DTD 5 ARTICLE IN PRESS YEXCR-06597; No. of pages: 12; 4C: 6 Experimental Cell Research xx (2005) xxx–xxx www.elsevier.com/locate/yexcr NF45/ILF2 tissue expression, promoter analysis, and interleukin-2 transactivating functionB Guohua Zhao, Lingfang Shi, Daoming Qiu, Hong Hu, Peter N. KaoT Division of Pulmonary and Critical Care Medicine, Stanford University Medical Center, 300 Pasteur Drive, Stanford, CA 94305-5236, USA Received 5 May 2004, revised version received 20 December 2004 Abstract NF45/ILF2 associates with NF90/ILF3 in the nucleus and regulates IL-2 gene transcription at the antigen receptor response element (ARRE)/NF-AT DNA target sequence (P.N. Kao, L. Chen, G. Brock, J. Ng, A.J. Smith, B. Corthe´sy, J. Biol. Chem. 269 (1994) 20691– 20699). NF45 is widely expressed in normal tissues, especially testis, brain, and kidney, with a predominantly nuclear distribution. NF45 mRNA expression is increased in lymphoma and leukemia cell lines. The human and murine NF45 proteins differ only by substitution of valine by isoleucine at amino acid 142. Fluorescence in situ hybridization localized the human NF45 gene to chromosome 1q21.3, and mouse NF45 gene to chromosome 3F1. Promoter analysis of 2.5 kB of the murine NF45 gene reveals that significant activation is conferred by factors, possible including NF-Y, that bind to the CCAAT-box sequence. The function of human NF45 in regulating IL-2 gene expression was characterized in Jurkat T-cells stably transfected with plasmids directing expression of NF45 cDNA in sense or antisense orientations. NF45 sense expression increased IL-2 luciferase reporter gene activity 120-fold, and IL-2 protein expression 2-fold compared to control cells. NF45 is a highly conserved, regulated transcriptional activator, and one target gene is IL-2. D 2005 Elsevier Inc. All rights reserved. Keywords: Transcriptional activator; RNA-binding protein; Nuclear factor of activated T-cells; Antigen receptor response element Introduction serve essential roles in development of lymphocytic immunity through their contributions to double-stranded NF45/ILF2 and NF90/ILF3 contribute to RNA gene DNA break repair and antibody and T-cell receptor diversity regulation at the levels of transcription, splicing, export, and [4–8]. The 110-kDa variant of NF90, termed NF110, translation. These proteins were isolated as principal contains a C-terminal RGG domain, and is a target for components of a Jurkat T-cell nuclear DNA-binding arginine methylation by protein arginine methyl transferase complex with affinity for the purine-rich antigen receptor 1 [9]. response element (ARRE)/NF-AT target DNA sequence NF45 and NF90 interact with DNA [1,2,10,11] and RNA in the IL-2 promoter. [1,2]. NF45 and NF90 stabilize [10,12–16]. NF90 contains 2 double-stranded RNA-binding the association between the Ku70, Ku80 lupus antoanti- domains and is thus related to other proteins that regulate gens, and the DNA-dependent protein kinase catalytic transcription (RNA helicase A; [17]), RNA editing (ADAR subunit, DNA-PKcs [3]. Ku70, Ku80, and DNA-PKcs each [18,19]), and translation (PKR [20]), and a zinc-finger nucleic acid binding motif, DZF. NF45 contains an N-terminal arginine- and glycine-rich domain at positions 3–22, and a B zinc-finger DZF domain homologous to NF90, at positions The nucleotide sequences for the human and murine NF45 genes have 104–338. been deposited in the GenBank database under GenBank Accession Numbers, human (AY099265), and murine (AF458249). NF45 and NF90 contribute to gene regulation at multiple T Corresponding author. Fax: +1 650 725 5489. levels. Transcriptional enhancement and repression have E-mail address: [email protected] (P.N. Kao). both been demonstrated for NF90 and NF110, depending on 0014-4827/$ - see front matter D 2005 Elsevier Inc. All rights reserved. doi:10.1016/j.yexcr.2004.12.030 ARTICLE IN PRESS 2 G. Zhao et al. / Experimental Cell Research xx (2005) xxx–xxx the promoter context [16,21,22]. Cotransfection of NF45 in Experimental methods these studies demonstrated augmented transcriptional acti- vation. Posttranscriptional message stabilization represents RNA and protein expression analysis an important level of regulation, in particular for cytokines such as IL-2 and IL-8. IL-2 mRNA is stabilized in activated Multiple tissue Northern blots (Clontech) were hybri- T-cells through specific interactions between NF90 and AU- dized sequentially with a 32P-labeled human NF45 probe rich sequences in the IL-2 3VUTR [23]. Furthermore, NF90/ (1046 bp of coding sequence), followed by a probe for ILF3 regulated the translocation of IL-2 mRNA from the glyceraldehyde-3-phosphate dehydrogenase (Clontech) for nucleus to the cytoplasm. The regulated export of IL-2 the human blot and for beta-actin (660 bp) for the mouse mRNA involves interactions of NF90/ILF3 with the nuclear blot, according to the manufacturer’s instructions [33]. karophyrin, exportin 5, and Ran GTPase [24]. NF90 and Intensities were quantitated by phosphorimaging (Cyclone, NF45 have also been identified as components of the Packard), and the normalized expression of NF45 was spliceosome [25,26] and NF90 has been demonstrated determined for each tissue. The expression of NF45/ILF2 to contribute to RNA splicing [15]. A role for NF90 and in human tissues and cell lines was also determined using NF45 in regulating translation was implied by the recovery Genechip 3.2 (Affymetrix, Santa Clara), and the results of these proteins in the ribosomal salt wash, as well as are presented with the permission of the corresponding the physical association with the double-stranded RNA- author (http://expression.gnf.org) [34]. For Western blot activated protein kinase, PKR, known to regulate transla- analysis, extracted proteins (40 Ag) were fractionated and tional initiation [13–15]. Furthermore, NF90 and NF45 detected with rabbit anti-NF45 serum which was affinity- copurified with translational elongation initiation factor 2 purified against recombinant NF45 protein using a alpha, beta, and gamma subunits [3]. Specific translational HiTrapk NHS-activated column (Amersham Bioscience) inhibition of acid beta-glucosidase and selected other liver [1]. mRNAs was mediated by translational control protein 80, which is identical to NF90/ILF3 [27]. Immunohistochemistry The highest levels of expression of NF45 and NF90 occur in the testes. NF45/ILF2 was shown to be regulated Normal adult male and female C57Bl/6 mice were by meiosis and to be associated with transcriptionally active sacrificed, and formalin-fixed, paraffin-embedded tissues chromatin [28]. Lopez-Fernandez et al. analyzed testis were sectioned. The sections were deparaffinized, rehy- development and demonstrated expression of NF45/ILF2 drated, and microwaved for 10 min in 0.01 M citrate buffer. and NF90/ILF3 in pachytene spermatocytes that had The endogenous peroxidase was blocked using 3% H2O2 completed meiosis. In adult testis, there was strong nuclear (5 min at RT). The sections were blocked with 5% normal expression of NF45/ILF2, except in the area of the XY donkey serum (Jackson Immunoresearch Lab) for 10 min at bivalent, which is transcriptionally silent. In addition to RT, and then incubated with primary antibody, rabbit anti- potential roles in regulating transcription, NF45 may NF45 antisera, generated, and affinity-purified against associate with RNAs in ribonucleoprotein complexes that recombinant NF45 protein (1:1500 dilution, 30 min at regulate delayed translation of mRNAs [29,30]. 378C). Detection was achieved using Biotin-SP-conjugated NF90 and NF45 represent host cellular factors that can donkey anti-rabbit IgG (1:400 dilution, Jackson Immuno- interact specifically with viral RNAs, including HIV TAR, research) followed by peroxidase-conjugated streptavidin adenovirus VA2, and hepatitis B virus RNA [12,31]. NF45 IgG (1:400 dilution, Jackson) and development with and NF90 bound to hepatitis B virus epsilon RNA and diaminobenzidine (Research Genetics). For a negative facilitated protein priming of the hepatitis B virus polyme- control, the slides were incubated with PBS instead of the rase. Behrens and coworkers demonstrated that binding of primary antibody. NF90 and NF45 to the 5Vand 3Vregions of bovine diarrheal virus RNA (a relative of human hepatitis C) mediated a Genomic sequence analysis of murine and human NF45 circular conformation of the viral genome that contributed to viral replication and translation [32]. A mouse genomic library prepared in bacterial artificial Our aim is to understand the function of NF45 (BAC) chromosome plasmids was screened using a full- in regulating gene expression. Towards this goal, we have length human cDNA for NF45, at Incyte Genomics. characterized the tissue expression and subcellular dis- Subclones of pBleoBAC11 were prepared by restriction tribution of NF45. We compare the genomic structures using HindIII, EcoRI, BamHI, and PstI enzymes, cloned of the murine and human NF45/ILF2 genes, and we into pBluescript II KS+ (Stragene), and positive clones characterize the CCAAT-box DNA sequence in the murine identified by Southern analysis using a human NF45 cDNA NF45 promoter that regulates basal transcription of NF45. were sequenced on both strands using an ABI prism big Functionally, we demonstrate that stable expression of dye terminator kit (Protein and Nucleic Acid Facility, NF45 augments T-cell transcription and expression of Stanford University). The DNA sequences were assembled IL-2. using MacVector and AssemblyLIGN software (Kodak). ARTICLE IN PRESS G. Zhao et al. / Experimental Cell Research xx (2005) xxx–xxx 3 Human genomic P1 clones selected by hybridization with UTR. The sequences of the oligonucleotides used as PCR the hNF45 cDNA were purchased from Incyte Genomics. primers to amplify the regulatory regions are available on The complete human NF45 genomic sequence was request. determined by assembling DNA sequence information from subclones of clone P1-17080 in pBluescript with Cell culture, transfections, and promoter analyses sequences from PCR amplifications of human genomic DNA using primers based upon the murine intron–exon Murine CRL-2053 cells (murine SV-40 transformed junction sequences and cloned into pCRII (Invitrogen).