters of two type I IFNs from Atlantic salmon. Both genes have the potential to encode IFN transcripts with either a short or a long 5′-untranslated region, apparently controlled by two distinct promoter regions, PR-I and PR-II, respectively. PR-I is located within 116 nucleotides upstream of the short transcript and contains a TATA-box, two interferon regulatory factor (IRF)-binding motifs, and a putative nuclear factor kappa B (NFkB)-binding motif. PR-II is located 469–677 nucleotides upstream of the short transcript and contains three or four IRF-binding motifs and a putative ATF-2/c-Jun element. Complete and truncated versions of the promoters were cloned in front of a luciferase reporter gene and analyzed for promoter activity in salmonid cells. Constructs containing PR-I were highly induced after treatment with the dsRNA poly(I:C), and promoter activity appeared to be dependent on NFkB. In contrast, constructs containing exclusively PR-II showed poor poly(I:C)-inducible activity. PR-I is thus the main control region for IFN-a/ẞ synthesis in salmon. Two pathogenic RNA viruses, infectious pancreatic necrosis virus and infectious salmon anemia virus, were tested for their ability to stimulate the minimal PR-I, but only the latter was able to induce promoter activity. The established IFN promoter-luciferase assay will be useful in studies of host-virus interactions in Atlantic salmon, as many viruses are known to encode proteins that prevent IFN synthesis by inhibition of promoter activation.
• The type I interferon (IFN) system plays a critical role in the innate immune defense against viruses in vertebrates. Virus-infected cells synthesize and secrete type I interferons (IFN-a/β), which circulate in the body and protect other cells from viral infection. The antiviral action is caused by binding of IFN-a/β to the type I IFN receptor resulting in activation of transcription of several hundred IFN-stimulated genes, some of which encode proteins that inhibit viral replication. The antiviral properties of at least three type I IFN-induced proteins are well established. These comprise dsRNA-activated protein kinase R (PKR), 2′,5′-oligo-adenylate synthetase and Mx proteins [1].
• Although the structures of the IFN-a and IFN-β promoters from human and mouse have been long known, the mechanisms involved in viral induction of type I IFNs have only recently been uncovered [2,3]. Of great importance has been the discovery of IFN super-producing blood cells called plasmacytoid dendritic cells (pDCs) and the realization that the
• Abbreviations
• Atlantic salmon interferon promoter
• mechanism of virus-mediated induction of IFNs is different in pDCs and other body cells [4,5]. Most nucleated cells of the body produce IFN-a/ẞ in response to recognition of dsRNA intermediates produced during viral replication. The main sensors of dsRNA are two intracellular RNA helicases (RIG-I and MDA5) [6-9], which, on binding of dsRNA, interact with the mitochondrial protein MAVS (also called IPS-1) [10,11]. This interaction leads to transcriptional induction of the IFN-ẞ gene through the co-ordinated activation of the transcription factors interferon regulatory factor 3 (IRF-3), nuclear factor kappa B (NFKB) and ATF-2/c-Jun heterodimer [2]. Infected cells secrete mainly IFN-ẞ in the initial phase of infection, but switch to IFN-a as a result of induction of IRF-7 synthesis during the subsequent amplification phase of the IFN response [12,13]. pDCs are specialized IFN producers and represent a major source of IFN-a in humans through activation of IRF-7 [14]. In pDCs, the main sensors of viral infection are Toll-like receptors (TLRs) expressed on the surface or in endosomes that recognize viral RNA or DNA. Human pDCs mostly express TLRs, which recognize ssRNA (TLR7 and TLR8) or dsCpG-rich DNA (TLR9) [15]. Recognition of viral nucleic acids by TLRs activates IRF-7, which transcriptionally activates multiple IFN-a genes [16,17]. A major difference between pDCs and other cell types is their capacity to constitutively produce relatively high concentrations of IRF-7 [18].
• Virus-induced expression of IFN-a and IFN-ẞ genes is mediated by regulatory sequences located within 200 bp upstream of the transcription start site of their promoters [19]. The IFN-ẞ promoter contains four positive regulatory domains (PRDs), which bind IRFs: mainly IRF-3 and IRF-7 (PRDI and PRDIII), NFKB (PRDII) and ATF-2/c-Jun (PRDIV) [20]. The promoters of IFN-a genes all contain DNA elements binding IRF members, notably IRF-3, IRF-5 and IRF-7, but they do not contain NFkB or ATF-2/c-Jun binding sites [21].
• In mammals and birds, IFN-a/ẞ genes are encoded by intron-lacking genes whereas IFN-λ genes possess a 4-intron/5-exon structure [22,23]. Recently, type I IFN genes of teleost fish were shown to possess a gene structure similar to IFN-λ genes, although their protein sequences are more similar to IFN-a than IFN-λ [24-28]. At present, little is known about the regulation of fish IFN genes, although the promoter of the zebrafish type I IFN gene was recently reported to contain one IRF-binding site and one NFKB-binding site [28]. The present work shows that type I IFN genes of Atlantic salmon show a rather unique organization of the promoter in comparison with mammals, birds and zebrafish. Atlantic salmon stimulated with the dsRNA polyinosinic polycytidylic acid [poly(I:C)] produces an IFN transcript with a short 5′-UTR called SasaIFN-a1, and another IFN transcript with a long 5′-UTR called SasaIFN-a2. In this work, we cloned two different Atlantic salmon IFN genes from genomic DNA that encode putative transcripts similar in sequence to SasaIFN-a1 and SasaIFN-a2. Surprisingly both genes apparently have the potential to produce both a short and long transcript because of the location of two separate promoter regions, one of which is present in the 5′-UTR of the long transcript. To perform functional analysis of the Atlantic salmon IFN promoter region, we fused the complete and truncated versions of the promoter region to a luciferase reporter gene and transfected it into Chinook salmon embryo (CHSE-214) or Atlantic salmon head kidney TO cells. Promoter activity was measured after stimulation with poly(I:C) or virus infection.
• Results
• Cloning of full-length type I IFN genes from genomic DNA
• A genome walking approach was used to clone a 1281-nucleotide sequence upstream of the SasaIFN-a1 transcription start site. This allowed design of primers that amplified genomic IFN sequences that expanded from -1281 of the promoter region (PR) to the polyA signal by PCR. Two full-length IFN genes, designated Sasa-IFN-A1 (A1 for short) and SasaIFN-A2 (A2), were identified in two different BAC clones (GenBank accession nos DQ354152 and DQ354153). A summary of nucleotide data on the Al and A2 gene is shown in Table 1. Both genes possessed the five-exon/four-intron structure found previously in fish type I IFN genes, although the intron sizes were somewhat different from those originally found in DNA from Atlantic salmon [26]. The joined exon sequences of Al and SasaIFN-a1 cDNA are completely identical, and the joined exon sequences of A2 and SasaIFN-a2 cDNA have only three nucleotide differences, possibly because they represent different alleles (Table 2). In contrast, Al diverges from SasaIFN-a2, with 10 mismatches, and A2 and SasaIFN-a1 diverge by 12 mismatches. This strongly suggests that Al encodes the SasaIFN-a1 transcript and A2 the SasaIFN-a2 transcript. Overall differences in Al and A2, including differences in promoter and intron regions (deletions, insertions and substitutions), confirm that they represent two different genes rather than allele variants (Table 1). Two pseudogenes were also identified in the screening of BAC
• clones, one having a premature stop codon (accession no. DQ354154) and one that appeared to be interrupted by a transposase gene (accession no. DQ354155). The pseudogenes were not investigated any further in this work.
• Analysis of the promoter regions
• The alignment of the 765-bp sequence regions upstream of the ORFs are very similar in the two genes except for 10 nucleotide substitutions and two insertions/deletions (Fig. 1). This was surprising because SasaIFN-a1 was originally identified as a short transcript (829 nucleotides) and SasaIFN-a2 as a long transcript (1290 nucleotides). We thus expected that the Al gene would encode a short transcript and A2 a long transcript. The present data indicate, however, that both genes have the potential to encode both transcripts.
• A total of six (in Al) or seven (in A2) IRF-binding elements (IRF-E) were identified in the 765-nucleotide region upstream of the putative transcription start site of SasaIFN-a1 (Fig. 1). The motifs conform to the GAAA(G/C)GAAA(T/C) consensus sequence [29] and were located at positions -63, -116, -376, -503, -545, -639, and -669 relative to the putative SasaIFN-a1 transcription start site. Interestingly, the IRF-E sequences at positions -116 and -545 were identical and probably bind the same IRF(s). In addition, we found two potential NFkB-binding sites, one in close proximity to the SasaIFN-a1 transcriptional start site (-80) and one more distant (-720) that appeared to be truncated in the A2 promoter. An ATF-2/c-Jun element, which is essential for activity of the human IFN-ẞ promoter, was found in the distal promoter region in close proximity to the IRF-E at position -557. Moreover, an atypical TATA-box was located at position -42 in both genes, and two CCAAT-boxes at positions -296 and -579 in the Al gene.
• In summary, both genes appear to possess two major regulatory regions:
• (a) promoter region I (PR-I) located within 116 nucle- otides upstream of the short transcript, containing a noncanonical TATA-box, two IRF-binding motifs and a putative NFkB-binding motif;
• (b) promoter region II (PR-II), located 469-677 nucle- otides upstream of the short transcript, containing three to four IRF-binding motifs and an ATF-2/c-Jun element.
• The putative salmon IFN promoters thus seem to have a unique feature, as PR-I controls the synthesis of a transcript with a short 5′-UTR and PR-II controls the synthesis of a transcript with a long 5′-UTR. Accordingly, the 5′-UTR of the long transcript in fact contains PR-I.
• Activity of the A1 and A2 promoters on poly(I:C) induction
• To study the activity of the promoters, we cloned full-length and deleted versions of the promoters in front of a promoterless luciferase reporter gene (Fig. 2A). From the Al gene the following constructs were made: pA1(-135), pA1(-202) and pA1(-333) containing only PR-I; and pA1(-747) and pA1(-1281) containing both PR-I and PR-II. From the A2 gene, the construct pA2(-275) containing only PR-II was made. The constructs were transfected into CHSE-214 cells or Atlantic salmon TO cells along with a constitutively expressed ẞ-gal standard (pJatLacZ) and then stimulated with poly(I:C) to induce IFN
• promoter activity. Figure 2 shows the luciferase activity from the different constructs relative to ẞ-gal measurements for poly(I:C)-stimulated or untreated CHSE-214 (Fig. 2B) or TO cells (Fig. 2C). All constructs were induced by poly(I:C) in both cell types. The overall higher promoter activity observed in CHSE-214 cells compared with TO cells is probably due to the fact that poly(I:C) was transfected into CHSE-214 cells, whereas it was applied extra- cellularly to TO cells. In CHSE-214 cells, the level of induction was highest for pA1(-1281), pA1(-747) and pA1(-202), and lowest for pA2(-275). This indicates that PR-I is most important for poly(I:C) induction in these cells. In TO cells, all constructs showed similar levels of relative luciferase activity after stimulation with poly(I:C). The main difference from CHSE-214 cells was that pA1(−1281), pA1(-747) and pA2(-275) all showed relatively high basal luciferase activity. Accordingly, the level of induc- tion was highest for pA1(-333), pA1(-202) and
• pA1(-135). The minimal promoter showing highest inducibility in TO cells was thus pA1(-135), containing only PR-I.
• The highly inducible minimal promoter construct, pA1(-202), and the full-length construct, pA1(-1281), were next compared for poly(I:C) induction in a time course study in CHSE-214 and TO cells (Fig. 3). In CHSE-214 cells, both promoter constructs were hardly induced at all at 12 h, but showed increasing luciferase activity at 24 h and 48 h after poly(I:C) treatment (Fig. 3). At 48 h, the minimal IFN promoter was induced more than 50-fold, whereas the full-length promoter was induced only 13-fold (Fig. 3A). The minimal promoter construct showed similar time kinet- ics in TO cells, whereas the pA1(-1281) construct showed hardly any induction at any of the time points (Fig. 3B).
• A dose-response curve for poly(I:C) induction of the minimal promoter construct was established. As little as 50 ng·mL¯¹ was sufficient to induce the
• promoter significantly (14-fold), and 500 ng·mL-1 poly(I:C) was sufficient to give maximal induction (50-fold) of the promoter (Fig. 4).
• Fig. 2. Analysis of IFN promoter activity in CHSE-214 and TO cells. (A) Salmon IFN promoter-luciferase constructs including the posi- tions of IRF-E, NFKB, and ATF-2/c-Jun sites relative to the tran- scription start site (+1). pA1 constructs are from the putative SasalFN-a1 promoter, and pA2 is the putative SasalFN-a2 promo- ter. (B) CHSE-214 or (C) TO cells were transiently transfected with the promoter constructs plus a ẞ-gal internal control vector in 24-well plates