The rate of transcription elongation plays an important role in the timing of expression of full-length transcripts as well as in the regulation of alternative splicing. In this study, we coupled Bru-seq technology with 5,6-dichlorobenzimidazole 1-b-D-ribofuranoside (DRB) to estimate the elongation rates of over 2000 individual genes in human cells. This technique, BruDRB-seq, revealed gene-specific differences in elongation rates with a median rate of around 1.5 kb/min. We found that genes with rapid elongation rates showed higher densities of H3K79me2 and H4K20me1 histone marks compared to slower elongating genes. Furthermore, high elongation rates had a positive correlation with gene length, low complexity DNA sequence, and distance from the nearest active transcription unit. Features that negatively correlated with elongation rate included the density of exons, long terminal repeats, GC content of the gene, and DNA methylation density in the bodies of genes. Our results suggest that some static gene features influence transcription elongation rates and that cells may alter elongation rates by epigenetic regulation. The BruDRB-seq technique offers new opportunities to interrogate mechanisms of regulation of transcription elongation.

Copy number variants (CNVs) resulting from genomic deletions and duplications and common fragile sites (CFSs) seen as breaks on metaphase chromosomes are distinct forms of structural chromosome instability precipitated by replication inhibition. Although they share a common induction mechanism, it is not known how CNVs and CFSs are related or why some genomic loci are much more prone to their occurrence. Here we compare large sets of de novo CNVs and CFSs in several experimental cell systems to each other and to overlapping genomic features. We first show that CNV hotpots and CFSs occurred at the same human loci within a given cultured cell line. Bru-seq nascent RNA sequencing further demonstrated that although genomic regions with low CNV frequencies were enriched in transcribed genes, the CNV hotpots that matched CFSs specifically corresponded to the largest active transcription units in both human and mouse cells. Consistently, active transcription units >1 Mb were robust cell-type-specific predictors of induced CNV hotspots and CFS loci. Unlike most transcribed genes, these very large transcription units replicated late and organized deletion and duplication CNVs into their transcribed and flanking regions, respectively, supporting a role for transcription in replication-dependent lesion formation. These results indicate that active large transcription units drive extreme locus-and cell-type-specific genomic instability under replication stress, resulting in both CNVs and CFSs as different manifestations of perturbed replication dynamics.

The mechanisms by which the p53 response is triggered following exposure to DNA-damaging agents have not yet been clearly elucidated. We and others have previously suggested that blockage of RNA polymerase II may be the trigger for induction of the p53 response following exposure to ultraviolet light. Here we report on the correlation between inhibition of mRNA synthesis and the induction of p53, p21 WAF1 and apoptosis in diploid human ®broblasts treated with either UV light, cisplatin or the RNA synthesis inhibitors actinomycin D, DRB, H7 and a-amanitin. Exposure to ionizing radiation or the proteasome inhibitor LLnL, however, induced p53 and p21 WAF1 without aecting mRNA synthesis. Importantly, induction of p53 by the RNA synthesis or proteasome inhibitors did not correlate with the induction of DNA strand breaks. Furthermore, cisplatin-induced accumulation of active p53 in repair-de®cient XP-A cells occurred despite the lack of DNA strand break induction. Our results suggest that the induction of the p53 response by certain toxic agents is not triggered by DNA strand breaks but rather, may be linked to inhibition of mRNA synthesis either directly by the poisoning of RNA polymerase II or indirectly by the induction of elongation-blocking DNA lesions.

Gene expression studies commonly examine total cellular RNA, which only provides information about its steady-state pool of RNA. It remains unclear whether differences in the steady-state reflects variable rates of transcription or RNA degradation. To specifically monitor RNA synthesis and degradation genome-wide, we developed Bru-Seq and BruChase-Seq. These assays are based on metabolic pulse-chase labeling of RNA using bromouridine (Bru). In Bru-Seq, recently labeled RNAs are sequenced to reveal spans of nascent transcription in the genome. In BruChase-Seq, cells are chased in uridine for different periods of time following Bru-labeling, allowing for the isolation of RNA populations of specific ages. Here we describe these methodologies in detail and highlight their usefulness in assessing RNA synthesis and stability as well as splicing kinetics with examples of specific genes from different human cell lines.