Genome-wide transcriptional effects of the anti-cancer agent camptothecin

Abstract

The anti-cancer drug camptothecin inhibits replication and transcription by trapping DNA topoisomerase I (Top1) covalently to DNA in a "cleavable complex". To examine the effects of camptothecin on RNA synthesis genome-wide we used Bru-Seq and show that camptothecin treatment primarily affected transcription elongation. We also observed that camptothecin increased RNA reads past transcription termination sites as well as at enhancer elements. Following removal of camptothecin, transcription spread as a wave from the 5'-end of genes with no recovery of transcription apparent from RNA polymerases stalled in the body of genes. As a result, camptothecin preferentially inhibited the expression of large genes such as proto-oncogenes, and anti-apoptotic genes while smaller ribosomal protein genes, pro-apoptotic genes and p53 target genes showed relative higher expression. Cockayne syndrome group B fibroblasts (CS-B), which are defective in transcription-coupled repair (TCR), showed an RNA synthesis recovery profile similar to normal fibroblasts suggesting that TCR is not involved in the repair of or RNA synthesis recovery from transcription-blocking Top1 lesions. These findings of the effects of camptothecin on transcription have important implications for its anti-cancer activities and may aid in the design of improved combinatorial treatments involving Top1 poisons.

Figure 1. Gene size is a major contributing factor to the effects of camptothecin on RNA synthesis.

Human fibroblasts were treated with 20 µM camptothecin for 45 min with 2 mM Bru added during the last 15 min of camptothecin treatment to label nascent RNA followed by Bru-Seq. (A), Long genes, such as TRIO, exhibit elongation defects, but not transcription initiation, after camptothecin treatment. (B), Short genes, such as BAMBI, show a relative increase of RNA synthesis following camptothecin treatment. (C), Effect of camptothecin on relative transcription as a function of gene size. Ratio of Bru-Seq signal of individual genes in camptothecin-treated over control cells as a function of gene size. Longer genes are inhibited preferentially over shorter genes. (D), The median length of genes induced >2-fold by camptothecin (919 genes) is 8,927 bp, whereas genes down-regulated >2-fold (1,145 genes) have a median length of 136,355 bp. The gene maps are from RefSeq Genes (UCSC genome browser).

Figure 2. Effect of camptothecin on transcriptional readthrough and synthesis of PROMPTs and eRNA.

As in Figure 1, human fibroblasts were treated with 20 µM camptothecin for 45 min with 2 mM Bru added during the last 15 min of camptothecin treatment to label nascent RNA followed by Bru-Seq. (A), Transcriptional readthrough of the termination site of the RHOB gene induced by camptothecin. (B), Enhanced initiation of the ASCC3 gene and coincident upregulation of divergent upstream PROMPT RNA. (C), Enhanced expression of eRNA from the 5’-upstream enhancer of FOS by camptothecin. (D), Camptothecin inhibits the transcription of the primary transcript of miRNA138-1. (E), Camptothecin induces transcription of the ncRNA MALAT1. (F), Camptothecin inhibits the transcription of a very long unannotated ncRNA on chromosome 2. The gene maps are from RefSeq Genes (UCSC genome browser).

Figure 3. Effect of camptothecin reversal on RNA synthesis.

(A), Aggregate view of RNA synthesis of genes larger than 100 kb in normal human fibroblasts with the genes aligned by transcriptional start sites (TSS). RNA synthesis recovers as a wave in a 5’-to-3’ direction following camptothecin removal with no apparent recovery of RNA polymerases stalled in the body of the genes. Elongation rates of the recovering transcription wave was estimated to be ~1.2 kb/min. (B), Wave of recovery of RNA synthesis can be seen advancing from the 5’-end of the CD44 gene with no apparent recovery in the body of the gene. The front of the transcription wave extended some 35 kb during the first 30 min recovery resulting in an elongation rate of about 1.2 kb/min. (C) Similar elongation rates after camptothecin removal were found for the MEISE1 gene. Color key: Blue, control (30 min Bru labeling); Yellow, Bru labeling during the last 15 min of a 45 min camptothecin treatment; Green, 45 min camptothecin treatment followed by a drug washout and 15 min of Bru labeling; Red, 45 min camptothecin treatment followed by a drug washout, 15 min incubation, and finally 15 min Bru labeling.

Figure 4. Effect of camptothecin reversal on RNA synthesis in Cockayne syndrome cells.

(A), Aggregate view of RNA synthesis of genes larger than 100 kb in CS-B cells with the genes lined up by transcriptional start sites (TSS) as in Figure 3. Elongation rates of the recovering transcription wave was estimated to be ~1.0-1.3 kb/min. Individual genes in fibroblasts from a CS-B individual showing similar recovery rates as in fibroblasts from a normal individual for (B), CD44 and (C) MEIS1. Color key as in Figure 3.

Figure 5. Pathway enrichment for genes following camptothecin treatment and reversal.

(A) Pathways represented by genes up-regulated at least 2-fold, and (B) down-regulated at least 2-fold following camptothecin treatment and recovery. Human fibroblasts were treated with 20 µM camptothecin for 45 minutes and incubated for the last 15 min with 2 mM Bru (“0 min”), incubated for 15 min with Bru following the removal of camptothecin (“15 min”) or incubated for 15 min with Bru following a 45 min treatment, a wash and a 15 min recovery (“30 min”). Enrichment analysis was performed using DAVID (david.abcc.ncifcrf.gov) and the numbers shown represents the p-values for enrichment.

Figure 6. Camptothecin preferentially inhibits large genes such as proto-oncogenes and anti-apoptotic genes, enhances the relative expression of small pro-apoptotic genes and activates the p53 response.

(A), Examples of large proto-oncogenes inhibited by camptothecin and showing no recovery (or slow recovery) following drug removal. (B), Examples of p53 target genes induced following camptothecin treatment. (C), Examples of large anti-apoptotic genes showing reduced relative transcription (left) and examples of small pro-apoptotic genes showing enhanced relative transcription following camptothecin treatment (right). (D), Model of mechanisms by which camptothecin may induce cell death or inhibit cell growth. Camptothecin triggers a p53 transcriptional response and selectively inhibits large proto-oncogenes and survival genes. The data is color coded where blue represents control (C), yellow represents 15 min Bru-labeling at the end of a 45 min camptothecin treatment with no recovery (“0 min”), green represents drug washout and 15 min Bru-labeling immediately after washout (“15 min”) and finally red represents labeling 15-30 minutes following washout (“30 min”).