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Hnrnpa1 Regulated IL6 Final Formated.Pdf Heterogeneous nuclear ribonucleoprotein A1 interacts with a 5’ flanking distal element of interleukin 6 and up-regulates its basal transcription Dongling Zheng1, Jenny Worthington2, John F. Timms2, Patricia Woo1 1Division of Infection and Immunity, 2EGA Institute for Women’s Health, University College London, London, WC1E 6JF *Running title: Regulation of IL-6 by HNRNPA1 To whom correspondence should be addressed: Prof Patricia Woo, Division of Infection and Immunity, 5 University Street, London, WC1E 6JF, UK. Tel: +442076796965. Fax: +442076796212. E-mail: [email protected] Keywords: IL-6, cis-regulation, SPR, HNRNP Background Transcription of interleukin-6 (IL- dependent on the presence of the sequence from 6) can be regulated by a promoter sequence -5307 to -5202 bp of the IL-6 gene. Thus, located -5 Kb upstream of its transcription start HNRNPA1 is a novel transcriptional regulator of site. IL-6 expression, acting via the 5’ flanking Result Heterogeneous nuclear ribonucleoprotein sequence of the gene. A1 and A2/B1 (HNRNPA1 and HNRNPA2/B1) show interaction with the upstream promoter Introduction element and regulates IL-6 promoter activity. Interleukin-6 (IL-6) is a key cytokine in both Conclusion HNRNPA1 is a novel innate and adaptive immune responses. transcriptional regulator of IL-6. Dysregulation of IL-6 signalling is implicated in Significance HNRNP proteins may play many disease processes characterised by chronic important roles during inflammation. inflammation and autoimmunity (1). It is a pleiotropic cytokine produced in numerous cell Summary types, but the primary sources are cells of the Interleukin-6 (IL-6) is an important pro- myeloid lineage (such as monocytes, inflammatory cytokine involved in many macrophages, B cells), epithelial, endothelial, autoimmune and inflammatory diseases. We and muscle cells. Its function includes promotion have shown previously that a region from -5307 of inflammation by induction of chemokines and to -5202 bp upstream of the IL-6 transcriptional adhesion molecules, but it also produces the start site is responsible for basal IL-6 gene interleukin 1 receptor antagonist (IL-1ra) and expression and that there were DNA binding inhibitor of metalloproteinases. It is in addition a proteins involved from EMSA and transient growth factor for a diverse population of cells expression experiments. Here we have combined and tissues including B-cells, T cells, endothelial surface plasmon resonance technology with cells, cardiac and skeletal muscle cells. It is also mass spectrometry analysis and identified referred to as a myokine in the literature on nuclear proteins bound to this region. muscle function and exercise. HNRNPA1 and HNRNPA2/B1 were found IL-6 gene transcription can be induced by consistently. EMSA supershift and chromatin other pro-inflammatory cytokines such as IL-1 immunoprecipitation assays confirmed the and tumour necrosis factor alpha (TNFα) in involvement of HNRNPA1, but not addition to other stimuli such as bacterial HNRNPA2/B1. Knocking down HNRNPA1 lipopolysaccharide (LPS). Functional cis- expression by siRNA resulted in reduced IL-6 regulatory elements described to date are transcriptional activity as assessed from transcription factor binding sites for NFκB (2), transfection experiments using reporter IRF-1 (3), AP-1 (4), C/EBP (5) and SP1 (6). constructs, mRNA and protein measurements. These cis-acting elements are all located within Overexpression of HNRNPA1 cDNA increased 1.2 kb upstream of the transcription start site IL-6 mRNA expression. This regulation was (TSS) of the IL-6 gene. A functional SNP at - 1 174 bp upstream of the IL-6 TSS (rs1800795) is flow cell. Empirically in the BIAcore technology, the most extensively studied SNP and showed 1 ng of a globular protein or 0.78 ng of a DNA association with diseases including systemic molecule bound at the surface gives a response onset juvenile arthritis (sJIA) (7,8), Systemic of 1000 RU (16). Lupus Erythematosus (SLE) (9), and For protein-DNA interaction analyses, 20 to cardiovascular disease (10-14). 200 ng/µL nuclear proteins were first incubated Previously we have reported that IL-6 gene with 10 ng/µL of poly [dI-dC], as a competitor transcription could be regulated beyond the 1 kb for non-specific protein binding to the DNA chip, 5’ flanking region and identified cis-acting in binding buffer containing 20 mM HEPES pH sequences as far as -5 kb upstream of the IL-6 7.6, 50 mM KCl, 5mM MgCl2, 1 mM EDTA, 40 TSS to be important for basal IL-6 gene ng/µL BSA and 0.05% P20 surfactant and then transcription (15). A specific region from -5307 applied to the DNA-immobilised chip at a rate of bp to -5152 bp was found to bind nuclear 1 µL/min. The sensorgrams were recorded proteins and reporter assays in HeLa cells automatically and adjusted by subtracting the showed higher IL-6 basal transcription activity. baseline response recorded immediately before In this report, we have applied surface plasmon the injection of each sample when only buffer resonance (SPR) technology and mass plus BSA was applied. The usage of BSA was to spectrometry analysis to identify the nuclear block non-specific binding sites on the DNA proteins that bind to this region. Further surface. A flow cell without immobilised DNA experiments confirmed the presence of served as a non-specific binding control. At the HNRNPA1 which was found to have a cis- end of each cycle, bound proteins were eluted by regulatory role in IL-6 transcription in cultured two-pulse injections of 0.05% SDS to regenerate cells. the chip. For protein recovery, all four flow cells on the chip were immobilised with IL6-155 Experimental procedures DNA and the bound proteins recovered to Cell culture and nuclear extraction-HeLa cells collection tubes by using the ‘Injection and were maintained in Dulbeco’s modified Eagle Recovery’ function of the BIAcore T100. medium (DMEM) supplemented with 10% fetal Multiple cycles and repeats were applied to bovine serum (FBS) in a humidified atmosphere obtain sufficient protein for mass spectrometry of 5% CO2 at 37°C. analysis. The recovered samples were Nuclear extracts were prepared from concentrated using a vacuum dryer and then exponentially growing HeLa cells following the resolved on a 10% Bis-Tris NuPAGE gel method described previously (15). The (Invitrogen) followed by silver staining using resuspension buffer contained 10 mM HEPES- SilverQuest Silver Staining Kit or colloidal KOH pH 7.9, 420 mM NaCl, 0.2 mM EDTA, Coomassie Blue (Invitrogen). 0.1 mM EGTA, 25% v/v glycerol, 1 mM DTT Mass Spectrometry analysis-Gel bands (stained and protease and phosphatase inhibitors. Protein with colloidal Coomassie Blue) were excised concentration was quantified using Bio-Rad and washed three times in 50% (v/v) acetonitrile, Bradford protein assay. dried in a vacuum centrifuge, reduced in 10 mM Surface Plasmon Resonance (SPR)-The SPR DTT in 5 mM ammonium bicarbonate pH 8.0 experiment was carried out using a BIAcore for 45 min at 50oC and alkylated with 50 mM T100 with Sensor chip SA (Biacore). The DNA iodoacetamide in ammonium bicarbonate for 1 fragment containing sequence from -5307 to - hr at room temperature in the dark. Gel pieces 5152 bp upstream of IL-6 TSS (IL6-155) was were washed twice in 50% acetonitrile, vacuum amplified using a forward primer 5’-biotin- dried, and then 50 ng sequence grade modified TGGCTCAGACATAGACCACTG-3’ and a trypsin (Promega, Southampton, UK) in 5 mM reverse primer 5’- ammonium bicarbonate was added to each dried TATTGTTCCAAGGGGTGCTG-3’, and gel piece. After allowing gel pieces to re-swell purified on Qiagen PCR columns. The SA chip for 5 min, 5 µL of 5 mM ammonium bicarbonate surface was activated by injection of 1 M NaCl was added and gel pieces were incubated at 37oC in 50 mM NaOH for 1 min three times. Biotin- for 16 hr. Tryptic peptides were extracted three labelled PCR product at 2 ng/mL in 0.5 M NaCl times with 50% (v/v) acetonitrile containing 5% was injected onto the chip surface at a flow rate (v/v) trifluoroactetic acid. Extracts from each gel of 5 µL/min for 30 min. Approximately 1,500 piece were pooled and vacuum centrifuged to RU (arbitrary resonance units) was achieved per dryness. Peptides were finally resuspended in 5 2 µL of 0.1% (v/v) formic acid and stored at -20oC labelled at the 3’ end using terminal prior to mass spectrometric analysis. deoxynucleotidyl transferase and biotin-11- Analysis of tryptic peptides from digested dUTP (Fermentas, Thermo Scientific). EMSAs bands was performed by nanoflow reversed- were performed using 10 nM biotinylated probes phase liquid chromatography-tandem mass incubated with 2 µg of nuclear proteins in 1x spectrometry (LC-MS/MS) on an LTQ Orbitrap binding buffer (8% Ficoll, 20 mM HEPES, 50 XL mass spectrometer (Thermo Scientific). mM KCl, 1mM EDTA, 0.5 mM DTT, 40 ng/µL Sample (5 µL) was injected onto a 300 µm i.d. x of poly [dI-dC] and 40 ng/µL BSA) for 30 min 5 mm C18 PepMap guard column (5 µm bead at 25°C. In experiments where competitor size, 100 Å pore size, LC Packings, Netherlands) unlabelled probes were added, reactions were and washed for 3 min with 95% solvent A (water pre-incubated with unlabelled probes in 100-fold + 0.1% FA) at a flow rate of 25 mL/min using an molar excess of the labelled probe at 25°C for 15 Ultimate 3000 system (Dionex). Reversed-phase min prior to the addition of the labelled probe. chromatographic separation was then carried out For supershift, nuclear proteins were pre- on a 75 mm i.d. x 250 mm C18 PepMap nano incubated with 2 µg of antibody for 30 min at LC column (3 µm bead size, 100 Å pore size; 25°C.
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