Summary DNA double-strand break (DSB) repair is mediated by multiple pathways. It is thought that the local chromatin context affects the pathway choice, but the underlying principles are poorly understood. Using a multiplexed reporter assay in combination with Cas9 cutting, we systematically measure the relative activities of three DSB repair pathways as a function of chromatin context in >1,000 genomic locations. This reveals that non-homologous end-joining (NHEJ) is broadly biased toward euchromatin, while the contribution of microhomology-mediated end-joining (MMEJ) is higher in specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 methyltransferase EZH2 reverts the balance toward NHEJ. Single-stranded template repair (SSTR), often used for precise CRISPR editing, competes with MMEJ and is moderately linked to chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway balance and guidance for the design of Cas9-mediated genome editing experiments.

Vertebrate Hox genes encode transcription factors operating during the development of multiple organs and structures. However, the evolutionary mechanism underlying this remarkable pleiotropy remains to be fully understood. Here, we show that Hoxd8 and Hoxd9, two genes of the HoxD complex, are transcribed during mammary bud (MB) development. However, unlike in other developmental contexts, their coexpression does not rely on the same regulatory mechanism. Hoxd8 is regulated by the combined activity of closely located sequences and the most distant telomeric gene desert. On the other hand, Hoxd9 is controlled by an enhancer-rich region that is also located within the telomeric gene desert but has no impact on Hoxd8 transcription, thus constituting an exception to the global regulatory logic systematically observed at this locus. The latter DNA region is also involved in Hoxd gene regulation in other contexts and strongly interacts with Hoxd9 in all tissues analyzed thus far, indicating that its regulatory activity was already operational before the appearance of mammary glands. Within this DNA region and neighboring a strong limb enhancer, we identified a short sequence conserved in therian mammals and capable of enhancer activity in the MBs. We propose that Hoxd gene regulation in embryonic MBs evolved by hijacking a preexisting regulatory landscape that was already at work before the emergence of mammals in structures such as the limbs or the intestinal tract.enhancers | TAD | mammalian development | mammary gland

DNA double-strand breaks are repaired by multiple pathways, including non-homologous end-joining (NHEJ) and microhomology-mediated end-joining (MMEJ). The balance of these pathways is dependent on the local chromatin context, but the underlying mechanisms are poorly understood. By combining knockout screening with a dual MMEJ:NHEJ reporter inserted in 19 different chromatin environments, we identified dozens of DNA repair proteins that modulate pathway balance dependent on the local chromatin state. Proteins that favor NHEJ mostly synergize with euchromatin, while proteins that favor MMEJ generally synergize with distinct types of heterochromatin. BRCA2 is an example of the former, which is corroborated by chromatin-dependent shifts in mutation patterns of BRCA2-/- cancer genomes. These results uncover a complex network of proteins that regulate MMEJ:NHEJ balance in a chromatin context-dependent manner.

17DNA double-strand break (DSB) repair is mediated by multiple pathways, including classical non-18 homologous end-joining pathway (NHEJ) and several homology-driven repair pathways. This is 19 particularly important for Cas9-mediated genome editing, where the outcome critically depends on the 20 pathway that repairs the break. It is thought that the local chromatin context affects the pathway choice, but 21 the underlying principles are poorly understood. Using a newly developed multiplexed reporter assay in 22 combination with Cas9 cutting, we systematically measured the relative activities of three DSB repair 23 pathways as function of chromatin context in >1,000 genomic locations. This revealed that NHEJ is broadly 24 biased towards euchromatin, while microhomology-mediated end-joining (MMEJ) is more efficient in 25 specific heterochromatin contexts. In H3K27me3-marked heterochromatin, inhibition of the H3K27 26 methyltransferase EZH2 shifts the balance towards NHEJ. Single-strand templated repair (SSTR), often 27 used for precise CRISPR editing, competes with MMEJ, and this competition is weakly associated with 28 chromatin context. These results provide insight into the impact of chromatin on DSB repair pathway 29 balance, and guidance for the design of Cas9-mediated genome editing experiments. 31 61Much less is known about the impact of chromatin on MMEJ and SSTR. Like HR, these pathways 62 require resection of the DNA ends to produce single-stranded DNA overhangs, but downstream of this step 63 the mechanisms and responsible proteins diverge (Chang et al., 2017; Scully et al., 2019; Yeh et al., 2019). 3It is thus possible that the local chromatin environment also modulates MMEJ and SSTR in unique ways, 65 but this has remained largely unexplored (Clouaire and Legube, 2019; Mitrentsi et al., 2020). 66One strategy to investigate the impact of local chromatin context on repair pathway balance is to 67 generate DSBs at various genomic locations with known chromatin states, and compare pathway utilization 68 across these locations (van Overbeek et al., 2016; Clouaire et al., 2018; Chakrabarti et al., 2019). However, 69 with such an approach it is difficult to separate the effects of chromatin context from the effects of sequence 70 context, because both vary simultaneously along the genome. Ideally, different chromatin contexts are 71 compared while the sequence context is kept fixed. 72Here, we report a strategy that effectively tackles these challenges in human cells. The strategy 73 consists of two parts. First, we developed a reporter that, when cut with Cas9, produces distinct "scars" 74 when repaired by either NHEJ, MMEJ or SSTR; high-throughput sequencing of these scars provides highly 75 accurate measurements of the relative activities of the three pathways. Second, we used a modification of 76 our TRIP method (Akhtar et al., 2013) to insert this reporter into >1,000 random genomic locations, tracking 77 each individual reporter in parallel by molecular barcoding. We thus systematically measured the r...

DNA double-strand breaks (DSBs) can be repaired through various pathways. Understanding how these pathways are regulated is of great interest for cancer research and optimization of gene editing. The local chromatin environment can affect the balance between repair pathways, but this is still poorly understood. Here we provide a detailed protocol for DSB-TRIP, a technique that utilizes the specific DNA scars left by DSB repair pathways to study pathway usage throughout the genome. DSB-TRIP randomly integrates a repair reporter into many genomic locations, followed by the induction of DSBs in the reporter. Multiplexed sequencing of the resulting scars at all integration sites then reveals the balance between several repair pathways, which can be linked to the local chromatin state of the integration sites. Here we present a step-by-step protocol to perform DSB-TRIP in K562 cells and to analyse the data by a dedicated computational pipeline. We discuss strengths and limitations of the technique, as well as potential additional applications to study DNA repair.

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The efficiency and outcome of CRISPR/Cas9 editing depends on the chromatin state at the cut site. It has been shown that changing the chromatin state can influence both the efficiency and repair outcome, and epigenetic drugs have been used to improve Cas9 editing. However, because the target proteins of these drugs are not homogeneously distributed across the genome, the efficacy of these drugs may be expected to vary from locus to locus. Here, we systematically analyzed this chromatin context-dependency for 160 epigenetic drugs. We used a human cell line with 19 stably integrated reporters to induce a double-stranded break (DSB) in different chromatin environments. We then measure Cas9 editing efficiency and repair pathway usage by sequencing the mutational signatures. We identified 67 drugs that modulate Cas9 editing efficiency and/or repair outcome dependent on the local chromatin environment. For example, we find a subset of histone deacetylase inhibitors that improve Cas9 editing efficiency throughout all types of heterochromatin (e.g., PCI-24781), while others were only effective in H3K27me3-marked regions (e.g., Vorinostat). In summary, this study reveals that most epigenetic drugs alter CRISPR editing in a chromatin-dependent manner, and provides a detailed guide to improve Cas9 editing more selectively at the desired location.