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Integration of CNS survival and differentiation by HIF2α

Hypoxia-inducible factor (HIF) 1α and HIF2α and the inhibitor of apoptosis survivin represent prominent markers of many human cancers. They are also widely expressed in various embryonic tissues, including the central nervous system; however, little is known about their functions in embryos. Here, we show that zebrafish HIF2α protects neural progenitor cells and neural differentiation processes by upregulating the survivin orthologues birc5a and birc5b during embryogenesis. Morpholino-mediated knockdown of hif2α reduced the transcription of birc5a and birc5b, induced p53-independent apoptosis and abrogated neural cell differentiation. Depletion of birc5a and birc5b recaptured the neural development defects that were observed in the hif2α morphants. The phenotypes induced by HIF2α depletion were largely rescued by ectopic birc5a and birc5b mRNAs, indicating that Birc5a and Birc5b act downstream of HIF2α. Chromatin immunoprecipitation assay revealed that HIF2α binds to birc5a and birc5b promoters directly to modulate their transcriptions. Knockdown of hif2α, birc5a or birc5b reduced the expression of the cdk inhibitors p27/cdkn1b and p57/cdkn1c and increased ccnd1/cyclin D1 transcription in the surviving neural progenitor cells. The reduction in elavl3/HuC expression and enhanced pcna, nestin, ascl1b and sox3 expression indicate that the surviving neural progenitor cells in hif2α morphants maintain a high proliferation rate without terminally differentiating. We propose that a subset of developmental defects attributed to HIF2α depletion is due in part to the loss of survivin activity.

Hypoxia-inducible factors (HIFs), including HIF1, HIF2 and HIF3, are known to function in oxygen homeostasis by regulating the genes responsible for glucose uptake and metabolism, erythropoiesis, angiogenesis, apoptosis and cell proliferation. 1 The HIFs are heterodimeric basic-helix-loophelix-PAS transcription factors that consist of an oxygenregulated a subunit and a constitutively expressed b subunit, which is also known as ARNT. 1 In normoxic conditions, both HIF1a and HIF2a are targeted for proteosomal degradation by prolyl hydroxylase and the von Hippel-Lindau (VHL) E3 ligase complex. 2 When cells are subjected to hypoxia, the HIF-a factors are stabilized and in turn associate with ARNT and activate target genes. 2 HIF3a lacks a conventional C-terminal transactivation domain and it is postulated to act as a negative regulator of hypoxia-inducible gene expression. 3 Despite their oxygen homeostatic functions in adult tissues, HIF-related pathways also have critical functions in embryos. Constitutive depletion of the mouse HIF1a gene (HIF1a À/À ) results in developmental arrest and embryonic lethality, which is characterized by neural tube defects, cardiovascular malformations, dysregulated erythropoiesis signaling, and marked cell death within the cephalic mesenchyme. [4][5][6] HIF2a null (HIF2a À/À ) embryos died as a result of inadequate blood vessel fusion, remodeling and impaired fetal lung maturation. 7,8 Inhibition of HIF2a expression also enhanced the generation of reactive oxygen species (ROS) and reduced transcription of primary anti-oxidant enzymes (AOEs), which in turn caused a syndrome of multiple-organ pathology. 9 Neural cell-specific depletion of HIF1a resulted in hydrocephalus accompanied by an increase in neuron cell apoptosis and vascular regression in the telencephalon of mutant mouse embryos. 10 Depending on the severity of hypoxia, hypoxic signals may induce different responses during cell death. For the proapoptotic pathway, HIF1a conspires with p53 and/or BNIP3 to promote apoptosis. 11,12 However, hypoxia can also induce an anti-apoptotic response by increasing the expression of the anti-apoptotic protein IAP2 and suppressing the expression of the pro-apoptotic protein Bax through a HIF1a-independent mechanism. 13 A recent study has shown that HIF2a may be involved in the anti-apoptotic properties of tumor cells. Inhibition of HIF2a promoted p53 activity and induced tumor cell death by disturbing cellular redox homeostasis and promoting the accumulation of ROS. 14 Survivin (Birc5) is the smallest member of the inhibitor of apoptosis proteins (IAPs) and contains a single baculovirus IAP repeat (BIR) domain and an extended C-terminal a-helical coiled-coil domain. 15 In addition to inhibiting apoptosis, survivin also has important functions in cell mitosis and cellcycle progression. 16 The mammalian survivin protein is widely expressed in embryonic cells, especially in neural progenitor cells, but it is barely detectable in quiescent adult cells. 17 During brain development, survivin is highly expressed in neural progenitor cells. Targeted deletion of survivin in neural precursor cells leads to massive apoptosis in the central nervous system (CNS) due to elevated caspase-3 and caspase-9 activities. 18 Interestingly, survivin is widely expressed in all kinds of malignant tumors, making it a potent target for cancer therapy. 15,19 There are multiple HIFa factors, including HIF1a, HIF2a and HIF3a, in zebrafish that are widely expressed during development. 20,21 Concurrent knockdown of these three factors leads to neural cell death and abrogates neuronal development, indicating that these HIF-a factors have critical roles in neural cell survival and differentiation. 20 Nevertheless, the authentic HIF-a factor responsible for the fates of CNS neuronal progenitor cells (NPCs) remains to be elucidated. Here, we demonstrate that of the three HIF-a factors, HIF2a has a major role in maintaining cell survival and promotes neural progenitor cell differentiation. HIF2a depletion caused massive cell death and abrogated neural cell differentiation due to aberrant expression of the survivin homologs (birc5a and birc5b). The surviving NPCs remained in the cell cycle. The defects that appeared in hif2a morphant embryos were rescued by ectopic injection of the birc5a or birc5b mRNA, suggesting that survivins act downstream of HIF2a to protect neural progenitor cells and promote neural differentiation. Chromatin immunoprecipitation assay revealed that HIF2a binds to both birc5a and birc5b promoters directly to modulate their transcriptions.

Results
HIF2a knockdown induces p53-independent apoptosis. There are multiple HIF-a factors, including HIF1a, HIF2a and HIF3a, in zebrafish that are widely expressed during development. 20,21 Concurrent knockdown of these three factors leads to neural cell death and abrogates neuronal development. 20 To clarify the particular HIF a factor that determines the fates of zebrafish CNS NPCs, we analyzed apoptotic events in individual hifa morphant embryos. We found that knockdown of hif2a by either of two distinct antisense morpholinos resulted in massive apoptosis at the 24-and 48-h post-fertilization (h.p.f.) stage (Figures 1a-c, gh and t). Conversely, knockdown of hif1a and hif3a either individually or concurrently did not increase the number of apoptotic cells (Figures 1d-f, i and t), indicating that HIF2a has a unique function in protecting embryonic cells against apoptosis.
In addition to the massive apoptosis, a morphological abnormality with small and acellular head was observed in hif2a morphants at the 48-h.p.f. stage (Figure 1h). During early stage of development, massive apoptosis was elicited by hif2a morpholino at the 12-h.p.f. stage before any morphological defect observed (Figures 1j and k). Moreover, the pcna expression in the anterior proliferative region was diminished at the 12-h.p.f. stage, possibly due to the considerable cell death (Figures 1l and m). It suggests that the gross developmental abnormalities observed in hif2a morphants is possibly caused by extensive cell death occurred in the early stage of development.
A similar anti-apoptotic function has been observed for the human HIF2a protein. siRNA-mediated knockdown of HIF2a promoted p53 activity in human tumor cells and induced cell death by disturbing the cellular redox homeostasis and prompting the accumulation of ROS. 14 To examine whether the hif2a morpholino-induced apoptosis in zebrafish embryos is mediated by p53 activity, we concurrently knocked down p53 and hif2a using translational morpholinos. Our results show that the depletion of p53 expression did not block the massive apoptosis induced by treatment with hif2a morpholinos (Figures 1n and o). Similarly, severe apoptosis was observed in p53 M214K mutant embryos after injection with a hif2a translation-blocking MO (Figures 1p and q), indicating that the HIF2a depletion-induced apoptosis is not an off-target effect of the morpholino and that it is mediated through a p53-independent pathway.
To verify whether the apoptotic cells observed in hif2a morphants are undifferentiated NPCs, embryos were first in situ hybridized with fluorescence nestin (nes) RNA probe followed by TUNEL assay. We found that most of apoptotic cells were colocalized with nes-positive cells (as labeled yellow in Figures 1r and s), indicating that the apoptotic cells in hif2a morphants are indeed NPCs.
Knockdown of hif2a abrogates CNS differentiation. We next examined the fate of the surviving NPCs in the hifa morphant embryos using the terminal differentiation marker elavl3. Knockdown of hif2a caused a dramatic reduction in  (Figures 4j-o). The colocalization of these two gene expression in the CNS of hif2a morphants (yellow in Figure 4o) suggesting that most of NPCs in these hif2a morphants were hyperproliferating cells.  Survivins function downstream of HIF2a to protect neural progenitor cells and promote neural differentiation. Survivin is the smallest member of the IAP family and has multiple functions in cell survival, mitosis and proliferation. Knockdown of the zebrafish survivin orthologues birc5a and birc5b causes profound neurodevelopmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. 22 Together, these results suggest that survivin functions in embryonic development. In various human cancer cells, survivin/BIRC5 transcription is upregulated as a result of increases in HIF1a. 23 This raises the possibility that the survivin orthologues in zebrafish are also affected by one of the HIF factors. Here, we analyzed the transcriptional activities of the survivin orthologues birc5a and birc5b in various hifa morphant embryos. In wild-type embryos, the survivin orthologues birc5a and birc5b and hif2a were all transcribed intensively in the CNS region (Figures 5a-c). Depletion of HIF2a resulted in a reduction in the transcription of both birc5a and birc5b (Figures 5d, e, h and i), indicating that hif2a correlates with survivin expression. Conversely, concurrent depletion of hif1a and hif3a did not affect the transcription of either birc5a or birc5b (Figures 5f-i). This suggests that zebrafish survivin orthologues are affected specifically by HIF2a but not by other HIF-a factors. To determine whether the survivin deficiency accounts for the HIF2a depletion-induced NPC apoptosis and aberrant neural differentiation, we examined NPC apoptosis and neural differentiation in birc5a and birc5b morphant embryos. Knockdown of either birc5a or birc5b caused considerable increases in apoptosis and morphological abnormality, which were similar to that observed in hif2a morphant embryos (Figures 6a, b, g, h, 7a-c and 8). Additionally, the birc5a and birc5b morphants exhibited much lower levels of elavl3 transcription compared with WT controls (Figures 6c, e and i-l). Likewise, birc5a and birc5b morphants exhibited higher levels of nes and pcna transcription compared with controls (Figures 9a-i). Moreover, the birc5a and birc5b morphant embryos had  9j-r). Together, these observations indicate that knockdown of either birc5a or birc5b results in the induction of NPC apoptosis and the inhibition of cell-cycle exit in the NPCs, which is similar to the phenotype observed in the hif2a morphant embryos. Moreover, ectopic treatment with the birc5a or birc5b mRNA not only successfully prevented hif2a MO-induced apoptosis and morphological abnormality (Figures 6m, n, 7d and 8), but also restored elavl3 expression to a nearly normal level (Figures 6o-r), indicating that the survivin orthologues indeed rescue the impairment in NPC development that is caused by HIF2a deficiency.
HIF2a binds to birc5a and birc5b promoters. Since depletion of HIF2a resulted in a reduction in the transcription of both birc5a and birc5b, it raises a possibility that HIF2a indeed controls the survivin orthologues through direct interactions with their promoters. We retrieved the upstream sequence of both bric5a and birc5b genes from UCSC genome website (http://genome.ucsc.edu/) and identified multiple hypoxia-response element (HRE) core sequences (A/GCGTG) in the promoter regions (Figure 10c). Similar multiple HRE core sequences were also identified in human and mouse BIRC5 promoters (Figure 10c). To determine if zebrafish HIF2a binds to the HRE specifically, oligonucleotides containing the HRE core sequence ACGTG were synthesized, biotin labeled and electrophoretic mobility shift assay (EMSA) was carried out (Figure 10a). When HIF2a was expressed alone or it was coexpressed with ARNT1a in COS-1 cells, a mobility complex was formed with wild-type HRE probe (lanes 3 and 4). This mobility complex was effectively competed by a 200-fold molar excess of the unlabeled HRE (lane 5). Conversely, the mobility complex was, however, unaffected by a 200-fold molar excess of the unlabeled mutated HRE (lane 6). These observations demonstrated the transfected HIF2a binds to HRE with a sequence specificity.
To examine whether HIF2a binds directly to birc5a and bric5b promoter in vivo, we immunoprecipitated crosslinked chromatin from adult brain tissue with a HIF2a-specific antibody. The precipitated chromatin was then analyzed by PCR using primer pairs that amplified modules spanning the birc5a and bric5b loci. The ChIP assay demonstrated that the module 5 of birc5a and modules 2 and 6 of birc5b were significantly enriched by the HIF2a antibody (Figures 10b  and c), suggesting that HIF2a binds directly to both birc5a and bric5b promoters to modulate their expressions.

Discussion
HIFa and survivin are prominent markers in many human cancers and have critical roles in cell apoptosis and proliferation. Previously, we found that depletion of all of the HIF a factors caused massive apoptosis in the brains of zebrafish embryos. 20 Here, we showed that of the three HIF-a factors, HIF2a is the factor that modulates neural progenitor cell growth and differentiation through its downstream effects on the survivin orthologues, Birc5a and Birc5b. Depletion of HIF2a expression caused reductions in both of the birc5a and birc5b transcripts, which in turn elicited massive apoptosis and abrogated neural progenitor cell differentiation. Although human survivin (BIRC5) and   (birc5a and birc5b) promoters are all enriched with multiple HREs, these survivin genes are modulated by different HIF a factors. In human cancer cells, BIRC5 transcription is modulated by HIF1a through its direct binding to the HRE in the proximate promoter region. 23 In zebrafish, we demonstrate that both birc5a and birc5b transcriptions are controlled by HIF2a, but not HIF1a. It is noteworthy that although zebrafish HIF1a does not involve in NPC growth and differentiation, it still have an essential role in other developmental events. Depletion of HIF1a expression abrogated erythropoiesis and reduced hemoglobin content (data not shown). It suggests that the sets of HIF1a-and HIF2a-modulated genes in human and zebrafish are slightly different due to the discriminative target selectivity of HIF1a and HIF2a factors cross species.  ATG-MO co-injected with 50 pg birc5a mRNA (n ¼ 167) and in the hif2a ATG-MO embryos co-injected with 50 pg birc5b mRNA (n ¼ 262). Ectopic injection of either birc5a or birc5b mRNA restored most hif2a morphants to a normal phenotype whereas birc5a mRNA had better rescuing capability It has been shown that HIFs have important roles in embryonic cell differentiation and proliferation. 24 Hypoxic preconditioning promotes embryonic stem cell neural differentiation and enhances cell survival through upregulating HIF1a and HIF2a. 25 However, the functions of these HIFs in cell differentiation still have not been elucidated. Here, we demonstrate that HIF2a depletion first causes massive NPC apoptosis and then stimulates the remaining NPCs proliferation. Nevertheless, these newly produced NPCs did not undergo final differentiation. Ectopic treatment with the birc5a or birc5b mRNA not only successfully prevented hif2a MO-induced apoptosis and morphological abnormality, but also restored neural differentiation. It suggests that the abrogation of differentiation is a secondary effect elicited by HIF2a depletion due to the loss of its downstream effectors, Birc5a and Birc5b.
Previous studies have demonstrated a close relationship between the hypoxia-signaling pathway and cell-cycle arrest in mammalian cells. For instance, either overexpression of the stable forms of HIF1a and HIF2a in murine cells or treatment of murine cells with hypoxia directly resulted in an increase in p27 transcription and cell-cycle arrest at the G1 phase. 26,27 Moreover, targeted depletion of HIF1a in chondrocytes resulted in decreased p57 expression, enhanced BrdU incorporation and cell death. 28 We demonstrate that the loss of survivin could be responsible for the reductions in p27 and p57 expression and the aberrant neural development that is observed in the hif2a morphant zebrafish embryos. Survivin also had a similar effect on cell differentiation in mouse erythroid development. Depletion of survivin from hematopoietic progenitor cells results in aberrant erythroid formation. 29 Likewise, earlier studies have demonstrated that depletion of zebrafish birc5a and birc5b transcription evoked profound neuro-developmental, hematopoietic, cardiogenic, vasculogenic and angiogenic defects. 22 In Drosophila, dying cells trigger compensatory proliferation through both initiator and effector caspase activities. 30 In apoptotic proliferating tissues, the initiator caspase Dronc coordinates apoptosis and compensatory proliferation through the Jun N-terminal kinase and p53 pathways, which in turn induce secretion of the mitogens Decapentaplegic (Dpp) and Wingless (Wg). 30,31 In apoptotic differentiating tissues, the effector caspases DrICE and Dcp-1 activate the hedgehog pathway to induce compensatory proliferation. 32 Hedgehog signaling triggers the reentry of cells that had previously exited the cell cycle. 32,33 In zebrafish embryos, knockdown of hif2a resulted in the loss of expression of the survivin orthologues, which in turn caused severe neural progenitor cell death, increased proliferation activity and abrogated neural differentiation. This phenomenon of cell death-associated proliferation and abrogation of differentiation in zebrafish embryos is analogous to what has been observed in Drosophila apoptosis-induced compensatory proliferation. We hypothesize that reentry into the cell cycle causes descendant cells to remain in the progenitor stage without further differentiating. These findings suggest that a key function of HIF2a in developing embryos is related to inhibition of neural progenitor cell death and maintenance of neural differentiation through the functions of downstream survivin orthologues (Figure 11). Depletion of HIF2a and the survivin orthologues causes massive neural progenitor cell death, which induces compensatory proliferation and abrogates neural differentiation.
It has been demonstrated that inhibition of human HIF2a promotes tumor cell death through p53 activation. HIF2a depletion promotes p53-mediated responses in clear cell renal cell carcinoma (ccRCC) by disrupting cellular redox homeostasis, which thereby permits ROS accumulation and DNA damage. 14 Although a similar level of p53 accumulation Figure 10 HIF2a binds to birc5a and birc5b promoter. (a) HIF2a binds to HRE as confirmed by EMSA. COS-1 cells were transfected with zHif2A-HA and zARNT1A-HA as indicated. Nuclear extracts were collected at 48 h post-transfection followed by gel mobility shift assays by using the HRE oligonucleotides as a probe. The asterisks in lanes 3, 4 and 6 indicate the specific HIF2-HRE complex formed. NE, nuclear extract; COS-1, nuclear extract of COS-1 cells without transfection as a control; Hif2A, nuclear extract of COS-1 cells transfected with pcDNA3-zHif2A-HA; Hif2A þ zARNT1A, nuclear extract of COS-1 cells co-transfected with pcDNA3-zHif2A-HA and pcDNA3-zARNT1A-HA. (b) HIF2a binds to the birc5a and birc5b promoters in brain tissues under physiological conditions. A chromatin immunoprecipitation (ChIP) assay was performed with polyclonal antibody against anti-HIF2a or preimmune serum as control. Chromatin was prepared from adult brain tissue and then immunoprecipitated using polyclonal antibody against anti-HIF2a or preimmune serum (IgG) as control. The immunoprecipitates were subjected to PCR using specific primers spanning the HREs in the birc5a and birc5b promoters (shown in panel c). The module 5 of birc5a and modules 2 and 6 of birc5b were significantly enriched by the HIF2a antibody. The rest modules failed to be amplified. A portion of chromatin solution (2.4%) was used as input. The primers used in ChIP assay were listed in Table 1. (c) Schematic representation of the birc5a (NM_194397), birc5b (NM_145195), human BIRC5 (NM_001012271) and mouse Birc5 (NM_001012273) promoters. The putative binding sites for HIF2, including HRE1 (ACGTG, black bars) and HRE2 (GCGTG, gray bars), are indicated. The position of the DNA modules analyzed in ChIP-PCR assays is indicated by the boxes. The amplified fragments in ChIP assays (panel b), including the module 5 in birc5a promoter and the modules 2 and 6 in birc5b promoter, are indicated by black boxes was also observed in hif2a morphant embryos (data not shown), we demonstrated that inhibition of p53 expression did not prevent massive apoptosis in hif2a morphant embryos. Similarly, HIF2a inhibition-induced massive apoptosis was also observed in the p53 mutant line. Instead, we demonstrated that the massive apoptosis observed in hif2a morphant embryos is caused by depletion of the survivin orthologues. Ectopic treatment with survivin mRNAs rescued the defects induced by HIF2a inhibition, indicating that survivins act downstream of HIF2a to maintain cell survival. It is noteworthy that expression of survivin/BIRC5 in ccRCC was inversely correlated with cancer-specific survival. 34 It will be interesting to examine the correlation between HIF2a and survivin expression in ccRCC patients. Our results have shed light on how cancer cells protect themselves through the HIF and survivin pathways.
RT-qPCR. Total RNA extracted from embryos using TRIzol (Invitrogen, Carlsbad, CA, USA) followed by DNase I treatment (Roche) was used for cDNA synthesis with Superscript-II (Invitrogen). Quantitative (q)PCR was performed using the Power SIBER Q-PCR Master Mix (BioNoVas, Toronto, Canada) in a Bio-Rad, iQ5 Gradient Real Time PCR System (Bio-Rad, Hercules, CA, USA). qPCR conditions were 951C for 3 min and then 40 cycles of 951C for 15 s and 601C for 1 min. The following primers were designed using the Primer Express Software  Duplicate mean values were calculated according to the Ct quantification method using b-actin transcript levels as the reference for normalization. Relative quantification was determined using the Ä Ä Ct method: relative expression ¼ 2 ÀDDCt .
Apoptosis detection. Zebrafish embryos at 24 h.p.f. were dechorionated and incubated for 30 min in 5 mg/ml acridine orange in E3 medium (5 mM NaCl, 0.17 mM KCl, 0.33 mM CaCl 2 and 0.33 mM MgSO 4 ). The embryos were subsequently washed repeatedly in E3 medium. For microscopic examination, embryos were transferred into 0.01% ethyl 3-aminobenzoate methanesulfonate (tricaine, Sigma). TUNEL assays were performed using an in situ cell death detection kit (POD, Roche) as described by the manufacturer. For combined fluorescent nes DIG in situ/ TUNEL staining, embryos were first in situ hybridized with fluorescence nes RNA probe followed by TUNEL assay. 39 The protocol of fluorescence in situ hybridization is as described above. The RNA probe was labeled by DIG. After conjugated with HRP anti-DIG antibody (Perkin-Elmer), the signal was detected by TSA Plus Fluorescein Solution (Perkin-Elmer). This was followed by TUNEL assay. The embryos were manually de-yolked and the images were captured on a Zeiss LSM 510 confocal microscope.
Chromatin immunoprecipitation. ChIP was performed by using Magna ChIP Protein A magnetic beads (Millipore, Billerica, MA, USA) according to the manufacturer's instructions. Briefly, adult brain tissues were crosslinked with formaldehyde and chromatin was isolated. The isolated chromatin was sonicated to an average size of about 200-300 bp. Protein A magnetic beads were incubated with antibody against HIF2a (ChIP grade rabbit polyclonal antibody purchased from GeneTex (Irvine, CA, USA), GTX103707) or control IgG at 41C for 1 h. Immunoprecipitation reactions were performed by incubating magnetic beadslinked antibody with the chromatin overnight at 41C. The immunoprecipitated chromatin complexes were washed with TE buffer and the protein-DNA crosslinks were reverse crosslinked by proteinase K digestion at 621C for 2 h followed by 951C for 10 min. The DNA was purified by spin columns and then eluted with elution buffer. Immunoprecipitated DNA and input DNA were used as templates for PCR amplification (30 cycles). The PCR primers used in ChIP assay are listed in Table 1.
Electrophoretic mobility shift assay. After COS-1 cells were transfected with the indicated plasmids for 48 h, nuclear extracts were prepared by minipreparation method as described previously. 40 The nuclear extracts were preserved at À701C until use. Oligonucleotide probe containing the HRE core sequence (5 0 -CATCGTCCTTCAAACGTGTAACTAAGTTCA-3 0 ) and mutant probe (5 0 -CATCGTCCTTCAAGTACATAACTAAGTTCA-3 0 ) was synthesized and labeled with 5 0 -end biotin (Purigo, Taipei, Taiwan). For each EMSA reaction, 20 mg of nuclear extracts from normal or transfected COS-1 cells was mixed with biotinlabeled probes and premixed binding reagents (Thermo, Waltham, MA, USA) and incubated at room temperature for 30 min. For competitive experiments, 50 Â normal and mutant probes were added. Unbound DNA probes were resolved from protein-DNA complexes by electrophoresis on a 10% polyacrylamide gel. After electrophoresis, DNA oligonucleotides were transferred onto a nylon membrane (Pall, Washington, NY, USA) and UV crosslinked. Biotin-labeled DNAs were detected by using the Lightshift Chemiluminescent EMSA Kit according to the manufacturer's instructions (Thermo).