Infection, Genetics and Evolution 24 (2014) 177–182

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Functional analysis of a highly conserved abundant larval transcript-2 (alt-2) intron 2 repeat region of lymphatic filarial parasites Moorthy Sakthidevi a, Sugeerappa Laxmanappa Hoti b, Perumal Kaliraj a,⇑ a b

Centre for Biotechnology, Anna University, Chennai, Tamil Nadu 600 025, India Vector Control Research Centre, Puducherry 605 006, India

a r t i c l e

i n f o

Article history: Received 9 May 2013 Received in revised form 12 March 2014 Accepted 17 March 2014 Available online 26 March 2014 Keywords: Gene regulation Transcription Silencer Intron DNA repeat Evolution

a b s t r a c t The filarial-specific protein abundant larval transcript-2 (ALT-2) is expressed exclusively in the infective larval stage (L3) and is a crucial protein for establishing immunopathogenesis in human hosts. The alt-2 gene has a conserved minisatellite repeat (29 or 27 bp) in intron 2 (IR2) whose significance within lymphatic filarial species is unknown. Here, we report the role of IR2 in the regulation of alt-2 gene expression using an in vitro model. Using electrophoretic mobility shift assays, we identified the presence of a putative nuclear protein binding region within IR2. Subsequent transient expression experiments in eukaryotic cell lines demonstrated that the IR2 downregulated the expression of a downstream luciferase reporter gene, which was further validated with RT-PCR. We therefore identify IR2 as a suppressor element that regulates L3 stage-specific expression of alt-2. Ó 2014 Elsevier B.V. All rights reserved.

1. Introduction Lymphatic filariasis is a neglected tropical disease that is prevalent in Asia and Africa. It is estimated that 120 million people are infected with lymphatic filariasis, and more than 1.3 billion people are at risk (WHO, 2012). Wuchereria bancrofti (Wb) and Brugia malayi (Bm) are the major causative agents of disease, leading to debilitating and disfiguring chronic manifestations, such as lymphedema, elephantiasis and hydrocele (Hotez et al., 2008). Like other tissue-dwelling parasites, lymphatic filarial parasites have complex life cycles, the third larval (L3) stage being infective. To establish pathogenicity in the human host, several genes, such as those encoding proteinases, proteinase inhibitors, secretory/excretory proteins and antioxidant proteins, are upregulated in the infective stage (Bennuru et al., 2009). Abundant larval transcript (ALT) proteins are secretory proteins that characteristically suppress Th1 immunity, allowing the parasite to survive in an altered Th2 environment (Hewitson et al., 2009; Maizels et al., 2004). In addition, alt genes are filarial nematode specific and are expressed abundantly; they account for 5% of the total transcript during the BmL3 stage (Gregory et al., 2000). As ALT is crucial for parasite immune evasion (GomezEscobar et al., 2005), it has been proposed as a vaccine candidate ⇑ Corresponding author. Tel.: +91 44 22350772; fax: +91 44 22350299. E-mail address: [email protected] (P. Kaliraj). http://dx.doi.org/10.1016/j.meegid.2014.03.017 1567-1348/Ó 2014 Elsevier B.V. All rights reserved.

for lymphatic filariasis. Bm-ALT, as a single antigen (Gregory et al., 2000) or as a multiple antigen in combination with other filarial proteins (Kalyanasundaram and Balumuri, 2011), has been shown to induce immunity in animal models. Immunization studies have shown that Bm-ALT-2 gives 74% immunoprotection in jirds (Gnanasekar et al., 2004). ALT immunogenicity has been better studied than other vaccine candidates against lymphatic filariasis (Dash et al., 2011). Among the 13 alt genes identified in the B. malayi whole genome sequence project, ALT-1 and ALT-2 proteins are immunologically well characterized in animal models (Blaxter et al., 2012). Although the molecular mechanism of the upregulation of alt in the L3 stage is still unexplored, the alt promoter is considered to be a key component in regulating expression (Gomez-Escobar et al., 2002). In addition, B. malayi alt-2 (Bm-alt-2) has tandem repeat units of 27 or 46 nt in intron 2 and intron 3, respectively. In our previous study on the genetic polymorphism of alt-2, we reported that the intron 2 tandem repeat (IR2) has 98% sequence identity between B. malayi and W. bancrofti (Sakthidevi et al., 2010). In contrast, the intron 3 tandem repeat (6 or 9 copies) is completely absent in W. bancrofti alt-2 (Wb-alt-2) (Sakthidevi et al., 2010). Furthermore, introns are known to have a functional role in regulating the expression of genes (Deitsch et al., 2001). We therefore explored the functional significance of the conserved IR2 as a modulator of gene expression, as well as its possible role in the stagespecific expression of alt-2 in filarial parasites.

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2. Materials and methods 2.1. Eukaryotic cell lines and nuclear protein extraction CHO-K1 cells were grown in Hanks’ F12 medium, Sf21 insect cells were grown in Grace’s Insect Medium (Invitrogen, Carlsbad, CA, USA) and MDA-MB231 and HepG2 cells (gift from Dr. Anjali A. Karande, IISC, Bangalore, India) were grown in Dulbecco’s Modified Eagle Medium (DMEM) (Invitrogen). All cell lines were grown in the presence of 10% fetal bovine serum (FBS). When the cells were 95% confluent, they were washed in 10 mM phosphate-buffered saline (PBS) (pH 8.0). An additional step of trypsin (0.1%) treatment was performed on adherent cell lines, such as CHO-K1, MDA-MB231 and HepG2, to detach them from the surface of the culture plate. Nuclear and cytoplasmic proteins were extracted from the harvested cells using the NE-PER Nuclear Protein Extraction Kit (Thermo Fischer Scientific, Rockford, IL, USA) by following the manufacturer’s protocol. The extracted nuclear and cytoplasmic proteins were quantified using the Micro BCA Protein Assay Kit (Thermo Fischer Scientific), according to the manufacturer’s procedure.

promoter regions (CMV or SV40 or EF-1a) were cloned in the pGL3Basic Vector at the KpnI and BglII sites, followed by Wb-alt-2 intron 2 or Wb-alt-2 Dintron 2 at the BglII and HindIII sites upstream of the luciferase gene (primers used are listed in Supplementary Table S1). The DNA construct with the promoter region and the luciferase gene was treated as a positive control for luciferase expression. Wb-alt-2 intron 2 constructs included the entire intron 2 region and 20–30 bp of the flanking exonic region. In the same manner, Bm-alt-2 intron 2 and Wb-alt-2 intron 2 (7 copies) DNA constructs were also cloned. Wb-alt-2 Dintron 2 was constructed by treating the alt-2 intron 2 PCR product with Tsp5091 endonuclease, which has multiple target sites within the 29- or 27-bp tandem repeat units. Thus, the resultant alt-2 Dintron 2 had only sequences other than the 29- or 27-bp repeat region (IR2) of the alt-2 intron-2. The schematic diagram for the various DNA constructs is shown in Supplementary Fig. S1. The deletion of the IR2 region to form alt-2 Dintron 2 was confirmed by DNA sequencing. All constructs were transformed in the DH5a strain and plasmids were extracted for transfection into cell lines. The purified plasmids were quantified by spectrophotometry at 280 nm. 2.4. Transient transfection in cell lines

2.2. Electrophoretic mobility shift assay (EMSA) EMSA, also referred as gel retardation assay is based on affinity electrophoresis and is used to study protein–nucleic acid interactions. The 29- or 27-bp intron 2 repeat (IR2) oligonucleotides for EMSA were biotin-labeled using a 30 -End DNA Labeling Kit (Thermo Fischer Scientific) following the manufacturer’s protocol. The single-stranded (ss) biotin-labeled oligonucleotides were denatured at 95 °C for 2 min. The biotinylated ssDNA probes were annealed by incubating at 58 °C (Tm) for 30 min to obtain doublestranded (ds) biotinylated DNA probes. The dsDNA biotinylated probes were purified using spin columns (Qiagen, Valencia, CA, USA). The biotinylated probe sequences corresponding to the IR2 of the alt-2 gene were Bm27R-F1-50 ATG TCA CAC ATT ACC TTA CTT ATG AAA-30 ; Bm27R-R1-50 TTT CAT AAG TAA GGT AAT GTG TGA CAT-30 (Bm-alt-2, 27-bp tandem repeats) and Wb29R-F150 ATG TCA CAC ATC TCA CCT TAC TTA TGA AA-30 ; and Wb29RR1-50 TTT CAT AAG TAA GGT GAG ATG TGT GAC AT-30 (Wb-alt-2, 29-bp tandem repeats). Oligonucleotides were ordered from MWG Biotech Pvt. Ltd (Bangalore, India). EMSAs were performed with the LightShift Chemiluminescent EMSA Kit; in each reaction, 25 fmol of the 29- or 27-bp IR2 biotinylated dsDNA probe was incubated with 10 lg nuclear protein extract for 30 min at 25 °C in 20 ll of binding buffer (100 mM Tris, 500 mM KCl, 10 mM DTT, pH 7.5) with 1 ll poly(dI–dC) (1 lg/ll). Here, the DNA copolymer, poly(dI–dC) was used as a non-competitive inhibitor. For competition experiments, a 200-fold molar excess of the unlabeled dsDNA probe was added to the mixture as a specific competitor before incubation. Control experiments were performed either with cytoplasmic protein or without nuclear protein. The DNA– protein binding reaction was stopped by adding 2 ll of 2.5 mM EDTA. The samples were electrophoresed on a 6% native polyacrylamide gel and were transferred (0.5 TBE, 50 V, 90 min) to a positively charged nylon transfer membrane (Thermo Fischer Scientific). The transferred membrane was developed using a chemiluminescent detection module (Thermo Fischer Scientific), in accordance with the manufacturer’s protocol. 2.3. Plasmid constructs for transient gene expression The pGL3-Basic Vector (gift from Dr. Nitish Mahapatra, IIT-Chennai, India) was used to construct the reporter plasmids for transient gene expression in animal cell lines. PCR products representing the

CHO-K1, MDA-MB231 and HepG2 cells (2  106) were grown individually in six-well cell culture plates (Thermo Fischer Scientific) for transient gene expression of the DNA constructs. After the cells had grown to 80% confluence, 2 lg of filter-sterilized plasmid constructs was co-transfected with 1 lg of pSPORT-b-gal (gift from Dr. Veena Pranaik, CCMB, Hyderabad, India) as an internal control by cationic liposome-mediated transfection with lipofectamine 2000 (Invitrogen). The transfected cells were incubated at 37 °C in a 5% CO2 incubator for 48 h. 2.5. Luciferase and galactosidase assay The influence of the 29- or 27-bp IR2 on gene expression was assessed using the luciferase reporter gene assay (Promega, Madison, WI, USA). Cells were harvested after 48 h of transient transfection and were washed in PBS before the addition of 500 ll of 25 mM Tris–HCl (pH 7.8). These cells were divided into two parts; one part was used for reporter gene assays, and the other part was stored in 80 °C for RNA isolation. After centrifugation at 7000 rpm for 5 min, the cells were resuspended in 200 ll of reporter lysis buffer. The cells were freeze-thawed in repeated cycles for efficient cell lysis, and cell debris was pelleted at 12,000 rpm for 2 min at 4 °C; 20 ll of supernatant was used to measure the luciferase activity with an automated luminescence microplate reader (BioTek Instruments Inc., Winooski, VT, USA), and the readings were measured in luminescence units. The b-galactosidase activity was assessed to normalize transfection efficiency. The sample supernatant used for the luciferase assay was also used in the bgalactosidase assay; 50 ll of 2 assay buffer containing 1 mg/ml of O-nitrophenyl-b-D-galactopyranoside substrate in phosphate buffer (pH 7.5) was added to an equal volume of cell lysate and incubated for 30 min at 37 °C. The reaction was stopped by adding Na2CO3 and the yellow color that developed was read at 420 nm. The value was represented as the mean ± SD of at least three independent experiments. 2.6. RNA isolation and RT-PCR Total RNA was isolated from the plasmid constructs in transiently transfected cell lines using TRIzol reagent (Invitrogen). Total cellular RNA was treated with RNase-free DNase (New England Biolabs, MA, USA) to remove DNA contamination, followed by heat inactivation of DNase. Total mRNA was reverse-transcribed to cDNA using the Pro-

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toScript First Strand cDNA Synthesis Kit (New England Biolabs), in accordance with the manufacturer’s protocol, and the cDNA was quantified spectrophotometrically. One microgram of cDNA was used for RT-PCR amplification using Phusion Hot Start High-Fidelity DNA Polymerase (Finnzymes, Pittsburgh, PA, USA). All of the RT-PCR reactions were performed as follows: pre-denaturation at 95 °C for 5 min; 30 cycles of denaturation at 95 °C for 45 s, annealing at 60 °C for 45 s, extension at 72 °C for 45 s, final extension at 72 °C for 5 min. The primers used for the amplification of the luciferase gene (1.8 kb) were 50 CCC AAG CTT ATG GAA GAC GCC AAA AAC ATA AAG 30 (forward) and 50 CGC GGA TCC TTA CAC GGC GAT CTT TCC GCC 30 (reverse). Primers 50 GAC GAG GCC CAG AGC AAG AGA GG 30 (forward) and 50 GCT CAT TGC CAA TGG TGA TGA CCT GG 30 (reverse) were used to amplify a 595-bp fragment of the b-actin gene to assess amplification efficiency. For semi-quantitative RT-PCR analysis, the PCR products were run on 1.2% agarose gel with a standard DNA marker (New England Biolabs). The corresponding integrated density value (IDV) for luciferase and b-actin was measured using gel analysis software (Bio-Rad). The IDV of the luciferase bands was then normalized with the IDV of the corresponding b-actin bands, and the IDV ratio was plotted. 2.7. Bioinformatics and statistical analysis MatInspector software (Cartharius et al., 2005) was used to predict the transcription factor (TF) binding sites in the IR2 region. The

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scores for TF binding sites were calculated with the MatInspector in-built program. This program calculates the score on the basis of the position-specific scoring matrix (Quandt et al., 1995). The difference in the relative luciferase activity, regulated by IR2 in different experimental groups, was analyzed by Student’s t-test. The data represent the means ± SD from three independent experiments. 3. Results and discussion 3.1. Evolutionary conserved IR2 region has nuclear protein binding sites The functional dissection of the IR2 region was initiated by a prediction analysis for TF binding sites using MatInspector software (Cartharius et al., 2005). The analysis predicted seven TF binding motifs within the IR2 region. Two transcriptional suppressor proteins, DeltaE and E4BP4, were predicted with high scores of 96 and 88, respectively (Fig. 1A). In addition, among the predicted TFs, E4BP4, CdxA and Nkx-2 are known to be highly conserved across species (Cowell, 2002; Guo et al., 2004; Junghans et al., 2004; Lints et al., 1993). In order to verify TF binding sites in IR2, we performed an electrophoretic mobility shift assay (EMSA) using nuclear extracts of mammalian (HepG2) and insect cell lines (Sf21). Well-characterized eukaryotic cell lines were used rather than filarial parasite

Fig. 1. Analysis of TF binding site(s) in the IR2 region of alt-2. (A) Sequence comparison of IR2 of Wb-alt-2 and Bm-alt-2 showing the predicted TF binding site by MatInspector. The gray shaded box underlying intron 2 marks the IR2 region in alt-2. The predicted score for binding of TF to the IR2 region is given in parentheses. (B) Confirmation of TF binding site in IR2 by EMSA. (+) Indicates presence of the component and () indicates absence. Lane 1: negative control without nuclear protein. Lane 2: 29-bp (Wb-alt-2 IR2) dsDNA probe incubated with HepG2 nuclear extract. Lane 3: 29-bp dsDNA probe incubated with HepG2 nuclear extract in the presence of a 200-fold molar excess of unlabeled 29-bp dsDNA probe. 4: 27-bp (Bm-alt-2 IR2) dsDNA probe incubated with HepG2 nuclear extract. Lane 5: 27-bp dsDNA probe incubated with HepG2 nuclear extract in the presence of a 200-fold molar excess of unlabeled 27-bp dsDNA probe. Lane 6: 29-bp dsDNA probe incubated with HepG2 cytoplasmic extract. Lane 7: 29-bp probe incubated with HepG2 cytoplasmic extract in the presence of a 200-fold molar excess of unlabeled 29-bp dsDNA probe. Lane 8: 29-bp DNA probe incubated with Sf21 nuclear extract. Lane 9: 29-bp dsDNA probe incubated with Sf21 nuclear extract in the presence of a 200-fold molar excess of unlabeled 29-bp dsDNA probe.

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for nuclear extracts because of limitations in identifying the TFs. The 29- or 27-bp biotin-labeled IR2 probe showed a mobility shift in the reactions containing the nuclear protein (Fig. 1B, lanes 2, 4 and 8), but not with the cytoplasmic extract (Fig. 1B, lanes 6 and 7). The band was not detected in the presence of a higher concentration of the unlabeled probe (Fig. 1B, lanes 3, 5 and 9), confirming that the observed shift was due to the specific binding of putative TF in the nuclear extract. In order to verify that the competitive binding of unlabeled probe to nuclear protein is sequence specific, we substituted the unlabeled probe with an irrelevant, 30-bp unlabeled probe (Supplementary Fig. S2). The EMSA result showed that the 29-bp unlabeled probe competed for the nuclear protein with the 29-bp labeled probe. Moreover, the 2-nt deletion in Bm-alt-2 IR2 (27 bp) did not affect the binding of putative TF (Fig. 1B, lane 4). As the mobility shift was observed with both HepG2 and Sf21 nuclear extracts, the putative TF might have a conserved DNA binding motif. However, we were not able to identify the specific TF binding to the IR2 probe. For example, monoclonal antibodies against computationally predicted E4BP4 failed to produce a

detectable supershift in EMSA (not shown). Therefore, this failure could be due either to a constraint in epitope recognition by monoclonal antibodies or to other unidentified TFs binding to the IR2 region, which could not be predicted by computational analysis. 3.2. IR2 downregulates expression of luciferase in transiently transfected cell lines After confirming the presence of putative nuclear protein binding site(s) in IR2 by EMSA, we examined the impact of the IR2 repeat region on gene expression. The lack of genetic tools to manipulate filarial parasites and developmental incompetency of filarial parasites in animal models are the major hindrance for studying gene regulation in the filarial parasites. Therefore, we selected the mammalian cell line system for transient gene expression studies because it is flexible for genetic manipulation and longitudinal studies are feasible. For transient gene expression analysis, the Wb-alt-2 intron 2 DNA sequence was placed between the commonly used constitutive promoter (CMV or SV40 or EF-1a) and the luciferase gene (Fig. 2A), whereas for the Wb-alt-2 Dintron

Fig. 2. Silencer activity of IR2 in transiently transfected cell lines. The DNA plasmid constructs used to reveal the role of IR2 in gene regulation is represented schematically. (A) Wb-alt-2 intron 2⁄ (6 copies) or (B) Wb-alt-2 Dintron 2 devoid of the IR2 region was placed between the constitutive promoter (CMV or SV-40 or EF-1a) and the luciferase gene. These constructs were transiently transfected in HepG2 (C), MDA-MB231 (D) and CHO-K1 (E) cell lines, and cells were harvested after 48 h for reporter gene assays. (F) Analysis of the effect of IR2 copy number and 2-nt deletion (Bm-alt-2 IR2) on IR2 silencer activity. In this experiment, Wb-alt-2 intron 2⁄⁄ (7 copies) or Bm-alt-2 intron 2⁄ (27bp repeats; 6 copies) was placed instead of Wb-alt-2 intron 2⁄ (6 copies) between the constitutive promoter CMV and the luciferase gene. The bar diagrams represent luciferase activity after normalization against b-galactosidase activity (internal control). The results are expressed in relative luminescence units and are the mean ± SD of three independent experiments performed in duplicate. ##Statistical significance at P < 0.001 and #P < 0.01.

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2 construct, the IR2 region was deleted (Fig. 2B). CMV or SV40 or EF-1a promoter with luciferase construct served as a positive control. Compared with the positive control, luciferase activity in the Wb-alt-2 intron 2 transiently transfected HepG2 cell lines decreased by 9-fold (P < 0.001) (Fig. 2C). In contrast, luciferase activity was unaltered in the Wb-alt-2 Dintron 2 transfected cell lines, suggesting that the suppressor-binding DNA element is indeed in the IR2 region of Wb-alt-2 intron 2. In addition, the results were similar in other mammalian cell lines, such as MDAMB-231 (P < 0.01) (Fig. 2D) and CHO-K1 (P < 0.001) (Fig. 2E), which displayed an 8- to 9-fold decrease in luciferase activity. These results suggest that the IR2 region might be a consensus DNA recognition site for TFs conserved among divergent species. Moreover, regardless of the presence of any constitutive promoter, IR2 exhibits repressor activity. Introns that affect heterologous gene expression, irrespective of promoter, are well documented in transgenic plants (Mascarenhas et al., 1990) and transgenic animals (Choi et al., 1991; Palmiter et al., 1991). The IR2 region in Bm-alt-2 differed from Wb-alt-2 by the IR2 copy number and a 2-nt deletion in the tandem repeat units of the Bm-alt-2 IR2 sequence (Sakthidevi et al., 2010). Therefore, we analyzed the influence of the IR2 copy number and the 2-nt deletion on silencer activity. Here, the HepG2 cell lines were transfected with the DNA constructs carrying Wb-alt-2 intron 2⁄⁄ (7 copies) or Bm-alt-2 intron 2⁄ (27-bp DNA repeat; 6 copies) instead of Wbalt-2 intron 2⁄ (6 copies). Upon transient gene expression, the Wbalt-2 intron 2⁄⁄ (7 copies) and the Bm-alt-2 intron 2⁄ (6 copies) con-

Fig. 3. Position-dependent negative regulation exhibited by IR2. The DNA plasmid constructs used to reveal the influence of the IR2 position on gene regulation are represented schematically. (A) Wb-alt-2 intron 2⁄ (6 copies) or (B) Wb-alt-2 Dintron 2 devoid of the IR2 region was placed downstream of the luciferase gene and 2 kb away from the constitutive promoter CMV. The bar diagrams represent luciferase gene expression in these constructs, which were transiently transfected in HepG2 cell lines. The luciferase activity was normalized against b-galactosidase activity (internal control). The results are expressed in relative luminescence units and are the mean ± SD of three independent experiments performed in duplicate. ##Statistical significance at P < 0.001.

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structs gave similar results to those achieved for Wb-alt-2 intron 2⁄ (6 copies) (Fig. 2F). These data show that the variation in copy number of the tandem repeats or the 2-nt deletion in Bm-alt-2 IR2 does not affect the silencer activity of IR2. In addition, this suggests that IR2 silencer activity upon gene expression is conserved within the lymphatic filarial species. Most silencer elements act in a position-independent manner (Ogbourne and Antalis, 1998) and are involved in an active repression mechanism; they are referred to as classical silencer elements (Ogbourne and Antalis, 1998). In recent studies of plasmodium var gene regulation, intron 1 acted as a silencer by base-pairing with the promoter (Deitsch et al., 2001; Frank et al., 2006) to restrict the expression of all of the multicopy var gene at once. However, there are silencers that act in a position-dependent manner with a direct passive repression mechanism (Carroll, 2005; Sone et al., 2011; Tanaka et al., 2001); they are referred to as negative regulatory elements (Ogbourne and Antalis, 1998). Therefore, to verify how IR2 exhibits negative regulation of gene expression, reporter plasmids were constructed with the alt-2 intron 2 or Dintron 2 downstream of the luciferase gene (Fig. 3A and B). Luciferase activity was low when the IR2 DNA sequences were placed between the promoter and the luciferase gene. Conversely, luciferase expression was unaltered when IR2 sequences were placed downstream of the luciferase gene (Fig. 3C). These results suggest that the IR2 functions as a non-

Fig. 4. IR2 silencer element blocks transcription of heterologous gene. DNA constructs in transiently transfected HepG2 cell lines used in the experiment in Fig. 2F were subjected to RT-PCR analysis. The PCR product of luciferase and b-actin (internal control) were run on agarose gel electrophoresis (A). Lane 1: negative control without template. Lane 2: CMV promoter construct. Lane 3: CMV + Wb-alt-2 intron 2⁄. Lane 4: CMV + Wb-alt-2 intron 2⁄⁄. Lane 5: CMV + Bm-alt-2 intron 2⁄. Lane 6: CMV + Wb-alt-2 Dintron 2. Here,  represents a 29- or 27-bp tandem repeat (IR2), 6 copies, and  represents IR2, 7 copies. The integrated density value (IDV) of the luciferase band is normalized with the IDV of the b-actin band, and the IDV ratio is plotted as a bar diagram (B). The results are the mean ± SD of three independent experiments. ##Statistical significance at P < 0.001 and #P < 0.01.

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classical, position-dependent silencer element. Therefore, as proposed for negative regulatory elements, IR2 could be involved in directing a passive repression mechanism (Gaston and Jayaraman, 2003), which requires further experimental validation.

3.3. IR2 acts as a transcriptional suppressor To investigate at which step of gene expression IR2 acts as a silencer, we performed semi-quantitative RT-PCR analysis with IR2 construct transiently transfected cell lines. The IDV of the luciferase bands was normalized with the internal control b-actin bands, and the IDV ratios were plotted. Compared with the promoter constructs, promoter + alt-2 intron 2 construct transfected cell lines showed 70% lower luciferase RNA transcript levels (P < 0.001–0.01) (Fig. 4A and B). The result was invariable with IR2 copy number (6 or 7 copies in Wb-alt-2) and 2-nt deletion in Bm-alt-2 (Fig 4A, lanes 3, 4 and 5). On the other hand, the luciferase RNA transcript level was restored in the promoter + alt-2 Dintron 2 constructs (Fig. 4A and B). These findings indicate that the reduced activity of luciferase is due to a deficit in the transcription process implemented by IR2 silencer elements. The present study demonstrates that the IR2 region has nuclear protein binding site(s) and acts as a transcriptional silencer element in a position-dependent manner. Moreover, B. malayi transcriptional profiling and expressed sequence tag analysis identified expression of alt-2 in the L3 stage that is not detectable in other stages (Bennuru et al., 2009; Gregory et al., 1997; Li et al., 2012). Therefore, we propose that IR2 might be involved in recruiting transcription repressor proteins for blocking, the promoter site or progression of the transcription preinitiation complex.

4. Conclusion The alt-2 gene is unique among the alt gene family for the presence of minisatellite tandem repeats in introns 2 and 3. Our previous study on the genetic polymorphism of alt-2 identified alt-2 IR2 as being highly conserved in B. malayi and W. bancrofti. Molecular characterization studies of IR2 demonstrate the presence of a nuclear protein binding site. Furthermore, IR2 acts as a transcription silencer in a position-dependent manner. Taken together, these findings indicate that IR2 could be acting as a silencer that represses the expression of L3-specific alt-2 in other life stages of lymphatic filarial parasites. ALT is a crucial protein for the parasite to establish infection in the human host. Thus, studying the regulation of alt-2 expression could broaden knowledge on filarial parasite genetics with respect to L3 stage-specific gene expression in establishing pathogenesis.

Acknowledgements This study was funded by Indian Council of Medical Research, Government of India and Sakthidevi was a recipient of Senior Research Fellowship from the Council for Scientific and Industrial Research, Government of India. We thank Dr. Prince R. Prabhu, Visiting faculty, Centre for Biotechnology, Anna University, Chennai, India for modifying the manuscript considerably.

Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j.meegid. 2014.03.017.

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