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Human Molecular Genetics Pages 609-617

Metaphase fragility of the human RNU1 and RNU2 loci is induced by actinomycin D through a p53-dependent pathway
Introduction
Results
   Sensitivity of RNU1 and RNU2 loci to actinomycin D
   U2 gene sequences required for actinomycin D-induced fragility
   Actinomycin D-induced RNU2 fragility requires p53 function
Discussion
Materials And Methods
Acknowledgements
References


Metaphase fragility of the human RNU1 and RNU2 loci is induced by actinomycin D through a p53-dependent pathway

Metaphase fragility of the human RNU1 and RNU2 loci is induced by actinomycin D through a p53-dependent pathway Adong Yu1, Arnold D. Bailey1 and Alan M. Weiner1,2,*

Departments of 1Molecular Biophysics and Biochemistry and 2Genetics, Yale University, 266 Whitney Avenue, PO Box 208114, New Haven, CT 06520-8114, USA

Received October 17, 1997; Revised and Accepted January 18, 1998

Infection of human cells with adenovirus 12 (Ad12), but not Ad2 or 5, induces four specific sites of metaphase chromosome fragility: 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). Significantly, each of these sites corresponds to a multigene family encoding a small, abundant structural RNA. We and others have shown previously that Ad12-induced fragility of the RNU2 locus requires U2 snRNA promoter elements, viral early functions and p53 function, but not viral replication or integration, Rb function or chromosomal sequences flanking the RNU2 locus. Remarkably, we now find that very low doses of actinomycin D (5-50 ng/ml) can phenocopy Ad12 infection: metaphase fragility of the RNU1 and RNU2 loci is induced specifically in the absence of virus, and induction also requires U2 promoter elements and p53 function. Concurrently, it has been found by others that treatment with cytosine arabinoside (araC), but not aphidicolin, can also phenocopy Ad12 infection. We propose that Ad12 infection, actinomycin D and araC all induce a similar or identical global damage arrest signal (perhaps a modification or altered conformation of p53) that preferentially interferes with metaphase condensation of the RNU1 and RNU2 loci. The RNU1 and RNU2 loci could be especially sensitive to this global signal either because specialized U snRNA transcription factors interact uniquely with the signal, or because the high concentration of short, active transcription units hinders chromatin condensation.

INTRODUCTION

Fragile sites are easily recognized as achromatic, incompletely condensed gaps in metaphase chromosomes, or more rarely as frank chromosomal breaks. A variety of evidence argues that fragile sites are recombinogenic on both an individual and evolutionary time scale; these sites appear to co-localize with recurrent cancer breakpoints (1), with preferential targets for integration of viral (2,3) and exogenous DNA (4), with boundaries of gene amplification (5), with sites of sister chromatid exchange (6-8) and with sites of chromosome translocations in primate lineages (1). Nonetheless, it is important to distinguish between physical fragility (assayed cytologically) and genetic instability (assayed as heritable DNA rearrangements). Although physical fragility might lead to genetic instability by exposing DNA to the recombination machinery, physical and genetic instability could both reflect an underlying defect which disrupts chromatin packing and facilitates recombination.

Infection of primary human cells at low multiplicity with adenovirus 12 (Ad12), but not Ad2 or 5, induces four sites of metaphase chromosome fragility (9), whereas both Ad12 and Ad2/5 induce generalized chromosome fragility at high multiplicity (9,10). Surprisingly, these four Ad12 `modification' sites co-localize with four tandemly repeated multigene families (11-14): U1 small nuclear RNA (snRNA) genes at 1p36 (the RNU1 locus), U2 snRNA genes at 17q21-22 (RNU2), moribund U1 snRNA pseudogenes at 1q12-q22 (PSU1), and 5S rRNA genes at 1q42-43 (RN5S). Although Ad12 efficiently induces fragility of the RNU1, RNU2 and PSU1 loci in several different cell lines (10,15-18), RN5S fragility may not be visualized readily except in primary cells (9).

Cytological co-localization of the four virally inducible fragile sites with four tandemly repeated multigene families suggested that the small RNA genes were necessary, and perhaps sufficient, for virally induced fragility. Indeed, an artificial tandem array of active, but not inactive, U2 snRNA genes creates a fully functional Ad12-inducible fragile site (16-18). Fragility, therefore, requires multiple copies of the minimal U2 snRNA transcription unit, but not chromosomal sequences flanking the RNU2 locus (19,20), nor other sequences from the 6.1 kb U2 repeat unit including the CT microsatellite (21) and the solo long terminal repeat (LTR) (19,22). These results support our working hypothesis that the four Ad12-inducible fragile sites co-localize with clustered, highly transcribed, multigene families encoding small RNAs because concentrated transcriptional activity somehow interferes with local chromatin packing (12).

The early E1 transforming region of Ad2, 5 and 12 consists of two independent transcription units, E1A and E1B. E1A encodes three related proteins by alternative splicing (the 13S, 12S and 9S mRNA products) while E1B encodes two unrelated proteins (19 and 55 kDa) in overlapping reading frames (for review, see ref. 23). Stable expression of the intact Ad12 E1 region induced constitutive fragility of the human RNU2 locus in a mouse somatic hybrid background (24), suggesting that viral early functions might be sufficient for fragility under certain circumstances. Other observations suggested that full Ad12-induced fragility might depend on an interaction between E1B 55 kDa and cellular p53. Mutations in the E1B 55 kDa protein substantially reduce, but do not abolish, Ad12-induced fragility in human cells (10). E1B 55 kDa is known to bind and repress the p53 transactivation domain (25,26; reviewed in ref. 27). E1B 55 kDa also associates with the viral E4orf6 34 kDa protein, which inhibits p53 transactivation (28) and repression (29), and relocalizes cytoplasmic E1B 55 kDa protein to the nucleus (30). In addition, Ad12 cannot induce fragility in cells lacking p53, but virally induced fragility can be rescued by expression of wild-type p53 or any of several mutants in the core DNA-binding domain (31). Finally, co-transfection of Saos-2 cells lacking p53 with E1B 55 kDa and p53 expression vectors, but not with either expression vector alone, efficiently induces RNU2 fragility (D.Liao, A.Yu and A.M.Weiner, in preparation).


Figure 1. Actinomycin D-induced fragility of the human RNU1 and RNU2 loci. (A) RNU2 locus after treatment with 50 ng/ml actinomycin D for 2 h prior to addition of colcemid. (B) RNU1 locus after treatment with 50 ng/ml actinomycin D for 2 h prior to addition of colcemid. (C) Control chromosomes 1 from HT1080 cells not treated with actinomycin D. (D) Control chromosomes 17 from HT1080 cells not treated with actinomycin D. All FISH in this and subsequent figures was performed as described previously (17,31). See Table 1 for quantitation.

Actinomycin D, a DNA intercalator best known as an inhibitor of transcription at high concentrations (>1 µg/ml), is also a potent inducer of p53 DNA binding and transactivation at concentrations as low as 30 ng/ml (32-35). Moreover, in the Chinese hamster cell line GMA32, actinomycin D not only triggers gene amplification, but induces a small number of specific metaphase fragile sites which apparently define the boundaries of the early amplicons (5). We therefore asked whether actinomycin D could induce specific metaphase fragile sites in human cells. To our surprise, actinomycin D phenocopies the effects of Ad12 infection, causing p53-dependent fragility of the RNU1 and RNU2 loci. These observations imply that actual DNA damage, or the perception of damage, may interfere locally or globally with metaphase chromatin condensation.

RESULTS

Sensitivity of RNU1 and RNU2 loci to actinomycin D

Coquelle et al. (5) observed a variety of specific fragile sites after treating the Chinese hamster line GMA32 with 100 ng/ml actinomycin D for 18 h, followed by an 8 h recovery and a final 2 h incubation with nocodazole. In exploratory experiments, we treated adherent human HT1080 fibrosarcoma cells at 30-50% confluence with a range of actinomycin D concentrations (0-100 ng/ml) for 2 h prior to addition of colcemid (0.1 µg/ml). After 3 h of metaphase arrest, cells were fixed, probed, and the RNU2 and RNU1 loci visualized by fluorescence in situ hybridization (FISH) as described (17). No effect on metaphase arrest was seen even at 100 ng/ml actinomycin D. The highest frequency of RNU2 fragility (>50% of the chromosomes 17) was seen with 50 and 100 ng/ml actinomycin D; maximal fragility was seen with 50 ng/ml actinomycin D, half-maximal at 10 ng ml, and quarter-maximal at 5 ng/ml (Fig. 1A, Table 1). The RNU1 locus behaved similarly, with >50% of the cells exhibiting some fragility at 50 ng/ml actinomycin D (Fig. 1B).

We previously observed that Ad12-induced fragility of the RNU1, RNU2 and PSU1 loci spans a broad range of chromosome morphologies from frank separation of chromosome arms to thinning of one or both chromatids; dislocation of one or both chromatid axes; and (most commonly) splitting, smearing, intensification or displacement of the RNU2 signal from the bulk chromosomal DNA as seen by 4',6'-diamidino-2-phenylindole (DAPI) staining (17,31). We often saw frank separations in earlier experiments with Ad12 (17), but more recently the fragile phenotypes have been more modest (31) as was the case in the meticulous original studies over three decades ago (9). Actinomycin D-induced fragility, like Ad12-induced fragility, also exhibits a range of morphologies. In our experience, no matter how we define `fragility', there will always be `background' that resembles weak fragility, and this `background' apparently varies from one cell line to another (Tables 0 and 0, compare HT1080 and Saos-2). We are confident of our results because we measure increments in fragility over background, are scoring >50 metaphases (Tables 0 and 0) and because we never see strong fragility in uninfected or untreated cells.

U2 gene sequences required for actinomycin D-induced fragility

To determine what U2 gene sequences were required for actinomycin D-induced fragility, we used a series of HT1080 derivatives containing artificial tandem arrays of intact, minimal, and transcriptionally disabled U2 snRNA genes (17). The intact arrays (iU2 series) contained tandem copies of the intact 6.1 kb U2 repeat unit. The minimal arrays (mU2 series) contained tandem copies of a minimal 834 bp gene segment spanning (from 5' to 3') the distal sequence element (DSE or enhancer), the proximal sequence element (PSE or TATA-equivalent element), the 188 bp coding region and the specialized 3' end formation signal (3' box). The disabled arrays contained tandem copies of a minimal U2 gene from which either the DSE ([Delta]DSE series) or both the DSE and PSE ([Delta]DSE [Delta]PSE series) had been deleted. In all constructs, the 188 bp U2 coding region was marked by the innocuous U87C point mutation in a phylogenetically variable position, allowing steady-state levels of U2 snRNA derived from the transgenes to be monitored by primer extension relative to the background of resident U2 snRNA (17).

Table 1. Concentration dependence of actinomycin D-induced RNU2 fragility in HT1080 fibrosarcoma
Actinomycin D RNU2 fragility
(ng/ml) (% of cells) (% of RNU2 loci)
0 4 1
5 16 12
10 33 25
50 62 48
100 52 40
At least 50 metaphases were examined for each datapoint. An RNU2 locus was considered fragile if the FISH signal was obviously fragmented, or was far more intense than most others in the field (31). At least two HT1080 lines are currently in circulation. Ours, which comes from the ATCC Type Culture Collection (kind gift of S.Bacchetti), is thought to be wild-type with a defect downstream of p21 (93; G.M.Wahl, personal communication). The other HT1080 line is a compound heterozygote (94-96).

Table 2. U2 gene sequence elements required for actinomycin D-induced RNU2 fragility
Cell line Repeat type Copy Fragility of artificial RNU2 locus Fragility of natural RNU2 loci
    no. (% of cells) (% of cells) (% of locus)
iU2 A37 intact 48 51 50 39
iU2 A41 intact 77 46 47 35
mU2 L48 minimal 8 18 61 46
mU2 [Delta]DSE25 [Delta]DSE 112 12 52 39
mU2 [Delta]DSE50 [Delta]DSE 31 6 62 40
mU2 [Delta]DSE [Delta]PSE42 [Delta]DSE[Delta]PSE 98 10 46 30
mU2 [Delta]DSE [Delta]PSE48 [Delta]DSE[Delta]PSE 78 3 67 50
At least 50 metaphases were examined for each datapoint. All cell lines are HT1080 derivatives as described (17). The solitary artificial U2 tandem array in each cell line was easily distinguished from the two resident RNU2 alleles by chromosome size, cytological location and/or signal strength. Metaphases in which both the artificial and natural RNU2 loci are fragile appear with the frequency expected if the fragility of each locus is independent.

Cells were grown as described (17) and treated with 50 ng/ml actinomycin D for 2 h before colcemid was added (Fig. 2, Table 2). In all cases, actinomycin D-induced fragility of the solitary artificial U2 array was compared with that of the two resident RNU2 alleles in the same metaphase as an internal control. A large tandem array of intact 6.1 kb repeat units (iU2 A37 and A41 cell lines) was as fragile as the resident RNU2 alleles. A small array of minimal U2 genes (mU2 L48) was a third as fragile as the iU2 A37 and A41 arrays, but this is most likely due to low copy number of mU2 repeats (17) rather than to loss of other sequences from the intact 6.1 kb repeat unit such as the CT microsatellite (21) and the solo LTR (19,22). Crippling transcription by deletion of the enhancer-like distal sequence element (mU2 [Delta]DSE50) reduced fragility nearly to background, although very high copy number (mU2 [Delta]DSE50) partially compensated for loss of the DSE. This suggests that actinomycin D-induced fragility depends on the total amount of transcription per array; a few strong promoters or many weak promoters seem to have comparable effects. Abolishing transcription by deletion of both the enhancer-like DSE and the TATA-like proximal sequence element (mU2 [Delta]DSE [Delta]PSE42 and mU2 [Delta]DSE [Delta]PSE48) almost completely abolished fragility regardless of copy number. Marginal fragility of some promoterless U2 genes could reflect cryptic promoters, random readthrough transcription or perturbation of normal chromatin structure by integration of foreign DNA.


Figure 2. U2 gene sequences required for actinomycin D-induced RNU2 fragility. (A) and (B) iU2 A37 and iU2 A41 lines with 48 copies and 77 copies of an intact 6.1 kb U2 repeat unit, respectively. (C) and (D) mU2 [Delta]DSE25 and [Delta]DSE50 lines, with 112 and 31 copies respectively of a minimal U2 gene from which the DSE has been deleted. (E) and (F) mU2 [Delta]DSE [Delta]PSE42 and [Delta]DSE [Delta]PSE48 lines, with 98 and 78 copies respectively of a minimal U2 gene from which both the DSE and PSE have been deleted. See Table 2 for quantitation. About 20% of the iU2 A37 arrays have generated what appear to be homogeneously banded regions (HBRs, in A, rightmost four chromosomes) suggesting that this particular array may be prone to physical breakage followed by breakage-fusion-bridge cycles (100).


Figure 3. Actinomycin D-induced RNU2 fragility requires p53 function. Chromosomes 17 from uninfected Saos-2 cells (A) or Ad12-infected Saos-2 cells (B). Cultures in (A) and (B) were not treated with actinomycin D. Chromosomes 17 from uninfected Saos-2 (C), mock electroporated Saos-2 (D) or Saos-2 transfected with an expression vector encoding wild-type p53 (E) or the R273H mutant (F). Cultures in (C), (D), (E) and (F) were all treated with 100 ng/ml actinomycin D. See Table 3 for quantitation.

Actinomycin D-induced RNU2 fragility requires p53 function

To determine whether p53 function is required for actinomycin D-induced RNU2 fragility, we first asked whether actinomycin D could induce RNU2 fragility in Saos-2 cells lacking p53 function (36). No RNU2 fragility above background could be detected (Table 3), although background fragility in Saos-2 cells (lacking both p53 and Rb) is typically higher than seen with the HT1080 fibrosarcoma (Fig. 1, Table 1). Next we asked whether transfection of Saos-2 with wild-type p53 or with the transcriptionally impaired hotspot mutant R273H (37-39) would restore actinomycinD-induced fragility of the RNU2 locus. Although electroporation raised background fragility further in Saos-2 from 10 to 16%, it is clear that both mutant and wild-type p53 substantially rescue actinomycin D-induced RNU2 fragility (Fig. 3, Table 3).

DISCUSSION

Ad12 induces four, and only four, sites of metaphase chromosome fragility in human cells (9,10). As all four sites (the RNU1, RNU2, PSU1 and RN5S loci) contain clusters of genes (or formerly active pseudogenes) encoding small, abundant, structural RNAs (12), we suggested that a high density of active transcription units retards metaphase chromatin condensation, and that Ad12 infection tips the balance between transcription and condensation so that full metaphase packing fails to take place. Consistent with this interpretation, we (17) and others (16,18) found that an artificial tandem array of active, but not inactive, U2 transcription units is sufficient to generate a new Ad12-inducible fragile site.

A variety of evidence implies that Ad12-induced fragility primarily reflects an interaction between the Ad12 E1B 55 kDa protein and cellular p53. E1B 55 kDa is known to bind and repress the p53 transactivation domain (25,26; reviewed in ref. 27). Ad12-induced fragility is substantially reduced, albeit not abolished, by mutations in the E1B 55 kDa protein (10). Ad12 cannot induce fragility in cells lacking p53, but expression of wild-type p53 or any of several mutants in the core DNA-binding domain rescues virally induced fragility (31). Most compellingly, co-transfection of Saos-2 cells lacking p53 with E1B 55 kDa and p53 expression vectors, but not with either expression vector alone, efficiently induces RNU2 fragility (D. Liao, A. Yu and A. M. Weiner, in preparation). Residual fragility induced by Ad12 virus lacking E1B 55 kDa function (10) might reflect the ability of the 12S- and 13S-encoded E1A proteins to inhibit the p53 transactivation function (40), the 13S-encoded E1A protein to relieve p53 repression (41) or the viral E4orf6 34 kDa protein to inhibit both p53 transactivation (28) and repression (29). For reasons discussed in detail elsewhere, Ad12-induced fragility is unlikely to involve the p53-dependent apoptotic pathway (31).

Table 3. Actinomycin D-induced RNU2 fragility requires p53 function
Cell line Treatment RNU2 fragility
    (% of cells) (% of RNU2 alleles)
Saos-2 none 8 4
Saos-2 Ad12 infection 9 5
Saos-2 10 ng/ml AMD 10 5
Saos-2 100 ng/ml AMD 9.5 6
Saos-2 none 10 4
Saos-2 mock electroporation 16 10
Saos2 mock assay (pCH110 only) 17 10
Saos2 mock assay (pCMV[beta] and pCH110) 19 10
Saos-2 p53 transfection, 100 ng/ml AMD 39 27
Saos-2 R273H transfection, 100 ng/ml AMD 35 24
Saos-2 cells were a kind gift of Dr W.-H. Lee (97). At least 100 metaphases were examined for each datapoint. Cells were transfected by electroporation as described in detail elsewhere (31) with a total of 20 µg of plasmid consisting of 18 µg of p53 expression vector and 2 µg of pCH110 [beta]-galactosidase expression vector (98). Cells were then grown for 16 h, treated with actinomycin for 2 h, arrested with colcemid for 3 h and harvested for FISH. Transfection efficiency was 30-40% as judged by [beta]-galactosidase color reaction (31,98). DNA was omitted in the mock electroporation; mock assays included either 20 µg of pCH110 (`pCH110 only') or 2 µg of pCH110 and 18 µg of pCMV[beta] (Clontech) in which the CMV promoter drives[beta]-galactosidase expression (`pCMV [beta]-gal and pCH110'). Constitutive (background) fragility of the RNU2 locus is typically somewhat higher in Saos-2 than in HT1080 cells, and is increased by electroporation. The p53 R273H hotspot mutant is transcriptionally impaired but not totally inactive (37-39,99). p53 expression vectors were a kind gift of Dr B. Vogelstein (91,92). AMD, actinomycin D.

Here we show that Ad12-induced metaphase fragility of the RNU1 and RNU2 loci can be phenocopied by treatment of human cells with low concentrations of actinomycin D (<30 ng/ml) which are sufficient to induce p53 (32-35) but are far below the concentrations (>1 µg/ml) required to inhibit DNA synthesis, rRNA synthesis (42) or transcription of typical mRNA precursors (43) that are many times larger than U1 and U2 snRNA (44-46). As is also true for Ad12-induced fragility (31), we find that actinomycin D-induced fragility requires an active U2 promoter (Fig. 2, Table 2) and p53 function (Fig. 3, Table 3), which can be supplied equally well by wild-type p53 or the transcriptionally impaired mutant R273H (37-39).

How might actinomycin D phenocopy, or counterfeit, Ad12 infection? The simplest unifying hypothesis is that actinomycin D and the Ad12 E1B 55 kDa protein both activate a p53-dependent DNA damage response pathway by causing similar, or functionally equivalent, changes in the levels, conformation, modification or subcellular localization of p53. p53 protein levels (47-49) and transcriptional transactivation (35,50) are strongly induced by DNA strand breaks but not by other DNA lesions (48), and need not be cell cycle dependent (51). Although actinomycin D stabilizes covalent complexes of topoisomerase I and II with DNA (52,53), it can also cause single and double strand DNA breaks at these sites in vitro (53,54). The observation that topo I intermediates trapped by camptothecin do not induce p53 or trigger the p53-dependent DNA damage response (48) further supports our proposal that actinomycin D induces p53 by generating DNA breaks, rather than by stabilizing covalent topoisomerase intermediates.

In mammals, four protein kinases are known to belong to the phosphatidylinositol-3 kinase (PI-3) superfamily; all four enzymes (FRAP, DNA-PK, ATM and ATR) are large (>2500 amino acids) and share a carboxy-terminal kinase domain (reviewed in ref. 55). Three of these enzymes-DNA-PK (DNA-dependent protein kinase), ATM (ataxia telangiectasia-mutated) and ATR (ataxia telangiectasia-related)-appear to be involved in signaling DNA rearrangements and cell cycle arrest. Induction of p53 by actinomycin D apparently requires the ataxia telangiectasia (AT) gene product (35,56). Curiously, DNA-PK is not required for the p53 response to ionizing radiation, UV radiation, or alkylating agents (49,50,57), although actinomycin D has, to our knowledge, not been tested. DNA-PK binds to broken DNA ends in complex with the two end-binding subunits Ku70 and Ku80 (reviewed in ref. 58), and can phosphorylate p53 in response to certain kinds of DNA damage (59). Thus single or double strand breaks induced by actinomycin D might activate DNA-PK or another member of the PI-3 protein kinase superfamily, which could in turn directly phosphorylate p53.

In concurrent work, MacArthur et al. (60) demonstrate that treatment of human cells with cytosine arabinoside (araC) but not aphidicolin (10) can also phenocopy the p53-dependent Ad12-induced metaphase fragility of the RNU2 locus. This remarkable coincidence leads us to propose that Ad12 infection, actinomycin D and araC all induce a similar or identical global damage arrest signal mediated by p53 that preferentially interferes with metaphase condensation of the RNU1 and RNU2 loci. Consistent with this proposal, araC damages DNA by functioning as a chain terminator (reviewed in ref. 61). The inability of aphidicolin (which inhibits DNA polymerases [alpha], [delta] and [epsilon]) to induce metaphase fragility of the RNU2 locus (10) suggests that DNA damage, or the perception of damage, is required to elicit this damage arrest signal; simple inhibition of DNA replication does not suffice. On the other hand, the major cause of araC toxicity may be oxidative stress not DNA damage (61), and araC does not induce p53 protein levels (30) although this does not exclude changes in post-transcriptional modification or subcellular localization.

Why might the RNU1 and RNU2 loci be especially sensitive to the postulated p53-mediated global damage arrest signal? Two simple, and potentially compatible, models come to mind. In the first, a high local concentration of short, active transcription units normally hinders or retards metaphase chromatin condensation, and condensation is perturbed further by the damage arrest signal; in the second, specialized U snRNA transcription factors interact uniquely with the damage arrest signal. Some evidence favors the first model. If metaphase chromatin packing were dominant over transcription, the cell would not go to great lengths to ensure mitotic shutdown of transcription: TATA box-binding protein (TBP) (62,63), RNA polymerase II transcription factors such as Oct-1 (64) and Sp1 (65), and TFIIIB (66,67) are inactivated by phosphorylation as metaphase approaches (for review, see ref. 68), and nascent transcripts are aborted (69). Thus the DNA damage arrest signal might prolong transcription into metaphase, or generate an aberrant mitotic `bookmark' identifying an active promoter region (70). Other evidence favors the second model. Although transcribed by RNA polymerase II, the specialized U snRNA promoter (71-73) and termination factors (74-76) are very different from those used to transcribe mRNA precursors, and could respond uniquely to a damage arrest signal. Indeed, the extraordinary sensitivity of the very short (<200 bp) U1 and U2 transcription units to mild UV irradiation might also reflect a specialized response, or heightened sensitivity, to DNA damage arrest signals (77-79).

In either model, p53 might work directly on the RNU1 and RNU2 transcription units to cause metaphase fragility. p53 interacts with many components of the basal RNA polymerase II transcription machinery (80) including the ERCC2 (xeroderma pigmentosum D), ERCC3 (xeroderma pigmentosum B) and p62 components of the basal factor TFIIH (39,81-84). The ERCC2 (XPD) and ERCC3 (XPB) helicases, as well as p62, belong to a common core of five subunits shared between the `repairosome' and the basal RNA polymerase II transcription factor TFIIH (85-88). Once alerted to DNA damage, either directly (89) or indirectly, p53 might then interact with the XPB (ERCC3) and/or XPD (ERCC2) helicases as part of either TFIIH or the repairosome. The connection between damage, repair, transcription and metaphase fragility is strengthened further by the observation that the RNU1 and RNU2 loci are constitutively fragile in cells having defects in the XPB (ERCC3) and/or XPD (ERCC2) helicases (31) or defects in the Cockayne syndrome B (CSB) protein that plays a role in transcription-coupled repair (D.Liao and A.M.Weiner, submitted).

Chromosome fragility is unlikely to be a unitary phenomenon, but rather to reflect many causes and mechanisms that ultimately have the same consequence: a weak spot in chromatin condensation and a predisposition to recombination or breakage. To date, the best studied fragile sites appear to be induced by defects in DNA replication (90), as might be expected if incomplete replication products interfere with proper chromatin condensation, or chromatin condensation is coupled to the completion of DNA replication. We find that a new class of fragile sites (RNU1, RNU2 and RN5S) requires transcription of the locus, p53 function, and can be induced by DNA-damaging reagents (actinomycin D and araC) or by defects in dual function transcription/repair factors (ERCC2/XPD, ERCC3/XPB and CSB). Investigation of these novel fragile sites caused by defects in transcription and/or repair, rather than in DNA replication, should help to shed additional light on the role of chromatin condensation in health and disease.

MATERIALS AND METHODS

Cells, growth conditions, transfection and FISH have been described previously (17) or elsewhere (31). The U1 probe was intact plasmid p5P2 containing a non-repetitive fragment from the U1 repeat unit (13). The U2 probe was intact plasmid 12L containing a 3.7 kb PvuII-HindIII fragment from the right end of the 5.8 kb U2 repeat unit cloned into pUC13 vector (19). p53 expression constructs (91,92) were the generous gift of B. Vogelstein. Saos-2 cells were kindly supplied by Drs S. Bacchetti and Wen-Hwa Li. Actinomycin D (95%, HPLC purified; Sigma) was dissolved in phosphate-buffered saline (PBS) at 1 mg/ml and stored cold in the dark.

ACKNOWLEDGEMENTS

We thank Dr Daiqing Liao for help with chromosome spreading and imaging; Dr Bert Vogelstein for wild-type and mutant p53 expression vectors; Dr Silvia Bacchetti for communicating results in advance of publication; and NIH awards GM31073 and GM41624 for support.

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*To whom correspondence should be addressed. Tel: +1 203 432 3089; Fax: +1 203 432 3047; Email: weiner@biomed.med.yale.edu

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T. Pavelitz, A. D. Bailey, C. P. Elco, and A. M. Weiner
Human U2 snRNA Genes Exhibit a Persistently Open Transcriptional State and Promoter Disassembly at Metaphase
Mol. Cell. Biol., June 1, 2008; 28(11): 3573 - 3588.
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