Adenovirus Type 12-Induced Fragility of the Human RNU2 Locus Requires p53 Function

Abstract

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 PSU1fragile 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 theRNU1 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.

Hot spots of DNA double-strand breaks and genomic contacts of human rDNA units are involved in epigenetic regulation

“…A high level of DSBs in rDNA could also be explained by extensive transcription of rDNA units, and perhaps an incomplete mitotic shutdown of transcription before condensation of the chromosomes. It has been described that adenovirus type 12 infection in human cells induces several fragile sites that may be linked through transcription and that persist into metaphase, thus interfering with chromatin packing and replication, leading to DNA breaks ( Li et al, 1998 ). More detailed analysis revealed that loss of Cockayne syndrome group B protein induces fragility in the same loci suggesting a link between basal transcription apparatus and fragility ( Yu et al, 2000 ).…”

Viral induction of site‐specific chromosome damage

“…The next step was to identify what cellular functions might also be involved. The finding by Weiner and his colleagues [30] that p53 function was also necessary was not surprising given the known interactions between p53 and the E1B 55 000 protein. However, virus-induced fragility was also observed in cells expressing a p53 mutant protein (R273H) that is partially defective for DNA binding and transactivation.…”

Differential alterations in metabolic pattern of the spliceosomal uridylic acid‐rich small nuclear RNAs (UsnRNAs) during malignant transformation of 20‐methylcholanthrene‐induced mouse CNCI‐PM‐20 embryonic fibroblasts

“…A translocation break point was observed within a large cluster of U1 and some tRNA genes in chr.1p36 in neuroblastoma 25. The U1 and U2 loci showed chromosomal fragility at metaphase due to infection of adenovirus serotype‐12, in a process that requires tumor‐suppressor gene p53 26, 27. Moreover, we have also previously reported, a differential alteration in metabolic pattern of UsnRNAs during development of premalignant lung lesions in benzoa(a)pyrene (BaP) or tea polyphenols‐treated mice and also in Sarcoma‐180 cells in vivo 28, 29.…”