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J Virol, May 1998, p. 4183-4191, Vol. 72, No. 5
Departments of Molecular Biophysics and
Biochemistry1 and of
Genetics,2 Yale University, New
Haven, Connecticut 06520-8114
Received 29 September 1997/Accepted 15 January 1998
Adenovirus type 12 (Ad12) infection of human cells induces four
chromosomal fragile sites corresponding to the U1 small nuclear RNA
(snRNA) genes (the RNU1 locus), the U2 snRNA genes
(RNU2), the U1 snRNA pseudogenes
(PSU1), and the 5S rRNA genes (RN5S). Ad12-induced fragility of the RNU2 locus requires U2 snRNA
transcriptional regulatory elements and viral early functions but not
viral replication or integration, or chromosomal sequences
flanking the RNU2 locus. We now show that Ad12 cannot
induce the RNU1, RNU2, or PSU1
fragile sites in Saos-2 cells lacking the p53 and retinoblastoma
(Rb) proteins but that viral induction of fragility
is rescued in these cells when the expression of wild-type p53 or
selected hot-spot mutants (i.e., V143A, R175H, R248W, and R273H)
is restored by transient expression or stable retroviral transduction.
We also observed weak constitutive fragility of the
RNU1 and RNU2 loci in cells belonging to
xeroderma pigmentosum complementation groups B and D (XPB and
XPD) which are partially defective in the ERCC2 (XPD) and ERCC3 (XPB)
helicase activities shared between the repairosome and the RNA
polymerase II basal transcription factor TFIIH. We propose a
model for Ad12-induced chromosome fragility in which interaction of p53
with the Ad12 E1B 55-kDa transforming protein (and possibly E4orf6)
induces a p53 gain of function which ultimately perturbs the RNA
polymerase II basal transcription apparatus. The p53 gain of function
could interfere with chromatin condensation either by blocking
mitotic shutdown of U1 and U2 snRNA transcription or by phenocopying
global or local DNA damage. Specific fragilization of the RNU1, RNU2, and PSU1 loci
could reflect the unusually high local concentration of
strong transcription units or the specialized nature of the U1 and U2
snRNA transcription apparatus.
Fragile sites are nonstaining gaps
in metaphase chromosomes caused by incompletely condensed chromatin or,
more rarely, by frank chromosome breaks. Fragile sites frequently
colocalize with recurrent cancer breakpoints (55), with
preferential targets for viral integration (62, 89), with
sites of sister chromatid exchange (19, 21; but see
reference 86), and with sites of chromosome
translocations in primate lineages (55). Although these
observations support the view that fragile sites are recombinogenic, it
is important to distinguish between physical fragility (as assayed
cytologically) and genetic instability (as assayed by rearrangements on
an individual or evolutionary time scale). Physical fragility might be
directly responsible for the observed genetic fragility, for example,
by rendering the DNA more accessible to the recombination apparatus,
but it is also possible that both physical and genetic fragility
reflect some other underlying phenomenon which facilitates
recombination and disrupts chromatin packing.
It has been known for 30 years that infection of human primary
embryonic kidney cells at a low multiplicity with adenovirus type 12 (Ad12), but not adenovirus 2 or 5, induces four sites of metaphase
chromosome fragility (94); at a higher multiplicity both
viruses induce generalized chromosome fragility (67, 94). The four Ad12 chromosome "modification" sites were subsequently found to colocalize with four tandemly repeated multigene families (6, 49, 50, 73): the human U1 small nuclear RNA
(snRNA) genes at 1p36 (the RNU1 locus), the U2 snRNA
genes at 17q21-22 (RNU2), an ancient family of U1
snRNA pseudogenes at 1q12-q22 (PSU1), and the 5S rRNA genes
at 1q42-43 (RN5S). Ad12 efficiently induces fragility of
the RNU1, RNU2, and PSU1 loci in
several different cell lines (4, 16, 20, 42, 67);
RN5S fragility has not been visualized except in primary
human embryonic kidney cells (94) and may be weak, cell type
specific, or abolished by immortalization. Colocalization of the four
Ad12-inducible fragile sites with highly transcribed, tandemly repeated
multigene families encoding small, abundant structural RNAs suggested
that concentrated transcriptional activity might somehow interfere with
local chromatin condensation (49); however, it was puzzling that none of the four modification sites appeared to be constitutively fragile in the absence of viral infection, and it was not known what
viral or cellular functions were required to induce fragility.
Cytological colocalization of the four virally inducible fragile sites
with four tandemly repeated multigene families strongly suggested that
the small RNA genes themselves were necessary and sufficient for
virally induced fragility. We (4) and others (20,
42) subsequently demonstrated that an artificial tandem array of
active, but not inactive, U2 snRNA genes creates a new Ad12-inducible
fragile site. Thus, Ad12-induced fragility of the RNU2 locus
requires the minimal U2 snRNA transcription unit but not the
chromosomal sequences flanking the RNU2 locus (44,
60) nor any other sequences from the 6.1-kb U2 repeat unit,
including the CT microsatellite (43) and the solo long
terminal repeat (45, 60).
The best-characterized fragile sites appear to be caused by defects in
DNA replication which interfere, either directly or indirectly, with
subsequent chromatin packing (reviewed in reference 79). Such chromatin-packing defects can be induced
by a variety of drugs, including aphidicolin (which directly inhibits
the DNA polymerases Stable expression of the E1 transforming region of Ad12 is sufficient
to induce constitutive fragility of the RNU2 locus in a
somatic-cell hybrid containing an intact human chromosome 17 in a mouse
background (15). The E1 region of both Ad12 and Ad2/5 consists of two independent transcription units, although the Ad2/5
system is far better studied. Alternative splicing of the Ad2/5 E1A
transcript generates 13S, 12S, and 9S mRNAs encoding related
multifunctional proteins; E1B encodes 19- and 55-kDa proteins in
overlapping reading frames. The E1A proteins bind pRB- and p300/CBP-family proteins (10). In addition, both the 12S-
and 13S-encoded E1A proteins can inhibit the p53 transactivation
(75), and the 13S-encoded E1A protein can relieve p53
repression (32). E1A-induced, p53-dependent apoptosis of
infected cells (88) is prevented by the E1B 55-kDa protein,
which inhibits p53 transactivation by binding to the N terminus of p53
(33, 92; reviewed in reference 7), and by the E1B 19-kDa protein, which shares
structural and functional homology with the cellular antiapoptotic
protein Bcl-2 (27, 63). Although encoded outside the E1
region, the E4orf6 34-kDa protein may modulate transformation by
inhibiting p53 transactivation (13) and repression
(58) or by relocalizing cytoplasmic E1B 55-kDa protein to
the nucleus (22).
Mutations in the Ad12 E1B 55-kDa protein but not in the 19-kDa protein
significantly reduced Ad12-induced fragility (67). Since the
Ad12 E1B 55-kDa protein also directly represses the p53 transactivation
domain (7, 33, 92), we wondered whether Ad12-induced
fragility of the RNU1 and RNU2 loci might reflect viral inactivation of p53 functions. We found, however, that the RNU1 and RNU2 loci are not constitutively fragile
in cells lacking p53 function and that Ad12 cannot induce fragility of
either locus in the absence of wild-type or mutant p53 function. Since
steady-state levels of U1 and U2 are within the normal range in cells
lacking p53, we conclude that fragility of the RNU1 and
RNU2 loci reflects an Ad12-induced p53 gain of function.
Cells, FISH, and recombinant DNA.
Saos-2, SBN, and SEN
osteosarcoma cells were obtained from W.-H. Lee and propagated as
described previously in Dulbecco's modified Eagle medium with 10%
fetal bovine serum at 37°C under 5% CO2 (9).
Epstein-Barr virus (EBV)-transformed lymphoblastoid cell lines GM5927
(normal), GM01855 (XPB), GM02285 (XPB), and GM02455 (XPD) were
purchased from the Coriell Institute and propagated in RPMI 1640 medium
with 20% fetal bovine serum. The fluorescent in situ hybridization
(FISH) protocol and U2 gene probe were as described previously
(4); U1 probes were as described by Lindgren et al.
(50). p53 expression constructs (5, 35) were the kind gift of B. Vogelstein.
Complementation assay for p53 function.
In preliminary
experiments with the pCH110 p53 function is required for Ad12-induced chromosome
fragility.
Analysis of metaphase chromosomes from a cell line
entirely lacking functional p53, the Saos-2 osteosarcoma (9,
82), revealed that the RNU2 locus was not
constitutively fragile in these cells nor could fragility be induced by
Ad12 infection (Fig. 1 and Table
1). Thus, although loss of p53 often
results in genomic and chromosomal instability, the induction of
fragility by Ad12 cannot simply reflect viral inactivation of p53
functions. Nor can these negative results be explained trivially by the
failure of Ad12 to infect p53-deficient cells; Saos-2 cells can be
efficiently infected with Ad12 (77), and most cells in our
infected cultures were dead within several days.
0022-538X/98/$04.00+0
Copyright © 1998, American Society for Microbiology. All rights reserved.
Adenovirus Type 12-Induced Fragility of the
Human RNU2 Locus Requires p53 Function
ABSTRACT
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
INTRODUCTION
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
, 
, and 
) and methotrexate (which affects
replication by causing an imbalance in the deoxynucleotide triphosphate
pools). Alternatively, packing defects can be caused by chromosomal
alterations such as the CGG triplet repeat expansion, which locally
retards DNA replication in the FRAXA form of fragile X syndrome
(28) and potentiates CpG hypermethylation (25).
In none of these instances is it clear why incomplete or delayed DNA
replication, or hypermethylation, would interfere with chromatin
condensation; the initiation of chromatin condensation might be coupled
to the completion of DNA replication, or independent cell cycle
controls might initiate chromatin condensation in late G2
phase regardless of whether DNA replication is truly complete. Here we
examine a very different kind of chromosome fragility which may be
caused by a defect in transcription rather than a defect in
replication.
MATERIALS AND METHODS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
-galactosidase expression vector
(Pharmacia) as described by Lim and Chae (46), we tested
Saos-2 transfection efficiencies over a range of electroporation voltages from 0.15 to 0.55 kV; 0.25 kV was optimal, with decreased efficiency at 0.15 kV and decreased survival at 0.55 kV. For the complementation assay, Saos-2 cells in 150-mm-diameter plates were
grown overnight in fresh medium to 30 to 50% confluence and then
infected with Ad12 for 1 h at a multiplicity of 10. Infected cells
were collected by trypsinization and resuspended in cold phosphate-buffered saline (PBS). Electroporation was performed at 0.25 kV and 960 µF, using 0.4-cm-diameter cuvettes and a Bio-Rad Gene
Pulser with a capacitance extender. For each electroporation, 107 cells were suspended in 0.6 ml of PBS containing 1.25%
dimethyl sulfoxide, and 20 µg of total DNA (18 µg of the p53 vector
and 2 µg of the pCH110 -galactosidase vector) was added to the
cell suspension. After electroporation, the cuvette was immediately chilled in ice, and the cell suspension was divided among four 100-mm-diameter plates containing 10 ml of Dulbecco's modified Eagle
medium. After 6 h the medium was replaced, and 16 h after transfection the cells were treated with Colcemid (0.1 µg/ml) for
3 h. Cells from three plates were then collected by
trypsinization, and metaphase spreads were prepared by the standard
techniques of hypotonic shock and methanol-acetic acid fixation
(4). The fourth plate was stained for -galactosidase as
described previously (46).
RESULTS
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Abstract
Introduction
Materials & Methods
Results
Discussion
References
View larger version (150K):
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FIG. 1.
Ad12-induced fragility of the human RNU2
locus requires p53 function. Representative metaphase chromosomes are
shown from the indicated cell lines with and without Ad12 infection.
Each RNU2-containing chromosome is represented by a pair of
images: the total DNA visualized by DAPI
(4',6-diamidino-2-phenylindole) staining is on the left, and the U2
arrays visualized by FISH and superimposed on the DAPI image are on the
right. One of the two RNU2 loci in Saos-2 cells is located
on a der(17) chromosome resulting from an unidentified translocation
distal to 17q22. (See Table 1 for quantitation.) Note that Ad12-induced
fragility covers a broad range of chromosome morphologies, including
(i) frank separation or thinning of one or both chromatids; (ii)
dislocation of the chromosome axis; and (iii) splitting, smearing,
obvious intensification, or displacement of the RNU2 signal
from the body of the chromosome as imaged by the DAPI stain
(4). As seen in the upper panel (+Ad12), the fragile
phenotype can be subtle: the RNU2 signal may coincide with
thinning of one (upper- and lower-right SBN chromosomes) or both
chromatids (upper- and lower-right SBN chromosomes); it may appear
immediately adjacent to the site of chromatid thinning (upper- and
lower-right SBN chromosomes); it may fail to coincide with the DAPI
stain (upper- SBN and lower-left SEN chromosomes); or the two
RNU2 signals may be displaced from normal lateral register
(lower- SBN and upper-left SEN chromosomes). These morphologies are
almost never seen in the absence of infection.
TABLE 1.
p53 dependence in Ad12-induced RNU2 fragility
Complementation assay for p53 function. Cells lacking p53 function usually undergo apoptosis when full or partial p53 function is restored; accordingly, SBN cells were more difficult to isolate, grew more slowly, and displayed decreased viability relative to SEN cells (9). To explore the role of additional p53 mutants in Ad12-induced RNU2 fragility, we therefore sought a more convenient transient-expression assay. The basic approach was to complement the defect in Saos-2 cells by transfection with p53 expression vectors; the key experimental decision was whether to transfect Saos-2 with p53 before Ad12 infection or to infect Saos-2 with Ad12 before p53 transfection. In addition, we also required that p53 transfection be very efficient, because only 3 to 5% of the cells normally produce scorable metaphases; the 20% transfection efficiency typically achieved by calcium phosphate precipitation would generate only 1% scorable metaphases, an unworkably low number. Since Saos-2 cells grow as an adherent monolayer, our initial strategy was to perform the entire assay without detaching the cells. We infected the monolayer by overlaying with Ad12 virus for 1 h, transfected with the wild-type p53 expression vector in the presence of a cationic lipid such as Lipofectin or Lipofectamine, and then washed out the excess cationic lipid before adding fresh medium. Unfortunately, all available cationic lipids caused high levels of speckled background in the FISH assay (42a); presumably this was due to nonspecific adsorption of biotinylated probe DNA on cellular components bearing residual cationic detergent. Use of higher concentrations of sonicated salmon sperm carrier DNA during hybridization failed to reduce this background, and we therefore resorted to electroporation.
Adherent Saos-2 cells were infected with Ad12 for 1 h, detached by trypsinization, electroporated, and replated (see Materials and Methods for details). Optimal electroporation conditions for Saos-2 (0.25 kV, 960 µF, PBS buffer containing 1.25% dimethyl sulfoxide) were established by a-galactosidase color assay (46), and
transfection efficiencies of 30 to 40% were routinely obtained. Most
cells survived the combined infection-transfection protocol, as
evidenced by reattachment within 6 h of replating. Cells were arrested with Colcemid 18 h after replating and harvested for FISH
3 h later. To ensure consistency and an adequate number of metaphases, 2 µg of the -galactosidase expression vector pCH110 was always cotransfected with 18 µg of the p53 expression vector, and
one of the four resulting plates was stained for -galactosidase.
In this transient assay, Saos-2 cells were infected with Ad12 at low
multiplicities (multiplicity of infection [MOI], 1 to 10) and then
transfected with expression vectors encoding wild-type p53, one of four
most common p53 hot-spot mutants (V143A, R175H, R248W, or R273H), or
the pCH110 -galactosidase expression vector alone. The p53 cDNA
expression vectors were driven by the cytomegalovirus promoter-enhancer; rabbit -globin splicing and polyadenylation signals ensured efficient expression (5, 35). As shown in Fig. 2 and summarized in Table
2, wild-type p53 and all four hot-spot
mutants complemented the Saos-2 defect, restoring RNU2 fragility to near-normal levels resembling those observed upon infection of HT1080 cells expressing resident p53. Although
>70% of all chromosomes 17 in transfected cells exhibit some
RNU2 fragility (either fragmentation or clear
intensification of the RNU2 signal), occasionally only
one sister chromatid exhibits fragility; thus, it is not unexpected
that a small number of chromosomes 17 (usually <20%) apparently
exhibit no RNU2 fragility at all.
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Defects in ERCC2 or ERCC3 function cause weak constitutive RNU2 fragility. U1 and U2 snRNA are RNA polymerase II transcripts, and U2 transcriptional regulatory elements are required for Ad12-induced fragility of an artificial U2 array (4, 20). Although originally identified as essential components of the DNA "repairosome," the excision repair cross-complementing ERCC2 and ERCC3 helicase proteins in fact belong to a common core of five subunits shared between the repairosome and the basal RNA polymerase II transcription factor TFIIH (14, 48, 53, 80). The ability of p53 to interact with three different subunits of TFIIH, including ERCC2, ERCC3, and p62 (40, 83), led us to ask whether the p53 gain of function induced by Ad12 might cause p53 to interact aberrantly with TFIIH.
ERCC3 is mutated in xeroderma pigmentosum complementation group B (XPB) (65) and ERCC2 in complementation group D (XPD) (78). We therefore examined RNU1 and RNU2 fragility in EBV-transformed XPB (ERCC3) and XPD (ERCC2) lymphoblastoid cell lines (Fig. 3 and Table 3). Although the weak phenotype was somewhat obscured in highly condensed late-metaphase chromosomes, a clear phenotype could be observed in the more extended chromosomes of early metaphase. The RNU1 and RNU2 loci were constitutively fragile in both XPB and XPD cell lines, compared with the low level of fragility observed for normal EBV-transformed lymphoblastoid control cells (Fig. 3 and Table 3). As discussed below, the weak phenotype may reflect the fact that the primary defects in xeroderma pigmentosum are in the repair pathway, and these defects may only mildly (or fortuitously) affect the transcriptional functions of ERCC2 and ERCC3.
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DISCUSSION |
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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We have shown that p53 function is required for Ad12-induced fragility of the human RNU2 locus. In one set of experiments (Fig. 1 and Table 1), Saos-2 cells lacking detectable p53 expression were stably transduced with retroviral constructs expressing wild-type p53 or the R273H mutant. In the other set of experiments (Fig. 2 and Table 2), Saos-2 cells were transfected with vectors expressing wild-type p53 or one of four selected mutants (V143A, R175H, R248W, and R273H). In both sets of experiments, Ad12-induced metaphase fragility of the RNU2 locus was substantially restored by the expression of wild-type p53 and each of the four mutants tested. Each of these four mutations falls within one of the four highly conserved regions of the core DNA-binding region of p53 designated II (V143A), III (R175H), IV (R248W), and V (R273H). The R175H, R248W, and R273H mutants are also hot-spot mutations at codons which account for >20% of all mutations thus far reported (26); V143 is not a hot spot, but it was included to provide a disabling mutation in region II. While all four mutants are partially defective in DNA binding, the degree of the defect would almost certainly depend on the particular cell, assay, and temperature used (18, 36). For example, transcriptional transactivation of the WAF1 promoter by R273H appears to be only slightly impaired in primary human fibroblasts (85) but is nearly abolished in Saos-2 cells (59). In contrast, transactivation by R273H is nearly normal when assayed on an hsp70 promoter driven by a p53 consensus binding site in K562 cells (93). The observation that all four p53 hot-spot mutants were as effective as wild-type p53 implies that DNA binding is not required or that weak binding is sufficient to rescue fragility.
Our data show that Ad12 induces a p53 gain of function. Steady-state levels of U1 and U2 snRNA are within the normal range in Saos-2 cells lacking p53 function (3a), and the same is presumably true in countless tumor cell lines that are partially or completely lacking p53 function. Thus, p53 cannot be required for U1 or U2 transcription, and Ad12 proteins (probably E1B 55-kDa protein, perhaps working together with the E1A and/or E4orf6 products as discussed below) must cause a p53 gain of function that specifically interferes with metaphase chromatin packing of the RNU1, RNU2, and PSU1 loci. Other p53 gains of function have been characterized (12, 47; reviewed in reference 41). In most cases, these p53 gain-of-function mutants have lost tumor suppressor functions but also contribute to tumor progression. Some of these mutants may simply be dominant negatives (8), while others represent a genuine gain of function (12, 47, 56).
Ad12-induced fragility is unlikely to involve the p53-dependent apoptotic pathway (87). First, fragilization of the RNU1, RNU2, and PSU1 loci is highly specific at low MOIs; generalized fragility or chromosome "pulverization" reminiscent of apoptosis is observed only at high multiplicities (67). Second, the human group C adenovirus dl1520 (a double mutant expressing no E1B 55K protein whatsoever) grows well on C33A cervical carcinoma cells expressing the R273H mutant, but not on U20S osteocarcinoma cells expressing wild-type p53 (7). Thus, R273H fully restores RNU2 fragility in the Saos-2 osteosarcoma cells, although it cannot support E1A-induced apoptosis in the C33A cervical carcinoma cells. Third, three of the p53 mutants that support Ad12-induced RNU2 fragility in our assay (V143A, R175H, and R248W) were completely unable to induce apoptosis when transiently expressed in primary human fibroblasts; the fourth mutant (R273H) induced apoptosis only weakly (85). Thus, mutants in the core DNA binding regions of p53 that affect transactivation (18, 59, 85) can still support Ad12-induced fragility but not p53-dependent apoptosis.
At least three Ad2/5 proteins are known to interact, physically or functionally, with p53: the E1A proteins encoded by the 12S and 13S alternatively spliced mRNAs, the E1B 55-kDa protein, and the E4orf6 34-kDa protein. The 12S- and 13S-encoded E1A proteins inhibit the p53 transactivation function, possibly by inducing oligomerization of the N-terminal region of the protein (75), whereas the 13S-encoded E1A protein can relieve p53 repression, apparently by dissociating the complex between the C-terminal region of p53 and the TATA-binding protein (32, 52). The Ad2/5 and Ad12 E1B 55-kDa proteins both repress transactivation by binding to the N-terminal region of p53 (33, 91, 92; reviewed in reference 36). The Ad2/5 E4orf6 34-kDa protein can block p53 transactivation by interfering with contact between the N-terminal region of p53 and TAFII31, a component of TFIID (13), and can also block p53 repression, most probably by binding to the C-terminal repression region of p53 (58).
Ad12-induced fragility of the RNU1 and RNU2 loci is significantly inhibited by mutations in the E1B 55-kDa protein but not in the E1B 19-kDa protein (67). Therefore, the simplest hypothesis to explain Ad12-induced metaphase fragility is that the Ad12 E1B 55-kDa protein binds p53 and induces a gain of function; residual fragility induced by the Ad12 E1B 55-kDa mutant virus might reflect inhibition of p53 transactivation and/or repression by E1A, E4orf6, or other adenovirus gene products. The Ad2/5 and Ad12 E1B 55-kDa proteins both interact with and stabilize p53 (24, 74), and both block p53 transactivation functions (91); however, the Ad12 55-kDa protein, unlike the Ad2/5 protein, does not form easily detected immunoprecipitable complexes with p53 and fails to sequester p53 in the cytoplasm (24, 74). On the other hand, the Ad5 E4orf6 34-kDa protein causes a dramatic reduction in steady-state p53 levels (13), and interaction with the E4orf6 protein can relocalize the predominantly cytoplasmic Ad5 E1B 55-kDa protein to the nucleus (22). These differences in the interaction of the Ad2/5 and Ad12 E1B 55-kDa proteins with p53 may account for the observation that Ad12 induces four specific sites of chromosome fragility, whereas Ad2/5 only induces generalized fragility at higher multiplicities (67, 94).
The XPB (ERCC3) and XPD (ERCC2) helicases belong to a common core of five subunits shared between the repairosome and the basal RNA polymerase II transcription factor TFIIH (14, 48, 53, 66, 80). p53 can interact with at least three subunits of TFIIH, including XPB (ERCC3), XPD (ERCC2), and p62; the N-terminal activation domain of p53 interacts with p62 (40, 90), and the C-terminal domain interacts with XPB and XPD (40, 83-85). Binding of the C-terminal domain of p53 (residues 319 to 393) to XPB (ERCC3) and/or XPD (ERCC2) is functionally significant since it can induce p53-dependent apoptosis in primary human fibroblasts (85). The ability of mutations in the XPB (ERCC3) and XPD (ERCC2) helicases to induce weak constitutive fragility of the RNU1 and RNU2 loci (Fig. 3 and Table 3) therefore supports a model (Fig. 4) in which the Ad12-induced p53 gain of function acts directly on the XPB (ERCC3) and/or XPD (ERCC2) helicases either as part of TFIIH or of the repairosome. The relatively weak constitutive phenotype of these particular xeroderma pigmentosum mutants may indicate that they are preferentially defective in repair; although the XPB and XPD helicases serve a dual role in repair and transcription, only a minority of XPD mutations cause the severe developmental defects that characterize Cockayne syndrome or trichothiodystrophy, and that may be indicative of defects in transcription (11, 38).
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We can imagine two very different models to explain Ad12-induced, locus-specific, metaphase chromosome fragility (Fig. 4). In the first model, the Ad12-induced p53 gain of function perturbs cell cycle regulation of U1, U2, and perhaps 5S transcription, causing transcription to persist into metaphase and to interfere directly with chromatin packing. Although it is often assumed that metaphase chromatin packing is dominant over transcription, there is good reason to believe that transcription must cease before proper metaphase chromatin packing can take place. Otherwise, it is difficult to explain why TATA binding protein (39, 69), RNA polymerase II transcription factors such as Oct-1 (68) and Sp1 (54), and TFIIIB (23, 88) are inactivated by phosphorylation as metaphase approaches (for a review, see reference 76) and nascent transcripts are aborted (72). The effect of p53 on the RNU1 and RNU2 transcription units might be mediated by interactions with the ERCC2 (XPD), ERCC3 (XPB), or p62 components of TFIIH as described above and/or with other components of the transcription machinery (36). In either case, it is tantalizing that TFIIH also contains a cdk7-cyclin H couple, but there is no direct evidence that TFIIH plays a role in the cell cycle regulation of RNA polymerase II transcription (70).
In the second model, the Ad12-induced p53 gain of function causes p53
to behave as though it had directly sensed (37) or had been
alerted to DNA damage (reviewed in references 34 and 36). Although most DNA damage causes a global block in cell cycle progression, certain kinds of DNA damage could conceivably block
local chromatin packing just as damage locally blocks transcription. For example, the Ad12-induced p53 gain of function might block chromatin packing by consolidating a transient or fortuitous complex between p53 and TFIIH or other transcription factors bound to the
specialized U1 and U2 snRNA transcription apparatus (see below). In
fact, new data strongly suggest that DNA damage, or the perception of
such damage, may be the key to understanding Ad12-induced metaphase fragility. Thus, DNA-damaging reagents such as
1--D-arabinylfuranosylcytosine (52a) and
actinomycin D that either cause or phenocopy DNA damage (92a) can efficiently induce p53- and
transcription-dependent fragility of the RNU2 locus. The
connection between transcription, repair, and fragility is further
supported by the observation that defects in the CSB protein (defective
in Cockayne syndrome complementation group B), which is involved in
transcription-coupled repair, can cause constitutive unpacking of the
RNU1, RNU2, and RN5S loci
(42a). The fact that transcription-coupled repair is normal
in primary Li-Fraumeni fibroblasts with homozygous p53 mutations but
global DNA repair is deficient (17) reinforces the idea that
a p53 gain of function and a CSB protein defect might be alternative
means of phenocopying DNA damage. We therefore propose that Ad12
infection, CSB protein mutations,
1--D-arabinofuranosylcytosine, and low concentrations of
actinomycin D all generate a similar or identical damage arrest signal
(perhaps a posttranscriptional modification, altered conformation, or
relocalization of p53) to which the actively transcribed
RNU1 and RNU2 loci are especially or uniquely
sensitive.
In both models, the preferential effect of Ad12 on metaphase condensation of the RNU1, RNU2, and PSU1 loci (49, 94) could be explained either by the unusually high local concentration of short, independent transcription units at each affected locus, by the specialized nature of the U1 and U2 snRNA promoter (3, 29, 64) and termination factors (30, 31, 57), or by the highly structured nature of the nascent U1, U2, and 5S RNA transcripts (42a). A high local concentration of strong transcription units could render chromatin packing especially sensitive to persistent transcription or to a global damage arrest signal. Alternatively, specialized U1 and U2 snRNA transcription factors could be required to interact with the p53 gain of function or to sense the global arrest signal. A third possibility is that specialized factors might be required to release polymerases whose progress has been blocked by secondary structure in the nascent RNA transcript (42a).
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ACKNOWLEDGMENTS |
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We thank David C. Ward, June Menninger, and Patricia Bray-Ward for cheerful instruction in FISH and for generous access to superb image capture and processing equipment in the early stages of this work; Wen-Hwa Li for graciously providing the SBN and SEN cell lines; Bert Vogelstein for wild-type and mutant p53 expression vectors; the Coriell Institute for prompt provision of GM cell lines; Silvia Bacchetti for communicating results in advance of publication; and Daiqing Liao and Arnold D. Bailey for critical comments on the manuscript.
This work was supported by NIH awards GM31073 and GM41624.
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FOOTNOTES |
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* Corresponding author. Mailing address: Department of Molecular Biophysics and Biochemistry, Yale University, 266 Whitney Ave., P.O. Box 208114, New Haven, CT 06520-8114. Phone: (203) 432-3089. Fax: (203) 432-3047. E-mail: weiner{at}biomed.med.yale.edu.
Present address: Department of Molecular Genetics and Microbiology,
University of Massachusetts Medical School, Worcester, MA 01605.
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Top
Abstract
Introduction
Materials & Methods
Results
Discussion
References
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[Abstract] [Full Text] -
Li, Z., Bailey, A. D., Buchowski, J., Weiner, A. M.
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