DNA topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the human HIF-1a gene locus

نویسندگان

  • Laura Baranello
  • Davide Bertozzi
  • Maria Vittoria Fogli
  • Yves Pommier
چکیده

Top1 inhibition by camptothecin (CPT) perturbs RNA polymerase II (Pol II) density at promoters and along transcribed genes suggesting an involvement of Top1 in Pol II pausing. Here, we demonstrate that Top1 inhibition favors Pol II escape from a promoter-proximal pausing site of the human HIF-1a gene in living cells. Interestingly, alternative splicing at exon 11 was markedly altered in nascent HIF-1a mRNAs, and chromatin structure was also affected with enhanced histone acetylation and reduced nucleosome density in a manner dependent on cdk activity. Moreover, CPT increases transcription of a novel long RNA (50aHIF1a), antisense to human HIF-1a mRNA, and a known antisense RNA at the 30-end of the gene, while decreasing mRNA levels under normoxic and hypoxic conditions. The effects require Top1, but are independent from Top1-induced replicative DNA damage. Chromatin RNA immunoprecipitation results showed that CPT can activate antisense transcription mediated by cyclin-dependent kinase (cdk) activity. Thus, Top1 inhibition can trigger a transcriptional stress, involving antisense transcription and increased chromatin accessibility, which is dependent on cdk activity and deregulated Pol II pausing. A changed balance of antisense transcripts and mRNAs may then lead to altered regulation of HIF-1a activity in human cancer cells. INTRODUCTION Human DNA topoisomerase I (Top1) regulate DNA superhelicity during fundamental DNA transactions by transiently cleaving and resealing DNA molecules (1,2). Top1 is the sole target of camptothecin (CPT) and close derivatives, which are anticancer drugs used in standard therapy of human ovarian, small-cell lung, and colorectal cancers (3). CPT interacts reversibly with its target preventing strand relegation and increasing the half-life of Top1-DNA cleavage complexes (Top1cc). A Top1cc can become lethal when it collides with DNA replication forks, leading to irreversible DNA breaks and the activation of S-phase checkpoint, G2 arrest and cell death (3). Although the established cellular effects of CPT are peculiar of DNA damage responses, CPT inhibition of Top1 occurs primarily in actively transcribed regions (2,3). In contrast to the replication-dependent effects, the transcription-dependent effects of Top1cc are not fully understood. Top1 is enriched in transcribed genomic regions as established by Top1 DNA cleavage sites (1–3) and chromatin immunoprecipitation (ChIP) (4). Top1 has been shown to activate gene transcription, and to bind to general transcription factors at promoters (5–7). A broad and general inhibition of transcription elongation is an immediate effect of CPT in cultured cells (3,8), likely due to the stalling of elongating RNA polymerases by Top1ccs (3,8) and/or by persistent transcription-generated DNA supercoils (9). Nevertheless, several groups have reported more specific effects at transcriptional levels for CPT. For instance, CPT can activate the initiation step of transcription (10) and the expression of selective genes in *To whom correspondence should be addressed. Tel: +39 51 2094282; Fax: +39 51 2094283; Email: [email protected] Published online 23 October 2009 Nucleic Acids Research, 2010, Vol. 38, No. 1 159–171 doi:10.1093/nar/gkp817 The Author(s) 2009. Published by Oxford University Press. This is an Open Access article distributed under the terms of the Creative Commons Attribution Non-Commercial License (http://creativecommons.org/licenses/ by-nc/2.5/uk/) which permits unrestricted non-commercial use, distribution, and reproduction in any medium, provided the original work is properly cited. at Penylvania State U niersity on Feruary 3, 2013 http://narrdjournals.org/ D ow nladed from human cells (11). In yeast, TOP1 gene deletion preferentially up-regulates the expression of telomereproximal genes by increasing core histone acetylation and chromatin accessibility (12). Strikingly, CPT-induced Top1ccs have immediate and specific effects on RNA polymerase II (Pol II). CPT triggers a high phosphorylation degree of the largest subunit (Rpb1) of Pol II (4,13,14), showing an effect on a critical step of transcription regulation. Hyperphosphorylation occurs selectively on Ser-5 residues of the conserved heptapeptide repeats of the carboxy-terminal domain (CTD) possibly mediated by Cdk7, component of TFIIH (13). Interestingly, a recent report showed that CPT can disrupt the large inactive P-TEFb complex, thus releasing a free active P-TEFb complex (containing the Cdk9 subunit), which can then contribute to CPTincreased phosphorylation of Pol II (15). A second immediate effect of CPT on Pol II has been reported by us previously, and can be correlated to the hyperphosphorylation of Rpb1 (4). Short cell treatments with CPT induce a redistribution of chromatin-bound Pol II along transcribed genes in human cancer cells, apparently by enhancing the escape of Pol II from promoterproximal pausing sites. Remarkably, this early specific CPT effect is independent from replication and replicative DNA damage (4). However, the release of Pol II from pausing into elongation remained to be established. Thus, to define the molecular mechanisms triggered by Top1ccs at transcribing regions, we have here investigated the CPT effects at transcription and chromatin levels at the hypoxia-inducible factor 1a (HIF-1a) gene locus in human cancer cells. HIF-1a is a transcription factor and a master regulator of the cell response to oxygen deprivation (16,17), and a target of anti-angiogenesis and anticancer agents (17). HIF-1a is subjected to post-translational regulation, as it is degraded at high oxygen tensions by an oxygen-mediated hydroxylation of conserved prolyl and asparaginyl residues, to which the von Hippel-Lindau protein (pVHL) E3 ligase binds targeting HIF-1a to proteasomal degradation (17). We selected to study the HIF-1a gene because previous reports demonstrated that CPT can markedly reduce HIF-1a protein accumulation in hypoxic cells in a manner independent from the hydroxylation pathway and from replicative DNA damage promoted by CPT (18,19). We here provide evidence that Top1ccs trigger the escape of Pol II from promoter-proximal pause site of the HIF-1a gene, activate a novel antisense transcript at the 50-end of the HIF-1a locus and induce a more accessible chromatin structure in a manner dependent on cyclin-dependent kinase activity. Thus, Top1 inhibition by CPT can result in a specific transcriptional stress leading to an imbalance of antisense and sense RNA ratios, which may alter the regulation of cancer-relevant factors such as HIF-1a. MATERIALS AND METHODS All methods and materials are fully described in the Supplementary Data. Cell lines and treatments Human HCT116, HCT116(top1 siRNA) (20), MRCV fibroblast, and Jurkat cells were cultured with standard techniques. Cells were maintained at 37 C in a humidified incubator containing 5% CO2 in air (normoxic conditions, 20% O2). Hypoxia treatments were in a cell chamber with 1% O2 at 37 C. All drug treatments were performed on exponentially growing cells at 37 C. In case of co-treatments, cells were first incubated with aphidicolin (3 mM) or DRB (50 mM) for 15min, or caffeine (5mM) for 30min, and then, CPT was added to the medium for the indicated time. RNA purification and primer-specific cDNA preparation Total RNA was purified with the acid phenol method. RNA pellets were resuspended in TE, and DNA was digested with DNAse I. Total RNA (1 mg) was used to prepare cDNA using random primers and SuperScript III (Invitrogen) as suggested by the manufacturer. When specific primers were used for reverse transcription with SuperScript III, conditions for the reaction were as follows: 5min at 65 C and 50min at 55 C. Negative controls were cDNAs prepared with no primer during reverse transcription reactions.

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DNA topoisomerase I inhibition by camptothecin induces escape of RNA polymerase II from promoter-proximal pause site, antisense transcription and histone acetylation at the human HIF-1α gene locus

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تاریخ انتشار 2009