Cloning and Characterization of LUN, a Novel RING Finger Protein That Is Highly Expressed in Lung and Specifically Binds to a Palindromic Sequence*

Dong ChuDagger , Naoki Kakazu§, Manuel J. Gorrin-Rivas, Hai-Ping Lu||, Mitsuhiro Kawata||, Tatsuo Abe§, Kunihiro UedaDagger , and Yoshifumi AdachiDagger **

From the Dagger  Laboratory of Molecular Clinical Chemistry, Institute for Chemical Research, Kyoto University, Gokasho, Uji, Kyoto 611-0011, Japan, the § Department of Hygiene, Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan,  Department of Surgery and Surgical Basic Science, Graduate School of Medicine, Kyoto University, Sakyo-ku, Kyoto 606-8507, Japan, and the || Department of Anatomy Kyoto Prefectural University of Medicine, Kamigyo-ku, Kyoto 602-8566, Japan

Received for publication, November 10, 2000, and in revised form, January 19, 2001




    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

We isolated cDNAs encoding a novel RING finger protein (LUN), the mRNAs of which were expressed at high levels in the lung. In situ hybridization revealed that LUN mRNAs were expressed in the alveolar epithelium of the lung. The LUN gene locus was assigned to chromosome 9p21, which contains candidate tumor suppressor genes associated with loss of heterozygosity in more than 86% of small cell lung cancers. We clarified that LUN is localized to the nucleus and reveals Zn2+-dependent DNA binding activity. The region from amino acids 51 to 374 of LUN is responsible for DNA binding. Furthermore, we identified a novel palindromic binding consensus (5'-TCCCAGCACTTTGGGA-3') for the LUN binding. Interestingly, this LUN binding palindromic sequence is found in the upstream transcriptional regulatory region of the E-cadherin gene and two intervening regions of the talin gene. Our results suggested that LUN might be an important trans-acting transcriptional regulator for lung cancer-associated genes including E-cadherin and talin genes.




    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

The RING1 finger motif is one example of a Zn2+ binding domain that is found in proteins from viruses to vertebrates and defines a superfamily of diverse proteins (1, 2). The motif itself is distinct from classic zinc finger motifs in terms of sequence homology, Zn2+ binding scheme, and three-dimensional structure (3-5). The RING finger motif appears to mediate both protein-DNA (6-8) and protein-protein interactions (2) and, in some cases, E2-dependent ubiquitination (9, 10). The RING finger proteins are thus involved in a variety of fundamental cellular roles such as gene regulation, oncogenesis, viral pathogenicity, embryogenesis, V(D/J) recombination, DNA repair, and signal transduction (1, 2).

There are a number of RING finger proteins with oncogenic potential, including the protein encoded by the breast cancer susceptibility gene BRCA1 (11), an acute promyelocytic leukemia-associated protein (PML) (12, 13), a RET finger protein (RFP) (14), c-Cbl and Bmi-1 proto-oncoproteins (15-17), and Mel-18, a nuclear DNA-binding protein isolated from melanomas (6). Among them, Mel-18 and Bmi-1 are highly homologous human oncogene products (18). Mel-18 possesses a tumor suppressor activity by acting as a negative regulator of transcription and is found in all tumor cells at increased levels (6, 7). Bmi-1 is morphogenic during embryonic development and hematopoiesis and cooperates with c-Myc in oncogenesis (16, 17). PML and RET finger protein (RFP) become oncogenic when found as fusion proteins resulting from chromosomal translocations (12-14). PML is found in a form fused to the retinoic acid receptor alpha  in patients with acute promyelocytic leukemia, and RET is comprised of the RET (RFP) finger protein fused to a tyrosine kinase domain. Human c-Cbl becomes oncogenic after deletion of the C-terminal region including the RING finger motif, which links the loss of a functional RING finger with tumorigenesis (15). Two predisposing mutations in BRCA1 result in deletion of the RING finger domain and a point mutation in one of the RING finger Zn2+ ligands (11, 19). Again, this links the loss of a functional RING finger with tumorigenesis.

In industrialized countries, lung cancer is a leading cause of cancer death. One of our research interests is to identify important RING finger family genes including oncogenes and tumor suppressor genes in lung alveolar cells. In this study, we employed the PCR approach and degenerate primers corresponding to the RING finger motif. Here, we describe the isolation and characterization of LUN, a new member of the RING finger proteins family that might be associated with tumorigenesis and/or tumor suppression. LUN mRNAs are expressed at high levels in alveolar epithelium of the lung. The LUN gene is localized to chromosome 9p21, which contains candidate tumor suppressor genes associated with loss of heterozygosity in more than 86% of small cell lung cancer. LUN is a 119-kDa nuclear protein and shows Zn2+-dependent and sequence-specific DNA binding ability. A novel palindromic binding consensus sequence for LUN51-374 was found in the upstream transcriptional regulatory region of the E-cadherin gene and introns of the talin gene. Our findings provide an important clue in the further study of the function of LUN and the mechanism of its involvement in tumorigenesis in small cell lung cancer (SCLC).


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of the LUN cDNA-- First-strand cDNA was prepared from human lung poly(A)+ RNA (CLONTECH). This cDNA was then used as a template for PCR with an oligo(dT) primer and two degenerate primers (5'-TG(T/C) CTI CA(T/C) TCI TT(T/C) TGC-3' and 5'-TG(T/C) TT(A/G) CA(T/C) AG(T/C) TT(T/C) TGC-3') homologous to an amino acid sequence (CLHSFC) that is conserved among several members of the RING finger family. The PCR products were hybridized with a second degenerate oligonucleotide (5'-CA(T/C) TCI TT(T/C) TGI AA(A/G) TCI TGC-3') homologous to an overlapping region of the RING finger domain (HSFCKSC). DNA from random positive clones was sequenced and compared with the GenBankTM data base. Two of the clones contained partial cDNAs for a novel RING finger protein, which we named LUN. The full-length human LUN cDNA was generated by 5'-rapid amplification of cDNA ends using a Marathon cDNA amplification kit (CLONTECH). The cDNA was reverse-transcribed from poly(A)+ mRNA from human lung, brain, the glioma cell line T98 or T-cell line Molt-4 using the cDNA synthesis primer provided with the kit. The first round of PCR was carried out using an adapter primer 1 and gene-specific reverse primer (5'-TAACTCGAGCACCAGCACGATAAAG-3'). The second round of PCR was performed using the first round gene-specific PCR amplification product as the template, adapter primer 2, and a nested gene-specific primer (5'-CAAAGATCTTTCATCTGCCGTAGTTG-3'). The cDNA amplified by 5'-rapid amplification of cDNA ends was then subcloned and sequenced.

Fluorescence in Situ Hybridization (FISH)-- Replication G-banded chromosomes were prepared from the phytohemagglutinin-stimulated lymphocytes from a normal male donor using the thymidine synchronization/bromodeoxyuridine release technique (20). Just before hybridization, the chromosome slides were denatured with 70% formamide in 2× sodium saline citrate at 70 °C for 2 min. The 3549-bp fragment (corresponding to nt 270-3818 of the LUN cDNA) was labeled with biotin-16-dUTP (Roche Molecular Biochemicals) by nick translation. The probes were denatured at 75 °C for 10 min and then mixed with an equal volume of hybridization buffer (20% dextran sulfate in 4× sodium saline citrate). The hybridization mixture was placed on the denatured chromosome slides. After overnight hybridization, signal detection was achieved in three amplification steps, one with avidin-fluorescein isothiocyanate (Vector), one with biotinylated anti-avidin D (Vector), and one with avidin-fluorescein isothiocyanate. Chromosomes were counter-stained with propidium iodide. FISH signals and the replicate G-bands for the same metaphase were detected under fluorescence microscopy (Nikon) through a B-2A filter and a UV-2A filter, respectively, and photographed separately. The precise assignment of the LUN gene to chromosomal bands was achieved by superimposing FISH signals over the G-banded chromosomes (21).

Northern Blotting Analysis-- Northern blots containing 2 µg of poly(A)+ RNA from several different adult human tissues (Invitrogen) were probed for expression of LUN mRNA as previously described (22). The probe, a 3549-bp fragment (corresponding to nt 270-3818 of the LUN cDNA), was random-primed with [alpha -32P]dCTP (220 TBq/mmol; Amersham Pharmacia Biotech) using a Megaprime random primer labeling kit (Amersham Pharmacia Biotech). Hybridization was performed at 42 °C with the radiolabeled LUN cDNA probe. After hybridization, the blots were washed at 65 °C for 10 min with 2× sodium saline citrate containing 0.1% SDS and for 30 min with 0.1× sodium saline citrate containing 0.1% SDS. The probed blots were subjected to autoradiography with intensifying screens at -80 °C.

In Situ Hybridization-- To generate probes for in situ hybridization, we cloned a 236-bp BamHI fragment (nt 1-236) and a 588-bp BamHI fragment (nt 270-857) of the LUN cDNA into the pBluescript II KS(-) vector (Stratagene), respectively. The plasmids were linearized with EcoRI and transcribed with T7 polymerase to yield an antisense RNA probe or linearized with NotI and transcribed with T3 polymerase to yield a sense RNA probe. Transcription reactions included digoxigenin-UTP (Roche Molecular Biochemicals) as a label. A human multi-tissue set was purchased from Novagen. Hybridization and high stringency washes were carried out according to established procedures (23). After deparaffinization and acetylation, digoxigenin-labeled probe was hybridized at a final concentration of 0.5 µg/ml. After the final high stringency wash, slides were washed successively in digoxigenin buffer (100 mM Tris-HCl, 150 mM NaCl, pH 7.5) and digoxigenin buffer containing 1.5% blocking reagent and reacted with alkaline phosphatase-conjugated anti-digoxigenin (1:500 dilution; Roche Molecular Biochemicals) in the dark. The slides were then washed in digoxigenin buffer and alkaline phosphatase buffer (100 mM Tris-HCl, 100 mM NaCl, 50 mM MgCl2, pH 9.0). The colorimetric reaction product was developed at room temperature for 18 h with 50 µg/ml 5-bromo-4-chloro-3-indolyl phosphate and 75 µg/ml nitro blue tetrazolium in alkaline phosphatase buffer.

DNA Transfection-- The BamHI to EcoRI fragment of the cDNA encoding LUN51-1045 was ligated into the BglII and EcoRI sites of the mammalian expression vector pEGFP-N1 (CLONTECH), which allows in-frame fusion with enhanced Aequorea victoria green fluorescent protein (GFP) under the control of cytomegalovirus immediate early promoter and contains SV40 polyadenylation signals (referred to as pGFP-LUN51-1045). HeLa cells were seeded on chamber slides and incubated for 16 h. Cells were then washed with Dulbecco's modified Eagle's medium twice and transfected with 1.4 µg of plasmid DNA by LipofectAMINE (Life Technologies)-mediated transfection in Dulbecco's modified Eagle's medium. A 3-fold volume of 15% fetal bovine serum in Dulbecco's modified Eagle's medium was added 3 h later. The transfection mixture was removed 21 h later, and the cells were maintained in Dulbecco's modified Eagle's medium containing 10% fetal bovine serum for an additional 24 h. At 48 h after transfection, confocal imaging analysis was performed using a Zeiss LSM510 laser-scanning microscope. For GFP visualization, an fluorescein isothiocyanate filter set was used.

Expression and Purification of Glutathione S-Transferase (GST) Fusion Proteins-- Segments of the LUN cDNA encoding aa 51-1045, and aa 51-374 were cloned into the BamHI and EcoRI/SmaI sites of the vector pGEX-2T (Amersham Pharmacia Biotech) to generate the plasmids pGST-LUN51-1045 and pGST-LUN51-374, respectively. Each plasmid produces an in-frame fusion of GST with LUN51-1045 or LUN51-374 (containing RING finger and leucine zipper coiled-coil domains). For isolation of GST fusion proteins, transformed bacteria were grown to early log phase and induced for 3 h with 0.1 mM isopropyl-1-thio-beta -galactoside. The resuspended bacterial pellet was then sonicated in lysis buffer (25 mM Tris-HCl, 1% Triton X-100, 1 mg/ml lysozyme, 5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, and 50 µM p-amidinophenylmethanesulfonyl fluoride hydrochloride, pH 8.0). The supernatant was then incubated with glutathione-Sepharose 4B beads (Amersham Pharmacia Biotech) for 12 h at 4 °C and washed with phosphate-buffered saline three times. Finally, the GST fusion proteins were eluted with elution buffer (50 mM Tris-HCl, 10 mM glutathione, pH 8.0). The GST fusion proteins were radiolabeled at their N-terminal amino group and epsilon -amino group of lysine residues using N-succinimidyl-3-(4-hydroxy-3,5-di[125I]iodophenyl) propionate (163 TBq/mmol; PerkinElmer Life Sciences) according to the procedure recommended by the manufacturer. The specific activities of 125I-labeled proteins were adjusted to ~3 × 105 cpm/pmol.

DNA Binding Assay-- Each 125I-labeled GST fusion protein (6 × 105 cpm, 2 pmol) was diluted in 600 µl of binding buffer A (10 mM HEPES-NaOH, 50 mM KCl, 1 mM 2-mercaptoethanol, 20% glycerol, pH 7.1) and then incubated with 15 µl of calf thymus double-stranded DNA-cellulose or single-stranded DNA-cellulose (Sigma) in the presence of 1 mM ZnCl2, 1 mM MgCl2, 1 mM CaCl2, 1 mM MnCl2, and/or 1 mM EDTA alone or in combination at 4 °C for 1 h with gentle shaking. After centrifugation, the pellets were washed three times with the same binding buffer A. The remaining radioactivity on the DNA-cellulose was measured by gamma -counting.

Construction of Genomic Library and Screening for LUN Binding Sequences-- Human genomic DNA was digested to completion with MboI. The resultant mixture of digestion products was ligated into the BamHI site of the plasmid pUC118 followed by electroporation into competent DH5alpha bacteria, which were then grown in mass liquid culture. Plasmid DNA was then extracted from the cultured bacteria and used for screening. The primary library, taken for the first round of screening, contained 5.9 × 106 clones, 80% of which contained genomic DNA inserts (data not shown). For screening, 20 µg of plasmid DNA from the library was mixed with GST-bound glutathione-Sepharose beads in binding buffer B (10 mM HEPES-NaOH, 50 mM KCl, 1 mM ZnCl2, 1 mM MgCl2, 1 mM 2-mercaptoethanol, 20% glycerol, pH 7.1). After incubation for 1 h at 4 °C, unbound DNA was recovered by centrifugation. Then, GST-LUN51-1045- or GST-LUN51-374-bound glutathione-Sepharose beads were added and incubated for 1 h at 4 °C. The beads were then washed five times with washing buffer (identical to the above binding buffer B except for 2% glycerol). The DNA was released from the beads by incubation for 10 min at 45 °C in a solution containing 1% SDS and used to transform DH5alpha . This screening was repeated twice to generate a more enriched library. After the third screening, 72 colonies were picked up, and plasmid DNA was extracted from each colony. The inserted genomic fragments were purified and sequenced.

Gel Mobility Shift Assay-- For preparation of a DNA probe, the synthetic oligonucleotide 5'-CCTGTAATCCCAGCACTTTGGGAGGCTGAGG-3' and its complementary oligonucleotide were end-labeled with T4 polynucleotide kinase in the presence of [gamma -32P]ATP (111 TBq/mmol; PerkinElmer Life Sciences), annealed, and purified with MicroSpin G-25 columns (Amersham Pharmacia Biotech). Each reaction mixture contained 8 pmol of 32P-labeled probe (~4 × 104 cpm) and 8 pmol of GST fusion protein in binding buffer C (10 mM HEPES-NaOH, 50 mM KCl, 1 mM ZnCl2, 1 mM MgCl2, 1 mM 2-mercaptoethanol, 20% glycerol, 5 µg/ml aprotinin, 0.5 µg/ml leupeptin, 0.5 µg/ml pepstatin, 50 µM p-amidinophenylmethanesulfonyl fluoride hydrochloride, pH 7.1) in a total volume of 80 µl. For competition assay, the binding reaction was carried out in the presence of a 200-fold molar excess of unlabeled probe added simultaneously with the labeled probe to the reaction mixture. For all assays, the protein was added last, and the reaction mixture was incubated at room temperature for 30 min. The reaction mixtures were resolved by nondenaturing electrophoresis through 5% polyacrylamide gels in running buffer (45 mM Tris borate, pH 7.8). Gels were dried and analyzed with a BAS2000 imaging analyzer (FUJI Film).


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Cloning of LUN cDNAs-- We sought to identify novel proteins of the RING finger family expressed in the lung. We used a PCR strategy with degenerate oligonucleotides to isolate partial cDNA clones of RING finger proteins using human lung cDNA as a template. Two of the partial cDNA clones isolated in this fashion encoded a novel RING finger protein, which we designated as LUN based on its unique expression pattern (see below). The cDNA clones isolated by PCR were subsequently used to generate 5'-overlapping clones by 5'-rapid amplification of cDNAs ends. Fig. 1A shows the complete nucleotide sequence and the predicted amino acid sequence of LUN cDNA. The LUN cDNA from lung and brain consisted of 3818 nt and contained a long open reading frame with one potential translational initiation codon (ATG) and in-frame upstream stop codons at the 5' end of the cDNA (GenBankTM accession number AB045732). The sequence surrounding this ATG codon is in a favorable context for translational initiation as defined by Kozak (24). We also isolated a 3623-bp LUN cDNA with deletion of nt 123-317, possibly due to alternative splicing from the lung, the glioma cell line T98, and T-cell line Molt-4 (GenBankTM accession number AB045733).




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Fig. 1.   Nucleotide sequence of human LUN cDNA and its deduced amino acid sequence. A, the complete nucleotide sequence is shown along with the corresponding translation of the deduced open reading frame. The region (nt 123-317), which is excluded by alternative splicing, is indicated by square brackets. The first boxed region corresponds to the RING finger motif, in which the conserved residues are circled. The region that is likely to adopt a leucine zipper and a coiled-coil structure is boxed with a dashed line in which hydrophobic amino acids occurring at the first position of the heptad repeat are indicated by italic letters and are dotted. The PEST sequences are underlined, putative bipartite nuclear localization signals are indicated by a dashed line, the stop codon is shown by an asterisk, and the polyadenylation signal is double-underlined. Two putative SUMO-1 conjugation sites (psi KxE) are boxed. The nucleotide sequences have been deposited in the GenBankTM data base under the accession numbers AB045732 and AB045733. B, schematic structure of the LUN protein. The amino acids are numbered below the box. The closed box corresponds to the RING finger (RF), which may form a Zn finger-like structure in LUN. The region that is likely to adopt a leucine zipper (LZ) and a coiled-coil structure (CC) is represented by a hatched box. The regions of PEST sequence are dotted, and the nuclear localization signal (NLS) is indicated by a striped hatched box. C, amino acid sequence alignments of the RING finger domains of LUN and other RING finger proteins. The positions of amino acid residues highly conserved in the family of RING finger proteins are highlighted with stippling.

During the course of this study, 5055-bp and 3264-bp human cDNA clones that matched our cDNA clones were reported by Zhou et al. (25) and Haluska et al. (26), respectively. However, the 5055-bp human cDNA isolated as a p53BP3 (p53-binding protein 3) cDNA (25) was inconsistent with our clones in the 5' end 1247 nt, which completely matched different human cDNA clones, NH0469M07 and RP11-422L5 (GenBankTM AC005037 and AC037455, respectively). On the other hand, although the 3264-bp human cDNA isolated as a Topors (topoisomerase I-binding RS protein) cDNA (GenBankTM AF098300) (26) lacked a 3'-untranslated region of 555 nt and polyadenylation signal, it matched our complete cDNA sequence (3818 and 3624 bp) almost exactly, except for a short deletion due to alternative splicing (Fig. 1A).

The predicted LUN protein consists of 1045 aa, with a calculated molecular mass of 119 kDa and pI 9.8. Sequence analysis revealed several distinctive features (Fig. 1, A and B). The most striking sequence of LUN is the RING finger motif (aa 103-141). The RING finger is one example of a Zn2+ binding motif defined by a conserved pattern of cysteine and histidine residues (C3HC4) that is found in a wide variety of proteins of diverse origins and functions (Fig. 1C). The region from aa 293-365 is likely to adopt a leucine zipper and a coiled-coil structure. The N-terminal region (aa 51-374) containing a RING finger motif, a putative leucine zipper, and a predicted coiled-coil structure revealed a Zn2+-dependent and sequence-specific DNA binding activity (see below). There are five PEST sequences at aa 13-28, 386-432, 490-509, 921-950, and 999-1024. Two putative bipartite nuclear localization signals are present near the middle of the protein, indicating that LUN may function in the nucleus. In addition, a region rich in serine and arginine residues and two putative small ubiquitin-like protein (SUMO-1) modification sites (psi KxE) (27, 28), which is modified in RanGAP1, Ikappa Balpha , PML, and p53, was found in the C-terminal half of the protein. The C-terminal regions (aa 456-731 and 456-882) were reported to interact with p53 (25) and topoisomerase I (26), respectively.

Chromosomal Localization of the Human LUN Gene-- To confirm the chromosomal mapping of the LUN gene, we performed FISH analysis on metaphase chromosomes from normal male lymphocytes using the 3549-bp LUN cDNA probe (corresponding to nt 270-3818). Several independent hybridizations exhibited a specific twin-spot signal on the short arm of a medium-sized chromosome, consistent with chromosome 9 on the basis of G-banding pattern (Fig. 2, A and B). No twin-spot signals were observed on other chromosomes. Fine analysis of 8 specifically hybridized chromosomes mapped the LUN gene to the p21 region of chromosome 9 (Fig. 2, C-E).



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Fig. 2.   Chromosome mapping of the LUN gene to chromosome 9p21 by FISH. A, metaphase spread hybridized with the 3549-bp (nt 270-3818) probe of LUN cDNA (arrow). B, the G-banding pattern of the same metaphase spread as seen in A. C and D, magnified micrographs of the FISH signals (C) and G-banding (D) on chromosome 9. The same probes were used as described in A. E, G-banded ideogram of chromosome 9. Each dot represents the twin-spot signal detected on chromosome 9. Comparison with the G-banding pattern resulted in assignment of the LUN gene to chromosome 9p21.

Expression of LUN mRNAs in Human Tissues-- As described in the cloning of LUN cDNAs (Fig. 1A), we cloned the 3.8-kb LUN cDNA from adult human lung and brain and the 3.6-kb cDNA from lung, T98 cell line, and Molt-4 cell line. Northern blotting analysis was performed to examine the expression of LUN mRNAs in adult human tissues (Fig. 3). Two LUN mRNAs were detected differentially among the tissues: a 3.8-kb mRNA in the brain, kidney, liver, lung, spleen, pancreas, and skeletal muscle and a 3.6-kb mRNA in the heart and lung. Human LUN mRNAs were most highly expressed in the lung. Moderate expression with variation was detected in the heart, brain, kidney, liver, spleen, and skeletal muscle. Little expression of LUN mRNA was detected in the pancreas. Thus, it was remarkable that the highest level of LUN expression occurred in the lung.



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Fig. 3.   Northern blotting analysis of human LUN mRNA. Several human tissues were analyzed by Northern blotting as described under "Experimental Procedures." Each lane contained ~2 µg of poly(A)+ RNA. The blots were hybridized with a 3549-bp probe (nt 270-3818) of the LUN cDNA. An arrow indicates the position of LUN mRNA of 3.8 or 3.6 kb due to alternative splicing. The positions of size markers (in kb) are indicated on the left.

Cellular Localization of LUN mRNA in the Lung-- As shown in Fig. 3, LUN mRNAs were expressed at much higher levels in the lung than in other tissues. To better localize the lung cells expressing LUN mRNA, we performed in situ hybridization with adult lung sections (Fig. 4). Hybridization with the antisense LUN probe revealed specific signals in the alveolar epithelium (Fig. 4A), whereas the sense probe did not show any signals in the lung tissue tested (Fig. 4C). In particular, not all, but a number of squamous cells (type I) and cuboidal cells (type II), revealed specific signals of LUN mRNA in the cytoplasm (Fig. 4B).



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Fig. 4.   Localization of LUN transcript in adult lung tissue. Sections of human lung tissue were hybridized with antisense (panels A and B) or sense (panel C) probes. The slides were reacted with the detection solution for 18 h as described under "Experimental Procedures" and then observed. Hybridization signals are visible as brown-colored reaction products. Panel B, higher magnification of panel A; hybridization signals over a limited number of squamous cells (type I) and cuboidal cells (type II) appear as brown products (arrows). Scale bars, 500-µm (panels A and C) and 200 µm (panel B).

Subcellular Localization of LUN Protein-- Because the LUN protein contains two putative bipartite nuclear localization signals (aa 616-645), it might function in the nucleus. To investigate the subcellular localization of LUN, HeLa cells were transformed with a plasmid expressing GFP alone or GFP-LUN fusion protein by DNA transfection. As shown in Fig. 5, cells expressing GFP-LUN exhibited nonhomogeneous fluorescent signals in the nucleus (panels A, C, and E). The nuclear localization pattern of LUN was similar to those of RING finger proteins, Mel-18, PML, RPT-1, and Vmw110 (6, 29-31). In contrast, the cells expressing GFP alone displayed diffuse fluorescence in both the cytoplasm and the nucleus (Fig. 5, panels B, D, and F). These results clearly indicated that LUN was localized to the nucleus.



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Fig. 5.   Subcellular localization of the GFP-LUN fusion protein in HeLa cells. HeLa cells were transiently transfected with a mammalian expression plasmid for LUN51-1045 fused to GFP, pGF- LUN51-1045 (panels A, C, and E) or a control plasmid expressing GFP, pGF (panels B, D, and F). 48 h after DNA-transfection, cells were fixed, illuminated, and observed under a fluorescence microscope (panels A and B). The same cells were also viewed by phase-contrast microscopy (panels C and D). Panels E and F show digitally merged fluorescent and phase contrast images. Magnification, ×400.

DNA Binding Property of the LUN Protein-- More than 80 RING finger proteins have been identified to date; however, the cellular functions of the RING finger are not well understood (2). Evidence has accumulated in recent years indicating that this motif is structurally diverse and is involved in protein-DNA interaction (6-8), protein-protein interaction (2-4), and E2-dependent ubiquitination (9, 10). To explore the ability of DNA binding of LUN by its RING finger, we prepared two kinds of recombinant LUN products, GST-LUN51-1045, which covers almost the full-length of the molecule, and GST- LUN51-374, which contains the RING finger and a leucine zipper and coiled-coil region (Fig. 6). After 125I-labeling, we examined the binding ability of GST-LUN51-1045 and GST-LUN51-374 to double-stranded or single-stranded DNA-cellulose in the presence or absence of mixed divalent cations (1 mM each of Zn2+, Mg2+, Mn2+, and Ca2+). As shown in Fig. 7A, both GST-LUN51-1045 and GST-LUN51-374 exhibited an ~10-fold higher level of binding to double-stranded DNA-cellulose in comparison with GST in the presence of mixed divalent cations, whereas little or no background binding was observed in the presence of 1 mM EDTA. Similar binding of GST-LUN51-1045 and GST-LUN51-374 to single-stranded DNA-cellulose, depending on divalent cations, was also observed (Fig. 7B). Thus, LUN, especially LUN51-374 containing the RING finger motif and a leucine zipper and coiled-coil region, was capable of binding to DNA.



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Fig. 6.   Expression and purification of GST fusion proteins. Bacterially expressed GST fusion proteins were purified by glutathione-Sepharose 4B. The purified GST-LUN51-1045 (lane 2), GST-LUN51-374 (lane 3), and GST (lane 1) were subjected to SDS-polyacrylamide gel electrophoresis and detected by Coomassie Brilliant Blue R-250 staining. Arrows indicate the expected positions for the purified proteins. Lane M contains molecular size standards.



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Fig. 7.   DNA binding characteristics of LUN. 125I-Labeled GST-LUN51-1045, GST-LUN51-374, and GST proteins (6 × 105 cpm/2 pmol each) were incubated with calf thymus double-stranded DNA (dsDNA)-cellulose (A) or single-stranded DNA (ssDNA)-cellulose (B) in binding buffer A (10 mM HEPES-NaOH, 50 mM KCl, 1 mM 2-mercaptoethanol, 20% glycerol, pH 7.1) in the presence or absence of mixed divalent cations M2+ (1 mM ZnCl2, 1 mM MgCl2, 1 mM MnCl2, and 1 mM CaCl2) at 4 °C for 1 h. After washing with the same buffer, the DNA-bound radioactivity was determined by gamma -counting. Data are shown as the means ± S.E. of duplicate determinations.

Because (i) divalent cations were required for DNA binding by LUN (Fig. 7) and (ii) Zn2+ is known to be necessary for autonomous folding of the RING finger motif (5), we investigated the effects of divalent cations on DNA binding of LUN in detail. As shown in Fig. 8A, the presence of a single metal ion (1 mM each) revealed background levels of double-stranded DNA binding of LUN, whereas double-stranded DNA binding was completely restored by the addition of 1 mM Zn2+ in combination with one of three other metal ions. Although the combinations of two of Mg2+, Mn2+, and Ca2+ exhibited low levels of DNA binding, these combinations without Zn2+ could not completely cover the DNA binding ability of LUN. Similar results were also obtained in single-stranded DNA binding assays (Fig. 8B). These results indicated that Zn2+ with either Mg2+, Mn2+, or Ca2+ is essential for the binding of LUN to DNA, probably by maintaining the structural integrity of the RING finger motif.



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Fig. 8.   Effects of divalent cations on LUN-DNA binding. 125I-Labeled GST-LUN51-1045 was incubated with double-stranded DNA (dsDNA)-cellulose (A) or single-stranded DNA (dsDNA)-cellulose (B) in binding buffer A (10 mM HEPES-NaOH, 50 mM KCl, 1 mM 2-mercaptoethanol, 20% glycerol, pH 7.1) in the presence of 1 mM ZnCl2, 1 mM MgCl2, 1 mM MnCl2, 1 mM CaCl2, and/or 1 mM EDTA alone or in combination at 4 °C for 1 h as described in Fig. 7. After washing, the remaining radioactivity on the DNA-cellulose was determined by gamma -counting. Data are shown as means ± S.E. of duplicate experiments.

Determination of LUN Binding Sequences from Human Genomic DNA-- To isolate human DNA elements capable of sequence-specific interaction with LUN, a library of MboI-digested small genomic DNA fragments was subjected to three consecutive cycles of in vitro binding selection with glutathione-Sepharose beads, which retained the GST-LUN51-1045 and GST-LUN51-374 fusion proteins. After the third selection cycle, 72 independent clones were isolated, and the inserted genomic fragments were sequenced. The average insert was 548 bp in length (range 47-1380 bp). Computer analysis of the sequences of the 72 clones revealed that 55 clones showed significant homologies with each other and were classified into three groups (Fig. 9). Each of the 25 binding clones in group A contained inverted copies of the 5-bp motif, 5'-TCCCA-3' and was capable of forming a palindromic structure with a 6-bp intervening loop (Fig. 9A). In contrast, each of the binding clones in groups B (22 clones) and C (8 clones) contained one copy of the 5-bp motif with a few bases of the other half of the palindromic structure (Fig. 9, B and C). These results clearly demonstrated that LUN is a sequence-specific DNA-binding protein like Mel-18, a RING finger-containing transcriptional negative regulator (7), and RAG-1, a RING finger-containing V(D/J) recombination mediator (32, 33). Furthermore, the consensus LUN binding sequence and its surrounding sequences encompass the well characterized cis elements, 5'-TTTGGGAG-3' (for Lyf-1 and Ik-2, mammalian lymphocyte-specific transcriptional regulators; Fig. 9, A and C) (34, 35), 5'-TCTAATCCC-3' (for Bcd, a Drosophila transcriptional regulator; Fig. 9, A and B) (36, 37), 5'-CTGGGAGT-3', and 5'-ACTCCCAG-3' (human erbB-2 promoter; Fig. 9A) (38). However, no homology was found in the binding sequences of Mel-18 and RAG-1 (7, 32, 33). To our surprise, the LUN binding palindromic sequence of 33 bp, 5'-GCCTGTAATCCCAGCACTTTGG-GAGGCTGAGGC-3', completely matched the sequence (-1934 bp from the transcription initiation site, nt 428-460 of GenBankTM D49685) within the upstream transcriptional regulatory region of the human E-cadherin gene (39) and two intervening regions (nt 3182-3208 and 5988-6013 of GenBankTM AF178081) of the human talin gene exons 2 and 8. 



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Fig. 9.   Definition of a consensus binding site for LUN. Fifty-five binding sequences for LUN obtained by three rounds of selection are shown. According to alignments, these sequences were categorized into three groups (A, 25 clones; B, 22 clones; C, 8 clones). The core sequences are indicated in boxes. Nucleotides in boldface represent the identity of a given position. Deletions are shown by gaps in the sequence. The boxed sequence indicates the palindrome-like binding consensus for LUN. To be considered the consensus, the threshold level of representation of a nucleotide at a given position was arbitrary set at >60%. In the case of deletions, adjacent fixed nucleotides next in sequence were considered the selected nucleotides.

For further verification of sequence-specific DNA binding of LUN, we performed gel mobility shift assay using a series of synthetic oligonucleotides corresponding to the binding consensus (C1-C5) and GST-fused recombinant LUN proteins. As shown in Fig. 10, a gel mobility shift assay revealed a retarded DNA complex with GST-LUN51-1045 or GST-LUN51-374 (panels B and C, lane 2), whereas the addition of GST had no effect (panel A, lane 2). The addition of a 200-fold excess of unlabeled identical oligonucleotide C1 or C2 abolished the band shift almost completely (panels B and C, lane 3 and 4). These results clearly indicated that LUN is capable of binding to the 22-bp palindromic sequences containing two copies of the 5-bp motif. Deletion mutant oligonucleotides C3 and C4 corresponding to each half-site of the palindromic structure also significantly competed with C1 binding, but this effect was not complete (panels B and C, lanes 5 and 6). Oligonucleotide C5 corresponding to the 5-bp motif was scarcely capable of competing formation of the retarded complex (panels B and C, lane 7). Thus, LUN had a much higher affinity to C1 oligonucleotide than to C5 oligonucleotide. These results, therefore, indicated that LUN has the propensity to bind to the palindromic structure containing inverted copies of the 5-bp motif, and the surrounding sequences of the 5-bp motif are indispensable for the interaction between LUN and DNA.



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Fig. 10.   Gel mobility shift analysis of the binding consensus for LUN. Identical amounts (8 pmol) of GST (panel A), GST-LUN51-374 (panel B), or GST-LUN51-1045 (panel C) were incubated with 32P-labeled 31-bp C1 probe (corresponding to the binding consensus for LUN; 4 × 104 cpm) in the presence or absence of a 200-fold excess of the indicated double-stranded DNA competitors (lanes 2-7). Lane 1 shows the binding reaction without fusion proteins. Free and LUN- complexed DNA fragments were separated on 5% nondenaturing polyacrylamide gels as described under "Experimental Procedures."



    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study, we isolated cDNAs encoding a novel RING finger protein, LUN, the mRNAs of which were expressed at high levels in the alveolar epithelium of the lung. The high level of LUN expression in the lung indicated that this molecule plays an important role(s) in the lung. We attempted to characterize the biochemical properties and functions of LUN. With regard to the cellular functions of LUN, it is important that several nuclear RING finger proteins, Mel-18, RAG-1, PML, RPT-1, RAD-18, and RING1, are involved in the regulation of transcription and chromatin structure (6, 7, 29, 30, 32, 33, 40, 41). The localization of a GFP-LUN fusion protein in punctate nuclear subdomains is consistent with the predicted nuclear function(s) of LUN. In fact, we clarified that the N-terminal RING finger-containing region (aa 51-374) of LUN specifically binds to a novel palindromic consensus sequence (5'-TCCCAGCACTTTGGGA-3') and genomic fragments containing the 5-bp core motif (5'-TCCCA-3') in a Zn2+-dependent manner. Furthermore, the LUN binding palindromic sequence and its surrounding sequence (5'-GCCTGTAATCCCAGCACTTTGGGAGGCTGAGGC-3') completely matched the sequence of the upstream transcriptional regulatory region (-1934 bp from the transcriptional initiation site) of the human E-cadherin gene (39) and two intervening regions (nt 3182-3208 and 5988-6013) of the human talin gene exons 2 and 8. E-cadherin is a Ca2+-dependent intercellular adhesion molecule and is closely associated with cancer metastasis and invasion (39, 42-44). Talin is a cytoskeletal protein that interacts with extracellular matrix components such as integrin to form focal adhesion (45, 46). Thus, LUN was proposed to be an important trans-acting transcriptional regulator of E-cadherin and talin genes in the alveolar epithelial cells during lung development, differentiation, and tumorigenesis.

In general, nuclear palindrome-binding proteins such as Maf, Jun, Fos, and PBP form leucine zipper-mediated homodimers and heterodimers that are important for their functions (38, 47-50). On the basis of the specific DNA binding data presented here, it seems possible that LUN forms a homodimer for palindromic sequence binding through its leucine zipper and coiled-coil region. Moreover, it is also possible that LUN forms several types of heterodimers with distinct DNA binding specificities because (i) many genomic LUN binding clones containing the half-site of the palindromic structure with the 5-bp core sequence were isolated and (ii) a p53 binding and topoisomerase I binding domain has been identified in the C-terminal half of the protein (25, 26). Interestingly, LUN contains two putative SUMO-1 modification sites in the p53 binding and topoisomerase I binding domain, and both p53 and topoisomerase I are modified with SUMO-1 in vivo (27, 51). Because SUMO-1 modification is known to affect the ability of the modified protein to interact with target proteins (27, 28, 51), the interaction of LUN with p53 and/or topoisomerase I, resulting in distinct DNA binding specificities, may be regulated through SUMO-1 modification.

We show that the human LUN gene was localized to chromosome 9p21. More than 86% of SCLC exhibit loss of heterozygosity at 9p21 (52), suggesting the existence of tumor suppressor genes within the lost region. Based on mapping of the LUN gene to 9p21 and its expression at high levels in alveolar epithelium, LUN is a candidate tumor suppressor gene related to SCLC. SCLC is distinct from other types of lung cancer accompanied by metastasis. Cancer metastasis and invasion are closely associated with cell-to-cell and cell-to-extracellular matrix adhesiveness. Down-regulation of E-cadherin has been reported to occur in various cancers including SCLC and other lung cancers (39, 42, 53) and to parallel with tumor progression toward a malignant invasive state (43, 44). As discussed above, LUN is capable of binding specifically to the transcriptional regulatory region of the E-cadherin gene and is expressed at high levels in the alveolar epithelium. The present results suggest a possible molecular mechanism of SCLC in which loss of the LUN gene would cause loss of trans-activation by LUN, resulting in transcriptional inactivation of the E-cadherin gene.

In this study, we isolated cDNAs encoding a novel RING finger protein, LUN, the mRNAs of which are expressed at high levels in alveolar epithelium of the lung. The LUN gene locus was assigned to the chromosome 9p21, which contains candidate tumor suppressor genes associated with loss of heterozygosity in more than 86% of SCLC. LUN localizes to the nucleus and binds specifically to a novel palindromic binding consensus (5'-TCCCAGCACTTTGGGA-3') in a Zn2+-dependent manner. The sequence from amino acids 51-374 of LUN is responsible for palindrome binding. The LUN binding palindromic sequence was found in the upstream transcriptional regulatory region of the E-cadherin gene and in two intervening regions of the talin gene, suggesting that LUN might be an important trans-acting transcriptional regulator for lung cancer-associated genes including E-cadherin and talin genes. The physiological and clinical significance of LUN remains unclear. However, the findings described in this report will be important for further intensive studies on transcriptional regulation of E-cadherin and talin genes, lung development, differentiation, and tumorigenesis.


    ACKNOWLEDGEMENTS

We thank Kazuko Wakai for technical assistance and Yuji Hara and Masayoshi Minakuchi for helpful discussions.


    FOOTNOTES

* This work was supported in part by grants-in-aid for Scientific Research on Priority Areas (to Y. A.) and Scientific Research B (to K. U.) from the Ministry of Education, Science, and Culture, Japan, by a grant from the Naito Foundation (to Y. A.), and by a grant from the Uehara Memorial Foundation (to Y. A.).The costs of publication of this article were defrayed in part by the payment of page charges. The article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

The nucleotide sequence(s) reported in this paper has been submitted to the GenBankTM/EMBL Data Bank with accession number(s) AB045732 and AB045733.

** To whom correspondence and reprint requests should be addressed. Tel.: 81-774-38-3222; Fax: 81-774-38-3226; E-mail: adachi@ scl.kyoto-u.ac.jp.

Published, JBC Papers in Press, January 25, 2001, DOI 10.1074/jbc.M010262200


    ABBREVIATIONS

The abbreviations used are: RING, really interesting new gene; kb, kilobase pair(s); bp, base pair(s) PCR, polymerase chain reaction; nt, nucleotides; aa, amino acids; FISH, fluorescence in situ hybridization; GFP, green fluorescent protein; GST, glutathione S-transferase; SUMO, small ubiquitin-related modifier; SCLC, small cell lung cancer.


    REFERENCES
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ABSTRACT
INTRODUCTION
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RESULTS
DISCUSSION
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