FTH1 expression is affected by promoter polymorphism and not DNA methylation in response to DHV-1 challenge in duck

Accepted Manuscript FTH1 expression is affected by promoter polymorphism and not DNA methylation in response to DHV-1 challenge in duck Qi Xu, Tiantian Gu, Ran Liu, Zhengfeng Cao, Yu Zhang, Yang Chen, Ningzhao Wu, Guohong Chen PII:

S0145-305X(17)30402-0

DOI:

10.1016/j.dci.2017.10.006

Reference:

DCI 3002

To appear in:

Developmental and Comparative Immunology

Received Date: 25 July 2017 Revised Date:

15 October 2017

Accepted Date: 15 October 2017

Please cite this article as: Xu, Q., Gu, T., Liu, R., Cao, Z., Zhang, Y., Chen, Y., Wu, N., Chen, G., FTH1 expression is affected by promoter polymorphism and not DNA methylation in response to DHV-1 challenge in duck, Developmental and Comparative Immunology (2017), doi: 10.1016/j.dci.2017.10.006. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

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FTH1 Expression Is Affected by Promoter Polymorphism and

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Not DNA Methylation in Response to DHV-1 Challenge in Duck

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Qi XU a, Tiantian GU a, Ran Liu b, Zhengfeng CAO a, Yu ZHANG a, Yang CHEN a,

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Ningzhao Wu a, and Guohong CHEN a,*

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a

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Yangzhou University, Yangzhou, Jiangsu, China

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b

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*Correspondence author. Email address: [email protected] (G.Chen)

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Jining animal husbandry and Veterinary Bureau, Jining, shandong, China

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Jiangsu Key Laboratory for Animal Genetic, Breeding and Molecular Design,

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Abstract Ferritin heavy polypeptide 1 (FTH1) plays a pivotal role in response to viral infections. FTH1 expression is modulated by various pathogens, but the regulatory

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mechanisms are unknown. We firstly construct duck hepatitis virus 1 (DHV-1)

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infection model, including morbid ducklings, non-morbid ducklings and control

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ducklings. Then the mRNA expression of duck FTH1 (duFTH1) was measured

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mRNA expression of duck FTH1 (duFTH1) in the liver and spleen after duck hepatitis

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virus 1 (DHV-1) infection using quantitative polymerase chain reaction (qPCR) and

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found that duFTH1 mRNA was down-regulated significantly in morbid ducklings

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(liver, P<0.01; spleen, P<0.05) compared with the control ducklings. We also found

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that duFTH1 expression was significantly higher in the spleen (P<0.01) and liver

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(P<0.05) of non-morbid ducklings than in morbid ducklings. Moreover, DNA

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methylation of the duFTH1 promoter was examined by bisulfite sequencing (BSP)

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and we found that the duFTH1 promoter was hypomethylated, the relative

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methylation was only 5.9% and 2.0% in the morbid ducklings and non-morbid

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ducklings, respectively. The promoter contained a -55 C/T mutation in 75% of

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non-morbid ducklings, and this polymorphism affected promoter activity. Further

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analysis suggested that this mutation altered the binding site of the transcription factor

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NRF1. Binding of NRF1 to the FTH1 promoter was confirmed by electrophoretic

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mobility shift assay(EMSA) analysis. Thus, our findings revealed the NRF1 was a

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negative regulator, and lossed of binding of NRF1 to duFTH1 promoter due to -55C/T

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mutation enhances duFTH1 expression in non-morbid ducks, which provided

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molecular insights into the effect of duFTH1 expression via promoter polymorphisms,

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but not DNA methylation, in response to DHV-1 challenge.

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Keywords: duck; Ferritin heavy polypeptide 1; polymorphism; DNA Methylation;

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duck hepatitis virus

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1. Introduction Ferritin is a ubiquitous and specialized protein involved in the intracellular

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storage of iron(Cozzi et al., 1990; Halliwell and Gutteridge, 1992); it is also a

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signaling molecule and an immune regulator(Coffman et al., 2009; Kim et al., 2012;

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Li et al., 2006; Recalcati et al., 2008). Ferritin heavy polypeptide 1 (FTH1)

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participates in cytokine signaling (Li et al., 2006) , adaptive immunity (Kim et al.,

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2012; Recalcati et al., 2008) , and cell death (Feng et al., 2012; Liu et al., 2012) .

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Previously, FTH1 was shown to be involved in the activation of IFN γ and IFN α/β

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signaling in triple negative breast cancer(Liu et al., 2014). Overexpression of FTH1

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also affected proliferation, apoptosis, and migration of neural progenitor cells in vitro

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(Feng et al., 2012). FTH1 was up-regulated in dendritic cells from hepatitis B surface

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antigen positive transgenic mice(Yao et al., 2011). An increase in FTH1 expression

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was detected in susceptible chickens infected with Marek’s disease virus(Xiao et al.,

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2015). In contrast, FTH1 was reduced in porcine alveolar macrophage cells infected

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with a highly pathogenic porcine reproductive and respiratory syndrome

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virus(Thanthrigedon et al., 2010). These findings suggest that FTH1 expression is

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altered differently by various pathogens.

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Ducks are considered as a feasible model of animal infection for the hepatitis B

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virus, which have attracted the interest of many researchers(Cova and Zoulim, 2004;

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Narayan et al., 2006; Tang et al., 2013; Xu et al., 2014b). In our previous study, we

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screened 70 differentially expressed genes using suppression subtractive hybridization

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cDNA libraries from 3-day-old ducklings infected with duck hepatitis virus 1 (DHV-1)

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and found that FTH1 was crucial (Chen et al., 2016; Xu et al., 2014a; Zhang et al.,

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2017). We also discovered that FTH1 mRNA was significantly up-regulated in the

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liver after DHV-1 injection. In our previous study, FTH1 expression peaked 4 hours

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post-infection, dropped progressively, and returned to basal levels after 24 h(Xu et al.,

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2014a). Several studies have indicated that ferritin is associated with hepatitis(Hagist

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et al., 2009; Lee et al., 2011; Nair et al., 2011; Yao et al., 2011). Hence, FTH1 also

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contributes to the immune response during hepatitis virus infection. However, the

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molecular mechanisms for transcriptional or post-transcriptional regulation of FTH1

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expression have not yet been elucidated. In this study, we examine DNA methylation and gene polymorphisms in the

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promoter region of duck FTH1. There was no evidence that DNA methylation of the

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FTH1 promoter region contributed to the regulation of FTH1 expression. Instead, our

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results reveal that FTH1 expression was regulated in ducks during DHV-1 challenge

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by a mutation that alters a NRF1 binding site.

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2. Material and Methods

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2.1. Ethics Statement

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All animal experiments were reviewed and approved by the Institutional Animal

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Care and Use Committee of Yangzhou University. Experiments were performed in

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accordance with the Regulations for the Administration of Affairs Concerning

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Experimental Animals of Yangzhou University (Yangzhou University, China, 2012)

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and the Standards for the Administration of Experimental Practices (Jiangsu, China,

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2008). All operations were performed according to recommendations proposed by the

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European Commission (1997), and all efforts were made to minimize suffering.

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2.2. Ducklings, viral infections, and sample collection

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The fifty 3-day-old ducklings were obtained from the Chinese Waterfowl

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Germplasm Resource Pool (Taizhou, China). Ducklings were determined to be free of

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DHV-1 by failure to amplify conserved regions of the DHV-1 3D gene using reverse

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transcription-polymerase chain reaction (RT-PCR)(Xu et al., 2014b) with primers

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shown in Table 1. The thirty ducklings were inoculated with 0.4 mL of allantoic liquid

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containing DHV-1 (ELD50 10-4.6/0.2ml) according to our earlier trials(Xu et al.,

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2014b) , which were verified with the specific bands of the conservative regions in the 4

ACCEPTED MANUSCRIPT DHV-13D gene as the above. And the other twenty ducklings were received normal

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saline (as uninfected controls). The two groups were maintained in separate rooms.

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The infected ducklings were monitored every 30 mins, and 30% (9/30) of the infected

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birds display typical symptoms of DHV-1 infection 24 h.p.i. (hours post-infection).

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They appeared depressed, ataxic and lack of appetite. When they show opisthotonus,

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the

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(intraperitoneal injection; 150 mg/kg) and killed by exsanguination. These birds was

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referred to as morbid ducklings. They were immediately anesthetized with sodium

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pentobarbital (intraperitoneal injection; 150 mg/kg) and killed by exsanguination.

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Spleen and liver tissues and the blood were obtained promptly. Tissues were

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snap-frozen in liquid nitrogen immediately and stored at -80°C until needed. 70%

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(21/30) of the infected ducklings without typical symptoms of DHV-1 (non-morbid

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ducklings) and the 20 uninfected control animals were also sampled.

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2.3. Quantitative real-time PCR to measure FTH1 mRNA

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Total RNA was extracted from spleens and livers of morbid and non-morbid

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ducklings using the TRIzol reagent per the manufacturer’s instruction (TaKaRa,

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Dalian，China), and 1 µg of each of RNA sample was reverse-transcribed (First Strand

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cDNA kit, TaKaRa, China). The first-strand cDNA (5 ng) was subjected to an ABI

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two-step RT-PCR system (Applied Biosystems 7500, USA) using the SYBR Premix

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Ex TaqTM (TaKaRa, Dalian, China). The reference gene, glyceraldehyde-3-

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phosphate dehydrogenase (GAPDH), was amplified with intron-spanning primers and

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used to normalize the cDNA concentrations. Quantitative qPCR programs for FTH1

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and GAPDH were: one cycle of 95°C for 5 min, 40 cycles of 95°C for 10 s and 60°C

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for 34 s of data collection, and one cycle for the melting curve analysis. The relative

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expression was calculated using the 2-∆∆Ct method. The GAPDH served as an internal

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reference gene, and the mean ∆Ct value of the control duckling within each group was

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used as the calibrator.

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2.4. Cloning of the duFTH1 flanking sequence by genome walking

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ACCEPTED MANUSCRIPT To obtain the sequence upstream of the duFTH1 gene, we performed genome

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walking PCR using a genome walking kit per the manufacturer's instructions

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(TAKARA, Dalian，China). Reverse primers were designed based the duFTH1 mRNA

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sequence (GenBank accession number: KC117386) and listed in Table 1. The forward

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primer was included in the kit. Genomic DNA of peripheral blood samples was

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extracted from issues using the Quick-gDNA™ MiniPrep Kit (Zymo Research Corp.,

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California, USA) and amplified by nested-PCR using the three sets of primers shown

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in Table 1. PCR products were sequenced directly by GenScript Co., Ltd. (Nanjing,

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China).

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2.5. Identification of the duFTH1 core promoter region using a dual-luciferase

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reporter assay

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Fragments of the duFTH1 5' flanking region starting at +143 bp and extending

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94, 155, 179, 288, 409, 630, and 909 bp upstream were prepared using primers with

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specific restriction sites (Table 1), then subcloned into the promoter-less luciferase

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reporter gene vector pGL3-Enhancer (Promega, Madison, USA), after being digested

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with KpnI and HindIII. This process generated a series of vectors containing

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progressive deletions of the duFTH1 promoter. The DF-1 cells (cell bank of Chinese

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Academy of Science, China), a spontaneously immortalized chicken cell line derived

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from 10 day old East Lansing Line (ELL-0) eggs, were cultured as described

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previously in complete growth medium—dulbecco’s modified eagle medium (GIBCO,

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USA) and 10% fetal bovine serum (GIBCO, USA) at 39℃ in a humidified 5%

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CO2/95% air incubator. These cells were seeded into 24-well plates. When cells

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reached 80% confluence, the cells were co-transfected with the different duFTH1

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promoter luciferase expression constructs and a Renilla luciferase plasmid using

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Lipofectamine 2000 (Invitrogen, California, USA) after 24 hours,. Luciferase

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activities in cell lysates were measured with a dual-luciferase reporter assay system

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(Promega, Madison, USA) according to the manufacturer’s instructions using a

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fluorospectrophotometer (Gene, Hong Kong, China). Firefly luciferase values were

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normalized using the Renilla luciferase activities.

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2.6. Bisulfite genomic sequencing of the duFTH1 promoter Genomic DNA of peripheral blood samples was extracted from morbid and

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non-morbid ducklings. The CpG island of the duFTH1 core promoter was predicted

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using online software (http://www.urogene.org/methprimer/index1.html), and primers

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were designed using MethPrimer, a program for designing bisulfite-conversion-based

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methylation PCR primers. Modified DNA was amplified by PCR using the two sets of

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primers shown in Table 1. These primers were specific for the converted DNA, but

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did not contain any CpG dinucleotides in their sequence; therefore, both methylated

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and unmethylated DNA could be amplified using the same primer sets. Each PCR

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mixture contained genomic DNA. PCR products were cloned and sequenced directly

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by GenScript Co., Ltd. (Nanjing, China).

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2.7. Identification of single nucleotide polymorphisms (SNPs) in the duFTH1 core

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promoter

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To identify SNPs in the duFTH1 core promoter, morbid and non-morbid

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ducklings were compared to detect polymorphisms. PCR products were amplified

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using DNA from the two group ducklings and sequenced directly by GenScript Co.,

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Ltd. (Nanjing, China). The obtained sequences were aligned using AlignIR (V2.0)

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software to screen for potential SNPs in the core promoter.

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2.8. Site-directed mutagenesis

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To assess the potential impact of SNPs on duFTH1 core promoter activity,

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transcription factor binding sites were predicted using Alibaba 2.1 and Jaspar

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databases. Transcription factor binding sites predicted to be altered by SNPs were

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selected for site-directed mutagenesis. Mutagenesis was carried out using the Fast

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Mutagenesis System (TransGen Bio, Beijing, China) and primers used for the

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mutagenesis procedures are shown in Table 1. After sequence verification, firefly and

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Renilla luciferase activities of the mutant constructs were measured as described 7

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above.

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2.9. Electrophoretic mobility shift assay Electrophoretic mobility shift (EMSA) and EMSA supershift assays to assess

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NRF1 binding to the FTH1 promoter were conducted using a commercial kit from

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Viagene Biotech, Co. (Changzhou, China) following the manufacturer’s protocol. The

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double-stranded DNA probe (5'-ACGCCGGCGCGCATGCGCGCGGTGA-3'; -46 to

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-70 nt; wild-type NRF1) containing three consecutive putative NRF1-binding sites

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was synthesized with and without a biotin label. DF-1 nuclear extracts (10 µg) and

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100-fold molar excess of unlabeled competing probes (including the mutated NRF1

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probe: 5'- ACGCCGGCGCGCATGTGCGCGGTGA-3', the mutated base is

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underlined) were mixed and incubated at room temperature for 20 min and then 15 µl

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of labeled probe (0.1 µmol) was added for 20 min. For the supershift assay, samples

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were incubated with 1 µl of anti- NRF1 polyclonal antibody (Abcam, Cambridge,

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England) for an additional 20 min at room temperature. Protein/DNA complexes were

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then isolated by non-denaturing polyacrylamide gel electrophoresis using a 6% gel

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and transferred onto a binding membrane. Protein/DNA complexes were analyzed

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using streptavidin–horseradish peroxidase and developed with an enhanced

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chemiluminescence system (Amersham Pharmacia Biotech/GE Healthcare, Uppsala,

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Sweden).

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2.10. Statistical analysis

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Each experiment was repeated at least three times and the data were analyzed

using independent sample t test (SPSS version 13).

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3. Results

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3.1. Differential expression of duFTH1 mRNA in morbid ducklings and non-morbid

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ducklings 8

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duck, the liver (Figure 1A) and spleen (Figure 1B) tissue were chosen to mRNA

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expression detection. Compared with the control ducklings, the transcript levels of

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duFTH1 mRNA was down-regulated very significantly in morbid ducklings in the

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two tissues (p < 0.01 or p < 0.05). However, duFTH1 mRNA showed the reverse trend

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in non-morbid ducklings that up-regulated significantly in spleen and down-regulated

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in liver(p < 0.05) (Figure 1). At the same time, we also found the expression of

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duFTH1 was very significantly higher in spleen of non-morbid ducklings than morbid

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ducklings (p < 0.01) , significantly higher in liver of non-morbid ducklings than morbid ducklings and in the (p < 0.05).

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3.2. Identification of the core transcriptional region of the duFTH1 promoter A partial duFTH1 gene fragment (1189 bp) was amplified by genome walking and

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sequence alignment confirmed that the fragment contained 1110 bp of the 5' flanking

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region and 79 bp of coding sequence. To investigate transcriptional and

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post-transcriptional mechanisms responsible for affecting duFTH1 expression, we

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first identified the transcription start site (TSS) by performing a BLAST search using

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the cDNA sequence of duFTH1 (GenBank accession number: KC117386) (Fig. 2A).

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To investigate if the duFTH1 promoter region was responsible for affecting FTH1

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transcription, we first determined the minimal sequences required for the

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transcriptional initiation of FTH1 expression. We generated promoter mutants by

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progressively deleting nucleotides from the 3' end and engineered these fragments

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into pGL-3 basic luciferase reporter vectors. Our results revealed that the minimal

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FTH1 promoter region consisted of the region from nt -486 to -35 (Fig. 2B).

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3.3. Comparison of duFTH1 core promoter methylation between morbid and

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non-morbid ducklings 9

ACCEPTED MANUSCRIPT A CpG island was predicted in the duFTH1 core transcriptional region and it included

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51 CpG sites. Bisulfite DNA sequencing was conducted to assess the extent of CpG

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island methylation in the duFTH1 promoter of morbid and non-morbid ducklings

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infected with DHV-1. Results indicated that this promoter was hypomethylated (Fig.

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3), the relative methylation of duFTH1 was only 5.9% and 2.0% in the morbid

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ducklings and non-morbid ducklings, respectively. These data suggested that duFTH1

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methylation was not involved in the effect of duFTH1 expression during DHV-1

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challenge.

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3.4. Identification and assessment of SNPs in the duFTH1 promoter of morbid

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ducklings

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We then identified potential cis-acting regulatory elements within the optimal

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FTH1 promoter region using Alibaba2.1 and Jaspar programs. Identified elements

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included multiple putative transcription factor binding sites, such as those for TBP,

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SRF, RXRβ, CREB, Ahr, C-Jun, NRF1, SP1, and E2F (Fig. 4A). Additionally, we

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sequenced this region using genomic DNA isolated from ducklings challenged with

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DHV-1. We identified 13 allelic variations (-55C/T, -67G/A, -155C/T, -238T/A,

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-239C/A, -250G/A, -265C/T, -266C/T, -284C/T, -339C/T, -357C/T, -386C/T, and

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-406C/T) in the essential promoter (Fig. 4B) and noted that two allelic variations

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(-55C/T, -67G/A) could alter NRF1 and Ahr transcription factor binding site,

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respectively. (Fig. 4C/D). Direct sequencing analysis revealed that the heterozygous

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(-55C/T) genotype was present in 75% (6/8) of non-morbid ducklings, whereas the

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homozygous allele (-55C/C) was present in 100% (8/8) and 25% (2/8) of morbid and

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non-morbid ducklings, respectively (Fig. 4E). The -67G/A allele was only present in

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one of the non-morbid ducklings. We also performed luciferase reporter gene assays

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using FTH1 promoter constructs (-144 to +145) containing either the wild-type (C) or

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mutant (T) allele to examine possible effects on promoter activity. We observed a

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significant difference in the transcriptional activity between the two constructs (Fig.

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4F). In contrast, -67G did not change the transcriptional activity when compared with

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the G allele (P>0.05). These data suggested that a promoter polymorphism regulates

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FTH1 expression by altering the NRF1 binding site. Next, to further strengthen the argument that FTH1 is a direct transcriptional

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target of NRF1, we performed a gel-shift competition assay using a biotin-labeled

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oligonucleotide spanning the potential NRF1-binding sequence as a probe. EMSA

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analysis was performed by competing unlabeled oligonucleotides (cold-NRF1, Fig.

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4G, lanes 3 and 4). Its mutant sequence was used as a negative control (mut-NRF1,

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Fig. 4G, lane 5). Consistent with the results of our reporter gene assay, competition

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with the NRF1 oligonucleotide dramatically eliminated specific binding in the DF-1

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cell nuclear extracts, whereas the corresponding mutated oligonucleotides (negative

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controls) did not. The specificity of NRF1 binding to the FTH1 promoter was

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confirmed by EMSA analysis. An antibody against NRF1 could supershift complexes

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on the oligonucleotide theoretically. However, because we used a polyclonal

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anti-NRF1 antibody, the supershifted bands were too large to run into the gel; thus, we

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only noted that the addition of anti-NRF1 dramatically eliminated specific binding of

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the oligonucleotide (Fig. 4H, lanes 3 and 4 compared to lane 1), while the unrelated

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antibody did not supershift these complexes (Fig. 4H, lane 5 compared to lanes 3 and

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4). Taken together, these data clearly demonstrate that FTH1 is a direct transcriptional

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target of NRF1 and that NRF1 might initiate the transcription of FTH1 by binding to

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the NRF1 transcription factor binding site in the FTH1 promoter.

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4. Discussion

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In this study, we examined the expression of FTH1 in the liver and spleen after

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DHV-1 infection to explore the function of the duck FTH1 gene. Expression of

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duFTH1 mRNA fluctuated significantly after infection. These results suggested that 11

ACCEPTED MANUSCRIPT FTH1 plays a role during DHV-1 infection. Research by Lee et al. (Lee et al., 2011)

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also suggested that FTH1 expression was down-regulated in the liver of pigs infected

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with hepatitis virus. In contrast, in non-morbid ducklings, duFTH1 mRNA expression

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was up-regulated significantly in the spleen and down-regulated in liver. The spleen,

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as an immunological organ, might be involved in the host’s defensive response against

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DHV-1. Consistently, ferritin expression also increases to protect ducklings from

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DHV-1 attack.

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Furthermore, we also found lower duFTH1 mRNA expression in the spleens and

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livers of morbid ducklings when compared to non-morbid ducklings (liver, P<0.01;

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spleen, P<0.05). In theory, DNA methylation inhibits gene expression. We assessed

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DNA methylation at the duFTH1 promoter of infected ducks and found that the

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promoters of both morbid and non-morbid ducklings were hypomethylated. Previous

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work found that FTH1 CpG was hypomethylated both in wild-type and Mbd5-/-

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mice(Tao et al., 2014). Similarly, V.F. Chekhun found that CpG islands in the FTH1

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promoter were hypomethylated when Guerin carcinoma cells were treated with

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doxorubicin or cisplatin(Chekhun et al., 2016). We did not identify any methylation

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changes in the FTH1 essential promoter region, indicating that promoter DNA

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methylation is not involved in the regulation of FTH1 expression.

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We also identified mutations in the core promoter of the FTH1 gene. Among

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these mutations, changes to the -55 nucleotide can affect the NRF1 binding site.

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Further, EMSAs demonstrate that NRF1 binds to the FTH1 promoter. Nrf1 and Nrf2

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are members of the Cap-N-Collar family of transcription factors (Biswas and Chan,

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2010). Previous reports demonstrated that Nrf2 regulated the human ferritin gene by

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binding to the antioxidant responsive element (ARE)(Huang et al., 2013; Jung et al.,

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2013; Pietsch et al., 2003). Recent studies showed that NRF1 negatively regulated the

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transcription of target genes(Lavigne et al., 2015; Wang et al., 2007; Zhang et al.,

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2006) and described the mechanism of Nrf1 transcriptional regulation(Tsujita et al.,

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2014). Nrf1 suppressed the expression of genes for nutrient and cysteine intake by

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ACCEPTED MANUSCRIPT binding to ARE/EpRE motifs in target genes as a heterodimer with small Maf (sMaf)

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proteins. Hence, Nrf1 acts as a negative transcriptional regulation factor to modify

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gene expression. Based on these previous studies, we speculate that low FTH1

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expression is due to the high frequency of the NRF1 binding site in morbid ducklings

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(-55C/C). Collectively, our findings strongly suggest that promoter polymorphisms,

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and not DNA methylation, are a major regulator of FTH1 expression. Our results

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reveal that the -55T allele of the FTH1 promoter region markedly enhanced FTH1

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transcription (Fig. 5).

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5. Conclusions

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In summary, we show that FTH1 expression during DHV-1 challenge was

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associated with promoter polymorphisms, but not with DNA methylation status. This

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study provides novel insights into the molecular mechanism responsible for regulating

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FTH1 transcription during DHV-1 challenge.

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Conflict of interest statement

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The authors have declared that no competing interest exists.

Acknowledgements

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This study was supported by the Natural Science Foundation of Jiangsu Province

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(BK20141275),

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China(2015BAD03B00), and the earmarked fund for Modern Agro-industry

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Technology Research System (CARS-43-3).

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Chekhun, V.F., Lozovska, Y.V., Naleskina, L.A., Borikun, T.V., Burlaka, A.P., Todor, I.N., Demash, D.V.,

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Figure Legends

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Fig.1. duFTH1mRNA expression in duckling tissue treated with DHV-1. Gene expression was

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determined by qPCR and was represented relative to GAPDH expression. Vertical bars represent

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the mean±S.D. (n=3). Significant differences relative to controls were indicated with * (P < 0.05)

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and ** (P < 0.01).

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Fig.2. The core transcriptional region of the duFTH1 promoter. (A) Schematic diagram of the 5'

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flanking region of the duFTH1 gene. Positions of the transcription start site (TSS) (+1 bp) and the

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first exon (blue) are depicted. (B) Reporter constructs of different lengths (pGL-Basic or M1-7)

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and pRL-TK, the internal control, were co-transfected into DF-1 cells. Firefly and Renilla

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luciferase activities in cell lysates were assayed 24 h after transfection and the relative luciferase

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values were calculated. Each value represents the mean±SEM of three biological replicates (**

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indicate P<0.01).

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Fig.3. Methylation patterns of the duFTH1 promoter in duckling challenged with DHV-1.(A1:

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morbid ducklings; A2: non-morbid ducklingsand uninfected duckling(B). Bisulfite sequencing

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analysis was performed to determine the DNA methylation profile of individual CpG sites.

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Amplified PCR fragments (-187 to +179), as indicated in the figure, were cloned, and 10

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independent clones were sequenced per fragment. Solid and open circles indicate methylated and

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unmethylated sites, respectively, and each row of circles is data from a single clone.

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Fig.4. Assessment of cis-acting transcriptional regulatory elements in the FTH1 promoter. (A)

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predicted using Alibaba2.1 and Jaspar programs. (B) Thirteen allelic variations were identified. (C,

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D) Allelic variations -55C/T and -67G/A alter the NRF1 and Ahr binding sites, respectively. The

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-67G and -55C SNPs have NRF1 and Ahr transcription factor binding sites, respectively. (E)

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Frequency distribution of the -55 alleles in morbid ducklings and non-morbid ducklings. (F) FTH1

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promoter sequences containing either the wild-type (C) or mutant (T) nucleotide were fused to the

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luciferase gene, and the relative luciferase activity was compared between constructs containing

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either the C or T nucleotide. Each value represents the mean±SEM of three replicates (** indicate

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P<0.01). (G) An electrophoretic mobility shift assay (EMSA) using DF-1 nuclear extracts

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incubated with either an excess of unlabeled consensus or mutant oligonucleotides of NRF1 as

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competitors. (H) EMSA supershift analysis was performed with anti-NRF1 and nuclear extracts

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from DF-1 cells.

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Fig.5. Plausible mechanism for NRF1 regulating FTH1 expression. Promoter polymorphisms, and

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not DNA methylation, may be the major regulator of FTH1 expression. Allelic variation of the

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FTH1 promoter may affect transcription activity by altering binding sites for the transcription

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factor Nrf1. (A) When the C nucleotide is present at the -55 locus, the NRF1 putative transcription

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factor binding site is present in the FTH1 promoter, and FTH1 expression is suppressed. (B) When

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the T nucleotide is present at the -55 locus, the NRF1 putative transcription factor binding site is

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absent in the FTH1 promoter, and FTH1 expression is facilitated.

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ACCEPTED MANUSCRIPT Table 1 Oligonucleotide primers used in the experiments Annealing temperature (°C)

Primer sequence (5′→3′)

ACAATGACCCAGCCTTAG CCACTGTATCTTCCCTTC

e FTH1-F e FTH1-R

AGATCGTGATGACTGGGAGAAT TCCAGGTAGTGAGTTTCGATGA

GAPDH-F GAPDH-R

TGCTAAGCGTGTCATCATCT AGTGGTCATAAGACCCTCCA ACGATCTGGTTTCTTGATGTCC TGCAGTTTCATCAGCTTCTCAG TCATCCCGGTCAAAGTAGTAGG

BFTH1-F1 BFTH1-R1

AGATCGATTTTATTGAGTTTAG ATTAATCTAACGATTAACGAC

BFTH1-F2 BFTH1-R2

TTGTGAGGGGAGGAGGAGGG TTTAACGCCTCGCAATCCTAATAAT

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Usage

DHV3D gene amplification

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RT-PCR RT-qPCR

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RT-PCR RT-qPCR

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1st BSP

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2nd BSP

CGGGGTACCGGCTATGGGGCGTAGGCACAAAT

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progressive deletions

MFTH1-F2

CGGGGTACCATGGCCCGAAGTTGTTCCAGTGT

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progressive deletions

MFTH1-F3

CGGGGTACCCTGCGATTCAGGCCCAGACCGAC

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progressive deletions

MFTH1-F4

CGGGGTACCCGGGGAGGTGCTGAGTCACGCT

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progressive deletions

MFTH1-F5

CGGGGTACCCGCTATAAAAGGCGCGGCGTCGA

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progressive deletions

MFTH1-F6

CGGGGTACCCAGCGCCGGCCCAGAGTGCGTC

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progressive deletions

MFTH1-F7

CGGGGTACCTTAGACGGAACCGGCCGCGCTC

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progressive deletions

MFTH1-R

CCCAAGCTTCGCTGGGATGAGATTGAGATGGAG

mutFTH1-F1 mutFTH1-R1

ACGCCGGCGCGCATGTGCGCGGTGACGT ACATGCGCGCCGGCGTGGCGGGAAACGG

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MFTH1-F1

Site-directed Mutagenesis

Note: MFTH1-R is the common downstream primer. Underlined sequences show the location of restriction sites used for subcloning. RT-PCR, reverse transcription PCR; RT-qPCR, reverse transcription quantitative PCR; BSP, bisulfite sequencing.

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ACCEPTED MANUSCRIPT Dear Editors,

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The highlights of this experiment are listed in two points: (1) The FTH1 expression was regulated in ducks during DHV-1 challenge by a mutation that alters a NRF1 binding site. (2) There was no evidence that DNA methylation of the FTH1 promoter region contributed to the regulation of FTH1 expression.

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Thank you and best regards.

Yours sincerely,

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Qi Xu, PhD

Key Laboratory of Animal Genetics ＆ Breeding and Molecular Design of Jiangsu province, Yangzhou University

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E-mail: [email protected]