Studying DNA Double-Strand Break Repair: An Ever-Growing Toolbox.

To ward off against the catastrophic consequences of persistent DNA double-strand breaks (DSBs), eukaryotic cells have developed a set of complex signaling networks that detect these DNA lesions, orchestrate cell cycle checkpoints and ultimately lead to their repair. Collectively, these signaling networks comprise the DNA damage response (DDR). The current knowledge of the molecular determinants and mechanistic details of the DDR owes greatly to the continuous development of ground-breaking experimental tools that couple the controlled induction of DSBs at distinct genomic positions with assays and reporters to investigate DNA repair pathways, their impact on other DNA-templated processes and the specific contribution of the chromatin environment. In this review, we present these tools, discuss their pros and cons and illustrate their contribution to our current understanding of the DDR.

Single-molecule imaging of transcription at damaged chromatin.

How DNA double-strand breaks (DSBs) affect ongoing transcription remains elusive due to the lack of single-molecule resolution tools directly measuring transcription dynamics upon DNA damage. Here, we established new reporter systems that allow the visualization of individual nascent RNAs with high temporal and spatial resolution upon the controlled induction of a single DSB at two distinct chromatin locations: a promoter-proximal (PROP) region downstream the transcription start site and a region within an internal exon (EX2). Induction of a DSB resulted in a rapid suppression of preexisting transcription initiation regardless of the genomic location. However, while transcription was irreversibly suppressed upon a PROP DSB, damage at the EX2 region drove the formation of promoter-like nucleosome-depleted regions and transcription recovery. Two-color labeling of transcripts at sequences flanking the EX2 lesion revealed bidirectional break-induced transcription initiation. Transcriptome analysis further showed pervasive bidirectional transcription at endogenous intragenic DSBs. Our data provide a novel framework for interpreting the reciprocal interactions between transcription and DNA damage at distinct chromatin regions.

Pervasive transcription read-through promotes aberrant expression of oncogenes and RNA chimeras in renal carcinoma.

Aberrant expression of cancer genes and non-canonical RNA species is a hallmark of cancer. However, the mechanisms driving such atypical gene expression programs are incompletely understood. Here, our transcriptional profiling of a cohort of 50 primary clear cell renal cell carcinoma (ccRCC) samples from The Cancer Genome Atlas (TCGA) reveals that transcription read-through beyond the termination site is a source of transcriptome diversity in cancer cells. Amongst the genes most frequently mutated in ccRCC, we identified SETD2 inactivation as a potent enhancer of transcription read-through. We further show that invasion of neighbouring genes and generation of RNA chimeras are functional outcomes of transcription read-through. We identified the BCL2 oncogene as one of such invaded genes and detected a novel chimera, the CTSC-RAB38, in 20% of ccRCC samples. Collectively, our data highlight a novel link between transcription read-through and aberrant expression of oncogenes and chimeric transcripts that is prevalent in cancer.

SETD2 is required for DNA double-strand break repair and activation of the p53-mediated checkpoint.

Histone modifications establish the chromatin states that coordinate the DNA damage response. In this study, we show that SETD2, the enzyme that trimethylates histone H3 lysine 36 (H3K36me3), is required for ATM activation upon DNA double-strand breaks (DSBs). Moreover, we find that SETD2 is necessary for homologous recombination repair of DSBs by promoting the formation of RAD51 presynaptic filaments. In agreement, SETD2-mutant clear cell renal cell carcinoma (ccRCC) cells displayed impaired DNA damage signaling. However, despite the persistence of DNA lesions, SETD2-deficient cells failed to activate p53, a master guardian of the genome rarely mutated in ccRCC and showed decreased cell survival after DNA damage. We propose that this novel SETD2-dependent role provides a chromatin bookmarking instrument that facilitates signaling and repair of DSBs. In ccRCC, loss of SETD2 may afford an alternative mechanism for the inactivation of the p53-mediated checkpoint without the need for additional genetic mutations in TP53.DOI: http://dx.doi.org/10.7554/eLife.02482.001.