Transcriptional regulation of human eosinophil RNases by an evolutionary- conserved sequence motif in primate genome
© Wang et al; licensee BioMed Central Ltd. 2007
Received: 17 April 2007
Accepted: 11 October 2007
Published: 11 October 2007
Human eosinophil-derived neurotoxin (edn) and eosinophil cationic protein (ecp) are members of a subfamily of primate ribonuclease (rnase) genes. Although they are generated by gene duplication event, distinct edn and ecp expression profile in various tissues have been reported.
In this study, we obtained the upstream promoter sequences of several representative primate eosinophil rnases. Bioinformatic analysis revealed the presence of a shared 34-nucleotide (nt) sequence stretch located at -81 to -48 in all edn promoters and macaque ecp promoter. Such a unique sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Gel electrophoretic mobility shift assay, transient transfection and scanning mutagenesis experiments allowed us to identify binding sites for two transcription factors, Myc-associated zinc finger protein (MAZ) and SV-40 protein-1 (Sp1), within the 34-nt segment. Subsequent in vitro and in vivo binding assays demonstrated a direct molecular interaction between this 34-nt region and MAZ and Sp1. Interestingly, overexpression of MAZ and Sp1 respectively repressed and enhanced edn promoter activity. The regulatory transactivation motif was mapped to the evolutionarily conserved -74/-65 region of the edn promoter, which was guanidine-rich and critical for recognition by both transcription factors.
Our results provide the first direct evidence that MAZ and Sp1 play important roles on the transcriptional activation of the human edn promoter through specific binding to a 34-nt segment present in representative primate eosinophil rnase promoters.
Human edn and ecp respectively encode eosinophil-derived neurotoxin (EDN) and eosinophil cationic protein (ECP), two of the four major proteins found in granules of human eosinophilic leukocytes. Their gene products belong to members of the human RNaseA superfamily, which comprises RNase1-13 [1–6]. The eosinophil RNases EDN and ECP are secreted to body fluid and have neurotoxic, helminthotoxic, and ribonucleolytic activities. Previously, we demonstrated that ECP enters neuroendocrine cells through protein-protein interactions with a granular protease, which in turn allows the cytotoxic ECP to inhibit growth of the target cells . In addition, the signal peptide of ECP is toxic to bacteria and yeast and induces expression of transforming growth factor α in human cells [8, 9]. Although both genes are expressed in eosinophils, edn, rather than ecp, can be extensively expressed in various tissues including liver, spleen, and kidney [10–12], whereas ecp is restrictively expressed in blood granulocytes. In terms of gene structure, edn and ecp are similar as they both contain an intron between a noncoding exon 1 and a coding exon 2. Each of these genes is translated from exon 2, and the sequence identity of the DNA in the coding region is 85% . It has been proposed that edn and ecp were evolved through a duplication event about 31 million years ago in the evolutionary lineage of New World and Old World monkeys . Gene duplication and subsequent functional divergence of duplicated genes is one of the important mechanisms for evolution of novel gene functions [15–18]. However, the regulatory motifs in promoter regions of duplicated genes are generally conserved during duplication events [19–21].
Previously, three important transcription factor binding sites, C/EBP, NFAT-1 and PU.1 were discovered in the promoter regions of both edn and ecp, and the non-translating exon 1 as well as intron 1 could enhance the promoter activities [22–26]. Although as high as 92% sequence identity was observed in the upstream 1-kb regions of human edn and ecp, much higher edn promoter activity was observed. Therefore, whether any sequence stretch located in the upstream regions of primate eosinophil rnases may govern regulatory transcription of ecp and edn was investigated employing cross-species sequence comparison, transcription element prediction and functional validation.
Primate edn and ecp promoters share high sequence similarity
We amplified and sequenced primate eosinophil rnase promoter fragments, -921 to -1  and -937 to -1 relative to the human edn and ecp transcription start site, respectively , from individual of six nonhuman primate species. These primate species represent independent genera from three great apes (P. pygmaeus, G. gorilla, P. troglodytes), one Old World monkey (M. fascicularis), and one New World monkey (S. sciureus). The sequence data have been submitted to GenBank (GenBank accession numbers DQ171721–DQ171729). Multiple sequence alignment by ClustalW showed that the identities among primate edn and ecp promoter sets were greater than 95% and 91%, respectively. In consistence with what Zhang and Rosenberg have characterized, the sequences of the 5' promoter regions of P. troglodytes (chimpanzee) edn and ecp determined in this study were identical to those reported in AF294016-AF294018 and AF294027-294028, respectively .
Comparative analysis reveals deletion of a 34-nt segment in the ecp promoter
Subsequently, we were able to distinguish within the 34-nt region two conserved sequence motifs "-74ACCCTCCCTC-65" and "-62GGCTCTGCTGGCCCA-48" (Fig. 1B). The Furthermore, primate eosinophil rnase promoter sequences corresponding to -921 to -1 of human edn and -887 to -1 of human ecp were analyzed by MEGA 3.1, and the phylogenetic relationship between ecp and edn promoter sequences among different species is was illustrated in Figure 1C. The results showed that the ecp and edn promoters branched when the 34-nt segment disappeared in some species, in agreement with the notion that ecp and edn were the most rapidly evolving coding sequences of primate genes . The phylogenetic tree of ecp and edn promoter sequences suggested another evolutional event by which deletion of the 34-nt sequence stretch might have occurred in the lineage Old Word monkey and hominoid.
Presence of the 34-nt segment enhances transcriptional activities
Multiple sequence alignment of the 34-nt segment in all primate edn and macaque ecp, revealed the existence of two well conserved sequence stretches, "-74ACCCTCCCTC-65" and "-62GGCTCTGCTGGCCCA-48". The combination of ConSite  and TESS (T ranscription E lement S earch S ystem)  suggested several putative transcription factor binding sites, including Sp1, MAZ, activator protein-2 (AP2) and liver factor A1 (LF-A1) located at (-73/-65), (-73/-67), (-68/-60) and (-69/-55), respectively. As shown in Figure 2C, the transcription activities of reporters containing mutant -74 to -65 region and mutant region -62 to -48 were similar to that of human edn with the 34-nt segment deleted, indicating that each of the two conserved regions, -74/-65 and -62/-48, contribute to the regulation of edn transcription.
Transcription elements for MAZ and Sp1 binding are present in the 34-nt segment
ConSite and TESS predictions revealed that transcription factors AP2, MAZ, Sp1, and LF-A1 might bind to the 34-nt segment. In addition, since hepatocyte nuclear factor-4 (HNF-4) was reported to be identical to LF-A1 , an HNF-4 consensus sequence was also used to compete wild type binding along with the consensus oligonucleotides representing the other four transcription factor binding motifs. The results showed that the formation of the major band shift was completely abolished only by the MAZ consensus oligonucleotide (Fig. 3B, lane 4), but not consensus Sp1, LF-A1, HNF-4, and AP2 oligonucleotides, with serum responsive element (SRE) and c-myb oligonucleotides were used as negative controls. However, the formation of the minor shift was diminished by MAZ and Sp1 consensus oligonucleotides (Fig. 3C, lanes 2 and 4). Furthermore, when the mutant MAZ (mMAZ) oligonucleotide containing GA box mutation was used as an unlabeled competitor, it did not compete with the major band shift (Fig. 3C, lane 3). Likewise, mutant Sp1 competitor (mSp1) containing GC box mutation was unable to compete with the minor band shift ether (Fig. 3C, lane 5). These EMSA competition experiments clearly demonstrate that the transcription factors MAZ and Sp1 are involved in formation of protein-DNA complex in the 34-nt sequence stretch.
MAZ binds to the 34-nt segment in vitro
The -73/-67 and -62/-52 regions in the 34-nt segment are important for MAZ binding
In this study, we examined further in vitro MAZ binding to the 34-nt segment employing DAPA with anti-SAF-1 against a rabbit zinc finger protein serum amyloid A activating factor-1 (SAF-1) homologous to human MAZ . We observed an enhanced MAZ signal was observed in the cells transfected with pcDNA3/MAZ (Fig. 5C), which indicated that rabbit anti-SAF-1 could be used to detect human MAZ in HepG2 cells. Furthermore, DAPA revealed that MAZ bound to the wild type 34-nt segment attached to the bead. However, the binding activity diminished when either the -74/-65 or -62/-48 region was mutated (Fig. 5D). These results were consistent with that of the EMSA scanning mutagenesis in which either the -74/-65 or -62/-48 mutation of the 34-nt segment led to decreased MAZ binding activity.
Transcription factor Sp1 also binds to the 34-nt segment
Competitive binding of Sp1 and MAZ in vitro
Sp1 and MAZ bind to the edn promoter in vivo
The available anti-MAZ could only recognize the DNA binding surface on MAZ such that it was not suitable for use in the ChIP assay. To overcome this problem, we developed a ChAP assay that did not require an antibody. For this assay, pcDNA/MAZ-6H was transiently transfected into HepG2 cells, and soluble chromatin was generated by sonication. The MAZ-bound chromatin was pulled down by nickel resin, and the DNA was amplified by PCR with edn promoter-specific primers. As expected, our modified method demonstrated in vivo MAZ binding to the human edn promoter (Fig. 8B). Moreover, the binding activity was dramatically reduced when mutant MAZ lacking of the DNA binding domain was transfected. As a control, non-specific binding was not detected when only the pcDNA was transfected into the HepG2 cells. Our results clearly support that both Sp1 and MAZ bind to the human edn promoter in vivo.
Sp1 and MAZ differentially regulate edn promoter activity in vivo
To further demonstrate the critical role that Sp1 plays in edn promoter activity, RNAi was used to deplete endogenous Sp1 in HepG2 cells. Two sets of double-stranded RNA oligonucleotides (Sp1/RNAi-1 and Sp1/RNAi-2) corresponding to human Sp1 were separately transfected into HepG2 cells. Western blot analysis of cell lysates collected 72 hrs after transfection revealed that both Sp1/RNAi-1 and Sp1/RNAi-2 effectively depleted endogenous Sp1 protein (Fig. 9C). As a control, β-actin was unaffected by RNAi treatment, indicating the specificity of the RNAi effect and that equivalent amounts of protein were loaded on the gel. Fig. 9D showed that cotransfection with Sp1/RNAi-1 or Sp1/RNAi-2 decreased the edn promoter activity to 40% and 50% relative to control, respectively. When the 34-nt region was deleted from the edn promoter, the effect of RNA interference on Sp1 was less apparent.
Taken together, our results demonstrate that the regulatory roles of MAZ and Sp1 on transactivation of human edn govern the differential expression of the duplicated eosinophil rnases and that binding between these transcription factors and the 34-nt region in the edn promoter determines its expression.
Both edn and ecp have been postulated to have emerged from a gene duplication event after divergence of Old World and New World monkeys, as well as the most rapidly evolving coding sequences of primate ribonuclease genes. Variance in their promoter sequences may imply that another key deletion or insertion event occurred before the separation of hominoids and Old World monkeys . Our observations as indicated in the putative phylogenic tree of the promoter regions of representative primate edn and ecp promoters (Fig. 1B and 1C) led to a hypothesis that deletion of the 34-nt sequence stretch might have occurred in the lineage of Old World Monkey and Hominoid.
Minor divergent segments upstream of the translation start site of edn and ecp
edn -584 CTCAAGTATGTGTAG -570
ecp -550 CTCAATTGTATGGAG -536
edn -372 TTCA TGTAC TTTG GTCA -356
ecp -338 TTCG TGTCA TTTA GTCA -324
edn -291 CAT CCAGAGTTTG GATCTA ACCA GC-267
ecp -257 CAC CCAGAGTCCA GATCCC ACCG GC-233
edn 24 GT CACAGCG CG GAG 37
ecp 24 GC CACAGCT CA GAG 37
edn 80 AGCA ATG GGGCAGCAA CT 97
ecp 80 AGCG ACA GGGCAGCAC CT 97
edn 132 AC GTT GCACACTTTGCA GACAGGAAGT A 159
ecp 132 AT GTG GCACACTTTGGG GACAGGAAGA A 159
The characteristic 34-nt sequence motif in the edn promoter enhanced the transcriptional activity in HepG2 (Fig. 2) as well as HL-60 clone 15 cells (Supplementary Fig. 1). EMSA with HepG2 nuclear extracts and 34-nt promoter probe revealed specific mobility shift patterns, suggesting that the 34-nt segment contains a region essential for transactivation of edn in HepG2 cells. When HepG2 nuclear extract bound to the wild type 34-nt probe, excess unlabeled wild type oligonucleotide, but not oligonucleotides containing site-specific mutations at -74/-65 or -62/-48, competed effectively for binding to the nuclear proteins. Thus, both conserved regions -74/-65 and -62/-48 were associated with the transcription factors, and elevation of the transcriptional activity in the cells was also verified by the reporter assay.
MAZ is a transcriptional factor with six zinc fingers that bind to a GA box (GGGAGGG) at the ME1a1 site [33, 38]. Its rabbit and mouse homologs are identified as SAF-1  and Pur-1, respectively. MAZ has also been shown to bind the upstream regions of various genes, including CD4 , NMDA receptor subunit type I , recombination activating gene-1 (RAG-1) , PTH-related peptide receptor , phenylethanolamine N-methyltransferase (PNMT) , serotonin 1a receptor , insulin I and II , nitric oxide synthase (NOS)  and p21 , and also is involved in RNA polymerase II termination  and adenovirus major late promoter activation . Our study has advanced further that MAZ binds the 34-nt region through the third and fourth zinc fingers, extending its effects on transcriptional activity. We were surprised that unlabeled EMSA competitors m10 to m17 did not appreciably compete the major band shift even though these competitors all contained the full-length GA box believed to be tightly bound by MAZ. Comprehensive EMSA scanning mutagenesis revealed that MAZ bound not only to the known GA box but also to another conserved site, CTCTGCTG, an apparently novel transcription factor binding motif. Thus, we postulate that some other cofactor protein(s) may bind to the CTCTGCTG motif to further enhance MAZ binding, and thus mutations at any position in this novel motif leads to decreased MAZ binding (Fig. 5). Interestingly, even in the absence of another protein(s), MAZ alone had strong binding activity because GST-MAZ bound the wild type 34-nt probe (Fig. 4B). Notably, MAZ always cooperates with another transcription factor, such as Sp1, to control promoter activity [35, 42, 43, 49, 50]. The discovery of two sites critical for MAZ binding is consistent with our luciferase assay results. As shown in Figure 2C, mutation in either the -74/-65 or -62/-48 region led to reduced luciferase activity almost equal to that of pGL3-edn without the 34-nt region. Furthermore, the ChAP assay showed that MAZ bound to the 34-nt region of the human edn promoter in HepG2 cells in vivo (Fig. 7C).
In addition to MAZ, the transcription factor Sp1 also bound the GA box (Fig. 6). In vivo Sp1 binding to the 34-nt sequence was also demonstrated by ChIP assay (Fig. 7A). Sp1 preferentially recognizes a guanidine nucleotide-rich sequence.  MAZ and Sp1 cooperatively regulate many genes such as NOS, PNMT, and those encoding serotonin 1a receptor and parathyroid hormone [42, 43, 45, 52]. Most studies report the binding site for MAZ near or partially overlapping that of Sp1.
In our study using HepG2 cells, cotransfection with MAZ DNA and reporter plasmid containing the edn promoter revealed that MAZ functioned as a repressor with respect to regulation of transcriptional activity in the presence of the 34-nt sequence region. Such repression effect was reversed when the 34-nt segment was removed, indicating that the 34-nt region served as the MAZ-responsive element in the edn promoter (Fig. 8A). In contrast, Sp1 is considered to play a role as an activator based on the observation derived from cotransfection with Sp1 DNA and the reporter plasmid containing the edn promoter (Fig. 8B). Previous studies have shown that MAZ and Sp1 recognize the same cis-elements in promoters and thereby regulate gene expression independently . Song et al. have found that MAZ and Sp1 associate with histone deacetylase and DNA methyltransferase, respectively, to regulate transcriptional activity .
In conclusion, we characterized the presence of an evolutionarily conserved 34-bp sequence in all primate genes encoding eosinophil-derived neurotoxin (edn) genes and only one gene encoding eosinophil cationic protein (ecp). Given the fact that primate edn and ecp are known to be products of gene duplication event,such a conserved 34-bp sequence motif constituted a region essential for transactivation of human edn in hepatocellular carcinoma cells. Our findings indicate that Sp1 as well as MAZ constitutively bind at 34-nt region of the edn promoter in HepG2 cells in vivo, and that the activity of the promoter primarily depends on competition between Sp1 and MAZ. Furthermore, our data reveal that Sp1 binds at the 34-nt region and activates edn gene expression, on the contrary, MAZ represses edn gene expression upon binding to the 34-nt region. The underlying molecular mechanisms about Sp1 and MAZ cooperation in governing edn promoter activity still remain to be further investigation.
Cell culture and transient transfection
Cells from non-human primates including Saimiri sciureus, Macaca fascicularis, Pongo pygmaeus, Gorilla gorilla and Pan troglodytes were purchased from Coriell Institute for Medical Research and their cultures maintained at Dr. Chung-I Wu's Laboratory (University of Chicago). All non-human primate cell cultures were maintained in alpha-modified minimum essential medium (α-MEM) plus 10% non-inactivated fetal calf serum (FCS). HepG2 cells were cultured in DMEM medium (Sigma) containing 10% heat-inactivated FCS. HL-60 clone 15 promyelocytic leukemia cells were cultured in RPMI-1640 medium with 10% fetal bovine serum supplemented with 25 mM HEPPSO (N-[2-hydroxyethyl]piperazine-N-[2-hydroxy-propanesulfonic acid] (Sigma), and maintained at pH 7.6. Exponentially growing cells at 95% confluence were trypsinized prior to genomic DNA purification employing the Wizard® Genomic DNA purification kit (Promega). All cells were cultured in a 37°C incubator under 5% CO2 in air. For transient transfections, the HepG2 and HL-60 clone 15 cell lines were treated with Transfast (Promega) and electroporation, respectively, according to the manufacturer's instructions.
RNA interference (RNAi) duplexes directed against Sp1 and irrelevant control RNAi (cat. no: 12935-100) were purchased from Invitrogen Life Technologies. The two targeted sequences used to silence Sp1 were 5'-GCAGACACAGCAGCAACAAAUUCUU-3' and 5'-GGAACAUCACCUUGCUACCUGUCAA-3'. Each RNAi was separately transfected into target cells using Lipofectamine 2000 according to the manufacturer's instructions (Invitrogen Life Technologies).
Amplification of Upstream Regions of Primate edn and ecp
The primer sets, #1/#2 and #3/#4, were designed based on sequences of the first exon and the upstream region of the corresponding human edn and ecp to amplify approximately 1-kb upstream fragments from the transcription start sites, respectively (Supplementary Table 1). Polymerase chain reaction (PCR) was conducted at 95°C for 5 min followed by 30 cycles of 95°C for 30 sec, 55°C for 30 sec, and 72°C for 90 sec. The desired fragments were purified and cloned into pGEM-T easy vector (Promega). The sequences were confirmed using automated DNA sequencing with the BigDye® Terminator V3.1 Cycle Sequencing kit (Applied Biosystems), and the reaction products were analyzed with a ABI PRISM® 3100 Genetic Analyzer.
The fragments of human edn and ecp promoters from nucleotides -1,000 to +297 were amplified using primer sets #5/#6 and #7/#8, respectively, from human genomic DNA to generate the luciferase reporter plasmids pGL3-edn and pGL3-ecp. The transcriptional start site of edn previously defined by Tiffany HL et al  to be located 23 nucleotides 3' from a consensus TATA box was used. That of the ecp was assumed to be at the correspondent site.
For mutant reporter plasmids, the edn Δ(-81/-48), edn-mut (-74/-65), edn-mut (-62/-48) and ecp+(-81/-48) were generated using primer sets #9/#10, #11/#12, #13/#14, #15/#16, respectively, by Quik-Change Site-Directed Mutagenesis (Stratagene). The reactions were conducted at 95°C for 5 min followed by 20 cycles of 95°C for 30 sec, 55°C for 30 sec and 72°C for 12 min. After PCR amplification, the products were treated with 2 U of Dpn I and separately transformed into E. coli Top10F'. The mutant edn and ecp promoter sequences were confirmed by DNA sequencing.
The pGEX/MAZ and pGEX/MAZΔF34 expression plasmids were kindly provided by Dr. Kazunari K. Yokoyama (Gene Engineering Division, Bioresource Center, Tsukuba Institute, RIKEN, 3-1-1 Koyadai, Tsukuba, Ibaraki 305-0074, Japan). The Sp1 expression plasmid, pcDNA3/Sp1, was obtained from Dr. Jacob Bar-Tana (Department of Human Nutrition and Metabolism, Hebrew University Medical School, Jerusalem, P.O.). The inserts of pGEX/MAZ and pGEX/MAZΔF34 were subcloned into pcDNA3 (Invitrogen Life Technologies) to generate pcDNA3/MAZ-6H and pcDNA3/MAZΔF34-6H. The sequences of each recombinant clone were determined by automated DNA sequencing.
Development of multiple indexing sequence alignment
A novel method of MISA based on interval-jumping searching algorithms and dynamic programming techniques was developed to identify characteristic features among the query sequences [53, 54]. The block-based pattern searching algorithms were designed to find short consensus motifs by a combination of hash encoding, quick sorting and interval-jumping techniques; this was to avoid unnecessary comparisons and to achieve approximate matching functions. In this module, each possible subsegment of the imported sequences was considered as a fundamental element, and its contents are encoded as a unique digital number and allocated in a corresponding interval for the following matching procedures. Due to the unique transformation and exact reconvertible features, the encoded numbers could be efficiently matched in an ordered and well-defined interval. According to a variable combination of parameter settings, we were able to identify the consensus motifs with different properties of pattern lengths, tolerance conditions, and occurrence frequencies and label each of the sequences. Once the consensus motifs were completely searched, a scoring matrix of pairwise distant measurements is constructed to select a referred indexed sequence prior to the multiple alignment procedures. The MISA algorithms are then implemented by performing dynamic programming and merging processing in accordance with the assigned reference sequence based on the indexed fundamental elements instead of single residues. If there are k indexed sequences under evaluation, the k pairwise alignments are further combined and transformed into a k-row multiple alignment indexing matrix through a multiple merging process obeying the principle "once a gap, always a gap" . Finally, the aligned overlapped consensus labels were combined as a representative segment to visually display the identified combinatorial patterns. To analyze primate eosinophil rnases in this paper, six edn and five ecp promoter sequences of approximately 1 kb length in FASTA format were input into MISA. Various combinations of fundamental pattern length, number of tolerant residues, and occurrence rate were set to scan along the full-length promoter sequences, and the results showed consistent performance for the conserved regions as well as the dominant portions of variation labeled in color-coded bars and dotted grey grids, respectively.
Phylogenetic analysis of DNA sequences
Primate ecp and edn promoter sequences were aligned using MEGA 3.1 software. Phylogenetic trees were generated by the neighbor joining method with bootstrap resampling (data resampled 100 times) to assess the degree of support for the phylogenic branching indicated by the optimal tree. The exact numbers of nucleotides of M. fascicularis edn,P. pygmaeus edn, G. gorilla edn, P. troglodytes edn, H. sapiens edn, M. fascicularis ecp, P. pygmaeus ecp, G. gorilla ecp, P. troglodytes ecp, and H. sapiens ecp used for analysis were respectively 921 bp, 923 bp, 921 bp, 921 bp, 921 bp, 931 bp, 883 bp, 887 bp, 887 bp, and 887 bp.
RNA isolation and RT-PCR
Total RNAs of HepG2 and HL-60 clone 15 cells were isolated by Trizol Reagent (Gibco) and the contaminant genomic DNA was further digested by RNase-free DNaseI (Promega). Two micrograms of total RNA and 0.5 μg oligo-dT were added in a sterile RNase-free microcentrifuge tube, heated at 70°C for 5 min to melt secondary structure within the template. A mixture containing 5 μl M-MLV 5× Reaction buffer (Promega), 1.25 μl 10 mM dNTP, 1 μl recombinant RNasin rib nuclease inhibitor (Promega), M-MLV RTase (Promega), and nuclease-free water were added to a final volume of 25 μl. The reverse transcription reaction was performed at 42°C for 1 hr in a thermal cycler (ASTEC, PC808). For each reaction, 5 nmole of forward and reverse primers (edn: 5'-CTCACTCCATGTCAAACCT-3', 5'-GCCGTTGATAATTGTTAAGGA-3', and ecp: 5'-TATGCAGACAGACCAGGATGG-3' and 5'-GGAACCACAGGATACCGTGGAGAA-3') and 80 ng cDNA were incubated with 1.25 units of Ex Taq™ (Takara), 6.25 nmole dNTPs and Ex Taq™ polymerase buffer. The PCR products were electrophoresed on a 2.0% agarose gel and stained with ethidium bromide.
Promoter activity assays
After forty-eight hours transfection with individual pGL3, pGL3-edn, pGL3-ecp reporter plasmids, the cells were washed twice with PBS and lysed by Passive Lysis Buffer (Promega). The lysates were centrifuged at 16,500 × g for 1 min at 4°C, and the supernatant was collected. The firefly and Renilla luciferase activities were measured by TD-20/20 luminometer (Victor), and the relative activity was calculated by simply dividing luminescence intensity obtained from the assay for firefly luciferase by that of Renilla luciferase.
Preparation of nuclear extract
Nuclear extracts were prepared by NE-PER nuclear extraction kit (Pierce). In brief, about 5 × 107 HepG2 cells were trypsinized, collected and suspended in 200 μl CER I buffer and incubated for 10 min on ice. After 11 μl CER II was added and incubated for 1 min on ice, the cytoplasmic and nuclear extracts were separated by centrifugation at 16,500 × g for 5 min at 4°C. Cytoplasmic extracts in the supernatant fraction were transferred into a clean microcentrifuge tube, whereas the pellets were resuspended by adding 100 μl ice-cold NER and the mixture was incubated for 40 min on ice. The mixture was finally centrifuged at 16,500 × g for 10 min at 4°C. Nuclear extracts in the supernatant fraction were transferred into a clean microcentrifuge tube and stored at -80°C until use. Concentrations of proteins in lysates were determined with BCA kit (Pierce) with bovine serum albumin (fraction V) as the standard.
Electrophoretic mobility shift assays (EMSA)
The oligonucleotide sequences used in this study are listed in supplementary Table 2. DNA annealing was performed by heating 10 μM of each complementary strand of the oligonucleotide to 95°C for 10 min, followed by cooling gradually to 25°C over a period of three hours. The oligonucleotides corresponding to wild type and mutant 34-nt segments were separately synthesized to span the -86 and -48 region of the human edn promoter. The wild type probe used for EMSA was end-labeled by incubation with T4 polynucleotide kinase and [γ-32P]ATP. The labeled probes were then individually purified by passing through a Sephadex G-25 spin column (GE Healthcare). Binding reactions were conducted with 1 μl [γ-32P]-labeled probe, 20 μg nuclear extract protein, 1 μg poly (dI-dC), 10 mM Tris-HCl (pH 7.5), 50 mM NaCl, 5 mM MgCl2, 1 mM EDTA, and 10% glycerol in a total volume of 20 μl, and the reactions were incubated at 30°C for 30 min. For competition experiments, the nuclear extracts were carried out with a 200-fold molar excess of double-stranded competitor oligonucleotide at 30°C for 30 min prior to the addition of radiolabeled probes. To identify the transcription factors constituting in the protein-DNA binding complexes, anti-Sp1 (PEP2, Santa Cruz Biotechnology) or anti-MAZ (Dr. Kenneth Marcu, State University of New York, Stonybrook, NY) was included in the binding reactions. The protein-DNA complexes were resolved by 6% non-denaturing polyacrylamide gel electrophoresis using 45 mM Tris, 45 mM boric acid, and 1 mM EDTA (pH 8.3). The gel was dried and exposed to X-ray film at -70°C using an intensifying screen.
Expression and purification of GST fusion proteins
pGEX/MAZ and pGEX/MAZΔF34 were introduced into E. coli BL21 (DE3), and synthesis of each fusion protein was induced by addition of 0.5 mM isopropylb-d-thiogalactopyranoside to the LB culture medium. Proteins were purified with glutathione-agarose beads according to the manufacturer's protocol (GE Healthcare).
DNA affinity precipitation assay (DAPA)
The oligonucleotide corresponding to -84 to -48 bp within the edn promoter as well as mutant oligonucleotides were individually biotinylated at the 5'-terminus and then annealed with their complementary strands. All reactions were carried out at 4°C with gentle rocking unless otherwise noted. DAPA was performed by incubating 400 μg of nuclear extract with 20 μg poly (dI-dC) in binding buffer (20 mM HEPES pH 7.6, 10 mM (NH4)2SO4, 1 mM EDTA, 1 mM dithiothreitol, 1% (w/v) Tween 20, 100 mM KCl) that was pre-cleaned with 50 μl streptavidin-agarose beads (GE Healthcare) for 1 hr, and then the supernatant was incubated with 2 μg of biotinylated DNA oligonucleotide for 4 hr or overnight. The protein-DNA complexes were incubated with 20 μl streptavidin-agarose beads for 1 hr prior to collection and washed five times with binding buffer containing 0.5% (w/v) Nonidet P-40. Sample buffer (2×) was added to the streptavidin-precipitated DNA-protein complex and subsequently boiled for 10 min. The proteins were resolved by 10% SDS-polyacrylamide gel electrophoresis followed by western blot detection with specific antibodies.
Chromatin immunoprecipitation assay (ChIP)
The ChIP assay kit and anti-Sp1 were purchased from Upstate Biotechnologies and used according to the manufacturer's instructions. Immunoprecipitations were performed at 4°C overnight with 5 μg primary antibody. Immune complexes were harvested with protein A-Sepharose beads (60 μl/precipitation) as described. Following immunoprecipitation, washing, and purification of DNA, the samples were dissolved in water and used as templates for PCR amplification using two primers: 5'-AAGTGGACTATGTACCAAT-3' and 5'-GGAACTGTTTAAATAAAGCA-3' to generate a 108-bp fragment. The reactions were conducted at 95°C for 5 min followed by 28 cycles of 95°C for 30 sec, 60°C for 30 sec and 72°C for 15 sec.
Chromatin affinity precipitation assay (ChAP)
An expression plasmid for His-tagged full-length MAZ, pcDNA3/MAZ-6H was transiently transfected into HepG2 cells. Two days after transfection, cells (1 × 107) were cross-linked with 1% formaldehyde in PBS for 10 min at 30°C. The treated cells were washed two times with ice-cold PBS, and the cell lysate was then collected with lysis buffer. The chromatin was fragmented by sonication to an average size of 1 kb and centrifuged at 16,000 × g at 4°C for 10 min. The supernatant was diluted fivefold with 1× binding buffer (500 mM NaCl, 20 mM Tris-HCl, pH 7.9) containing protease inhibitors and mixed well with 50 μl salmon sperm DNA-saturated nickel resin slurry (GE Healthcare) for 1 hr. The resin was precipitated followed by washing two times with 1 ml binding buffer and two times with 1 ml wash buffer (50 mM imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9). Protein-DNA complexes were eluted from the His-binding resin with 100 μl of elution buffer (1 M imidazole, 0.5 M NaCl, 20 mM Tris-HCl, pH 7.9) two times. The protein-DNA crosslinks were reversed by heating at 65°C overnight, and proteins were digested with proteinase K (0.5 mg/ml) at 55°C for 1 hr. DNA was purified by phenol-chloroform extraction and ethanol precipitation. DNA from the complexes was analyzed by PCR amplification using the same primer pair for ChIP assay.
After electrophoresis, the proteins were transferred onto a nitrocellulose membrane (GE Healthcare). The membrane was incubated in 3% BSA at 25°C for 1 hr prior to incubation overnight with anti-Sp1 or anti-SAF-1 (Dr. Alpana Ray, Department of Veterinary Pathobiology, University of Missouri, Columbia, MO), followed by secondary antibody HRP-conjugated IgG (1:5000, Jackson ImmunoResearch) for 2 hr. The target proteins were visualized by the ECL system (Pierce).
All experiments were performed at least three times, each in duplicate. The data are expressed as the means ± S.E. For comparison, the data were analyzed using the Wilcoxon Rank Sum test. The cutoff for statistical significance was set as P < 0.05.
We thank Drs. P.-C. Huang, L.-Y. Lin, W.-G. Chou, H.-Y. Chang, C.-Y. Lan, W.-Y. Chow and Y.-K. Lai for their critical comments and discussions about this manuscript. This work was supported by research grants NSC 95-2627-B-007-003, VGHUST96-G2-02-1, and National Tsing Hua University Structural Proteomic Center for Drug Discovery grant 95N2517E1 to M. D.-T. Chang, NSC95-2221-E-019-032 and Center for Marine Bioscience and Biotechnology (CMBB), National Taiwan Ocean University grant to T.-W. Pai, and NSC 96-2752-B-007-003-PAE to Dr. Ping-Chiang Lyu.
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