Sequence-specific DNA Binding Activity of RNA Helicase A to the p16INK4a Promoter*

Sanna Myöhänen and Stephen B. BaylinDagger

From the Johns Hopkins Oncology Center, Baltimore, Maryland 21231

Received for publication, May 24, 2000, and in revised form, October 4, 2000



    ABSTRACT
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

p16INK4a is frequently altered in human cancer, often through epigenetically mediated transcriptional silencing. However, little is known about the transcriptional regulation of this gene. To learn more about such control, we initiated studies of proteins that bind to the promoter in cancer cells that do, and do not, express the gene. We identify RNA helicase A (RHA) as a protein that binds much better to the p16INK4a promoter in the expressing cells. RHA has not previously been characterized to manifest sequence-specific DNA interaction but does so to the sequence 5' CGG ACC GCG TGC GC 3' in the p16INK4a promoter. The Drosophila homologue to RHA, maleless (Mle), functions in the fly for 2-fold activation of male X-chromosome genes. In our experimental setting, RHA induces a similar modest up-regulation of the p16INK4a promoter that is dependent upon its sequence-specific interaction. Mle colocalizes with hyperacetylated H4Ac16 on the X-chromosome and some autosomal loci. The decreased binding of RHA to p16INK4a in our cells, where the gene is transcriptionally inactive, is associated with decreased amounts of RHA that immunoprecipitate with acetylated lysine antibodies. Finally, we show RHA to be a cellular substrate for caspase-3, which decreases its sequence-specific binding to p16INK4a by cleavage of the N terminus. Thus, we have identified a new protein interaction with the p16INK4a promoter that involves an important protein for transcriptional modulation. This interaction is decreased in cancer cells, where this gene is aberrantly transcriptionally silent.



    INTRODUCTION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Defects in the Rb-p16INK4a-cyclin D pathway are one of the most commonly found abnormalities in a wide variety of cancer cells. Dysregulation of this pathway either through loss of Rb or p16INK4a or amplification of cyclin D1 causes abnormal cell cycle regulation at the G1/S checkpoint (1). Whereas Rb is commonly mutated within the coding region (1), gene deletions and transcriptional silencing are preferred mechanisms for p16INK4a loss in tumor cells (2). The transcriptional silencing is associated in most tumors with abnormal hypermethylation that extends over the proximal promoter and the 5' untranslated region of the gene both in primary tumors and in cell lines (2). Several studies have established that loss of p16INK4a function is an early event in tumor progression (3, 4), and it has been postulated that loss of p16INK4a expression is essential for tumor growth (5, 6).

Despite the widespread alterations of p16INK4a in tumorigenesis, little is known about the molecular events that control its transcriptional expression and that might predispose tumor cells to abnormal transcriptional silencing of the gene. We have thus initiated a search for specific DNA-binding proteins whose function, if compromised, might contribute to events leading to such abnormal regulation of the p16INK4a gene promoter, including hypermethylation of the region. In this paper, we describe a sequence-specific differential binding of human RNA helicase A (RHA)1 to the p16INK4a promoter sequences in cells with unmethylated versus methylated endogenous p16INK4a genes. RHA is a homologue of maleless (Mle), a dosage compensation gene in Drosophila (7). Mle, along with msl-1, msl-2, and msl-3, is needed for the hyperactivation of the single male X-chromosome in flies (8), and this complex colocalizes with hyperacetylated H4Ac16 on the X-chromosome and in some autosomal loci (9). In this regard, in our studies, RHA appears to have a potential modulatory role for p16INK4a transcription.


    EXPERIMENTAL PROCEDURES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Gel Shift Analysis-- Nuclear proteins from culture lines were extracted according to Dignam et al. (10). Protein amounts were measured using the Bradford assay (Bio-Rad). All oligonucleotides were from Life Technologies, Inc. Binding reactions were performed in 1× binding buffer (50 mM KCl, 10 mM Tris-HCl, pH 7.9, 1 mM EDTA, 10% glycerol, 5 mM MgCl2, 1% Nonidet P-40, 1 mM dithiothreitol, 10 ng/ml pepstatin, 100 ng/ml leupeptin, 100 ng/ml aprotinin, and 1 mM phenylmethylsulfonyl fluoride) for 15 min on ice in a volume of 20 µl. Each reaction contained, in addition, 1 µg of poly(dA)·poly(dT) (Amersham Pharmacia Biotech) as a nonspecific competitor. Nuclear extracts were preincubated with nonspecific and specific competitors for 10 min on ice before addition of labeled oligonucleotide. Gel shifts were run for 2 h in 1× low salt, tris-acetate EDTA (6.75 mM Tris-HCl, pH 7.9, 1 mM EDTA, 33 mM sodium acetate, pH 7.9) on a 5% nondenaturing polyacrylamide gel.

Southwestern Analysis-- Nuclear proteins (30 µg) were separated on a 7.5% denaturing polyacrylamide gel. Proteins were transferred to an ImmobilonTM (Millipore) membrane, which was treated with 6 M guanidine HCl in binding buffer (10 mM Hepes, pH 7.9, 1 mM EDTA, 1 mM dithiothreitol, 50 mM KCl) for 5 min. The membrane was then denatured by treatment with decreasing concentrations of guanidine-HCl (3, 1.5, and 0.75 M) in binding buffer and finally with binding buffer alone. Each step was for 5 min at room temperature. The membrane was blocked in 5% milk in binding buffer overnight at 4 °C and then incubated with 100,000 cpm of concatemerized B4 oligonucleotide (see Table I) in the binding buffer for 1 h at 4 °C and washed three times for 5 min each with cold binding buffer.

DNA Affinity Purification and MALDI-MS Protein Identification-- Concatemerized B4 molecules (see Table I) with a biotin label in one end were attached to streptavidin-coated magnetic beads (Dynal AB). Total nuclear protein fraction from cell line NCI-H69 (0.5 × 109 cells) was mixed with nonspecific competitor poly(A)·poly(T) (60 ng/ml) and oligo B4M1 (2 ng/ml; see Table I) in 1× binding buffer with proteinase inhibitors. After 50 min of incubation on ice, the mixture was centrifuged for 10 min at 17,000 × g, and the supernatant was added to the streptavidin-coated magnetic beads. The binding reaction was incubated for 10 min at room temperature with rotation. Beads were washed two times with binding buffer and eluted with 0.4 M KCl in binding buffer. Eluted proteins were separated on a 7.5% SDS-PAGE gel and Coomassie stained. Selected proteins were excised and analyzed by MALDI-MS at the Keck Foundation (Yale University, New Haven, CT).

Plasmid Constructs-- For transfection studies, the ATG site initiating translation of the p16INK4a was mutated to a BamHI site with polymerase chain reaction mutagenesis using an oligonucleotide, 5' AAA GGA TCC GCT GCT CCC CGC CGC CCG CT 3'. p16INK4a promoter constructs of 0.9 kb (EcoRI-digested region) or 2.0 kb (HindIII-digested region) upstream of the ATG were cloned in pGL3 luciferase vector (Promega). Mutation in the RHA binding site described under "Results" was introduced by polymerase chain reaction mutagenesis with the oligonucleotide 5' CTG GCT GGT CAC CAG AGG GTG GGG CGG ACC GAG TGC GCT C 3' (with the underlined base representing the site where C in the original sequence is mutated to A (mutant oligo B4M1; see Table I)). The polymerase chain reaction oligonucleotide included an endogenous BstEII site in the 5' end, allowing the replacement of a BstEII-BamHI fragment in the wild-type constructs. All constructs were verified by sequencing. An expression plasmid containing the RHA cDNA in pcDNA3 was a gift from Dr. Che-Gun Lee.

Transfections-- One day before transfections 2 × 105 U1752 cells were plated and the next day transfected with 1 µg of wild-type or mutant p16INK4a promoter-luciferase constructs and 8 µl of LipofectAMINETM (Life Technologies, Inc.). In cotransfection experiments, 1 µg of luciferase construct was transfected along with 1 µg of RHA expression plasmid or with vector plasmid alone. Transfections were incubated for 4 h. NCI-H69 cells were transfected with 1 µg of plasmids and 2.5 µl of DMRIE transfection reagent (Life Technologies, Inc.) for 4 h. A renilla luciferase plasmid (Promega) was used to control for transfection efficiency, and luciferase activities were measured at 24 h using the Dual Luciferase kit (Promega). All transfections were done in triplicate, and results are shown from three independent experiments.

TnT Reactions-- The RHA cDNA in the pRSET plasmid was a kind gift from Dr. Che-Gun Lee. Deletion constructs AvaIII and XhoI were prepared from the full-length cDNA by removing amino acid regions 841-1270 and 693-1052, respectively, from the C-terminal end of the protein. 1 µg of plasmid DNA was transcribed and translated using the Quick Coupled Transcription/Translation system from Promega. 5 µl of the reaction was analyzed on SDS-PAGE gels or in a gel shift assay.

Drug Treatments-- Trichostatin A (TSA) treatments were for indicated periods with 500 nM TSA (Wako). Control reactions were treated with EtOH. The caspase-3-specific inhibitor DEVD (final concentration 50 µM) was added 1 h before addition of TSA.

Western Blotting and Immunoprecipitations-- Western blotting was performed using standard protocols (ECL, Amersham Pharmacia Biotech). Antibodies and protein-A-agarose were purchased from Santa Cruz Biotechnology (Sp1 PEP2, Sp3 D-20, Sp4 V-20), except for topoisomerase, which was from TopoGEN, Inc. RHA antiserum was a kind gift from Dr. Che-Gun Lee. Immunoprecipitations with acetylated lysine antibodies were performed as described in the product manual (New England Biolabs) and washed five times with phosphate-buffered saline before loading on a 7.5% SDS-PAGE gel.


    RESULTS
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

Putative Sp1 Elements at the p16INK4a Promoter Bind Sp1 and Sp3 in Vitro-- With respect to differences in normal and abnormal control of p16INK4a transcription, we first chose to examine Sp1 binding activities for the p16INK4a promoter. Such sequences have been proposed for a role in protection of CpG islands from methylation (11-13). We extracted nuclear proteins from several cell lines, representing tumors of different tissue types, and with or without p16INK4a hypermethylation-associated transcriptional silencing, to establish whether any gross differences were detected in binding of Sp1 family proteins (14) to p16INK4a sequences in gel shift assays. The double-stranded oligonucleotides used in the binding studies are described in Table I. Gel shift analysis with all three putative Sp1 elements from the proximal p16INK4a regulatory region showed strong binding of three specific proteins (a, b, and c in Fig. 1, A and B). Using Sp1- and Sp3-specific antibodies in supershift analysis (data not shown), we identified two Sp3-specific shifted complexes (b and c) and one Sp1 specific shift (a) at each site (Fig. 1, A and B). We did not detect any quantitative differences in binding of these two proteins between different culture lines with or without methylation of the endogenous p16INK4a. Furthermore, all cell lines also had high levels of expression of both Sp1 and Sp3 by Western analysis (data not shown).


                              
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Table I
Oligonucleotides used in gel shift analyses
The top three lines of the table show p16INK4a promoter sequence oligonucleotides used for initial gel shift analyses to characterize protein binding patterns to the region. Putative Sp1 sites are underlined, and the position of sites are shown in Fig. 1C as A, B, and C. The seven lines in the bottom portion of the table show oligonucleotides used in gel shift analyses to characterize the DNA binding specificity of a newly defined factor. B4 is the sequence identified as the specific binding site (located at B in Fig. 1C) and used as the concatemerized probe to affinity-purify the factor. The M1 through M5 derivatives contain mutations of the B4 sequence and were used in competition studies to assess binding specificity. The Sp1 oligonucleotide was used to compete away binding of Sp1 family proteins in some gel shift studies.



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Fig. 1.   . Gel shift analyses for Sp1 binding site sequences in the p16INK4a proximal promoter with proteins from various cancer cell lines. A, gel shift with labeled probe p16C (Table I and panel C). Cell lines are as follows: lane 1, U1752 (human squamous cell lung carcinoma); lane 2, NCI-H69 (human small lung carcinoma); lane 3, NCI-H727 (lung carcinoid); lane 4, NCI-H82 (human variant small cell lung carcinoma). M, methylated endogenous p16INK4a gene; U, unmethylated endogenous p16INK4a gene. Shifted complexes are as follows: a, Sp1; b and c, Sp3. The major band below c represents a nonspecific complex. Without nuclear extract, no bands were seen above the migration position for the free probe at the bottom of the gel (as shown directly in lane F of Fig. 2A). B, gel shift with labeled probe p16B (Table I and panel C). Cell lines are as follows: lane 1, U1752; lane 2, NCI-H69; lane 3, H157 (human large cell lung carcinoma); lane 4, H2O9 (human small cell lung carcinoma); lane 5, DuPro (human prostate carcinoma); lane 6, LnCaps (human prostate carcinoma); lane 7, H727; lane 8, H82. M and U are as in A. The arrow denotes the position of the novel complex not detected by probe p16C in A or probe p16A (data not shown; Table I). Complexes a, b, and c are as in A. The major complex below c is nonspecific as denoted in A. C, schematic of the p16INK4a proximal promoter region. A, B, and C designate the positions of the oligonucleotide probes used in panels A and B, and ovals are the positions of the putative Sp1 binding sites in the promoter. The arrow designates the position of the putative transcription start sites, and ATG designates the translation start site.

Detection of a Protein with Decreased Binding Activity to p16INK4a Regulatory Region in Cell Lines with an Epigenetically Silenced p16INK4a Gene-- Although protein extracts from all cell lines had uncompromised binding of Sp1 and Sp3 factors to p16INK4a-specific sequences, one of the oligonucleotides produced a distinctive pattern of shifted complexes (Fig. 1B). Oligonucleotide p16B, located at -76 to -48 nucleotides from the translation start site (Fig. 1C, position B), shifted a protein that was not recognized by Sp1, Sp3, or Sp4 antibodies in supershift analysis (data not shown), and there were marked differences between the relative binding activities for this complex for the different cell lines used in the study. The complex was markedly diminished in each cell line with a hypermethylated endogenous p16INK4a gene (Fig. 1B, arrow). This decreased binding was especially evident by comparing the intensity of the novel band to that for the Sp3 band (Fig. 1B, c). These differences were even more clearly apparent in gel shifts in which we used an unlabeled Sp1 consensus oligonucleotide to compete the shifted complexes due to Sp1 or Sp3 binding (Fig. 2A). Although some binding was detectable for the novel complex in every cell line, binding activity was strongest in the NCI-H69 cell line and other cell lines with an unmethylated and a highly expressed p16INK4a gene and much weaker in cell lines with a hypermethylated, epigenetically silenced p16INK4a gene (Fig. 2A).



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Fig. 2.   Gel shift analyses with labeled probe p16B and various competitor probes. A, competition with 500× Sp1 oligonucleotide (Table I). Lanes are as follows: 1, H157; 2, H2O9; 3, U1752; 4, NCI-H69; 5, H727; 6, H82; 7, DuPro; 8, LnCaps; and F, free probe. M and U are as in Fig. 1. The arrow denotes the position of the retained novel complex seen in Fig. 1B, and the band below the arrow is a nonspecific complex. B, gel shift competition with the panel of B4 oligonucleotides shown in Table I. Lanes are as follows: -, protein and no competitor; B4, the wild-type sequence for binding of the novel complex; 1, B4M1; 2, B4M2; 3, B4M3; 4, B4M4; 5, B4M5. Note that whereas all of the competitor oligonucleotides variably compete the fastest migrating, nonspecific complex, only wild-type B4 fully competes the novel complex (arrow). C, Southwestern analysis with concatemerized B4 probe. Protein extracts were from the following cell lines: lane 1, U1752; lane 2, NCI-H69; lane 3, T47D (human breast carcinoma); lane 4, H2O9; lane 5, DuPro; lane 6, LnCaps. Molecular mass marker positions are shown at the left.

Characterization of the DNA Sequence for the p16INK4a-binding Protein-- By testing a series of oligonucleotides surrounding the Sp1 consensus sequence (data not shown), we determined the minimal binding sequence, 5' CGG ACC GCG TGC GCT G 3' (sequence B4 in Table I), that was able to compete away the novel complex. This sequence overlaps with the 3' end of the Sp1 site in oligonucleotide p16B (Table I) and extends downstream. We next designed a panel of mutant oligonucleotides to test the sequence specificity of the interaction (Table I). Interestingly, all the mutations inside the minimal sequence affected the binding, and most of them abolished it altogether (Fig. 2B). Importantly, mutation in the overlapping Sp1 consensus site, which abolished Sp1 binding, did not eliminate binding of the newly detected protein (data not shown). Using the information from these competition studies, we constructed a concatemerized B4 oligonucleotide. This labeled, concatemerized probe was used in a Southwestern analysis to determine the size of the unknown DNA-binding protein. We did not detect any nuclear proteins binding to this probe from cell lines with an epigenetically silenced p16INK4a gene (Fig. 2C), whereas a single 130-kDa protein band was detected from cells that had a transcriptionally active p16INK4a gene (Fig. 2C).

DNA Affinity Purification and MALDI-MS Analysis Identified the Protein as RNA Helicase A-- We used a concatemerized double-stranded wild-type sequence probe, B4 (Table I), labeled at one end with biotin, to purify the DNA binding activity. To increase the stringency we added an excess of an oligonucleotide containing a single base pair mutation, B4MI (Table I), and a nonspecific homopolymer poly(dA)·poly(dT) to the binding buffer. Nuclear proteins from NCI-H69 cells were preincubated in the presence of competitors, and specific proteins binding to the recognition sequence were separated using streptavidin-coated magnetic beads. After extensive washing, DNA binding activity was eluted using increasing salt concentrations. Presence of the binding activity at each step was detected by gel shift analysis (Fig. 3A). Approximately equal amounts of two major proteins (130 and 160 kDa) were purified in the binding fraction eluted with 0.4 M KCl as detected by Coomassie staining of an SDS gel (Fig. 3B). Analysis of these proteins by MALDI-MS identified both proteins as human RNA helicase A, a homologue of the Drosophila dosage compensation protein, maleless (Mle). Both of these molecular mass forms of human RHA have been recognized by other investigators although the determinants of the size differences are not known (15, 16).



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Fig. 3.   Affinity purification of the protein. Nuclear proteins from the NCI-H69 cell line were incubated (see "Experimental Procedures") in buffer containing a biotin-labeled, concatemerized B4 oligonucleotide for the wild-type RHA binding sequence with a mutant RHA binding site as competitor (oligonucleotide B4M1; see Table I). Bound protein was isolated with streptavidin-coated magnetic beads. A, specific DNA binding activity of purification fractions as monitored by gel shift assays with B4 as the labeled probe (the arrow marks the position of the specific complex). Lanes are as follows: 1, initial incubation mixture on ice; 2, supernatant following centrifugation of the mixture before addition of beads; 3, unbound supernatant fraction following incubation with beads; 4 and 5, supernatants from first and second washes, respectively, of beads with binding buffer; 6, supernatant from elution of beads with 0.4 M KCl (note elution of the specific binding protein); 7, supernatant from final elution with 1 M KCl. B, proteins eluted in washes from A as detected by SDS-PAGE and Coomassie staining. Lanes are as follows: 1 and 2, washes 1 and 2, respectively, with binding buffer; 3, elution with 0.4 M KCl. Arrows indicate the 160- and 130-kDa proteins that were analyzed by MALDI-MS.

RHA Binds to the p16INK4a Promoter-- To verify that RHA indeed was the protein that produced the original shifted complex, we tested binding of RHA proteins, produced from an RHA cDNA (Fig. 4D) in a rabbit reticulocyte TnT lysate system, to the original p16INK4a-specific oligonucleotide, p16B (Table I) using gel shift analysis. The lysate contained some endogenous RHA that was detected by anti-RHA antiserum, but we were able to produce exogenous RHA in higher quantities (Fig. 4A, lane 2). As shown in Fig. 4B, lane 2, this produced a much stronger complex in the gel shift than for the TnT reaction using a control template (Fig. 4B, lane 1). To further show that RHA indeed can specifically bind to the p16INKa regulatory region, we transfected an RHA expression plasmid and a vector control into cultured cells and detected binding activity by gel shift analysis. Expression of the exogenous RHA but not of the vector alone caused an increase in the specific complex detected by gel shift (Fig. 4C). This increase in binding can be seen in both cell lines with or without a methylated endogenous p16INK4a gene (data not shown), but the relative increase in the shift was larger in the cell line, NCI-H69 (Fig. 4C), that initially contained more endogenous binding and has an unmethylated endogenous gene.



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Fig. 4.   TnT reactions in a rabbit reticulocyte system for RHA cDNA constructs. A, Western blots for RHA produced from the cDNA templates are shown as follows: lane 1, luciferase control construct; lane 2, full-length RHA construct; lane 3, the AvaIII deletion construct shown in D; lane 4, the XhoI deletion construct shown in D. Molecular mass marker positions are shown at the left. B, gel shift analyses of the proteins in the lanes as shown in A with B4 as the labeled probe. The arrow denotes the position of the specific complex for RHA. C, gel shift analyses, using B4 as the labeled probe, of proteins from NCI-H69 cells transfected with either a control vector plasmid (lane 1) or an expression vector containing the full-length RHA cDNA (lane 2). The arrow depicts the specific complex. D, schematic presentation of the human RHA structure with the amino acid numbers. The sites for interaction with known proteins (1, 24) are shown at the top of the graph, and the restriction enzyme deletions of the cDNA constructs analyzed in A and B are shown below the graph. The arrow depicts the site of a consensus caspase-3 cleavage sequence. The shaded blocks represent (in order from the N terminus) two double-stranded RNA binding sites (region of amino acids 1-200 approximately), six conserved helicase domains (amino acids ~400 to ~600), and a RGG cysteine-rich domain (amino acid ~1200).

The p16INK4a Sequence-specific Binding Domain of RHA Is Located in the N-terminal End of the Protein-- To determine what domains of RHA are needed for its sequence-specific binding to the p16INK4a promoter region, we deleted, from the RHA cDNA, portions encoding for the C-terminal end of the protein, translated these plasmids in TnT reactions (Fig. 4A, lanes 3 and 4), and tested DNA binding activity using gel shift analysis (Fig. 4B, lanes 3 and 4). Both deletion constructs AvaIII, lacking amino acids 841-1270 from the C-terminal end of the protein, and XhoI/1, lacking the conserved helicase domains five and six (amino acids 693-1052), retained full binding activity. Thus, the binding activity resides in the N-terminal end, which contains the two double-stranded RHA binding domains (15).

Binding of RHA Modulates p16INK4a Transcription-- We tested whether RHA can modulate activity of the p16INK4a promoter in transient transfection assays. p16INK4a promoter-luciferase constructs containing 0.9 or 2.0 kb of wild-type p16INK4a promoter sequence upstream from the ATG site (Fig. 1C) and with or without a mutation in the RHA binding site were transfected into U1752 cells. The basal expression of the mutant constructs was consistently 30% less than that for the wild-type constructs at 24 h after transfection (Fig. 5A). In addition, cotransfection studies with a plasmid expressing human RHA produced a 1.5-fold increase in luciferase activity of the wild-type construct at 24 h, whereas activity from the mutant construct did not show this increase (Fig. 5B).



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Fig. 5.   Activity of p16INK4a promoter-luciferase reporter constructs transiently transfected into U1752 cells. A, activity of reporter constructs containing sequences either 0.9 kb (lanes 1 and 2) or 2 kb (lanes 3 and 4) upstream of the ATG site (Fig. 1C) in the p16INK4a promoter. The upstream regions are either wild type (white bars) or mutant (shaded bars) for the RHA binding site as outlined under "Experimental Procedures." The activity of the wild-type construct is normalized to 100%, and all results are normalized for transfection efficiency as per "Experimental Procedures." The error bars represent ± S.E. for the average of three separate assays. B, identical luciferase activity assays for the 0.9 kb p16INK4a promoter-luciferase constructs (wild type = white bars; RHA binding mutant = shaded bars) in cells transiently transfected with a control vector plasmid (lanes 1 and 3) or the plasmid containing full-length RHA cDNA (lanes 2 and 4). The white and shaded bars are as in A. All results are compared with activity in the wild-type control-transfected cells, which is normalized to 100%, and the error bars are ± S.E. for an average of three separate experiments.

Differences in the Sequence-specific Binding Activity of RHA Are Associated with Posttranslational Modifications-- We next wanted to identify factors that might affect the sequence-specific binding of RHA and be determinants of differences in this activity between the cell lines studied. We first sequenced RHA coding regions from the cell line U1752, which has a low sequence-specific binding activity and a methylated p16INK4a gene, to detect any mutations that might contribute to lowered activity of RHA. However, no mutations were found (data not shown). In addition, no overall differences were seen in the total amounts of RHA protein between cell lines by Western analysis (see Fig. 6). The identification of the 130-kDa protein under the denaturing conditions of the Southwestern analysis suggested that RHA is able to bind to the p16INK4a region without a partner protein. It then seemed most likely, from all of the above data, that binding differences between cell lines might be a result of differences in posttranslational modification of the RHA.

Because RHA has been shown to interact with the transcriptional coactivator cyclic AMP response element-binding protein (17), which has acetylase activity, one possible modification of RHA is acetylation. To evaluate the role of acetylation in the RHA binding to the p16INK4a promoter, we first treated U1752 and NCI-H69 cells with TSA, a specific histone deacetylase inhibitor (18), for 18 h and analyzed DNA binding by gel shift assay. As shown in Fig. 6A, TSA treatment of U1752 cells did not affect the DNA binding activity of RHA to a p16INK4a-specific oligonucleotide (lanes 1 and 2). However, in NCI-H69 cells, TSA treatment caused a marked decrease in the sequence-specific DNA binding of RHA (Fig. 6A, lanes 3 and 4). A complete time course indicated that this decrease became apparent 12 h after TSA administration (data not shown). Interestingly, immunoblot analysis of total proteins with RHA-specific antibodies revealed a difference in isoforms of RHA between control-treated and TSA-treated NCI-H69 cells (Fig. 6B, lanes 3 and 4), which also appeared only after 12 h of TSA treatment (data not shown). This indicated to us that TSA treatment caused a shift in either posttranslational modification or degradation of the protein in NCI-H69 cells; this was not seen in U1752 cells (Fig. 6B, lanes 1 and 2).



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Fig. 6.   Effects of TSA treatment on RHA binding and protein forms and immunoprecipitation of protein from U1752 and H69 cells with acetylated lysine antibodies. A, gel shift analyses of the extracts using the B4 oligonucleotide as the labeled probe. The arrow denotes the position of the RHA specific complex. In A, B, and C the lanes are as follows: 1, U1752 + EtOH (18 h); 2, U1752 + TSA (18 h); 3, NCI-H69 + EtOH; 4, NCI-H69 + TSA (18 h). Note the decreased RHA binding in TSA-treated NCI-H69 cells in A. B, total RHA protein as analyzed by Western blots with anti-RHA antibody (upper panel) and anti-topoisomerase antibody (lower panel) for a loading control. C, immunoprecipitation of the same protein extracts shown in B with acetylated lysine antibodies. The immunoprecipitates were analyzed by Western blots of SDS-PAGE gels probed with the RHA antiserum. The nonspecific band at the bottom of the gel serves as a loading control. Note the presence of a smaller molecular mass form of RHA in TSA-treated NCI-H69 cells in B and C. Molecular mass marker positions are shown to the left in B and C.

We next analyzed control- and TSA-treated samples by immunoprecipitations with acetylated lysine antibodies. Immunoreactive RHA could be immunoprecipitated from both NCI-H69 and U1752 cells (Fig. 6C, lanes 1 and 3) with acetylated lysine antibodies. However, the amount of RHA complexed with acetylated lysine antibodies was considerably less in U1752 cells (a cell line with reduced RHA binding activity to the p16INK4a-specific sequence) than in NCI-H69 cells. Yet, both cell lines contain the same steady state amount of total RHA (Fig. 6B, lanes 1 and 3). In NCI-H69 cells the smaller isoform of RHA produced by TSA treatment was brought down less efficiently with acetylated lysine antibodies than was the full-length form (Fig. 6C, lane 4). In U1752 cells no change was seen in RHA complexed with acetylated lysine antibodies after TSA treatment (Fig. 6C, lane 2).

It was interesting that TSA induced a change in the form of RHA associated with the decreased binding in NCI-H69 cells. In addition to its direct inhibition of histone deacetylases, TSA has been shown recently to have later, and indirect, effects on apoptosis associated with induction of caspase activity (19). In the NCI-H69 cells, the new form of RHA seen after TSA treatment was estimated to be 145 kDa, 15 kDa smaller than the full-length (160 kDa) RHA. Analysis of the RHA amino acid structure revealed the sequence EEVD, a putative caspase-3 cleavage site, in the N-terminal portion of the molecule between the two previously identified double-stranded RNA binding domains (Fig. 4D). Cleavage at this site would cut a 15-kDa fragment off the N-terminal portion of the RHA molecule. To test whether TSA might be inducing such cleavage, we incubated NCI-H69 cells with the caspase-3 specific inhibitor DEVD before addition of TSA. This inhibitor substantially reduced formation of the 145-kDa TSA-specific form of RHA and also substantially reversed the TSA-induced decrement in RHA binding to the p16INK4a promoter (data not shown). These findings further localize the region of the protein responsible for the binding to the far N terminus of the molecule.


    DISCUSSION
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES

In the present study we have defined a sequence, 5' CGG ACC GCG TGC GC 3', in the context of the p16INK4a promoter region, that specifically binds RNA helicase A. We show that this binding activity of RHA is decreased in tumor cell lines with an epigenetically silenced p16INK4a gene. This decrement appears to be associated with decreased amounts of RHA that immunoprecipitate with acetylated lysine antibodies. In an experimental setting RHA can induce a modest up-regulation of the p16INK4a promoter, and this modulatory activity is dependent upon the sequence-specific interaction of RHA with the promoter. Finally, we show that RHA is a substrate for caspase-3 and that sequence-specific binding of RHA to the p16INK4a promoter is sensitive to caspase cleavage.

Transcriptional Activity of RHA-- The transcriptional modulatory activity of RHA in our study may best be considered in the context of defined functions of Mle, the RHA homologue in Drosophila (20). In the male fly, this protein functions, together with MSL proteins, to ensure 2-fold increase in transcription of the single male X-chromosome. Although Mle is only transiently or loosely associated with the complex, it may be the essential factor in the process by recruiting the other proteins (20).

The precise manner in which RHA modulates transcription is not known. RHA is a member of the DEAH-box DNA/RNA helicases (15), and it has previously been shown to have unwinding activity toward both RNA and DNA, suggesting a role in DNA replication and transcription (21). The sequence-specific binding for RHA that we demonstrate is the first described for this protein. This binding activity appears to reside within the region containing the two previously characterized, nonsequence-specific, double-stranded RNA binding domains at the far N-terminal end of the molecule. A glycine-rich RGG domain in the C-terminal end has been suggested to have a role in single-stranded DNA/RNA binding. Neither the double-stranded RNA binding domains nor the RGG domain is needed for the unwinding activity (15).

The transcriptional role of RHA in mammals has not yet been well characterized. In mice, knockout of the gene coding for this protein was recently shown to be embryonically lethal (22). However, the only characterized potential functions of human RHA, to date, are nuclear export and stability of retroviral RHAs (23, 24). Our studies now indicate a role for this helicase in transcriptional competence of specific genes. All of the properties mentioned above for the protein could be important in this process, and the actual degree of transcriptional modulation in our experimental setting may tell only a small part of the story. The helicase activity of the protein and the ability to target other proteins to a transcriptional complex could facilitate the transcription of genes such as p16INK4a. For example, it has been shown that RHA has a role in cAMP-dependent transcriptional activation through direct interaction with the transcriptional coactivator cyclic AMP response element-binding protein (17) and that RHA can directly interact with RNA PolII (17, 25).

Posttranslational Modulation of RHA Binding and Differences in the Process between Tumor Cell Types-- The differences that we have found between tumor cell lines in the sequence-specific DNA binding activity of RHA appear to be mediated by steady state differences in signal transduction involving the acetylation status of the protein or proteins complexed with it. From our experiments it seems probable that acetylation helps regulate the sequence-specific DNA binding of RHA. The amounts of RHA brought down with anti-acetylated lysine antibodies were much higher from NCI-H69 cells, which have a high sequence-specific DNA binding activity of RHA, than from U1752 cells, which have much lower binding activity. Coactivator cyclic AMP response element-binding protein, with which RHA can complex, has a histone acetyltransferase activity, but it has also been shown to modify nonhistone proteins such as p53 (26). Acetylation of specific lysine residues in the p53 protein has been shown to be important in induction of the sequence-specific binding activity (26).

Colocalization of Msl complexes in the fly with H4Ac16 indicates that X-chromosome dosage compensation is closely linked to chromatin structure and histone acetylation. Mle, the homolog of RHA, also colocalizes with H4Ac16 in distinct regions on autosomes (9). It is not surprising then that, in higher eukaryotes, RHA has functions in transcriptional activation associated with the status of protein acetylation.

It remains to be determined how RHA binding and its modulation by translational modification mediate the biological roles of the p16INK4a gene. p16INK4a, by controlling the functional status of Rb in the cyclin D-Rb pathway, plays a critical role for cell cycling. Interestingly, each of the tumor cell lines that we found to have increased binding activity of RHA have a mutant Rb gene and a marked increase in p16INK4a expression, probably in response to the loss of Rb function in cyclin D-Rb pathway check point control (1). RHA could play a role in this increased expression. Furthermore, in normal cells, p16INK4a expression is up-regulated in cells entering the quiescent state of senescence (27). Interestingly, we have shown that RHA is a substrate for caspase activity. Perhaps, caspase-mediated abrogation of RHA binding to the promoter is involved with an alternative signal for decreasing p16INK4a expression in cells programmed for apoptosis or increasing expression of the gene in arrested cells entering senescence. This area may be a rich one for further exploration.

Does RHA Binding Play a Role in Aberrant Transcriptional Silencing and Methylation of the p16INK4a Promoter?-- We originally began the current study looking for factors that might render the p16INK4a promoter region sensitive to aberrant hypermethylation during tumorigenesis. Indeed, we detected the novel RHA interaction partly because binding of this protein to the p16INK4a promoter is reduced in tumor cells of different types that have an endogenously hypermethylated and epigenetically silenced p16INK4a gene. How might the RHA interaction influence the methylation events? Interestingly, the binding site for RHA at the p16INK4a promoter is located just downstream from, and partially overlaps with, a classical Sp1 consensus sequence. Several studies on the APRT gene have suggested that Sp1 elements can protect CpG islands from methylation (11-13). However, it is now clear that the Sp1 protein alone is not enough to keep CpG islands free from methylation because Sp1-knockout mice do not hypermethylate CpG islands (28), and this site alone cannot protect transgene CpG islands from methylation (29).

The close overlap of the RHA binding site with an Sp1 site might suggest interaction between RHA and Sp1 in this region. Both transcription and DNA methylation could be influenced by such interaction. Recently, another helicase-like transcription factor was shown to interact with Sp1 and Sp3, and it was suggested that this protein, HLTF, might provide chromatin opening to facilitate transcriptional activation by Sp family members (30). We have shown in this study that RHA binding can also positively modulate transcriptional activity of p16INK4a constructs in transient assays. Perhaps RHA makes the p16INK4a promoter more accessible to transcriptional activators like Sp1 and thus helps render it protected from CpG island methylation.

We have made some initial attempts to see whether binding of RHA is important to keep p16INK4a sequences free of methylation. We stably introduced the wild-type and mutant p16INK4a promoter-luciferase constructs used in the present studies into DuPro and Du145 cells in which the endogenous p16INK4a gene is methylated and unmethylated, respectively (31). We followed six independent wild-type and mutant clones in both cell lines for over 20 passages but observed no methylation in any of our clones (data not shown). In these experiments loss of RHA binding did not then increase the probability of methylation at the exogenous p16INK4a promoter.

Whereas these results could rule out a role for RHA in p16INK4a promoter methylation patterns in cancer, our results may not address this issue for a number of reasons. First, the chromosomal context of the endogenous gene may be essential for any role of RHA. Second, the initial hypermethylation of p16INK4a and loss of expression during tumorigenesis may require a selective pressure closely linked to a functional Rb gene. In cancers of multiple types, p16INK4a is only altered, including promoter hypermethylation, when a wild-type Rb gene is present (1).

Active Rb has been shown by some workers to produce negative transcriptional pressure on the p16INK4a promoter (32). This potential role for active Rb is also evident from recent studies where transcriptional silencing and hypermethylation of p16INK4a provides an early step toward cellular immortalization by allowing cells to escape mortality check point "M0" (6, 33). Importantly, this hypermethylation only occurs if the Rb gene and/or protein is present in a functional state (33). Both of the cell lines into which we introduced our constructs have reduced Rb function, either because the Rb gene is mutant or because chronic loss of p16INK4a expression leads to phosphorylation and inactivation of the Rb protein (31). Thus, neither cell line may have the setting to induce initial methylation and silencing of the exogenously introduced p16INK4a promoter constructs, regardless of RHA function.


    ACKNOWLEDGEMENTS

We thank Tammy Means for help in preparation of the manuscript. We thank Dr. Che-Gun Lee for RHA expression plasmids and for RHA antiserum. We thank Erica Seiguer, Jessa Jones, and Jennifer Hackett for technical assistance and Dr. Margaret Johns, Dr. Beth Cameron, and Dr. James Herman for their advice.


    FOOTNOTES

* This work was supported in part by a Public Health Service Grant (R01 CA43318) from the NCI, National Institutes of Health.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.

Dagger To whom correspondence should be addressed. Tel.: 410-955-8506; Fax: 410-614-9884; E-mail: sbaylin@jhmi.edu.

Published, JBC Papers in Press, October 18, 2000, DOI 10.1074/jbc.M004481200


    ABBREVIATIONS

The abbreviations used are: RHA, human RNA helicase A; MALDI-MS, matrix-assisted laser desorption ionization mass spectrometry; SDS-PAGE, SDS-polyacrylamide gel electrophoresis; kb, kilobase(s); TSA, trichostatin A; TnT, transcription and translation.


    REFERENCES
TOP
ABSTRACT
INTRODUCTION
EXPERIMENTAL PROCEDURES
RESULTS
DISCUSSION
REFERENCES


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