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INTRODUCTION |
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.
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EXPERIMENTAL PROCEDURES |
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.
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RESULTS |
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.
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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.
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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.
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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).
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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.
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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.
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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 |
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.