54, 529 –541 (1998) GE985536
Analysis of the Human RAD51L1 Promoter Region and Its Activation by UV Light Lan Peng, Michael C. Rice, and Eric B. Kmiec1 Kimmel Cancer Center/Jefferson Center for Biomedical Research, Thomas Jefferson University, 700 East Butler Avenue, Doylestown, Pennsylvania 18901-2697 Received May 27, 1998; accepted August 10, 1998
The human REC2/RAD51B gene (HGMW-approved symbol RAD51L1) encodes a 350-amino-acid protein with regional homologies to members of the RAD52 epistasis group. It is induced by DNA-damaging agents, and the overexpression of this gene product causes G1/S cell cycle arrest. In this report, the promoter region, containing the UV-responsive element, is revealed. Deletion analyses of a 1699-base fragment at the 5* end of the human REC2/RAD51B cDNA identified a 116-base sequence that appears to be responsible for radiation induction. This fragment contains many DNA sequences that have been identified in the promoter regions of other radiation-inducible genes in yeast and humans. Within this region are “consensus” binding sites for both the AP2 and the p53 proteins that may act to regulate the expression of the human REC2/RAD51B gene. Five putative transcripts have been identified from regions 5* of the promoter element that splice near the ATG translation start site. None of the transcripts contain the UV-inducible element nor the consensus transcription factor binding sites. © 1998 Academic Press
The capacity of the cell to respond to DNA damage is a critical aspect of its life cycle. During cell growth, DNA is exposed to agents that can induce genetic mutations. These mutagenic sources include ultraviolet light (UV), ionizing radiation (IR), environmental carcinogens, and cytostatic drugs, and the cell must be able to deal with such insults by repairing or replacing the damaged DNA. Fortunately, the evolutionary process has enabled efficient DNA repair. At the center of the response are a series of genes whose expression is induced by the damage signal, an as-of-yet undefined message. Among the genes responding to the signal are those that act directly on the lesion and those that regulate the cell division rate (Bates and Vousden, 1996; Friedberg et al., 1995). In lower eukaryotes, genes that deal with DNA dam1 To whom correspondence should be addressed. Telephone: (215) 489-4903. Fax: (215) 489-4922. E-mail: [email protected]
age are broadly categorized into three groups, although exceptions to each class do exist. This classification is based on phenotype and epistasis analyses of various cell mutants from organisms such as Saccharomyces cerevisiae (Friedberg et al., 1991). The first group, excision repair, contains a group of genes that serve to remove adducts from photodamaged DNA, and organisms lacking one or more of these gene functions are sensitive to UV light. A second group includes the genes involved in postreplication repair or error-prone repair. The genes of the third group, known as the recombinational repair group, are recognizable by their sensitivity to ionizing radiation. Mutations in these genes are defective in the repair of broken DNA strands (usually double strands). Interestingly, these mutants often display dichotomous sensitivity to UV light and ionizing radiation—at some level. We have been interested in the DNA repair/recombination pathways of Ustilago maydis and have been analyzing the genetics and biochemistry of genes involved in these metabolic pathways. Three mutants from this fungus have been isolated and represent an example of each of the classes defined above (Holliday, 1967). Of particular interest has been the rec2 mutant, which displays a phenotype sensitive to both ionizing and UV radiation. The rec2 mutant was isolated originally in a screen for UV sensitivity, and the REC2/ RAD51B gene2 was cloned by complementing the UV sensitivity of the rec2 mutant (Bauchwitz and Holloman, 1990). Other experimental evidence (Ferguson et al., 1997) indicates that REC2/RAD51B is responsible for a significant amount of repair to DNA damaged by ionizing radiation. By screening human and mouse libraries, Rice et al. (1997) isolated, cloned, and characterized a gene based on homology to the Ustilago REC2/RAD51B gene. The human gene has regional homology to human RAD51 and is likely to be a member of the RAD52 epistasis group. The expression pattern of human REC2/ RAD51B is diverse among many tissues, unlike the related repair protein human RAD51, the expression of 2 The HGMW-approved symbol for the gene described in this paper is RAD51L1.
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PENG, RICE, AND KMIEC
which appears limited to testis, ovaries, thymus, and spleen. Among the most significant observations was the IR inducibility of the human REC2/RAD51B gene in normal primary foreskin fibroblasts. After treatment with IR, the human REC2/RAD51B transcript was found to be induced about four- to fivefold in a dose-dependent fashion. Hence, it is possible that human REC2/RAD51B may respond to DNA damage initiated by either form of radiation—a phenotype similar to that displayed by Ustilago (Rubin et al., 1994). To explore the radiation inducibility more directly, we cloned a DNA sequence upstream of the transcriptional start site of the human REC2/RAD51B gene by walking in a 59 direction. We have identified a section of DNA that is 1699 bp in length and contains a TATA box binding element and a series of elements presumed to be involved in responding to DNA damage caused by both UV radiation and IR in lower eukaryotes. Of particular interest is an element located near the extreme 59 end of our fragment. The site is similar to the socalled “p53 binding location” described for several genes whose expression is dependent on p53 (el-Deiry et al., 1993; Miyashita and Reed, 1995; Hollander et al., 1993). In this report, we describe the human REC2/ RAD51B promoter elements, the activation of these elements by UV light, and a possible relationship to p53 binding regulating the UV response. MATERIALS AND METHODS Cloning of the human REC2/RAD51B gene promoter. The human REC2/RAD51B promoter was cloned from a library of uncloned adapter-ligated genomic DNA fragments using PromoterFinder DNA Walking Kits (Clontech Laboratories, Inc.). Each kit contains five “libraries” of uncloned adapter-ligated genomic DNA fragments that constitute a pool of specially prepared DNA fragments. The first or primary PCR used the outer adapter primer (AP1) provided in the kit and an outer, gene-specific primer (GSP1), 59-CAGACGGTCACACAGCTCTTGTGATAA-39, designed based on the known rec2 cDNA sequence. The primary PCR mixture was then diluted and used as a template for a secondary or “nested” PCR using the nested adapter primer (AP2) and a nested gene-specific primer (GSP2), 59-ACCCACTCGTTTTAGTTTCTTGCTAC-39; this produced specific 213-, 258-, and 830-bp PCR products from the PvuII, ScaI, and DruI libraries. We subcloned these three different-sized PCR products into the pCRII vector using T-A cloning (Invitrogen), sequenced them, and named them HP1, HP2. and HP3. All of them overlap, based on the known HP3 sequence. We designed another group of gene-specific primers, GSP3, 59-TAGAGAGAGAGAGAGAGCGAGACAG-39, and GSP6, 59-GTCGACCACGCGTGCCCTATAG-39, using the same method mentioned above. From these primers we obtained an 869-bp PCR product from the SspI library and subcloned this fragment into the pCRII vector using T-A cloning. Each construct was sequenced to confirm the position of insert. DNA sequencing. The sequence reaction was performed with the PRISM reaction DyeDeoxy Terminator Cycle Sequence Kit, which is a single-tube formulation of the DyeDeoxy terminator reaction chemistry for sequence analysis on the Applied Biosystems Model 373A and 377 DNA sequencing system. By computer analysis with the GCG software package (version 9.1), the sequences of the mouse and human rec2 promoters were screened for restriction sites and for homologies to transcription factor binding sites in the GenBank database. Determination of 59 ends of transcripts by 59 RACE. 59 RACE was performed using a Marathon cDNA amplification kit (Clontech). In brief, about 4 mg of poly(A) RNA from H1299 cells was treated with
RNase-free DNase for 2 h at 37°C to remove the genomic DNA and reverse-transcribed with MMLV reverse transcriptase using random hexamers. The second-strand cDNA was synthesized with Escherichia coli DNA polymerase I from this single-strand cDNA, and the Marathon cDNA adapter was ligated. The product was subjected to PCR. Amplification conditions for the primer set RACE-GSP/AP1 were denaturation at 94°C for 5 s, annealing at 70°C for 2 min, and synthesis at 68°C for 2 min. The temperatures for the nested primer set RACE-NGSP/AP2 were 94°C for 5 s, 70°C for 2 min, and 68°C for 2 min. The PCR products were separated by electrophoresis in a 1% agarose gel and purified using the Gel Extraction Kit (QIAEX). Respective DNA fragments were cloned into the T vector (Friedberg et al., 1995), and the nucleotide sequence was determined. Luciferase constructs and transient transfection. Polymerase chain reaction was used to generate specific portions of the human REC2/RAD51B promoter (Fig. 4A) Eight different reactions were run for each set of primers (each pair of primers contains BglII and HindIII as linkers). The BglII/HindIII fragments of the 56 flanking region of the human REC2/RAD51B gene were inserted between the BglII and HindIII sites of the pGL2-Basic vector (Promega) containing luciferase as a reporter gene. For transfection, 3.3 3 105 Hct116 and H1299 cells were plated on six-well tissue culture plates for 18 –24 h prior to transfection by DOSPER liposome (Boehringer Mannheim). The cells were treated with 0, 10, 20, and 30 J/m2 doses of UV radiation (UV Stratalinker 2400 Stratagene) 48 h after plating and were harvested 24 h later for luciferase activity. The human Rec2/Rad51B gene promoter–Luc construction (pLP 1699-Luc) consisted of 1699 kb of genomic sequences linked to the luciferase report gene. When we transiently cotransfected H1299 and Hct1176 cell lines with equal amounts of an expression vector encoding a wildtype p53 cDNA (gift from Dr. E. Mercer, Thomas Jefferson University), the luciferase activity decreased 10- and more than 20-fold, respectively. Luciferase assay. To measure luciferase activity of the transfected cells, the Luciferase Reporter Gene Assay System (Boehringer Mannheim) was used according to the manufacturer’s instructions. Briefly, cells were washed twice with PBS, lysed on the plate with 13 lysis buffer (250 ml/well) for 15 min at room temperature, scraped, and spun down (12,000g) for 30 s at 4°C. Protein concentration was measured by the Micro BCA Protein Assay Reagent Kit (Pierce), supernatant from lysed cells was added to luciferase substrate (100 ml), and the light output was measured immediately at room temperature with a scintillation counter (Beckman LS 5801). The baseline background activity in each tube was measured prior to addition of sample, and the activity of the pGL2-Basic vector (Promega) alone served as a negative control. Statistical analysis was conducted using MS Excel III. Electrophoretic mobility shift assay (EMSA). EMSAs were performed using the Bandshift Kit (Pharmacia Biotech). Briefly, DNAbinding reactions were performed in a volume of 40 ml with 2 mg poly(dI– dC) and 12% glycerol in binding buffer (pH7.9). Six micrograms of recombinant p53 cell lysate or nuclear extracts from HCT116 cells (Andrews and Faller, 1991) was preincubated for 10 min at 25°C followed by the addition of 10,000 cpm of 32P-labeled 345-bp DNA sequence in the reaction mixture. The binding reaction was allowed to proceed for 20 min at 25°C, and the bound complexes were separated from the unbound probe on low-ionic-strength 6% polyacrylamide gels with recirculating Tris–acetate–EDTA buffer containing 7 mM Tris–HCl, 3 mM sodium acetate, and 1 mM EDTA. In competition experiments, increasing concentrations of unlabeled DNA sequence were added to the reaction mixtures before the addition of the radiolabeled DNA sequence probe for 10 min; this was followed by incubation with the labeled probe as described above. Immunoprecipitation. Equivalent amounts (20 mg) of Sf9 recombinant p53 cell lysate were cleared of nonspecific binding by incubation with 20 ml of protein A agarose beads for 1 h at 4°C in an orbital shaker. After preclearing, 2 mg of monoclonal anti-p53 antibodies (a combination of 1 mg of DO-1 [Santa Cruz Biotechnology] and 1 mg of Pab421 [Oncogene Science]) was added and incubated at 4°C for 12 h in the shaker followed by precipitation with protein A agarose beads
RADIATION-INDUCIBLE PROMOTER OF HUMAN RAD51L1 as previously described. Supernatants were collected; 10 ml of the supernatant solution was used for EMSA with the 350-bp probe as described above.
Promoter Region Organization and Determination of Transcription Initiation Site A section of DNA located upstream of the coding region of the human and mouse REC2/RAD51B genes was cloned using the Promoter Finder DNA Walking Kit (Clontech Laboratories, Inc.). This approach was taken to determine whether sequence elements known to be involved in the response to DNA damage were present. Earlier work had revealed that the human REC2/RAD51B gene was induced at both the RNA and the protein levels by UV and ionizing radiation (Rice et al., 1997; Havre et al., 1998). A fragment comprising 1699 nucleotides was cloned and the DNA sequence determined for the human upstream region (Fig. 1). Within this 1699-bp fragment, however, a series of sequences that are common to promoter elements of genes known to respond to DNA damage was found. These sequences are underlined and catalogued in Fig. 1 but are assembled in Table 1. A large number of upstream response sequences (URS) found in DNA repair genes from yeast are present. These elements comprise genes responding to UV damage (Rad2 and RAD16) and ionizing radiation (RAD51 and Rhp51). These two classes of genes fall into two different repair categories, nucleotide excision repair and recombinational repair (Friedberg et al., 1991). A clustering of these sequences and others is observed within 100 bases of the transcription start site surrounding the TATA box. Of particular interest is the presence of a consensus p53 binding site located near the far distal end of the isolated promoter, a sequence that spans 49 nucleotides and comprises five 8-base elements having greater than 75% identity to a generalized consensus p53 binding site (Miyashita and Reed, 1995; el-Deiry et al., 1992). This site and other sequences known to be involved in the RNA polymerase II-mediated transcription homology to binding sites for AP-2, AP1, Ets-1, Sp1, and LBP-1, as well as C-Myc and C-Myb, were also identified (Fig. 2). To identify transcripts from the human REC2/ RAD51B gene, the 59-RACE system (Clontech) was employed. A primer, homologous to the region between positions 260 and 284 downstream of ATG, was synthesized. Using reverse transcriptase, five reaction products that corresponded to five transcripts whose locations are indicated in Fig. 3A were observed. Each transcript contains G-G-C adjacent to the ATG start site. The sequence for the cDNA, reported previously (Rice et al., 1997), is matched perfectly with the downstream coding region. In the 59 direction (upstream), three of the transcription start sites are located in a 62-bp region, separated from the downstream ATG by 3699 nucleotides. These were designated P2A(11),
P2B(133), and P2C(143). The fifth transcript emanates from a 97-bp region 4305 nucleotides 59 of the ATG and 447 nucleotides from the 62-bp site (P311). The nucleotide sequence for all transcripts is provided in Fig. 3B. The three upstream regions harboring the transcription start sites are underlined, and the exact base at which a transcript begins is identified by arrows. Putative “TATA boxes” are encased in the figure, although it is not yet clear if all of them are active. The boxed regions represent potential 59 promoter regions for each putative transcript. Based on the position of the one closest to the ATG and the strength of that region of the promoter (see below), we believe that the one located at (254) is active. A number of other groups have used this system to identify transcripts from particular genes (see Adolph et al., 1997, and references therein). Although our results indicate the presence of five transcripts, because of potential pausing or inaccurate termination of the RT-PCR process, the three emanating from the exon 2 (?) region could have the same start site. Recently Cartwright et al. (1998) reported multiple transcripts for this gene as well. Subcloning of the Human REC2/RAD51B Promoter Specific PCRs using one fixed and one variable primer generated constructs with varying lengths of the putative human REC2/RAD51B promoter. The resulting fragments were inserted into a vector containing the luciferase gene. The position of each deletion was such that these fragments would serve as the promoter for luciferase gene expression (Fig. 4A). Plasmids containing 245, 345, 830, 1050, 1354, 1492, 1583, and 1699 bp of the human REC2/RAD51B promoter were created and positioned at the 59 end of the luciferase gene. Each of these plasmid constructs was transfected into HCT116 cells at equimolar concentrations using liposome (DOSPER)-mediated gene transfer. HCT116 cells are particularly useful in these studies because they contain allelic copies of wildtype p53 (Take et al., 1996). The transfection efficiency of each cell line was normalized based on control experiments using intact luciferase and b-gal expression vectors (data not shown). After transfection, the luciferase activity was quantified by light emission at 562 mm. As seen in Fig. 4B, the expression of luciferase was influenced by the length of the upstream human REC2/RAD51B promoter sequence. In fact, the shortest construct (pLP 245-luc) is significantly stronger in controlling the expression of the reporter gene. All of the deleted vectors provide a higher level of expression than the full-length sequence, albeit to somewhat different degrees. Furthermore, pLP 245-luc produces a fivefold higher expression level than the control SV40 promoter, whereas pLP 1699 is noticeably lower in promoting luciferase expression. Of particular interest is the observation that the levels with expression of the pLP 1699 and pLP 1583, constructs containing adjacent sequences, are quite different. The major se-
FIG. 1. Nucleotide sequence of the 1699 bases immediately upstream of the human REC2/RAD51B transcription start site. The DNA sequence of the UV-inducible region is presented with underlined segments that correspond to previously identified sequences that are present in radiation-responsive genes. The definitions and references of the underlined sequences are presented in Table 1.
RADIATION-INDUCIBLE PROMOTER OF HUMAN RAD51L1
TABLE 1 Homology between Human REC2/RAD51B Upstream Sequences and URSs of Yeast DNA Repair Genes Gene MAG PHR1 RAD2 RAD51 DDR48 RNR2 RNR3 Rhp51 RAD16 Consensus PHR1 RAD2 RAD23 Consensus rhp51 RAD51 RAD54 RNR3 Consensus
2215 2103 2109 2169 2157 2271 2322 2374 2467 2233 2213 2309
GTAGGTCGAA CGAGGAAGCA CGAGGAAGAA CGTGGAGGCA CGTGGTGGGA CGAGGATGAC CGTGGTTGAT CGAGGTCGCA CTAGGTAGCA GTAGGTGTTA CTAGGTAACA CATGGTTGCC CGTGGTNGAA A A CC
2103 2110 2166 2295
CGAGGAAGCAGT CGAGGAAGAAAA GGAGGCATTAAA GGTGGCGAAATT GGAGGARRNANA C T C
2290 2260 2215 2256 2429
CGTT_CCCTAT CCTA_CCCTAA CGTTACCCTAT CGTTACCCAAT CGGTTGCCATG CGTTACCCTAT
quence element present in pLP 1699, and not present in pLP 1583, corresponds to a consensus p53 binding site (el-Deiry et al., 1992). Hence, essential elements for basal level gene expression appear to be contained in the 245-bp human REC2/RAD51B promoter fragment with perhaps cryptic suppression sites located throughout the region. Induction of Promoter Activity by UV Light HCT116 cells were transfected with the full-length promoter construct and the cells subsequently irradiated with UV light at 254 mm. After 24 h, luciferase gene expression was determined. For comparison, the same construct was transfected into H1299 cells, which
Rec2/Rad51B promoter location
2892 2892, 2924 2924 29 2964 21097, 2246 2218 2947, 2924, 2892, 2246 2924, 2892 21504, 2473, 2466 2687, 2678 2246
(39) (33) (33) (25, 28, 33, 34, 39) (1, 4) (39, 36) (39, 36) (12, 20) (39, 40) (22) (22) (39) (5)
2892 21259 2554, 2720 2116, 2665, 21211, 21620
21350, 273 2539 281, 21333 2357 889, 2244
(34) (23, 38)
(1) (9) (12)
are known to be null for active p53 protein (Chen et al., 1996). As shown in Fig. 5A, a significant stimulation occurred only in HCT116 cells. At each of the three dosages of UV irradiation, an increase in luciferase expression was observed, with a maximal response (six- to eightfold) observed at 20 J/m2. The reduction in response at 30 J/m2 is likely due to the progressive loss of cell viability. The result was quite different in H1299 cells, in which induction was not found to occur at any of the radiation levels tested. In fact, a reduction in response was found to occur as the dosage was increased. Among several possibilities, two are most likely. Either UV response in the human REC2/ RAD51B promoter is dependent on the presence of p53 or H1299 cells become uniquely UV sensitive after
FIG. 2. Transactivating factor binding sites. The positions of various transcription factor binding sites are indicated by the appropriate symbol. A “TATA” box element is located at (254). A common transcription start site is indicated at (23) and the translated region (ATG start) is at (11).
PENG, RICE, AND KMIEC
FIG. 3. Five putative transcripts are identified for human REC2/RAD51B. (A) The positions of the five transcripts of human REC2/ RAD51B are indicated by a black and an open rectangle. The dotted lines between these rectangles indicate regions of the DNA sequence that are not found in the transcripts. The sequence of the transcripts is presented at the bottom along with the 59 end. The designations and numbers in parentheses to the left of the 59 end refer to each individual transcript and the change in length of the transcript relative to the farthest upstream start site (11). The position of the start codon is represented by three dots. The human REC2/RAD51B “promoter-element” (1699 bases) is also positioned relative to the transcribed sites. (B) The 1699-nucleotide sequence containing one transcription start site (2) and the distal 59 region containing the other four start sites (2) are presented. The dotted line represents 2.0 kb of DNA separating the inducible promoter (1699) region and the upstream sections containing the other start sites. The boxed areas represent the promoter region immediately 59 of a transcription start site, which is designated by a P.
transfection. Cell viability assays on H1299 after exposure to 10 or 20 J/m2 were conducted, however, and no appreciable cell death was seen, while at 30 J/m2 loss of cell viability paralleled that of the HCT116 cells. Previous data (Rice et al., 1997) suggest that human REC2/RAD51B gene expression is inducible in primary foreskin fibroblasts, and identical experiments in these cells produce results as described above. Another series of experiments was carried out in which the full-length and truncated human REC2/ RAD51B promoter constructs were employed. These constructs were transfected at equimolar concentrations into either HCT116 or H1299 cells. The results, presented in Fig. 5B, reveal induction by UV light to the same extent in the full-length construct in HCT116 cells (Fig. 5A), while H1299 cells did not support elevated levels of luciferase gene expression (Fig. 5C). Furthermore, constructs lacking the first 116 nucleotides, 59 distal to the transcription start site, did not demonstrate UV responsiveness in HCT116 cells. In H1299 cells, no induction by exposure to UV light is observed with any of the constructs tested. These results coupled with the observation that induced expres-
sion was found only in HCT116 cells suggest that the putative p53 binding site, contained within the 116-bp sequence, may be important for human REC2/RAD51B induction by radiation. The possibility that p53 was interacting with the distal end of the human REC2/RAD51B promoter was addressed directly by EMSA. For convenience and optimal assay readout, we used the 345-bp fragment, containing the p53 binding site, as the DNA substrate. The radiolabeled probe was incubated with cell lysates containing recombinant p53, and the shift in electrophoretic mobility was measured as described under Materials and Methods. As shown in Fig. 6A, a definite retardation in probe mobility is observed. Subsequent experiments revealed that this complex was disrupted by competing unlabeled specific DNA sequences but not by nonspecific ones. Furthermore, preincubation of the p53 cell lysate with monoclonal antibodies (see Materials and Methods) prohibited the binding to the p53 sequence. Since HCT116 cells contain wildtype p53, we utilized cell-free extracts from this source to measure binding of the 345-bp fragment. As shown in Fig. 6B (lane 2) the migration of radiolabeled probe is
RADIATION-INDUCIBLE PROMOTER OF HUMAN RAD51L1
altered after incubation with the extract as judged by a gel mobility shift assay. Incubating specific unlabeled 345-bp fragment (lane 4) removes over 80% of fragment
binding, whereas incubation with nonspecific unlabeled DNA reduces binding by less than 10% (lane 3). Hence, taken together, these data suggest that the
PENG, RICE, AND KMIEC
FIG. 4. A region of DNA between 21699 and 21583 contains a negative regulator for the human REC2/hRAD51B gene. (A) Diagram of the promoter deletion constructs of the human REC2/RAD51B gene spliced to the reporter gene, luciferase. (B) The expression of luciferase is measured by production of cpm/50 mg total protein and is averaged over five experiments. As a negative control pLP-0 containing only the full 1699-bp promoter element is plotted, whereas pSV40-luc contains the luciferase gene under the control of the SV40 early promoter (Boehringer Mannheim). The numbers after pLP indicate the length of the 59 human REC2/RAD51B sequence.
345-bp fragment from the 59 distal end of the human REC2/RAD51B promoter has the capacity to bind wildtype p53 with specificity. To demonstrate directly that the UV response element resides within the last 345 bases of the human REC2/RAD51B promoter, we cloned the fragment from this region adjacent to the 245-bp fragment that had previously demonstrated basal level regulation of luciferase gene expression. The fragment was ligated to the 245-bp promoter–luciferase construct. The plasmid was transfected into HCT116
and treated with UV light at 20 J/m2, and the level of luciferase induction was measured. Figure 7A displays the results demonstrating that the presence of the “p53 site” reduces the level basal activity of the 245-bp promoter construct. The stimulation by UV light, however, requires the presence of the distal sequence element. Since H1299 does not harbor functional wildtype copies of p53 and the full-length promoter construct pLP 1699 was active in these cells, the prediction is that the transient expression of wildtype p53 in these cells would
RADIATION-INDUCIBLE PROMOTER OF HUMAN RAD51L1
FIG. 5. Human REC2/RAD51B promoter element is induced by UV light in HCT116 cells. (A) The intact pLP-1699 plasmid was transfected into either HCT116 cells or H1299 cells, and the cells were exposed to the indicated levels of UV light and harvested for measuring luciferase expression 24 h later. The data represent information from four experiments. (B) The assays were conducted in HCT116 cells as described in (A) except that the indicated plasmid construct was used. (C) The assays were conducted as described in (B) except that H1299 cells replaced HCT116 cells as the host line.
reduce the level of luciferase expression from pLP 1699luc. This can be tested directly. An experiment was carried out in which pLP 1699-luc and a p53 expression plasmid were cotransfected into H1299 cells. As a control, the expression vector lacking p53 was used. As seen in Fig. 7B, the cotransfection of plasmid expressing wildtype p53 reduced the expression of pLP 1699-luc by 20fold at each of the levels tested. The control plasmid had no noticeable effect on luciferase activity. In addition, expression of luciferase from pLP 1583 was unaffected by the expression of p53; pLP 1583 lacks the putative p53 binding site. Taken together, these results reinforce the potential role of p53 in the suppression of the human REC2/RAD51B promoter.
In this study, we have cloned and characterized a promoter region of the human REC2/RAD51B gene that responds to UV light. The promoter region is 1699 nucleotides in length and contains a series of DNA sequences found in other radiation inducible genes. Some elements are found in the upstream region of RAD23 and RAD16 from S. cerevisiae, a group of genes involved in nucleotide excision repair that are inducible by UV light. Similarly, upstream elements from the yeast genes RAD51, RHP51, and RAD54 are present. These genes are induced by ionizing radiation. Other sequences include RNR3-UAS and PHR1-URS,
PENG, RICE, AND KMIEC
FIG. 6. Human p53 protein binds to an element within the 1699 promoter region. (A) The 345-bp DNA fragment used to construct pLP345 served as a probe to assay for specific binding of p53. The radiolabeled probe was found to be band-shifted in the presence of the protein and blocked by both specific competition DNA (345-bp DNA fragment) and anti-p53 monoclonal antibody. Nonspecific DNA refers to the fragment from the promoter region (2649 to 869) not containing the putative p53 binding site. (B) The 345-bp fragment was incubated with an extract from HCT116 cells. Lane 1, no addition; lane 2, HCT116 cell extract added with nonspecific (2649 to 869) DNA competitor; lane 4, HCT116 cell extract with specific (345-bp) competitor. Bound refers to the area on the gel where retardation of the DNA probe is most prominent. Free refers to either untreated DNA or unbound DNA.
elements representative of inducible genes from yeast (Friedberg et al., 1991). Hence, by simple alignment, the 1699-nucleotide sequence representing the promoter of human REC2/RAD51B appears to have assembled elements capable of responding to DNA damage caused by radiation. The promoter region also contains sequence elements that are sites for transcription factor binding. Multiple AP1 and AP2 sites are present as well as LBP1 sites, which are most abundant. This last element is found in the 59 untranslated leader region and functions in basal promoter activity (Jones and Prakash, 1991). There are five transcripts identified thus far from the human REC2/RAD51B gene. Each of them contains a G-G-C element adjacent to the start codon. For one transcript, this sequence is the only untranslated region while the other four contain sequences that come
from two upstream nests. A region that is 3699 bp upstream of the ATG harbors three transcript start sites contained within a 54-base region. We have tentatively termed this region an untranslated exon (exon 2). An upstream (454 bases) element harbors the other transcription start site (exon 1). The region that contains the UV response element is spliced out of any transcript and thus may act purely as a regulatory element. The RHP51 gene from Schizosaccharomyces pombe has been shown to produce three transcripts of different sizes (Muris et al., 1993). Further characterization of RHP511 revealed that two transcription start sites were responsive to DNA damage (Jang et al., 1996). Additionally, the three transcripts were regulated differentially during the cell cycle, and the level peaked at the G1/S border. DNA damage can induce a number of mammalian genes of which the GADD genes
RADIATION-INDUCIBLE PROMOTER OF HUMAN RAD51L1
FIG. 7. The UV response element resides near the 59 end of the 1699 construct. (A) A plasmid, pLP345-245-luc, consisting of 345 bp from the 59-most distal end of the 1699 construct and 245 bp immediately adjacent to the luciferase gene, was transfected into H1299 or HCT116 cells. One group of each cell line was irradiated with 20 J/m2 of UV light, and, 24 h later, the level of luciferase gene expression was measured. As controls, the same experiment was carried out on cells transfected with pLP245-luc. (B) Plasmid constructs pLP1699-luc or pLP1583-luc were transfected into H1299 cells in conjunction with either pLPO(V) or a plasmid capable of expressing wildtype p53 (see Materials and Methods). After 24 h, the level of luciferase expression was measured as described above and is presented as the average of three experiments.
are best characterized (Fornace et al., 1989). GADD45 has radiation-responsive regulatory regions in the proximal promoter region and within introns (Hollander et al., 1993). A dependence on p53 binding to the intron segment was found to initiate the response to DNA damage, a response similar to that observed in human REC2/RAD51B. Cartwright et al. (1998) recently confirmed the presence of multiple transcripts from the human REC2/RAD51B gene. The utilization of a particular transcript either may be in response to a different DNA insult or may be tissue-specific. We have found evidence for only one transcript upon induction in primary foreskin fibroblasts (Rice et al., 1997). Hence a distribution in tissue may be a more likely explanation. As deletions progressively generate shorter fragments, a consistent pattern of reporter gene (luciferase) expression was noted. The elimination of 116 nucleotides from the ultimate 59-distal end reduced expression 10- to 15fold while a region between (2)1354 and (2)1830 was found to support lower levels of expression. A region containing only 245 bases of the promoter was as effective as any other construct in regulating luciferase expression. The low level of expression found in pLP 1699luc may be due to the presence of a p53 binding site located within the first 116 bp at the 59 end. The reduc-
tion of expression when pLP 1354-luc, pLP 1050-luc, or pLP 830-luc is used may reflect the presence of a regional suppressor sequence that is overcome by upstream sequence roughly between 21354 and 21492. We should note that all of the constructs with the exception of pLP 1699-luc supported luciferase expression to a greater extent than the early SV40 promoter that was used as a control in the assays. Based on the present results, we suggest that this element resides within 116 bases at the extreme 59 end of the 1699-bp fragment. In every assay, the regulation of gene expression was examined under conditions of DNA damage. This region alone appeared to enable the UV induction of luciferase expression. Using a variety of doses, we determined that 20 J/m2 was optimal to mediate a 10- to 15-fold induction of pLP 1699-luc. Based on sequence alignment and database searches, the single known element contained in the 116-base region is a p53 binding site (el-Deiry et al., 1992). The site is 49 bases in length and is 75% homologous to the established p53 binding site. Surprisingly, a second p53 binding site with similar levels of homology (75%) is located between nucleotides (2)111 and (2)66 within 18 bases 59 of the TATA box. In the absence of the upstream p53 site, the downstream site does not, however, confer UV inducibility.
PENG, RICE, AND KMIEC
Further support for the importance of p53 in UV response comes from three experimental results. First, band-shift experiments demonstrate specific binding by p53 to the upstream element. Second, the UV-inducible response is found only in cell lines containing wildtype p53. Cell lines HCT116 and H1299 were selected because the p53 status in these lines was established. At present, we do not know how mutant p53 proteins act on this promoter. Experiments, for example, in HeLa cells, yielded high levels of expression but no UV inducibility (L. Peng, unpublished results). Hence, we chose cell lines that were either wildtype for p53 or null for p53. Finally, the overexpression of p53 in H1299 cells (null for p53) inhibited luciferase expression only in pLP 1699-luc, the single construct containing the upstream p53 binding site. p53 has been identified as a transcription factor that can both induce (Bates and Vousden, 1996; Israeli et al., 1997) and repress (Ko and Prives, 1996) gene expression. Both actions are through the binding of a specific sequence in the promoter region of the target genes. The inhibition or repression activity is often mediated indirectly by the induction of inhibitor proteins such as p21Waf1 (Waldman et al., 1995). An interesting gene known as PAG608 (Israeli et al., 1997) has strikingly similar effects on the cell cycle compared to human REC2/RAD51B. Both are induced by DNA damage dependent on wildtype p53. Both proteins contain DNA binding domains, and the overexpression of either leads to apoptosis. In the case of human REC2/ RAD51B, however, cell lines lacking wildtype p53 or those null for p53 also enter an apoptotic pathway when human REC2/RAD51B, coupled with DNA damage, is overexpressed. Taken together, the results suggest that p53 or a factor enabled by p53 suppresses human REC2/ RAD51B expression. For example, only low levels of human REC2/RAD51B mRNA are found in HCT116 cells, while H1299 cells have naturally higher levels of REC2/RAD51B message. After DNA damage induced by UV light, p53 may be required for the onset of promoter function. This “dual-opposing” personality of p53, or its negative regulation role, may reflect an alteration in the protein or in the activity of a cellular factor that modifies p53. Hence, under normal cell growth conditions, human REC2/RAD51B is actively suppressed in some cells, but upon DNA damage, REC2/RAD51B plays a role in cellular response. It is possible, therefore, that normal growth patterns, controlled by p53, would be disrupted by the presence of REC2/RAD51B. However, with extensive DNA damage caused by irradiation, p53 may release the suppression of human REC2/RAD51B, enabling the cell to enter programmed cell death. This mechanism would prevent the inheritance of mutations to daughter cells after replication and cell division.
ACKNOWLEDGMENTS We thank Dr. Bill Holloman (Cornell University) and Dr. Ramesh Kumar (Kimeragen, Inc., Newtown, PA) for helpful comments on the manuscript. We are also grateful to members of the Kmiec laboratory for technical advice and critical reading of the paper. This work was supported by a grant from Kimeragen, Inc. (Newtown, PA).
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