Title: A <scp>HIF</scp> – <scp>LIMD</scp> 1 negative feedback mechanism mitigates the pro‐tumorigenic effects of hypoxia
Abstract: Research Article21 June 2018Open Access Source DataTransparent process A HIF–LIMD1 negative feedback mechanism mitigates the pro-tumorigenic effects of hypoxia Daniel E Foxler Daniel E Foxler orcid.org/0000-0003-3753-0278 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Katherine S Bridge Katherine S Bridge orcid.org/0000-0003-1516-1459 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author John G Foster John G Foster orcid.org/0000-0002-8591-0833 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Paul Grevitt Paul Grevitt orcid.org/0000-0001-8224-1315 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Sean Curry Sean Curry Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author Kunal M Shah Kunal M Shah orcid.org/0000-0002-4508-9606 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Kathryn M Davidson Kathryn M Davidson orcid.org/0000-0001-7922-2367 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Ai Nagano Ai Nagano Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Emanuela Gadaleta Emanuela Gadaleta orcid.org/0000-0002-5740-6293 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Hefin I Rhys Hefin I Rhys orcid.org/0000-0002-7385-9014 The Francis Crick Institute, London, UK Search for more papers by this author Paul T Kennedy Paul T Kennedy orcid.org/0000-0001-5668-9132 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Miguel A Hermida Miguel A Hermida orcid.org/0000-0002-2633-4119 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Ting-Yu Chang Ting-Yu Chang orcid.org/0000-0002-0561-2757 Institute of Microbiology and Immunology, National Yang Ming University, Taipei City, Taiwan Search for more papers by this author Peter E Shaw Peter E Shaw orcid.org/0000-0002-2598-4283 Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author Louise E Reynolds Louise E Reynolds orcid.org/0000-0001-6075-1808 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Tristan R McKay Tristan R McKay orcid.org/0000-0002-9128-9115 School of Healthcare Science, Manchester Metropolitan University, Manchester, UK Search for more papers by this author Hsei-Wei Wang Hsei-Wei Wang Institute of Microbiology and Immunology, National Yang Ming University, Taipei City, Taiwan Search for more papers by this author Paulo S Ribeiro Paulo S Ribeiro orcid.org/0000-0002-6020-6321 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Michael J Plevin Michael J Plevin orcid.org/0000-0003-2057-8291 Department of Biology, University of York, York, UK Search for more papers by this author Dimitris Lagos Dimitris Lagos orcid.org/0000-0003-0637-281X Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, York, UK Search for more papers by this author Nicholas R Lemoine Nicholas R Lemoine orcid.org/0000-0001-8675-058X Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Prabhakar Rajan Prabhakar Rajan orcid.org/0000-0001-8064-9878 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Trevor A Graham Trevor A Graham orcid.org/0000-0001-9582-1597 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Claude Chelala Claude Chelala orcid.org/0000-0002-2488-0669 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Kairbaan M Hodivala-Dilke Kairbaan M Hodivala-Dilke orcid.org/0000-0002-2859-749X Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Ian Spendlove Ian Spendlove orcid.org/0000-0002-7480-3768 Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author Tyson V Sharp Corresponding Author Tyson V Sharp [email protected] orcid.org/0000-0003-1861-7984 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Daniel E Foxler Daniel E Foxler orcid.org/0000-0003-3753-0278 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Katherine S Bridge Katherine S Bridge orcid.org/0000-0003-1516-1459 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author John G Foster John G Foster orcid.org/0000-0002-8591-0833 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Paul Grevitt Paul Grevitt orcid.org/0000-0001-8224-1315 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Sean Curry Sean Curry Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author Kunal M Shah Kunal M Shah orcid.org/0000-0002-4508-9606 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Kathryn M Davidson Kathryn M Davidson orcid.org/0000-0001-7922-2367 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Ai Nagano Ai Nagano Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Emanuela Gadaleta Emanuela Gadaleta orcid.org/0000-0002-5740-6293 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Hefin I Rhys Hefin I Rhys orcid.org/0000-0002-7385-9014 The Francis Crick Institute, London, UK Search for more papers by this author Paul T Kennedy Paul T Kennedy orcid.org/0000-0001-5668-9132 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Miguel A Hermida Miguel A Hermida orcid.org/0000-0002-2633-4119 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Ting-Yu Chang Ting-Yu Chang orcid.org/0000-0002-0561-2757 Institute of Microbiology and Immunology, National Yang Ming University, Taipei City, Taiwan Search for more papers by this author Peter E Shaw Peter E Shaw orcid.org/0000-0002-2598-4283 Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author Louise E Reynolds Louise E Reynolds orcid.org/0000-0001-6075-1808 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Tristan R McKay Tristan R McKay orcid.org/0000-0002-9128-9115 School of Healthcare Science, Manchester Metropolitan University, Manchester, UK Search for more papers by this author Hsei-Wei Wang Hsei-Wei Wang Institute of Microbiology and Immunology, National Yang Ming University, Taipei City, Taiwan Search for more papers by this author Paulo S Ribeiro Paulo S Ribeiro orcid.org/0000-0002-6020-6321 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Michael J Plevin Michael J Plevin orcid.org/0000-0003-2057-8291 Department of Biology, University of York, York, UK Search for more papers by this author Dimitris Lagos Dimitris Lagos orcid.org/0000-0003-0637-281X Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, York, UK Search for more papers by this author Nicholas R Lemoine Nicholas R Lemoine orcid.org/0000-0001-8675-058X Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Prabhakar Rajan Prabhakar Rajan orcid.org/0000-0001-8064-9878 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Trevor A Graham Trevor A Graham orcid.org/0000-0001-9582-1597 Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Claude Chelala Claude Chelala orcid.org/0000-0002-2488-0669 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Kairbaan M Hodivala-Dilke Kairbaan M Hodivala-Dilke orcid.org/0000-0002-2859-749X Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Ian Spendlove Ian Spendlove orcid.org/0000-0002-7480-3768 Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK Search for more papers by this author Tyson V Sharp Corresponding Author Tyson V Sharp [email protected] orcid.org/0000-0003-1861-7984 Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK Search for more papers by this author Author Information Daniel E Foxler1,‡, Katherine S Bridge1,‡, John G Foster1, Paul Grevitt1, Sean Curry2, Kunal M Shah1, Kathryn M Davidson1, Ai Nagano1, Emanuela Gadaleta1, Hefin I Rhys3, Paul T Kennedy1, Miguel A Hermida1, Ting-Yu Chang4, Peter E Shaw2, Louise E Reynolds5, Tristan R McKay6, Hsei-Wei Wang4, Paulo S Ribeiro5, Michael J Plevin7, Dimitris Lagos8, Nicholas R Lemoine1, Prabhakar Rajan1, Trevor A Graham5, Claude Chelala1, Kairbaan M Hodivala-Dilke5, Ian Spendlove2 and Tyson V Sharp *,1 1Centre for Molecular Oncology, Barts Cancer Institute, Queen Mary University of London, London, UK 2Faculty of Medicine and Life Sciences, University of Nottingham, Nottingham, UK 3The Francis Crick Institute, London, UK 4Institute of Microbiology and Immunology, National Yang Ming University, Taipei City, Taiwan 5Centre for Tumour Biology, Barts Cancer Institute, Queen Mary University of London, London, UK 6School of Healthcare Science, Manchester Metropolitan University, Manchester, UK 7Department of Biology, University of York, York, UK 8Centre for Immunology and Infection, Hull York Medical School and Department of Biology, University of York, York, UK ‡These authors contributed equally to this work *Corresponding author. Tel: +44 (0)20 7882 3848; E-mail: [email protected] EMBO Mol Med (2018)10:e8304https://doi.org/10.15252/emmm.201708304 PDFDownload PDF of article text and main figures. Peer ReviewDownload a summary of the editorial decision process including editorial decision letters, reviewer comments and author responses to feedback. ToolsAdd to favoritesDownload CitationsTrack CitationsPermissions ShareFacebookTwitterLinked InMendeleyWechatReddit Figures & Info Abstract The adaptive cellular response to low oxygen tensions is mediated by the hypoxia-inducible factors (HIFs), a family of heterodimeric transcription factors composed of HIF-α and HIF-β subunits. Prolonged HIF expression is a key contributor to cellular transformation, tumorigenesis and metastasis. As such, HIF degradation under hypoxic conditions is an essential homeostatic and tumour-suppressive mechanism. LIMD1 complexes with PHD2 and VHL in physiological oxygen levels (normoxia) to facilitate proteasomal degradation of the HIF-α subunit. Here, we identify LIMD1 as a HIF-1 target gene, which mediates a previously uncharacterised, negative regulatory feedback mechanism for hypoxic HIF-α degradation by modulating PHD2-LIMD1-VHL complex formation. Hypoxic induction of LIMD1 expression results in increased HIF-α protein degradation, inhibiting HIF-1 target gene expression, tumour growth and vascularisation. Furthermore, we report that copy number variation at the LIMD1 locus occurs in 47.1% of lung adenocarcinoma patients, correlates with enhanced expression of a HIF target gene signature and is a negative prognostic indicator. Taken together, our data open a new field of research into the aetiology, diagnosis and prognosis of LIMD1-negative lung cancers. Synopsis This study identifies the tumour suppressor LIMD1 as a facilitator of an adaptive cellular response to hypoxia. Under hypoxic conditions, LIMD1 expression is induced, facilitating degradation of HIF, limiting HIF transcriptional activity and mitigating its pro-tumorigenic effects. The LIMD1 promoter contains a hypoxia responsive element (HRE) and its expression is induced under hypoxic conditions driving increased HIF degradation via the PHD/VHL axis. HIF protein stability and transcriptional activity are reduced under chronic hypoxic conditions by LIMD1. Aberration of the LIMD1-dependent hypoxic negative feedback loop causes larger and more vascularised tumours to grow in vivo. LIMD1 is lost in 47% of adenocarcinoma driving a hypoxic gene signature that serves as a negative prognostic indicator. Introduction The HIF family of transcription factors are heterodimeric proteins formed of a HIF-α and HIF-β subunit (Wang et al, 1995). HIF-α is regulated by intracellular oxygen levels; at physiological oxygen tension (normoxia), two highly conserved proline residues within the oxygen-dependent degradation domain of the HIF-α subunit (P402/564 on HIF-1α; P405/531 on HIF-2α) are hydroxylated by prolyl hydroxylase domain (PHD) proteins. Hydroxylated HIF-α is then recognised and ubiquitinated by the von Hippel–Lindau (VHL) E3 ubiquitin ligase complex, resulting in its degradation by the 26S proteasome (Salceda & Caro, 1997; Maxwell et al, 1999; Jaakkola et al, 2001; Foxler et al, 2012). Under low oxygen (hypoxic) conditions, the hydroxylase activity of the PHD enzymes is inhibited; HIF therefore escapes hydroxylation and degradation to initiate a transcriptional programme of cellular response and adaptation to hypoxia. Under conditions of chronic hypoxia, a negative regulatory feedback loop is initiated whereby free oxygen from inhibited mitochondrial respiration leads to overactivation of PHDs, causing HIF-α degradation and a desensitised hypoxic response (Ginouves et al, 2008). However, neoplastic cells survive under conditions of chronic tumour hypoxia by inhibiting the degradation of HIF (Bertout et al, 2008). This is exemplified by VHL mutations in clear cell renal carcinomas, leading to sustained HIF-α expression and activity (Rechsteiner et al, 2011). In non-small-cell lung cancer (NSCLC), deregulation of the HIF negative feedback loop is far less characterised, even though HIF-α protein expression is implicated as a poor prognostic indicator (Giatromanolaki et al, 2001; Kim et al, 2005). The lung tumour suppressor protein LIMD1 is a member of the Zyxin family of adaptor proteins, initially characterised as signal transducers (Kadrmas & Beckerle, 2004) shuttling between the cytoplasm and nucleus. LIMD1 loss has been identified in lung, breast, head and neck squamous cell carcinomas, and adult acute leukaemia (Sharp et al, 2004, 2008; Spendlove et al, 2008; Ghosh et al, 2010b; Liao et al, 2015), and its decreased expression in diffuse large B-cell lymphoma has clinical significance to patient prognosis and disease classification/stratification (Xu et al, 2015). Limd1-knockout mice have increased lung tumour numbers and volume and decreased survival rate compared to Limd1-expressing control mice when either challenged with a chemical carcinogen or cross-bred with KrasG12D mice (Sharp et al, 2008) validating its critical role in normal cellular homeostasis. Furthermore, it has been reported that silencing of LIMD1 in multidrug-resistant colorectal carcinoma cells increased their chemosensitivity in vitro (Chen et al, 2014). As a scaffold protein, LIMD1 exerts multiple tumour-suppressive functions depending on its binding partners. Basal LIMD1 gene expression is under the control of PU.1, a member of the Ets family of transcription factors (Foxler et al, 2011). LIMD1 can repress cell cycle progression through pRb-dependent and pRb-independent inhibition of E2F (Sharp et al, 2004) and regulates Hippo signalling by binding to LATS, causing sequestration of the Hippo kinase complex (Das Thakur et al, 2010; Codelia et al, 2014; Jagannathan et al, 2016). LIMD1 is also part of the Slug/Snail complex that regulates E-cadherin transcription (Ayyanathan et al, 2007; Langer et al, 2008) in addition to facilitating centrosomal localisation of BRCA2 to prevent aberrant cellular proliferation (Hou et al, 2016). Our recent work has shown that LIMD1 is a critical effector of microRNA (miRNA)-mediated gene silencing, a process generally considered to be a global tumour-suppressive mechanism (James et al, 2010; Bridge et al, 2017). LIMD1 forms complexes with PHD2 and VHL to post-translationally repress HIF-1α protein levels and therefore HIF-1α-mediated gene activation (Foxler et al, 2012; Zhang et al, 2015). However, the pathophysiological link between this mechanistic role of LIMD1 within the PHD-LIMD1-VHL HIF regulatory complex and cancer is unknown. Here, we report that LIMD1 expression is upregulated in hypoxia, through a functional HIF-1α-specific hypoxic response element (HRE) within the CpG island in its promoter. LIMD1 facilitates HIF-1α protein degradation under hypoxic conditions by maintaining the PHD2/VHL/HIF-1α degradation complex, thereby reducing HIF-1α-driven gene activation. Utilising an RNAi-mediated knockdown-rescue system, we have identified that inhibition of hypoxia-driven increase in LIMD1 expression causes HIF-1α protein stabilisation and HIF target gene activation. In vivo, inhibition of hypoxia-driven LIMD1 expression results in larger and more vascularised xenograft tumours. Finally, our data provide a molecular mechanistic insight into clinico-pathological data indicating that LIMD1 loss or haplo-insufficiency correlated with elevated HIF-1α-driven gene expression within lung tumours is associated with poorer patient prognosis. Results LIMD1 is a HIF-1-responsive gene Homeostatic signalling pathways often have in-built self-regulatory feedback mechanisms to attenuate their activation (Yosef & Regev, 2011). With this in mind, we hypothesised that LIMD1 might be a HIF target gene as well as a component of the degradation complex. We therefore assessed endogenous LIMD1 expression in a panel of cell lines exposed to 1% O2 (henceforth referred to as hypoxia), including transformed/immortalised lines (A549, HeLa, HEK293 and U2OS), non-transformed small airway epithelial cells (SAEC) and primary human dermal fibroblasts (HDF). We observed an increase in LIMD1 mRNA and protein expression in all cell lines in hypoxia when compared to atmospheric oxygen (20% O2, herein referred to as normoxia) using PHD2 as a positive control and PHD1 as a hypoxia non-responsive gene (Figs 1A–C, and EV1A and B; Stiehl et al, 2006). Figure 1. LIMD1 expression is regulated by hypoxiaThe indicated panel of cell lines was exposed to either normoxia (20% O2) or hypoxia (1% O2) for up to 48 h prior to RNA and protein extraction. A, B. (A) LIMD1 mRNA and (B) protein levels were increased following hypoxic exposure. C. Densitometric analysis of (B). D. The LIMD1 promoter contains a hypoxic response element responsible for HIF binding and transcriptional activation of LIMD1. Three predicted HRE elements were individually deleted within the context of the wild-type LIMD1 promoter-driven Renilla luciferase. E. Reporter constructs in (D) were expressed in U2OS cells and exposed to hypoxia for the indicated time-points. Luciferase activity was then assayed and normalised to firefly control. Data are displayed normalised to the normoxic value for each construct. Deletion of the third HRE present within the LIMD1 promoter (ΔHRE3) inhibited hypoxic induction of LIMD1 transcription. F, G. (F) Sequence alignment and (G) sequence logo of LIMD1 promoters from the indicated species demonstrate that the HRE3 consensus sequence is highly conserved. Data information: Unless otherwise stated, data shown are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, ***P < 0.001, according to the Student's t-test (A) or Holm–Šidák post hoc tests, comparing time-points within each cell line (A and C) or comparing the VO group to every other genotype within each time-point (E), following significant main effects/interactions of a mixed-model ANOVA. See Appendix Table S1 for a summary of statistical analysis. Source data are available online for this figure. Source Data for Figure 1 [emmm201708304-sup-0004-SDataFig1.pdf] Download figure Download PowerPoint Click here to expand this figure. Figure EV1. LIMD1 expression is induced in hypoxia via a HIF-1 hypoxia-responsive element (HRE) A. Small airway epithelial cells (SAEC) and human dermal fibroblasts (HDF) were incubated in hypoxia for up to 48 h, total protein extracted and analysed by Western blot. B. Densitometric analysis of LIMD1 protein expression in (A), normalised to β-actin loading control and 0-h hypoxic time-point. C. PHD2 mRNA expression is upregulated by hypoxia. A549, HEK293, HeLa and U2OS cells were incubated in hypoxia for up to 48 h, RNA extracted and PHD2 mRNA expression analysed by qRT–PCR. D. PHD1 mRNA expression is unchanged by hypoxia. qRT–PCR analysis of PHD1 mRNA in the indicated cell lines, as in (C). E–G. The indicated cell lines were incubated in normoxia or 4- or 24-h hypoxia, total protein extracted and PHD2, HIF-1α or HIF-2α protein expression quantified by densitometry and normalised to β-actin loading control. H. The LIMD1 promoter contains three putative HRE elements. The 2-kb upstream region of the LIMD1 promoter was scrutinised in silico for predicted HIF binding sites using MatInspector software as previously described (Foxler et al, 2011). I. A 2-kbp region of the LIMD1 promoter was cloned into a pGL4 Renilla luciferase plasmid, and a series of 10 consecutive internal deletions were constructed (Δ1–10); the positions of the three in silico HRE elements are indicated. J. Reporter constructs in (I) were expressed in U2OS cells and exposed to hypoxia for 24 h. Data shown are normalised to vector only (VO) control. The Δ3 mutation inhibited hypoxic induction of LIMD1 transcription compared to the other nine analysed; significance values for all other constructs are omitted for clarity. Data information: Unless otherwise stated, data shown are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, n.s. = not significant, according to the Student's t-tests (B; comparing means to the theoretical value of 1) or Holm–Šidák post hoc tests, comparing time-points within each cell line, following significant main effects/interactions of a mixed-model ANOVA (C–E). See Appendix Table S6 for a summary of statistical analysis. Source data are available online for this figure. Download figure Download PowerPoint In silico analysis of the LIMD1 promoter identified three putative hypoxic response elements (HRE 1–3; Fig EV1H; Foxler et al, 2011). To assess their functionality, we used a LIMD1 promoter-driven luciferase reporter construct, spanning 1990-bp upstream from the LIMD1 transcriptional start site [as predicted by the RefSeq NM_014240.2, which corresponds to nucleotides 45634323-6323 on the primary chromosome 3 ref assembly NC_000003.11 (Foxler et al, 2011)] and encompassing all three predicted HRE elements (Fig 1D). Within this construct, we created a series of ten consecutive small internal deletions within the CpG Island that have previously been identified as containing transcriptional regulatory elements (Foxler et al, 2011; Fig EV1I). These reporter constructs displayed ~ threefold induction of wild-type LIMD1 promoter activity in hypoxia compared to normoxia. However, deletion of the 31-bp Δ3 region that encompasses the predicted HRE3 ablated any hypoxia-induced increase in luciferase activity (Fig EV1J). Furthermore, internal deletion of the three predicted HREs confirmed HRE3 to be the active hypoxia-responsive element within the LIMD1 promoter (Fig 1E). The position and sequence of this HRE is also highly conserved, further supporting its functional importance (Fig 1F and G). We next determined which HIF-α paralogue was involved in LIMD1 regulation by combining the LIMD1 promoter-driven luciferase reporters (Foxler et al, 2011) with shRNA-mediated knockdown of HIF-1α and HIF-2α. Depletion of HIF-1α, but not HIF-2α, prevented induction of LIMD1 expression in hypoxia (Fig EV2A). This finding was corroborated by ChIP and EMSAs, which further demonstrated HIF-1 binding to the LIMD1 promoter (Fig 2A and B). siRNA-mediated depletion of HIF-1α reduced LIMD1 protein and mRNA expression under hypoxic and, to a lesser extent, normoxic conditions in all cell lines examined (Figs 2C and D, and EV2B–E). LIMD1 depletion did not affect HIF1A or HIF2A mRNA expression, with the exception of an observed increase in HIF2A mRNA in HeLa cells under hypoxic conditions (Fig EV2F–I). The decrease in LIMD1 expression in normoxia following si-HIF-1α demonstrates that HIF-1 activity is required for LIMD1 expression in normoxia, an observation that has been previously described for other genes (Pillai et al, 2011). Furthermore, under hypoxic conditions HIF preferentially binds to gene loci that are already transcriptionally active to further activate their expression (Xia & Kung, 2009). Thus, these data show that under hypoxic conditions, HIF-1 binds the LIMD1 promoter to increase its expression. Click here to expand this figure. Figure EV2. LIMD1 hypoxic induction occurs via HIF-1 and facilitates formation of a hypoxic HIF-1 degradation complex A. HIF-1 but not HIF-2 is responsible for the hypoxic increase in LIMD1 expression. HIF-1α or HIF-2α was knocked down in U2OS cells using transient shRNA-expressing plasmids. The wild-type LIMD1 promoter-driven firefly luciferase was co-transfected with a Renilla normalisation plasmid into these cells and exposed to up to 24-h hypoxia. Resultant luciferase activity was assayed and normalised to Renilla. Data are displayed normalised to the normoxic value for each shRNA knockdown line. B–D. Hypoxic induction of LIMD1 is impaired upon knockdown of HIF-1α. Western blot analysis for the indicated proteins from HeLa, A549 and SAEC transfected with the indicated siRNA (40 nM) for 48 h prior to exposure to hypoxia for 24 h. E. siRNA-mediated depletion of HIF-1α but not HIF-2α reduces LIMD1 expression in both normoxia and hypoxia. qRT–PCR analysis of LIMD1 mRNA from HeLa cells transfected with the indicated siRNA (40 nM) and maintained in normoxia (20% O2) or exposed to hypoxia (1% O2) for 24 h. F–I. Knockdown of HIF-1α but not HIF-2α significantly reduces LIMD1 mRNA expression in both normoxia and hypoxia. qRT–PCR analysis of the indicated mRNA from cellular extracts in (B) and Fig 2C. J. LIMD1 endogenously complexes with PHD2, VHL, HIF-1α and HIF-2α. Western blot analysis of endogenous LIMD1 immunoprecipitated from A549 cells in either normoxia or following 24-h hypoxia. Data information: Unless otherwise stated, data shown are mean ± SEM, n = 3, *P < 0.05, **P < 0.01, according to Holm–Šidák post hoc tests, comparing siRNA treatment within each time-point, following significant main effects/interactions of a mixed-model ANOVA (A) or Holm–Šidák-corrected one-sample Student's t-tests (E to I; comparing means to the theoretical value of 1). See Appendix Table S7 for a summary of statistical analysis. Source data are available online for this figure. Download figure Download PowerPoint Figure 2. LIMD1 is a HIF-1-responsive gene A. HIF-1 binds to the LIMD1 promoter. Chromatin immunoprecipitation assay (ChIP) of endogenous HIF-1α from paraformaldehyde cross-linked U2OS cells exposed to 16-h hypoxia, followed by qRT–PCR analysis of the indicated gene promoters. B. EMSA of nuclear extracts from U2OS cells exposed to normoxia (lanes 1 and 5) or 16-h hypoxia identified that HIF-1α but not HIF-2α bound the LIMD1 HRE consensus sequence, causing a band supershift (ss). Wild-type unlabelled oligo probes that encompass the LIMD1 or PHD2 HRE were used as controls to compete out the ss, and probes where the HRE sequences have been mutated (mLIMD1/mPHD2) were used to show specificity for HRE binding. C. siRNA-mediated depletion of HIF-1α but not HIF-2α reduces LIMD1 expression in both normoxia and hypoxia. qRT–PCR anal