Characterization of the human α9 integrin subunit gene: Promoter analysis and transcriptional regulation in ocular cells.

Experimental Eye Research xxx (2015) 1e18

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Characterization of the human a9 integrin subunit gene: Promoter analysis and transcriptional regulation in ocular cells line Duval a, 1, Karine Zaniolo a, 1, Steeve Leclerc a, Christian Salesse a, b, Ce rin a, b, * Sylvain L. Gue ^pital du Saint-Sacrement, Centre de Recherche FRQS du CHU de Qu Centre Universitaire d'Ophtalmologie-Recherche, Axe M edecine R eg en eratrice, Ho ebec, Qu ebec, Canada b Departement d'Ophtalmologie, Facult e de M edecine, Universit e Laval, Qu ebec, QC, Canada a

a r t i c l e i n f o

a b s t r a c t

Article history: Received 11 November 2014 Received in revised form 26 January 2015 Accepted in revised form 2 March 2015 Available online xxx

a9b1 is the most recent addition to the integrin family of membrane receptors and consequently remains

Keywords: Integrin Alpha9 Promoter Gene Extracellular matrix Transcription factor Tenascin

the one that is the least characterized. To better understand how transcription of the human gene encoding the a9 subunit is regulated, we cloned the a9 promoter and characterized the regulatory elements that are required to ensure its transcription. Transfection of a9 promoter/CAT plasmids in primary cultured human corneal epithelial cells (HCECs) and uveal melanoma cell lines demonstrated the presence of both negative and positive regulatory elements along the a9 promoter and positioned the basal a9 promoter to within 118 bp from the a9 mRNA start site. In vitro DNaseI footprinting and in vivo ChIP analyses demonstrated the binding of the transcription factors Sp1, c-Myb and NFI to the most upstream a9 negative regulatory element. The transcription factors Sp1 and NFI were found to bind the basal a9 promoter individually but Sp1 binding clearly predominates when both transcription factors are present in the same extract. Suppression of Sp1 expression through RNAi also caused a dramatic reduction in the expression of the a9 gene. Most of all, addition of tenascin-C (TNC), the ligand of a9b1, to the tissue culture plates prior to seeding HCECs increased a9 transcription whereas it simultaneously decreased expression of the a5 integrin subunit gene. This dual regulatory action of TNC on the transcription of the a9 and a5 genes suggests that both these integrins must work together to appropriately regulate cell adhesion, migration and differentiation that are hallmarks of tissue wound healing. © 2015 Elsevier Ltd. All rights reserved.

1. Introduction Integrins function as cell surface adhesion receptors that are responsible for cell/cell and cell/extracellular matrix interactions (Hynes, 1992; Katz and Yamada, 1997; Margadant et al., 2011). They constitute the link between the outside and the inside of the cell and can transduce and modulate bi-directional signals to regulate many cellular mechanisms such as adhesion, migration, proliferation, differentiation, and apoptosis (Hynes, 1992; Ye et al., 2011). Structurally, they are type I trans-membrane glycoproteins composed of an alpha and a beta subunit, which are non-covalently

^pital du Saint-Sacrement, Centre de * Corresponding author. CUO-Recherche, Ho bec, QC, Canada. Recherche du CHA, Que rin). E-mail address: [email protected] (S.L. Gue 1 Equally contributed to this study and should thus be considered as co-first authors.

linked. In human, 18 a and 8 b subunits have been identified, which can form 24 distinct aeb heterodimers (Camper et al., 1998; Hynes, 2002; Katz and Yamada, 1997; Sheppard, 2000). They play important functions in a wide range of physiologic and pathologic processes including embryonic development, wound healing, inflammation, and tumorigenesis (Hynes, 1992; Kinashi, 2012; Ramsay et al., 2007; Ruoslahti and Pierschbacher, 1987; Zhang and Wang, 2012) by recognizing diverse ligands in the extracellular matrix or through their interactions with other cells (Alam et al., 2007). Among the 24 integrins described in humans, the a9 subunit is one of the least studied. Discovered in 1993, the a9 subunit was shown to associate only with the b1 subunit (Palmer et al., 1993). Integrin a9b1 is normally expressed in both adult and embryonic tissues and in many cell types such as airway epithelial cells (Weinacker et al., 1995), muscle cells (Hoye et al., 2012; Palmer et al., 1993; Stepp and Zhu, 1997b; Stepp et al., 1995; Yokosaki

http://dx.doi.org/10.1016/j.exer.2015.03.001 0014-4835/© 2015 Elsevier Ltd. All rights reserved.

Please cite this article in press as: Duval, C., et al., Characterization of the human a9 integrin subunit gene: Promoter analysis and transcriptional regulation in ocular cells, Experimental Eye Research (2015), http://dx.doi.org/10.1016/j.exer.2015.03.001

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C. Duval et al. / Experimental Eye Research xxx (2015) 1e18

et al., 1994), neutrophils, hepatocytes (Palmer et al., 1993; Taooka et al., 1999), endothelial cells (Palmer et al., 1993; Staniszewska et al., 2007; Tarui et al., 2001; Vlahakis et al., 2005) and corneal epithelial cells (Stepp et al., 1995). This integrin is also expressed in several tumor tissues and correlates with tumor growth and metastasis (Allen et al., 2011; Gupta et al., 2012; Jones et al., 2006; Majumder et al., 2012; Ramsay et al., 2007). Previous studies have revealed changes in the expression and distribution of corneal and epidermal integrins in wound healing (Haapasalmi et al., 1996; Juhasz et al., 1993; Larjava et al., 1993), notably a9b1, which is not expressed in healthy central corneal epithelia and becomes upregulated during wound repair (Breuss et al., 1995; Haapasalmi et al., 1996; Pajoohesh-Ganji and Stepp, 2005; Pal-Ghosh et al., 2004; Stepp and Zhu, 1997b). Recent studies using conditional knockout of the a9 subunit confirmed that a9 is a critical contributor to the re-epithelialization process (Singh et al., 2009). However, the molecular mechanisms behind this process are not fully understood. The a9 deficiency in mice results in a defective lymphatic development and death due to a respiratory failure shortly after birth (Huang et al., 2000). The a9b1 integrin is widely expressed and it can bind a large number of ligands. A variety of extracellular matrix proteins, including tenascin-C (TNC) (Yokosaki et al., 1998, 1994), fibronectin (Liao et al., 2002; Shinde et al., 2008), osteopontin (Smith et al., 1996; Yokosaki et al., 1999), thrombospondin-1 (Staniszewska et al., 2007), several membrane proteins such as members of ADAMs family (Bridges and Bowditch, 2005; Eto et al., 2002; Lafuste et al., 2005; Tomczuk et al., 2003; Zhang and Wang, 2012), vascular cell adhesion molecule 1 (Taooka et al., 1999), and growth factors (VEGF-A, -C, -D, NGF) (Staniszewska et al., 2008, 2007; Vlahakis et al., 2007) have been reported to interact with integrin a9b1. Yet it remains unclear why such an extended range of ligands can bind to this integrin. In contrast to many integrins that recognize the ArgeGlyeAsp (RGD) sequence (Ruoslahti, 1996), a9b1 does not depend on this common motif (Eto et al., 2000). Some binding sequences in a9b1 ligands have already been identified, such as the EIIIA segment of fibronectin (Bazigou et al., 2009; Liao et al., 2002) and the thrombin cleaved form of osteopontin through the SVVYGLR sequence, which is close to the RGD site (Smith et al., 1996). The wide distribution, the diversity of ligands, the specialized functions and the lethal phenotype of this subunit knockout all suggest that the a9b1 integrin is an essential factor in the living cell. Many studies have shown that a9b1 is important in both physiological and pathological mechanisms, and the many functions of this integrin are now widely investigated. However, and unlike for other integrin subunit genes, the molecular mechanisms underlying expression of the a9 integrin subunit remain unknown and no data that describe the transcriptional regulation of a9b1 at the gene level have been published so far. In this study, we investigated the mechanisms that govern a9 expression in different human cells including corneal epithelial cells as well as uveal melanoma cell lines that express this integrin subunit to different levels. We demonstrated the presence of both positive and negative regulatory elements along the a9 promoter that are important for its transcription and positioned the basal a9 promoter to within 118 bp from the a9 mRNA start site. The regulatory influence mediated by these elements was found to be determined by their interaction with the transcription factors Sp1, NFI and c-Myb both in vitro and in vivo. Most of all, culturing human corneal epithelial cells in the presence of the a9b1 ligand TNC increased a9 transcription but simultaneously decreased expression of both the fibronectin binding integrin subunit a5 and its only ligand fibronectin. These results suggest that very precise regulation of the expression of these receptors must be required to

appropriately modulate mechanisms, such as tissue wound healing, that require adhesion, migration and differentiation of epithelial cells. 2. Materials and methods This study was also conducted in accordance with our institution's guidelines and the Declaration of Helsinki. The protocols were approved by the hospital and the University Committees for the Protection of Human Subjects. 2.1. Cell culture and matrix production Human corneal epithelial cells (HCECs) and human corneal fibroblast cells (HCFCs) were cultured in DME-F12 and fDME, respectively, as recently described (Zaniolo et al., 2013) (refer to Supplementary methods for details). These cells were isolated from normal eyes supplied by the National Eyes Bank from the CHU de bec (QC, Canada) and were cultured in the presence of an Que irradiated mouse Swiss 3T3 (i3T3) fibroblast feeder layer (20,000 i3T3 per cm2 irradiated at 6000 rad) to prevent further proliferation. Human skin fibroblast (HSFCs) and epithelial (keratinocyte) cells (HSECs) were co-cultured along with i3T3 as previously described (Bisson et al., 2014, 2013; Masson-Gadais et al., 2006) (refer to Supplementary methods for details). Human uveal melanocytes (UVM) were isolated and grown in supplemented F12 medium according to a previously published procedure (Hu et al., 1993). The uveal melanoma cell lines (T115, T142, T143) were cultured from the primary tumors of three different patients diagnosed with this type of cancer as described previously (Beliveau et al., 2000) in DMEM (Dulbecco's modified Eagle's medium, Sigma, St. Louis, MO, Canada) supplemented with 10% FetalClone II serum (HYclone) and 0.002% v/v gentamicin (Invitrogen). All cells were grown at 37 C under 8% CO2. The tissue-engineered, 3D human corneal matrix that has been used as a biomaterial on which corneal epithelial cells were cultured was produced following the self-assembly approach (Carrier et al., 2008; Proulx et al., 2010) as recently described (Lake et al., 2013). Briefly, corneal fibroblasts from a 26-year old donor (Fbb26) were seeded and cultured in fibroblast growth medium supplemented with 50 mg/ml ascorbic acid (Sigma) for 35 days. Ascorbic acid allows fibroblasts to secrete and lay down their own ECM (in this case, designated as ECM/Fbb26) (Carrier et al., 2008). 2.2. Plasmid constructs and oligonucleotides A 1.5 kb fragment bearing the 50 flanking sequence of the human a9 gene was synthesized and cloned into the pUC19 vector (Invitrogen) (Codon Devices, Cambridge, MA, USA). Then, a 1.3 kb fragment (positions 1 to 1188 relative to the major mRNA start site identified in the present study) out of the 1.5 kb fragment cloned above was inserted upstream the chloramphenicol acetyltransferase (CAT) reporter gene into the unique SphI site of the pCATbasic vector (Promega, Madison, WI). Unidirectional deletion of this fragment was carried out with exoIII and mungbean Nuclease (Promega, Madison, WI, USA). The pCATbasic/a9-1188 was digested with Bsu36I (50 overhang) and SphI that left a fourbases 30 overhang that protected this end from exonuclease III activity, allowing downstream unidirectional digestion of the a9 promoter from the Bsu36I 50 overhang site. After blunt-ending and ligation, plasmids that bear derivatives from the a9 promoter all shared the same 30 end (at position þ111 relative to the 50 start site identified in this study) but different 50 termini (50 positions: 593, 478, 333, 203, 118, 17). Three additional derivatives were made by digestion with Bsu36I (ITGA9(693)),



SacI (ITGA9(þ13)) and SacII (ITGA9(þ55)). The cDNAs encoding each of the four human NFI isoforms (NFIA, -B, -C and -X) were initially synthesized and cloned into the plasmid pTXB1 (New England Biolab, Ipswich, USA) by Codon Devices Inc. (Cambridge, USA). Each NFI-encoding cDNA was then sub-cloned into the pLenti6V5A vector (a kind gift of Dr François Boudreau, partement d'Anatomie et de Biologie Cellulaire, Faculte de De decine, Universite de Sherbrooke, Sherbrooke, QC, Canada), Me their transcription being ensured by the CMV promoter. The double-stranded oligonucleotides used for the competition assays in the EMSAs contained the high-affinity binding sites for the transcription factors Sp1, Stat-1, AP-1, E2F1, NFI, NF-kB, and AP-2. They were chemically synthesized using a Biosearch 8700 apparatus (Integrated DNA Technologies, Inc., Coralville, IA, USA). Their DNA sequences are listed in Supplementary Table 1. 2.3. Transient transfections and CAT assays HCECs and the T115 uveal melanoma cell line were grown to near 70% confluence. The a9-CAT-recombinant plasmids were then transfected by lipofection using Lipofectamine 2000 (Invitrogen) with T115 cells (this procedure proved to be easier to conduct and yields better transfection efficiencies with this cell line) or by electroporation with the NEON electroporation device (Invitrogen) following the manufacturer's instructions, with HCECs. The pLenti6V5A derivatives were transfected into T115 UM cells by lipofection. Lipofectamine 2000 transfected plates (35 mm) received 1.5 mg of the test plasmid and 0.5 mg of the human growth hormone (hGH) encoding plasmid pXGH5, a kind gift of David D. Moore (Department of Molecular and Cell Biology, Baylor College of Medicine, Houston, TX, USA), whereas electroporation transfected plates received 15 mg and 5 mg of the test and pXGH5 plasmids, respectively. Cells were harvested at 48 h post-transfection and CAT activities were determined and normalized to the amount of hGH secreted into the culture media and assayed using a kit for quanal, Que bec). The titative measurement of hGH (Medicorp, Montre value presented for each test plasmid transfected corresponds to the mean of at least three separate transfections performed in triplicate. Student's t-test was performed for comparison of the groups. Differences were considered statistically significant at P < 0.05. All data are expressed as mean ± SD. 2.4. Nuclear extracts and EMSA (electrophoretic mobility-shift assay) Nuclear extracts were prepared from all cell types cultured to mid-confluence (70% coverage of the culture flasks) and dialyzed against DNaseI buffer [50 mM KCl, 4 mM MgCl2, 20 mM K3PO4 pH 7.4, 1 mM b-mercaptoethanol (Bio-Rad Laboratories, Mississauga, ON, Canada) and 20% (v/v) glycerol], as described in Roy et al. (1991). EMSAs were then conducted as described previously (Gaudreault et al., 2009) by incubating nuclear proteins with a 5’ 32 P-end-labeled HindIII/SmaI, 132 bp DNA fragment bearing most of the basal promoter of the a9 gene from position 117 (HindIII site) to þ12 (SmaI site) and labeled at its HindIII site. Briefly, 5 104 cpm labeled probe was incubated with either 5 or 10 mg of crude nuclear proteins from each type of cells in the presence of 0.5 mg of poly(dIdC)-(dI-dC) (Amersham Biosciences, Piscataway, NJ, USA) and 75 mM KCl in buffer D [10 mM Hepes pH 7.9, 10% v/v glycerol, 0.1 mM EDTA, 0.5 mM DTT (dithiothreitol; SigmaeAldrich Canada, Oakville, ON, Canada) and 0.25 mM phenylmethylsulfonyl fluoride (PMSF; SigmaeAldrich Canada)]. When indicated, the unlabeled 230 bp a9 fragment, or double-stranded oligonucleotides bearing various binding sites for known transcription factors (Supplementary Table 1) were added as competitors (10-, 25- and

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50-fold molar excesses) during the assay. DNAeprotein complexes were next separated by gel electrophoresis through 8% native polyacrylamide gels run against Tris-glycine buffer (50 mM Tris, 2.5 mM EDTA, 0.4 M glycine) at 4 C. Gels were dried and autoradiographed at 80 C to reveal the position of the shifted DNAeprotein complexes generated. 2.5. DNaseI footprinting A 262-bp DNA fragment containing the a9 promoter sequence from position 333 to 593 was 50 -end-labeled and used as a probe in DNaseI footprinting. DNaseI digestion was performed in buffer A (50 mM KCl, 20 mM K3PO4 pH 7.4, 1 mM MgCl2, 1 mM bmercaptoethanol, 20% glycerol) by incubating 3 104 cpm labeled probe with 20 mg of a heparin-Sepharose enriched (Guerin et al., 1993) nuclear extracts prepared from human placentas at term bec, Que bec, (kindly provided by Dr Yves Tremblay, CHU de Que Canada) and known to express a high level of a9 integrin subunit (Tabata et al., 2007). Further analysis of the DNaseI digested products on polyacrylamide sequencing gels was performed as described previously (Robidoux et al., 1992). 2.6. Western blots Sample buffer (63 mM Tris PH8.0, 10% glycerol, 2% SDS, 0.0025% bromophenol blue, 300 nM b-mercaptoethanol) was added directly to cells cultures and a rubber policeman was used to collect the cells. Western blots were conducted as described (Larouche et al., 2000). For detection of the a9 integrin subunit, a mouse monoclonal antibody against human integrin subunit a9 incubated overnight at 4 C (1:1000, ab55395 from Abcam, Cambridge, MA, USA) and a peroxidase-conjugated AffiniPure Goat secondary antibody against mouse IgG (1:2000 dilution; Jackson ImmunoResearch Laboratories Baltimore, PA, USA) were used. The labeling was revealed using ECL Plus Western Blotting Detection Reagents Kit (Amersham). 2.7. Indirect immunofluorescence Biopsies from human corneas were embedded in frozen tissue embedding medium (OCT compound, Tissue-Tek, Bayers Canada, Etobicoke, ON, Canada), frozen in liquid nitrogen, and stored at 70 C until use. Indirect immunofluorescence assays were performed either on tissue cultured cells (HCECs, UVM, T115, T142 and T143) grown on glass coverslips as previously described (Masson-Gadais et al., 2006), or on ethanol-fixed (10 min at 20 C) cryosections (5 mm) with native corneas. Sections were incubated for 45 min with a primary antibody directed against the a9b1 integrin (MAB20782, Chemicon) used at optimal dilution of 1:33 in phosphate-buffered saline (137 mM NaCl, 2.7 mM KCl, 6.5 mM Na2HPO4 and 1.5 mM KH2PO4) containing 1% bovine serum albumin. The conjugated secondary antibody (rabbit anti-mouse IgG (H þ L) conjugated with Alexa-fluor® 488 (1:400; Molecular Probes)) was incubated for 30 min. Cell nuclei were also labeled with Hoechst reagent 33258 (1:100; Sigma Chemicals) following immunofluorescence staining. Tissue samples were then observed with an epifluorescence microscope (Eclipse E600; Nikon). They were photographed with a numeric CCD camera (AxioVision). Negligible background was observed for controls (primary antibodies omitted). 2.8. Gene expression profiling All microarray analyses were conducted by the gene profiling service of the molecular genetic platform (Genetiquemoleculaire.


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bec, QC, Canada). Briefly, com) from the CUO-Recherche (Que cyanine 3-CTP labeled cRNA targets were prepared from 25 ng of total RNA isolated from all types of cells using the Agilent OneColor Microarray-Based Gene Expression Analysis kit (Agilent Technologies Canada Inc., Mississauga, ON, Canada). Then, 600 ng cRNA were incubated on a G4851A SurePrint G3 Human GE 8 60 K array slide (60,000 probes, Agilent Technologies). Slides were then washed, stained and scanned on an Agilent SureScan Scanner and data further analyzed using the ArrayStar V4.1 (DNASTAR, Madison, WI) software for scatter plots and generation of the heat maps of selected genes of interest as recently described (Landreville et al., 2011; Molloy-Simard et al., 2012). All data generated from the array were also analyzed by RMA (‘Robust Multiarray Analysis’) for background correction of the raw values. They were then transformed in log2 base and quantile normalized before a linear model was fitted to the normalized data in order to obtain an expression measure for each probe set. The number of replicate samples for HCECs, HSECs, HCFCs, HSFCs, UVM, T115, T142 and T143 were respectively 8, 4, 3, 3, 3, 3, 2 and 2. All microarray data presented in this study comply with the Minimum Information About a Microarray Experiment (MIAME) requirements. The gene expression data have been deposited in NCBIs Gene Expression Omnibus (GEO, http://www.ncbi.nlm.nih.gov/geo/) and are accessible through GEO Series accession number GSE 62075. 2.9. Chromatin immunoprecipitation assays (ChIP) ChIP analyses were conducted using the Zymo-Spin™ ChIP kit (Zymo Research, Irvine, CA, USA) on T115 and T143 cells grown to 80% confluence and chromatin immunoprecipitated with 1 mg (5 mg were used for Sp1) antibodies against the transcription factors cMyb, Sp1 and NFI as previously reported (Gaudreault et al., 2007; Ouellet et al., 2006). Incubation was also performed with a mouse antibody against IgG2a Fc (Chemicon, Temecula, CA) as a negative control. The resulting DNA was analyzed by PCR using pairs of primers spanning the a9 gene promoter region that also bears the FP1 and FP2 elements from position 585 to 451 (ITGA9/FPs/F and ITGA9/FPs/R; Supplementary Table 1) and the basal a9 promoter from position 121 to þ14 (ITGA9/basal/F and ITGA9/basal/ R; Supplementary Table 1). As a negative control, each ChIP sample was also subjected to PCR using primers (p21-F and p21-R; Supplementary Table 1) specific to a region located ~2 Kbp upstream from the human p21 promoter. Cycle parameters were the following: denaturation 90 C, 10 s; annealing 70 C, 30s, with 30 cycles. 2.10. Quantitative PCR (qRT-PCR) Quantity and quality of total RNA from UVM, HCEC, T115, T142 and T143 cells was assessed using an Agilent Technologies 2100 bioanalyzer and RNA 6000 Nano LabChip kit (Agilent Technologies). RNAs were used for qPCR and gene profiling analyses only if their RNA integrity number (RIN) was greater than 7 over 10. Reverse transcription was performed using High Capacity cDNA Reverse Transcription Kit random hexamer primers following the manufacturer's protocol for synthesis the first strand cDNA (AB applied biosystems, Foster City, CA, USA). Equal amounts of cDNA were run in quadruplicate and amplified in a 20 ml reaction containing 10 ml of 2 Brillant III Ultra-Fast SYBR Green QPCR Master Mix (Agilent Technologies), 250 nM of upstream and downstream primers, and 25 ng (2.5 ng was used for HCECs grown on TNC) of cDNA target. No-template controls were also used as recommended. The mixture was incubated at 95 C for 3 min, and then cycled at 95 C for 10 s and at 60 C for 20 s 40 cycles using the QIAGEN Rotor-Gene Q real-time cycler. Amplification efficiencies were validated and

normalized to the actin mRNA transcript and quantity of target genes were calculated according to a standard curve. The primers were designed using Primer3 (v.0.4.0) and are listed in Supplementary Table 1. 2.11. Primer extension 32 A P 50 -end-labeled oligonucleotide (50 -CATCCC0 CAGCCGGCCGC-3 ) was used for extension by AMV reverse transcriptase (Promega) with poly(Aþ) RNA (50 mg) isolated from UVM, HCEC and HCFC cells essentially as described (Lachance et al., 1990). The extended products were analyzed by electrophoresis on a 8% polyacrylamide sequencing gel. Autoradiography was performed using Kodak XAR-5 films in the presence of intensifying screens at 80 C.

2.12. Lentivirus production and cell transduction The details regarding the production of lentiviruses that overexpress Sp1 shRNAs are provided in Supplementary methods. Briefly, HSEC and HaCaT cells were plated in 6-well plates at a density of 350,000 cells per well and incubated overnight at 37 C. The medium was removed and 700 ml of the virus suspension was added to the cells (overnight at 37 C) along with 1.3 mL of fresh medium and Hexadimethrine bromide (Polybren) (Sigma) at a final concentration of 4 mg/mL. When the plenti6/V5-U6 lentiviral vector was selected for the transduction (in HaCaT cells) blasticidine (Sigma) was added to the culture medium at a concentration of 10 mg/ml 48 h following virus transduction and cells were allowed to grow for an additional 48 h. Expression and DNA binding of Sp1 was finally determined in the blasticidin-resistant HaCaT cell population. 2.13. Statistical analyses Student's t-test was performed for comparison of the groups in both transfection and qRT-PCR analyses. Differences were considered to be statistically significant at P < 0.05. All data are also expressed as mean ± SD. 3. Results 3.1. Expression of the a9 gene in human ocular cells Gene profiling on microarrays was first exploited in order to monitor a9 gene expression in a variety of tissue cultured human cells. We selected both primary cultured, normal cells (HCECs, HSECs, HCFCs, HSFCs and UVM) as well as a few cancer cell lines. As we have in hand a large array of uveal melanoma cell lines, we selected some (T115, T142, T143) that we previously characterized in detail in other published works (Landreville et al., 2011; MolloySimard et al., 2012; Mouriaux et al., 2015). These included primary cultures of corneal epithelial cells (HCECs), corneal fibroblast cells (HCFCs), uveal melanocytes (UVM), and a number of uveal melanoma (UM) cell lines cultured from the primary tumors of patients diagnosed with this type of cancer (T115, T142, T143). Expression of a9 was also monitored in skin epithelial cells (HSECs) and skin fibroblast cells (HSFCs) for comparison purpose. Examination of the pattern of alpha integrin subunit genes expressed by these cells revealed that they all transcribed low to moderate levels of the a9 gene (linear signals normalized to the internal control b2microglobulin (B2M) ranging from 0.0025 to 0.0398; Fig. 1A and B). The highest normalized level of a9 expression was observed in UM cell lines T115 (ratio of normalized signal (RNS): 0.0398 ± 0.0243), T143 (RNS: 0.0171 ± 0.0099) and UVM (RNS:



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Fig. 1. Expression of the a9b1 integrin in primary cultured cells and UM cancer cells. A) Heatmap representation of all a integrin subunit genes expressed by primary cultured cells (HCECs, HSECs, HCFCs, HSFCs and UVM) and uveal melanoma cell lines (T115, T142 and T143). The color scale used to display the log2 expression level values is determined by the Hierarchical clustering algorithm of the Euclidian metric distance between genes. Genes indicated in dark blue correspond to those whose expression is very low whereas highly expressed genes are shown in orange/red. B) Ratio of the a9 linear signals normalized to those of B2M. C) qPCR analysis of a9 expression in cells used on Panel A. Data are presented as the ratio of a9 mRNA copy number over that of the GAPDH. Standard deviation is provided. N.D.: not detectable. D) Western blot analysis of a9 expression in UVMs, HCECs, T115, T142 and T143 cells. Actin expression was monitored as a normalization control. E) Immunofluorescence analysis of a9b1 expression (in green) in primary cultured UVM and HCECs and uveal melanoma T115, T142 and T143 cells (left column). Expression of a9b1 was also monitored in native human cornea (bottom panel). Insets: negative controls in which the primary antibody was omitted. Nuclei were counterstained with Hoechst 33258 reagent and appear in blue. E: epithelium; S: stroma. Scale bar: 20 mM. Right column: phase contrast micrographs of the cells used for immunofluorescence analyses. Scale bar: 50 mM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

0.0100 ± 0.0029) whereas the lowest levels were observed in primary cultures of HCECs (RNS: 0.0023 ± 0.0056), HCFCs (RNS: 0.0032 ± 0.0019) and HSFCs (RNS: 0.0025 ± 0.0003). In comparison, all epithelial cells (corneal and skin) expressed very high levels of the av (average RNS: 0.1989 ± 0.0806), a3 (average RNS: 0.1476 ± 0.1061) and a6 (average RNS: 0.2207 ± 0.1966) subunits whereas fibroblasts (both corneal and skin) had high levels of av (average RNS: 0.1097 ± 0.0076) and a11 (average RNS: 0.2265 ± 0.0773). On the other hand, only very low levels of the leukocyte integrins aD (average RNS: 0.00018 ± 0.00016) and aL (average RNS: 0.00024 ± 0.00025) were found to be expressed by all these cells, which is consistent with the inability to detect

significant levels of these integrin subunits in tissues other than the cells from the immune system (Lim and Hotchin, 2012; Mabon et al., 2000). The variations in a9 expression observed by microarrays between primary cultured cells and UM cell lines were also validated by qPCR (Fig. 1C). Both Western blot (Fig. 1D) and indirect immunofluorescence (Fig. 1E) analyses confirmed the expression of the a9 integrin subunit, although to different levels, in UVM, HCECs, T115, T142 and T143 cells, as well as in the epithelial cells from the limbal area of native, human corneas (Fig. 1E, lower panel), therefore validating the data from the gene expression profiling on microarrays. There are discrepancies between expression level of the a9 mRNA (which is clearly lower in HCECs than in T115 cells;


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Fig. 1AeC) and its encoded a9 protein (higher in HCECs than in T115 cells; Fig. 1D). Many different mechanisms may be raised to explain why expression of any given protein does not match that of its corresponding mRNA transcript as both mRNA translation and the protein translated from the mRNA are known to be heavily regulated in any given cell by mechanisms, such as glycosylation, phosphorylation, ubiquitinylation, that will in turn alter the halflife of that protein. Meanwhile, the differences noted in the intensity of immunofluorescence labeling between T115, T142 and T143 in the IF but not in Western blot may simply rely on the possibility that a9 receptor internalization is greater in T142 than in T115 and T143 cells, despite a similar level of protein expression as suggested from the Western blot data. 3.2. Mapping of the a9 transcription initiation site and analysis of the 50 -flanking region As no previous study ever reported the identification of the a9 mRNA start site, we next used a 17-nucleotide primer complementary to a region from the a9 mRNA that overlaps the first ATG (from position 14 to þ3 relative to the ATG) to conduct primer extension analyses on RNAs isolated from primary cultures of UVM, HCECs and HCFCs in order to position the major a9 transcription initiation site. The results presented in Fig. 2A indicate that both UVM and HCECs have a major transcription start site located 38 nucleotides upstream from the ATG initiating codon (38) and a minor site at position 32. However, the major mRNA start site is located further upstream at position 112 in HCFCs (Fig. 2B and Supplementary Fig. 1). Besides this predominant 112 site, a number of minor, alternative sites also appeared to be used by HCFCs to transcribe the a9 gene, including the 32 site identified in UVM and HCECs. Because it is clearly the most potent and 50 -upstream located site, we recognized the 112 position as the major a9 mRNA start site and used it as a reference for the remaining of this study. In order to identify putative target sites for transcription factors (TFs) that may be important for expression of the a9 gene in cultured human cells, we subjected a segment from the a9 gene extending up to approximately 1.2 Kbp upstream from the a9 mRNA start site to a search with the TFSEARCH program. Target sites for 17 different TFs that can potentially bind the a9 promoter were therefore identified using this program (Fig. 3A). Among them, multiple target sites for the members of the Sp1, C/EBP, CREB, AP-2, NFI and p300 transcription factors were identified close to the a9 mRNA start site. Members from the NFI family are of a particular interest in that they have been reported to function either as transcriptional activators or repressors of many genes, including integrin genes (Gaudreault et al., 2008; Gingras et al., 2009; Laniel et al., 2001). We next examined the pattern of expression for each of these TFs in the different cell types assayed (HCECs, HSECs, HCFCs, HSFCs, UVM, T115, T142 and T143 cells) that also express the a9 gene to different levels by searching the microarray data files used for generating Fig. 1. As shown on Fig. 3B, some of these TFs, such as NFIC, c-Ets 1, CREB-5, AP-2b, AP-2d, AP-2ε, C\EBPε, Sox-5 and GATA-1 are either not expressed or only barely detectable in all the cell types examined and are thus not worth paying them too much attention. The low a9 expressing cells (HCECs, HCFCs, HSFCs) distinguish themselves from those that moderately express that gene (UVM, T115, T142 and T143 cells) by their lower level of expression of the positive TFs Sp1/Sp3, the AP-1 constituting subunit JunD and reduced levels of NF-kB2 therefore suggesting that these TFs might represent important positive regulators of a9 expression. On the other hand, expression of the TFs NFIA, the AP-1 constituting subunits Fra-1 and Fra-2, p300, c-Ets 2, AP-2g, C/EBPd, c-Myc and RUNX-1 is more elevated in low a9 expressing cells

suggesting that they might act as negative regulators of this gene in these cells. 3.3. Basal a9 gene transcription is ensured by the 17/118 region of the a9 promoter To precisely delineate the position of the regulatory elements required for ensuring basal expression of the a9 gene, recombinant plasmids bearing the CAT reporter gene fused to different segments from the a9 gene promoter and 50 -flanking sequences (Fig. 4A) were transfected into HCECs and T115 cells that, based on the microarray (Fig. 1A) and qPCR analyses (Fig. 1C) express low and moderate levels of a9, respectively. Transfection of the plasmids ITGA9(þ55), ITGA9(þ13) and ITGA9(17) that contain the a9 promoter sequence from 30 position þ111 to 50 positions þ55, þ13 and 17 relative to the transcriptional start site yielded very low, barely detectable CAT activities upon transfection of T115 cells (Fig. 4B). However, extending the a9 promoter to position 118 (in plasmid ITGA9(118)) caused a dramatic increase in CAT activity indicating clearly that basal a9 promoter activity is ensured by the sequences located between positions 17 to 118. Examination of the 17/118 promoter segment indicates that it bears at least five high affinity binding sites for Sp1 and a putative site for NFI (Supplementary Fig. 1). Extending further the a9 promoter by 85 bp to position 203 (in ITGA9(203)) strongly repressed basal promoter activity (Fig. 4B). This repression was partly released with plasmid ITGA9(333). A very similar pattern of alternative repression/activation directed by the a9 promoter was also observed in HCECs (Fig. 4C). Consistent with the data from the microarrays, all transfected plasmids yielded CAT activities much lower in HCECs (peak CAT activity yielded by plasmid ITGA9(118) of 46 ± 34 in HCECs compared to 711 ± 377 in T115 cells), a 15-fold difference consistent with that observed between HCECs and T115 by microarray analyses (17-fold difference; Fig. 1). Interestingly, ITGA9(1188) that also bears the most extended a9 promoter, directed a CAT activity (228 ± 138) that was approximately 3-fold lower than that encoded by the basal a9 promoter bearing plasmid ITGA9(118) in a9-expressing T115 cells (711 ± 377). On the other hand, CAT activity directed by ITGA9(1188) was more than 17-fold lower than that of ITGA9(118) when transfections were conducted in HCECs, which is consistent with the lower level of a9 expression observed in HCECs by gene profiling and qPCR (Fig. 1B and C). Collectively, these results indicate that multiple positive regulatory sequences are present on the a9 promoter fragments extending from positions 17 to 118 and 203 to 333 whereas potent repressor elements are present between positions 118 to 203 and 693 to 1188. They also suggest that regulatory elements required to ensure cell-specific expression of a9 are present between positions 333 and 1188. The regions from the a9 promoter with the most potent repressor activity are located between positions 118/203 and 478/593. Examination of the 118/203 sequence with TFSEARCH identified only a single putative binding site for the positive TF Sp1 (Supplementary Fig. 1). However, subjecting the 478/593 to a similar search identified putative target sites for c-Myc, GATA, Sox-5, c-Myb and NFI (Supplementary Fig. 1). In order to demonstrate whether any of these TFs is interacting with this upstream segment from the a9 promoter, we 50 -end labeled a 262-bp DNA fragment containing the sequence from position 333 to 593 and used it as a probe in DNaseI footprinting. As we could not obtain enriched nuclear extracts in sufficient amounts from HCECs and T115 monolayer cells to conduct these experiments, we turned our attention toward the use of human placenta as a source of nuclear proteins (kindly provided by Dr. Yves Tremblay from the bec, Que bec, QC, Canada), this tissue being CHU de Que


G A T C

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G C C G T C G G C G G G C C C C G C G G C C C G C G C G C T C G C G C C C T G C T C G C C G G C

3’

Fig. 2. Primer extension analysis of the a9 gene transcriptional start site. PolyAþ RNAs isolated from primary cultures of UVM, HCECs (panel A) and HCFCs (panel B) were primerextended using a 32P-50 -end-labeled oligonucleotide extending from position 14 to þ3 relative to the ATG. Lanes G, A, T and C represent the sequence of the template clone using the same primer. The position of the most selected start site is indicated as þ1 for both UVM, HCECs (panel A) and HCFCs (panel B) and is positioned on the a9 sequence relative to the first ATG. Minor, alternative start sites (ASS) are also indicated. Numbers in parentheses indicate the position of the start sites relative to the ATG.

demonstrated to express the a9b1 integrin (Tabata et al., 2007). Incubation of the labeled probe with nuclear proteins from human placenta identified two areas from the a9 promoter that are partially protected from digestion by DNAseI (Fig. 5A and B). Target sites for GATA and c-Myb are present in the first of these protected sites (identified as FP1 and located from position 529 to 555) whereas no typical transcription factor target site could be identified in the second protected site (designated FP2 and located from position 557 to 583; Supplementary Fig. 1) besides a binding site for NFI that is found nearby the 50 end of the FP2 site. To verify whether c-Myb and/or NFI proteins interact in vivo with the area from the a9 promoter that bears the FP1 and FP2 sites (GATA genes were excluded as they are not expressed to significant levels in any of the cell types examined), chromatin immunoprecipitation (ChIP) assays were conducted using antibodies directed against each of these TFs. ChIP was also conducted using an antibody against Sp1 as target sites for this transcription factor are present nearby the FP1 and FP2 elements. As shown on Fig. 5C, antibodies against these transcription factors all enriched the a9 promoter sequence containing both the FP1 and FP2 regions to very identical levels in T115 cells (TF/input ratios of 2.4-, 2.6- and 3.1 for c-Myb, Sp1 and NFI, respectively) indicating that they are bound to this genomic area

in vivo. When ChIP was conducted on T143 cells that express higher levels of a9 than T115 cells (Fig. 1), all three proteins again enriched the a9 promoter, but with signal intensities different from those yielded by T115 cells. Indeed, normalization of the PCR amplification products resulting from the a9 promoter segment immunoprecipitated by each antibody to the input chromatin control indicated a clear reduction in the binding of c-Myb and Sp1 (TF/ input ratios of 0.4 and 1.4, respectively), whereas binding of NFI was clearly the predominant event occurring in this region of the a9 promoter in T143 cells (TF/input ratio: 3.2; Fig. 5C). In contrast, none of these antibodies could enrich a region located ~2 Kbp upstream from the p21 promoter that is used as a negative control for the ChIP assay (Ouellet et al., 2006). Western blot analyses confirmed the expression of Sp1, c-Myb and NFI in both T115 and T143 UM cells although clearly visible changes in the electrophoretic mobility of both c-Myb and NFI that may also be related to their altered pattern of promoter occupancy, are observed in T143 cells (both these transcription factors have been shown to be subjected to post-translational modifications, such as phosphorylation, ubiquitination or glycosylation (Duval et al., 2012; Feikova et al., 2000; Kanei-Ishii et al., 2004), which may explain the change in their pattern of migration on gel) (Fig. 5D). Consistent with the very


8


A.

-1188

-1000

-750

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c-Ets Sox-5 p300 p300 c-Myc Oct-1 NFI NFI AP-2 NFI GATA 1-2 c-Myb p300 GATA 1-2 Nkx-2.5 Nkx-2.5 C/EBP c-Myb AP-1 AP-1 C/EBP RUNX-1 C/EBP Nkx-2.5 c-Myb GATA 1-2 GATA 1-2 c-Ets Sox-5 CREB Sp1 AP-1 p300 GATA 1-2 Sp1 GATA 1-2 GATA 1-2 Oct-1 AP-1 c-Ets Sp1 Nkx-2.5

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c-Ets AP-2 NF-κB Sp1 NFI Sp1 Sp1 Sp1 NFI AP-2 Sp1 Sp1 AP-2 Sp1 Sp1 NFI

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T143 α9 Sp1 Sp3 NFIA NFIB NFIC NFIX c-Jun c-Fos FosB Fra-1 Fra-2 JunB JunD p300 c-Ets 1 c-Ets 2 NF-κB1 NF-κB2 CREB-1 CREB-3 CREB-5 AP-2α AP-2β AP-2δ AP-2γ AP-2ε C/EBPα C/EBPδ C/EBPε C/EBPγ c-Myc c-Myb Oct-1 RUNX-1 Sox-5 GATA-1 GATA-2 NKX2.5 +2,7

+15,6

B2M GOLGA1 Fig. 3. Putative transcription factor binding sites along the a9 promoter. Schematic representation of the human a9 promoter and 50 -flanking sequence. Potential binding sites for a variety of transcription factors (Sp1, AP-1, AP-2, NF-kB, CREB, NFI, etc …) are indicated, along with the position of the transcriptional start site (þ1). Negative values are positions relative to the mRNA start site. B) Heatmap representation of the transcriptional profiles of all the TFs for which a putative target site was identified in panel A. Microarray data for the housekeeping genes b2-microglobulin (B2M) and golgin subfamily A member 1 (GOLGA1) that are expressed respectively to very high and low levels in all types of cells are also shown. The data presented were extracted from the microarray data files used for generating Fig. 1.

low level of a9 expression noted in HCECs, no c-Myb and only very low expression of Sp1 could be observed at the protein level in these primary cultured cells whereas they express low but detectable amounts of NFI. 3.4. The transcription factors Sp1, Sp3 and NFI bind the a9 basal promoter Search for putative TF binding sites using the TFSEARCH program identified target sites for five distinct TFs in the a9 basal promoter (up till position 150): Sp1 (8 sites), NFI (4 sites), AP-2 (3

sites), NF-kB (1 site) and c-Ets (1 site) (Fig. 3A and Supplementary Fig. 1). We therefore conducted competition and supershift experiments in EMSA to determine which of these TFs bind the basal a9 promoter in vitro. Incubation of a 50 end-labeled DNA probe covering the entire basal a9 promoter from position 118 to þ13 with nuclear proteins from HCECs revealed the formation of only two DNAeprotein complexes of low electrophoretic mobility (Fig. 6A, lane 2). The formation of both complexes was entirely prevented when an unlabeled, double-stranded competitor oligonucleotide bearing the high affinity binding site for the TF Sp1 was added (Fig. 6A, compare lane 3 with lane 2) but not when using a



9

Fig. 4. Transfection of the a9 gene promoter in T115 cells and HCECs. A) Schematic representation of recombinant constructs bearing the CAT reporter gene fused to various segments from the human a9 gene promoter. Numbers indicate position relative to the a9 mRNA start site (indicated by a curved arrow). B and C) CAT activities measured following transfection of the a9 constructs shown in panel A in the uveal melanoma cell lines T115 (panel B) and HCECs (panel C).

similar oligomer bearing the sequence of the NFI site (Fig. 6A, lane 4). Addition of a polyclonal antibody directed against the TF Sp1 almost entirely supershifted the most intense DNAeprotein complex (SSC; Fig. 6B, lane 7) but had no influence on the weakest, faster migrating complex, whose formation was, however, supershifted by the addition of an antibody against the TF Sp3 (which recognizes the same target sequences as Sp1) (Fig. 6B, lane 8). The simultaneous addition of the Sp1 and Sp3 antibodies completely supershifted both the strong and weak DNAeprotein complexes (Fig. 6B, lane 9) whereas neither an antibody against NFI nor NF-kB had any influence on their formation (Fig. 6B, lanes 10 and 11). That there is no residual DNAeprotein complex visible when both Sp1 and Sp3 are competed in vitro (Fig. 6A, lane 3) is a clear indication that Sp1/Sp3 binding predominates over the binding of any of the other putative TFs that have target sites in the basal a9 promoter despite the fact that they are all expressed by HCECs (Fig. 3B). As there are four putative target sites for the members of the NFI

family of TFs in the basal a9 promoter, we wished to verify if NFI proteins could indeed interact in vitro with any of these sites. We therefore incubated the 118/þ13 a9 promoter labeled probe with a Carboxymethyl-(CM)-Sepharose enriched preparation of NFI. As shown on Fig. 6C (lane 13), incubation of the enriched preparation of NFI with the a9 promoter labeled probe yielded a single, intense DNAeprotein complex in EMSA. The formation of this complex was entirely prevented when an unlabeled, double-stranded competitor oligonucleotide bearing the high affinity binding site for NFI was added (Fig. 6C, compare lane 14 with lane 13) but not by a similar oligomer bearing the sequence of the Sp1 site (Fig. 6C, lane 15). ChIP analyses conducted on both T115 and T143 cells confirmed the occupancy of the basal a9 promoter by both Sp1 and NFI in vivo (the more diffused and weaker signals resulted from the difficulty in conducting qPCR on the immunoprecipitated genomic DNA that covers the basal a9 promoter primarily due to the very high content in G-C residues present in the basal a9 promoter) (Fig. 6D). Among


10


A.

C

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G G G T C A A C A C T C T A T T C G A C A T C C T C G

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Fig. 5. In vitro DNaseI footprinting and in vivo ChIP analysis of the a9 gene distal promoter region. A) A labeled probe bearing the 333 to 593 distal region from the a9 gene promoter was incubated with 20 mg heparin-sepharose enriched nuclear proteins from human placenta and subjected to DNaseI digestion (P). Two distinct sites (FP1 and FP2) protected from DNaseI digestion are indicated and precisely positioned along the a9 promoter sequence used as a probe. C, labeled probe subjected to DNaseI digestion without addition of nuclear proteins; G, Maxam and Gilbert G sequencing ladder. B) Same reactions as on panel A but ran for 5 h (rather than 2.5 h) on the sequencing gel in order to obtain a better separation on the FP1 protected site. The DNaseI digestion products whose formation is reduced by the addition of nuclear proteins are indicated by a black dot. C) In vivo ChIP analysis of c-Myb, Sp1 and NFI binding to the area from the a9 promoter that spans both the FP1 and FP2 DNaseI protected sites (ITGA9 FP1/FP2 region) and conducted in T115 and T143 cells. IgG: negative control that uses a mouse antibody against IgG2a Fc; Neg: qPCR reaction conducted in the absence of DNA. D) Western blot analysis of c-Myb, Sp1 and NFI expression in HCECs, T115 and T143 cells. Actin expression was monitored as a normalization control.

all the cell types examined in this study, T115 cells are those that also express the lowest levels of NFI transcripts (refer to Fig. 3B). We therefore transfected T115 cells with the plasmid ITGA9(118) along with derivatives of pLenti6V5A that express each of the human NFI isoforms to determine whether NFI proteins truly act as repressors of a9 basal promoter activity. As a negative control, T115 cells were also co-transfected with empty pLenti6V5A. As indicated on Fig. 6E, co-expression of each of the NFI isoforms caused a significant reduction in the promoter activity driven by the a9 basal promoter, the most potent repression being observed with NFIC and NFIX (2.6- and 2.7-fold repression, respectively relative to T115

cells transfected with the empty vector). Recently, we transduced both primary cultured HSECs and the immortalized human keratinocyte cell line HaCaT (Schurer et al., 1993) with lentiviruses (either plenti6/V5-U6 (pLenti) or pNLSIN-GFP (pNL) derivatives) encoding different shRNAs (Sp1-1 to Sp1-4; see Supplementary Table 1) directed against human Sp1 in order to investigate whether this transcription factor contributes to the expression of the gene encoding the catalytic subunit of the human telomerase (hTERT) (Bisson et al., 2014). Both the pLenti and pNL-shSp1 expressing lentiviruses were then found to be very effective at suppressing expression of Sp1 in these cells, as revealed


P

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HSEC pNL CTLpNL shSp1-3 pNL shSp1-4

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(copy number α9 /GAPDH)

(copy number α9 /GAPDH)

Fig. 6. EMSA and ChIP analyses of Sp1 and NFI binding to the a9 basal promoter. A) A end-labeled probe covering the basal a9 gene promoter from position 118 to þ13 was incubated with nuclear proteins from HCECs in either the absence (HCEC) or presence of a 10-fold molar excess of double-stranded, unlabeled oligonucleotides bearing the high affinity binding sites for Sp1 and NFI. Formation of DNA/protein complexes was then monitored by EMSA on a 8% native polyacrylamide gel. The position of the Sp1 and Sp3 DNAeprotein complexes is indicated along with that of the free probe (U). B) The 118/þ13 labeled probe used in panel A was incubated with nuclear proteins from HCECs in the presence of antibodies directed against the TFs Sp1, Sp3, NF-kB and NFI. Formation of DNA/protein complexes was then monitored by EMSA as in panel A. The position of the Sp1 and Sp3 DNAeprotein complexes is indicated along with that of supershifted complexes (SSC) and the free probe (U). P: labeled probe with no proteins added. C) The 118/þ13 labeled probe used in panel A was incubated with a CM-Sepharose enriched preparation of NFI in either the absence (CM sep) or presence of a 10-fold molar excess of unlabeled NFI or Sp1 oligonucleotides. The position of the NFI complex is indicated as well as that of the free probe (U). P: Labeled probe with no proteins added. D) In vivo ChIP analysis of Sp1 and NFI binding to the area from the a9 gene that also spans the basal a9 promoter (ITGA9 118/þ13 region) and conducted in T115 and T143 cells. IgG: negative control that uses a mouse antibody against IgG2a Fc; Neg: qPCR reaction conducted in the absence of DNA. E) The plasmid ITGA9(118) was co-transfected in HCECs and T115 cells either with the empty pLentiU5V6 vector (empty) or with pLentiU5V6 derivatives that encode high levels of each of the four NFI isoforms (NFIA, -B, -C and -X). CAT activity is expressed as (%CAT/ 4 h/100 mg proteins)/ng hGH. F) Influence of suppressing Sp1 on a9 gene expression was monitored by qPCR in both HSECs and HaCaT cells transduced with recombinant lentiviruses (pNL shSp1-1 and pNL shSp1-3 in HSECs; pLenti shSp1-1 and pLenti shSp1-2 in HaCaT cells) that each express a shRNA against the Sp1 mRNA transcript. Cells were also transduced either with an empty lentivirus (with pLenti CTL-) or with a lentivirus that encodes a shRNA against the luciferase gene (in pNL CTL-) as negative controls. Data are presented as the ratio of a9 mRNA copy number over that of the GAPDH. *: Values considered to be statistically significant from those obtained with the negative controls pNL CTLand pLenti CTL- (P value < 0.001). 50

by EMSA and Western blot analyses (Bisson et al., 2014). To demonstrate the contribution of Sp1 to the expression of a9, we used these Sp1-suppressed HSECs and HaCaT cells and examined the a9 mRNA transcript by qPCR. Total RNA was isolated from HSECs and HaCaT cells transduced either with a negative control

(empty lentivirus (with pLenti CTL-) or a lentivirus that encodes a shRNA against the luciferase gene (in PNL CTL-)) or with lentiviruses that express various shRNAs directed toward the Sp1 transcript (Sp1-1 to Sp1-4). As shown on Fig. 6F, a marked reduction in the expression of the a9 mRNA transcript was observed in both


12


Fig. 7. Influence of TNC on the expression of a9 and the growth properties of HCECs. A) Scatter plots of log2 of signal intensity from 60,000 different targets covering the entire human transcriptome of HCECs grown on BSA (y-axis) plotted against HCECs grown on tenascin-C (TNC; x-axis) at confluence. B) Heatmap representation of all a and b integrin subunit genes expressed by HCECs cultured from the eyes of two different donors of 44- (HCEC44y) and 52-years old (HCEC52y) and grown to confluence on BSA or TNC. C, D) qPCR (panel C) and Western blot analysis (panel D) of both a5 and a9 expression in HCECs grown on BSA or TNC. qPCR data are presented as the ratio of a9 mRNA copy number over that of the GAPDH. Actin expression was monitored as a normalization control for the Western blot analysis. E) Phase-contrast images of HCECs seeded at 3.5 104 cells/cm2 on BSA, FN



HSECs and HaCaT cells when they are transduced with any of the shSp1-encoding lentiviruses (data are compared to cells transduced with the empty lentiviral vectors as negative controls (pNL CTL- and pLenti CTL-)). Consistent with our previous results, the Sp1 shRNA that turned out to be the most effective in HaCaT cells was that bearing the Sp1-2 sequence, which caused a 20-fold reduction in the amount of a9 transcript (Fig. 6F). With a dramatic reduction of 32,300-fold, the shSp1-1 shRNA proved particularly effective at suppressing a9 gene transcription in HSECs. Taken together, these results demonstrate the critical function played by Sp1 in the basal transcription of the a9 gene. 3.5. Expression of the a9 gene is influenced by tenascin-C in corneal epithelial cells We previously demonstrated that ECM components such as fibronectin and laminin, profoundly influence, either positively or negatively, the transcription directed by the promoter of various integrin subunit genes in vitro (Gaudreault et al., 2007; Gingras et al., 2009; Vigneault et al., 2007). However, no such analysis has ever been conducted on TNC, the primary ligand of the a9b1 integrin. We therefore examined whether expression of the a9 subunit gene changes between HCECs grown on BSA or on tissue culture plates coated with TNC by gene profiling on microarray. A scatter plot analysis of the 60,000 different transcripts contained on the arrays indicated clearly that HCECs grown on TNC-coated plates have patterns of expressed genes very distinctive from those yielded by HCECs grown solely on BSA as revealed by the dispersion of the normalized signals that appear as a cloud of dots on Fig. 7A. A total of 735 genes have a 2-fold or more expression variation unique to HCECs grown on BSA paired against the expression profile of HCECs grown on TNC. Clustering of the in vivo microarray data for all a-integrin genes in HCECs grown at confluence on BSA or on TNC into a heatmap revealed that expression of a9 increases when HCECs are grown on TNC (1.8-fold increase) whereas that of a5 (and also a2) decreases (2.2-fold reduction) (Fig. 7B; two different cultures of HCECs (HCEC 44y and 52y) were examined by microarray). Apart from the b2 subunit, no comparable alteration was observed for the expression of the b integrin's subunits (Fig. 7B, bottom). The increased and reduced expression noted respectively for the a9 and a5 genes when HCECs are grown on TNC were further validated by qPCR at the transcriptional level (Fig. 7C). However, the alterations observed at the transcriptional level did not translate into corresponding changes at the protein level as no significant alteration was observed for both a9 and a5 in Western blot analyses between HCECs grown on BSA or TNC for these integrin subunits (Fig. 7D). Basal epithelial cells from the central cornea normally rest on a basement membrane essentially made out of collagen type IV and LM that contains very little FN and no TNC, which are however produced only when the corneal surface is damaged (Kang et al., 1999; Murakami et al., 1992; Stepp and Zhu, 1997b). We have shown that ECM components such as FN, LM and collagen type IV profoundly influence the growth properties of rabbit corneal epithelial cells (Stepp and Zhu, 1997b). We therefore examined whether TNC also alter the adhesive and migratory properties of HCECs when seeded on culture plates coated with this ECM component. As a positive control, HCECs were also plated on FNcoated culture plates or on tissue-engineered corneal stromas

13

produced when stromal fibroblasts from human corneas are grown in the presence of ascorbic acid such as described previously (Lake et al., 2013) (ECM/Fbb26). As shown on Fig. 7E, both FN and ECM/ Fbb26 considerably improved proliferation of HCECs over the 7-day culture period relative to cells grown solely on BSA (HCECs grown on FN and ECM/Fbb26 covered approximately 85% and 95%, respectively, of the culture wells when seeded at 3.5 104 cells/cm2 whereas they cover approximately 50% of the culture wells on BSA). Although all cells attached and spread on TNC 7 days after plating, they covered only 50- to 55% of the culture well (that is identical to HCECs grown on BSA). In addition, HCECs had a less differentiated phenotype with much smaller cells when grown on FN or ECM/ Fbb26 but not when cultured on BSA or TNC. As we previously determined the pattern of expression of the putative TFs interacting with the promoter and 50 -flanking sequence of the a9 gene in HCECs (Fig. 3), we then examined whether any of them seeks its expression altered when these cells are grown on TNC. Of the 17 TFs identified in Fig. 3, only c-Myb had its expression significantly altered (7-fold increase) when HCECs are grown on TNC (Fig. 7F) suggesting that c-Myb might positively contribute to the TNmediated increased expression of the a9 gene. 4. Discussion Tissue repair requires adhesion of proliferating cells to the basement membrane (BM) and cell migration to cover the wound (Gaal, 2002) which in turn needs ECM synthesis and assembly. The ECM is a complex, cross-linked structure of proteins and polysaccharides that organizes the geometry of normal tissues. Fibronectin, laminin, and tenascin are ECM adhesion proteins that have been identified as potential wound healing agents because of their cell-attachment, migration and differentiation properties (see Humphries et al. (1989); Hynes (1986); Ruoslahti (1988) for reviews). Tenascin expression correlates with development and wound healing. Among integrins, a2b1, a8b1, a9b1, avb3, and avb6 have been reported to participate in the adhesion of epithelial cells to tenascin (Joshi et al., 1993; Liao et al., 2002; Prieto et al., 1993). However, among all those that seek their expression deregulated during corneal wound healing, only a9b1 recognizes tenascin as its ligand. Here, we investigated the mechanisms that govern a9 expression in different human cells including corneal epithelial cells as well as uveal melanoma cell lines that express this integrin subunit to varying levels. We demonstrated that both positive and negative regulatory elements are present in the basal and 50 flanking region of the human a9 gene. In vitro DNaseI footprint and EMSAs, and in vivo ChIP analyses collectively demonstrated the binding of the transcription factors c-Myb, Sp1, Sp3 and NFI to different regions of the a9 promoter. Data from primer extension analyses provided evidence that transcription of the a9 gene may initiate at different positions from one cell type to another. Indeed, primary cultures of HCFCs initiate transcription of the ITGA9 gene at a major site located 74 bp upstream from that located for both UVM and HCECs. Beside these two major mRNA start sites, multiple minor initiation sites were also identified in each cell type examined. This result is not surprising considering that: i) no TATA box is present in the a9 promoter, and ii) the basal a9 promoter has a particularly elevated content in GC residues (81%). Analysis of the a9 promoter sequence identified three short sequences, at positions þ4, 17 and 52, that

and TNC, or on a tissue-engineered reconstructed corneal stroma (ECM Fbb26) and left to grow for 7 days. Magnification: 4 (left column) and 10 (right column). Scale bar: 20 mM. F) Heatmap representation of the transcriptional profiles of the TFs for which a putative target site was identified in the promoter and 50 -flanking region of the a9 gene (Fig. 3) between HCECs grown on BSA or on TNC. The only gene whose expression is deregulated by more than 2-fold is indicated (arrowhead). Microarray data for the housekeeping genes b2-microglobulin (B2M) and golgin subfamily A member 1 (GOLGA1) are also presented.


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almost entirely fit (one mismatch in each) that of the prototypical Initiator element (Inr; T/TC/CANT/TA/TC/C). Interestingly, the a9 basal promoter also contains three sequences that perfectly match that of BRE elements (C/CG/AG/GCGCC) (Lagrange et al., 1998). The close proximity of these regulatory elements near the a9 mRNA start site is consistent with their typical organization in GC-rich core promoters. Our DNA binding analyses suggested that the transcription factor c-Myb interacts with a regulatory region from the a9 promoter that also bears both the FP1 and FP2 elements, and which accounts for the repression of the a9 gene transcription in both primary cultured (HCECs) and cancer cells (T115). ITGA9 is not the only integrin gene whose transcription is regulated by c-Myb. Indeed, c-Myb has been shown to participate to the expression of the integrin subunit a4 by promoting a4 gene transcription through competition with the transcriptional repressor ZEB, a process that also requires the cooperation of the transcription factor Ets (Postigo et al., 1997). c-Myb has also been demonstrated to positively regulate the expression of the aX integrin subunit gene (Rubio et al., 1995). Despite the fact that c-Myb is generally viewed as a transcriptional activator, yet a role for this transcription factor as a repressor is now well documented. Indeed, besides the presence of both an N-terminal DNA-binding domain and a central transactivation domain, c-Myb also bears a C-terminal domain involved in transcriptional gene repression (Sakura et al., 1989), which confers to c-Myb the ability to function either as an activator or a repressor of gene expression. c-Myb can repress gene expression either by having its DNA target site overlapping that of other, more potent transcriptional activators (Nakagoshi et al., 1989) or general transcription factors such as TFIID (Mizuguchi et al., 1995), or by sequestering other TFs that positively regulate transcription of the target gene, as has been shown to occur with MyoD (Kaspar et al., 2005). In addition, post-translational modifications such as SUMOylation and phosphorylation have been reported to suppress the trans-activating properties of c-Myb (Bies et al., 2013; Matre et al., 2009). However, that transcription of the a9 gene is negatively regulated by c-Myb through its interaction with the distal negative regulatory element is unlikely as c-Myb expression was coordinated with an increased expression of the a9 gene in HCECs grown on TNC suggesting that c-Myb is a positive regulator of a9 gene transcription in HCECs. These results also suggest that other, yet unidentified TFs must account to the repressive influence mediated by the distal FP1/FP2 bearing negative regulatory element. It is particularly interesting to note that binding of a4b1 and a5b1 integrins to their ligand fibronectin preserves the expression of transcription factors, including c-Myb, that are associated with primitive stem cell maintenance (Dao and Nolta, 2007). In turn, fibronectin expression has been shown to be induced by c-Myb in embryonic kidney and neuroblastoma cells (Tanno et al., 2010). C-Myb was also found to participate in the expression of other ECM components, notably collagen type I (Kopecki et al., 2007). A role for c-Myb in the structural organization of the ECM is further highlighted by the fact that it also regulates expression of MMP1 and MMP9, two metalloproteinases that play very important functions in the ECM remodeling occurring during wound healing of the cornea (Knopfova et al., 2012). TNC has been suggested to be involved in corneal development, differentiation, and proliferation of stem cells primarily because it is widely expressed in the preterm cornea but then seeks its expression restricted to the limbal area of child and adult corneas (Maseruka et al., 2000). Among the many ligands recognized by the a9b1 integrin, TNC is particularly interesting in that its expression was postulated to be coordinated with that of its corresponding integrin receptor during corneal wound healing (Stepp and Zhu, 1997b). The results presented in this study somehow validate this

hypothesis at the mRNA but not at the protein level. Indeed, despite significant changes in the mRNA expression of both a5 and a9 when HCECs are grown on TNC, no difference in the expression of a5 and a9 proteins were observed. This discrepancy may rely on the fact that cell surface integrin receptors may have a much longer turnover than their corresponding mRNAs. Therefore, a much longer culture time might be required when these cells are grown on TNC before one can appreciate the alterations observed for both a9 and a5 at the mRNA level. Meanwhile, the antibody used to monitor a5 expression in Western blot is directed against the heterodimeric a5b1 integrin. As there is no simultaneous increase but rather a near 2-fold reduction (average RNS: 0.0122 on BSA vs 0.075 on TNC) in the expression of the b1 subunit at the mRNA level when HCECs are grown on TNC (Fig. 7B), a significant alteration in the total amount of the a5b1 integrin might consequently be difficult to detect. Studies in TNC knockout mice revealed that both skin and corneal wounds appear to have reduced fibronectin expression and that corneal healing defects occur after suture injuries (Forsberg et al., 1996; Mackie and Tucker, 1999; Matsuda et al., 1999). TNC has been shown to interfere with fibronectin action (ChiquetEhrismann et al., 1988) by altering the fibronectin-promoted adhesion and spreading of chick embryo fibroblasts (Fischer et al., 1997). Our results also support this observation as HCECs cultured on TNC did not grow as quickly as when they are cultured on fibronectin or on tissue-engineered reconstructed corneal stromas (Fig. 7E). Although this result contrasts with previous studies that reported a reduced cell adhesion and migration of HCECs during corneal wound healing when TNC is lacking, it is however consistent with other in vitro analyses that also reported a negative effect of TNC on corneal fibroblast adhesion and migration (Schmidinger et al., 2003). Most importantly, Filenius et al. noted the complete lack of adhesion of corneal epithelial cells on TNC- but not on fibronectin-coated tissue-culture plates (Filenius et al., 2003). Therefore, the positive influence on wound healing often reported for TNC may simply rely on its ability to positively alter expression of fibronectin, over which corneal epithelial cells and stromal fibroblasts attach and spread with a much greater extent than on TNC (Doane et al., 2002; Schmidinger et al., 2003), rather than on a direct positive action of TNC on cell adhesion and migration. Therefore, TNC-mediated adhesive and anti-adhesive activities (Chiquet-Ehrismann et al., 1988; Doane et al., 2002; Fischer et al., 1997; Murphy-Ullrich, 2001) may be dictated by specific variables such as the precise composition of the underneath ECM or on the cellular context (Stepp and Zhu, 1997b). It is noteworthy that our analysis of the a9 promoter region also revealed that the more distant negative regulatory element from that gene is predominantly occupied in vivo by NFI in T143 cells, which also express that gene to a lower level than T115 cells. In vertebrates, the NFI family includes four genes: NFIA, -B, -C and -X. The first 200 amino acids from the N-terminal region of NFI are rigorously conserved in all four isoforms and act as a DNA binding domain that can bind the 50 -TGGA/C(N)5GCCAA-30 consensus element often located in close proximity of binding sites for other transcription factors in gene promoter regulatory regions (Gronostajski, 1986; Nagata et al., 1983; Roulet et al., 2000, 2002). However, unlike other transcription factors such as Sp1 and Sp3, that are unquestionably recognized as TFs that positively influence gene transcription, members from the NFI family have been reported to be as efficient as repressors (Laniel et al., 2001; Nakamura et al., 2001) than activators (Gao et al., 1996) of gene transcription. We previously demonstrated that both the a5 and a6 integrin subunit genes are also negatively regulated by members of the NFI family (Gaudreault et al., 2008; Gingras et al., 2009). Both NFIA and NFIX bear a clearly defined repression domain (reviewed in



Gronostajski (2000)). However, rather than acting as true repressors, NFI proteins more frequently repress gene transcription by competing for the availability of their promoter target sites with other, more potent, positive regulatory proteins that have their DNA binding site located nearby or overlapping those of NFI. That target sites for other positive regulatory TFs are present very close or overlapping with each one of the putative NFI target sites identified in the a9 promoter sequence further corroborate the ‘competitive’ nature of NFI as a mechanism to suppress a9 gene expression. This is particularly noticeable in the a9 basal promoter as it bears four distinct NFI sites within a near 100 bp crowded area that also contains six putative target sites for Sp1/Sp3, three sites for AP-2 and single sites for c-Ets and NF-kB (Supplementary Fig. 1). In such instance, the balance in the ratio of NFI against these positively acting transcription factors then becomes particularly critical in order to dictate the level to which the a9 gene is to be transcribed in any given cell. In support of this, the severe reduction (and in some instance, the complete suppression) in the transcription of the a9 gene observed when Sp1 expression is knockeddown through RNAi might have resulted from the lack of the Sp1 positive regulatory influence combined to the facilitated access of the negatively-acting NFI to its multiple target sites in the a9 basal promoter that are, as mentioned above, closely located to the Sp1 sites. Meanwhile, post-translational alteration of some of these TFs rather than variation in their absolute level of expression at the protein level may also dictate whether any such given TF will interact efficiently or not with its target sequence in DNA. In addition to phosphorylation, NFI is also known to be posttranslationally modified through O-glycosylation, both alterations being suggested to somehow alter its regulatory function (Duval et al., 2012; Jackson and Tjian, 1988; Kawamura et al., 1993; Mukhopadhyay and Rosen, 2007; Reifel-Miller et al., 1994). One particularly interesting feature emerging from the analysis of the microarray data is the heterogeneity of AP-1 subunitsencoding genes that are expressed in primary cultured cells. Indeed, all Jun (c-Jun, JunB and JunD) and Fos (c-Fos, FosB, Fra-1 and Fra-2) subunits are expressed to varying levels in all the cell types examined (HCECs, HSECs, HCFCs and HSFCs). On the other hand, UM cell lines predominantly express either c-Jun or JunB as their Jun subunit, and c-Fos as their Fos subunit. Among all the transcription factors that can potentially participate to restrict expression of the a9 gene in primary cultured cells but not in UM cell lines are the AP-1 constituting subunits Fra-1 and Fra-2. Indeed, microarray analyses indicated that both Fra-1 and Fra-2 genes are predominantly expressed in HCECs, HSECs, HCFCs and HSFCs that also express low a9 levels. A role for the AP-1 constituting subunits Fra1 and Fra-2 in the repression of gene transcription is well documented (Luther et al., 2014; Rutberg et al., 1997). Therefore, and as for NFI, cell-specific expression of distinct AP-1 isoforms may also contribute to lower the expression of the a9 integrin subunit gene in primary cultured cells. 5. Conclusions While the massive secretion of fibronectin by stromal fibroblasts and epithelial cells remains the earliest and most prominent alteration in the ECM occurring during wound healing (Berman et al., 1983; Kang et al., 1999; Murakami et al., 1992), changes in the secretion of TN is also a hallmark of that process as it also increases beneath the migrating epithelial cells 6 days after corneal damage (Stepp and Zhu, 1997a). The results presented in this study bridge the molecular mechanisms that control basal regulation of ITGA9 gene transcription to the coordinated appearance of the a9b1 ligand tenascin during wound healing of the cornea. In a study that we published last year (Lake et al., 2013), we evaluated the

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impact of culturing HCECs on a complex, tissue-engineered corneal stroma. Data from mass spectroscopy analyses provided evidence that the reconstructed stroma is rich in various ECM components, including tenascin. However, the influence of the complex stromal ECM on a5 and a9 integrin subunit gene expression was found to be exactly the opposite (increased expression of a5 and reduced a9 expression) of that reported in the present study for tenascin (reduced a5 expression and increased a9 expression). These very interesting results therefore suggest that the negative influence exerted by tenascin on the adhesion/migration properties of HCECs reported in the present study is somehow completely abolished by other, most probably more abundant components from the ECM that are present in the more complex, tissue-engineered corneal stroma. However, the particularly complex relationship in the regulatory networks modulated by the different components from the ECM and their very distinctive influences on the many properties (adhesion, migration and proliferation) of the cells that adhere and grow on it will require additional works to precisely outline their contribution to tissue wound healing. Acknowledgments The authors would like to thank Solange Landreville (CUObec) for providing the UVM microarray Recherche, CHU de Que annotation files, and Lucie Germain (Centre de recherche en orgarimentale de l'Universite Laval/LOEX, CHU de nogenese expe bec) for providing the annotation files for HSECs and HSFCs. Que This study was supported by a grant from the Natural Sciences and Engineering Research Council of Canada (NSERC) to S.L.G. (grant bec Ocular tissue bank and the Uveal #138624-2012). The Que seau Melanoma Infrastructure are financially supported by the Re de la vision from the Fonds de recherche du de recherche en sante bec e Sante (FRQS). Que Appendix A. Supplementary data Supplementary data related to this article can be found at http:// dx.doi.org/10.1016/j.exer.2015.03.001. References Alam, N., Goel, H.L., Zarif, M.J., Butterfield, J.E., Perkins, H.M., Sansoucy, B.G., Sawyer, T.K., Languino, L.R., 2007. The integrin-growth factor receptor duet. J. Cell. Physiol. 213, 649e653. Allen, M.D., Vaziri, R., Green, M., Chelala, C., Brentnall, A.R., Dreger, S., Vallath, S., Nitch-Smith, H., Hayward, J., Carpenter, R., Holliday, D.L., Walker, R.A., Hart, I.R., Jones, J.L., 2011. Clinical and functional significance of alpha9beta1 integrin expression in breast cancer: a novel cell-surface marker of the basal phenotype that promotes tumour cell invasion. J. Pathol. 223, 646e658. Bazigou, E., Xie, S., Chen, C., Weston, A., Miura, N., Sorokin, L., Adams, R., Muro, A.F., Sheppard, D., Makinen, T., 2009. Integrin-alpha9 is required for fibronectin matrix assembly during lymphatic valve morphogenesis. Dev. Cell. 17, 175e186. Beliveau, A., Berube, M., Rousseau, A., Pelletier, G., Guerin, S.L., 2000. Expression of integrin alpha5beta1 and MMPs associated with epithelioid morphology and malignancy of uveal melanoma. Invest. Ophthalmol. Vis. Sci. 41, 2363e2372. Berman, M., Manseau, E., Law, M., Aiken, D., 1983. Ulceration is correlated with degradation of fibrin and fibronectin at the corneal surface. Invest. Ophthalmol. Vis. Sci. 24, 1358e1366. Bies, J., Sramko, M., Wolff, L., 2013. Stress-induced phosphorylation of Thr486 in cMyb by p38 mitogen-activated protein kinases attenuates conjugation of SUMO-2/3. J. Biol. Chem. 288, 36983e36993. Bisson, F., Paquet, C., Bourget, J.M., Zaniolo, K., Rochette, P.J., Landreville, S., Damour, O., Boudreau, F., Auger, F.A., Guerin, S.L., Germain, L., 2014. Contribution of Sp1 to telomerase expression and activity in skin keratinocytes cultured with a feeder layer. J. Cell. Physiol. (Epub ahead of print), PMID: 24962522. Bisson, F., Rochefort, E., Lavoie, A., Larouche, D., Zaniolo, K., Simard-Bisson, C., Damour, O., Auger, F.A., Guerin, S.L., Germain, L., 2013. Irradiated human dermal fibroblasts are as efficient as mouse fibroblasts as a feeder layer to improve human epidermal cell culture lifespan. Int. J. Mol. Sci. 14, 4684e4704. Breuss, J.M., Gallo, J., DeLisser, H.M., Klimanskaya, I.V., Folkesson, H.G., Pittet, J.F., Nishimura, S.L., Aldape, K., Landers, D.V., Carpenter, W., et al., 1995. Expression of the beta 6 integrin subunit in development, neoplasia and tissue repair


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