ranscription of protein-coding and noncoding genes requires RNA polymerase II (RNAPII), which synthesizes RNA transcripts complementary to the DNA template strand. The presence of DNA lesions in the template strand causes stalling of elongating RNAPII (RNAPIIo), which leads to genome-wide transcriptional arrest 1-3 . It is essential that cells overcome this arrest and restore transcription. The transcription-coupled repair (TCR) pathway efficiently removes transcription-blocking DNA lesions through the proteins CSB, CSA and UVSSA [4][5][6] . Inactivating mutations in CSB and CSA cause Cockayne syndrome (CS), which is characterized by severe neurological dysfunction related to persistent RNAPII arrest at DNA lesions 2,7 .The sequential and cooperative actions of CSB, CSA and UVSSA recruit TFIIH to DNA damage-stalled RNAPII to initiate DNA repair 6 . In addition to protein-protein contacts, efficient transfer of TFIIH onto RNAPII requires ubiquitylation of a single lysine on the largest subunit of RNAPII (RPB1-K1268), which is essential for efficient TCR 2 . This DNA damage-induced modification of RNAPII is dependent on cullin-ring type E3-ligases (CRLs) and is strongly decreased in CSA-deficient cells 2 , which indicates that the CRL4 CSA E3 ligase complex drives RNAPII ubiquitylation.CSB binds to DNA upstream of RNAPII 8 (Extended Data Fig. 1a) and recruits the CRL4 CSA complex through an evolutionarily conserved motif in its carboxy terminus 6 . However, how the activity of CRL4 CSA ubiquitin ligase is specifically directed towards the K1268 site remains to be elucidated. Results A CRISPR screen identifies ELOF1 as a putative TCR gene.To identify unknown TCR genes, we performed a genome-wide CRISPR screen in the presence of the compound illudin S, which induces transcription-blocking DNA lesions that are eliminated by TCR 9 . RPE1-iCas9 cells were transduced with the pLCKO-TKOv3 library, which contains 70,948 single guide RNAs (sgRNAs) targeting open reading frames 10 , and cultured for 12 population doublings, after which sgRNA contents were analysed (Extended Data Fig. 1b).Using a false-discovery rate (FDR) cut-off of 0.01, we found 104 sensitizer hits and 18 hits conferring resistance to illudin S. The strongest resistance was conferred by guide RNAs (gRNAs) targeting PTGR1, which is in line with its known role in bioactivating illudin S 11 (Fig. 1a and Extended Data Fig. 1c). Nine known core TCR genes, including CSB, CSA and UVSSA, but also genes connected to transcription recovery after UV irradiation (HIRA 12 , DOT1L 13 and STK19 (ref. 14 ) (Fig. 1a,b), were required for illudin S tolerance. Consistent with known effects of illudin S on replication 9 , we found the 9-1-1 complex, translesion synthesis and sister-chromatid cohesion components (Fig. 1b). Our screen also identified the ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation Yana van der Weegen 1,10 , Klaas de Lint 2,10 , Diana van den Heuvel

Head-and-neck squamous cell carcinomas (HNSCCs) are relatively common in patients with Fanconi anemia (FA), a hereditary chromosomal instability disorder. Standard chemo-radiation therapy is not tolerated in FA due to an overall somatic hypersensitivity to such treatment. The question is how to find a suitable alternative treatment. We used whole-exome and whole genome mRNA sequencing to identify major genomic and transcriptomic events associated with FA-HNSCC. CRISPR-engineered FA-knockout models were used to validate a number of top hits that were likely to be druggable. We identified deletion of 18q21.2 and amplification of 11q22.2 as prevailing copy-number alterations in FA HNSCCs, the latter of which was associated with strong overexpression of the cancer-related genes YAP1, BIRC2, BIRC3 (at 11q22.1-2). We then found the drug AZD5582, a known small molecule inhibitor of BIRC2-3, to selectively kill FA tumor cells that overexpressed BIRC2-3. This occurred at drug concentrations that did not affect the viability of untransformed FA cells. Our data indicate that 11q22.2 amplifications are relatively common oncogenic events in FA-HNSCCs, as holds for non FA-HNSCC. Therefore, chemotherapeutic inhibition of overexpressed BIRC2-3 may provide the basis for an approach to develop a clinically realistic treatment of FA-HNSCCs that carry 11q22.2 amplifications.

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The cohesin complex regulates higher order chromosome architecture through maintaining sister chromatid cohesion and folding chromatin by active DNA loop extrusion. Impaired cohesin function underlies a heterogeneous group of genetic syndromes and is associated with cancer. Here, by using synthetic lethality CRISPR screens in isogenic human cell lines defective of specific cohesion regulators, we mapped the genetic dependencies induced by absence of DDX11 or ESCO2. The obtained high confidence synthetic lethality networks are strongly enriched for genes involved in DNA replication and mitosis and support the existence of parallel sister chromatid cohesion establishment pathways. Among the hits, we identified the chromatin binding, BRCT-domain containing protein PAXIP1 as a novel cohesin regulator. Depletion of PAXIP1 severely aggravated cohesion defects in ESCO2 defective cells, leading to mitotic cell death. PAXIP1 promoted the global chromatin association of cohesin, independent of DNA replication, a function that could not be explained by indirect effects of PAXIP1 on transcription or the DNA damage response. Cohesin regulation by PAXIP1 required its binding partner PAGR1 and a conserved FDF motif in PAGR1. Similar motifs were previously found in multiple cohesin regulators, including CTCF, to mediate physical interactions with cohesin. PAXIP1 co-localizes with cohesin on multiple genomic loci, including at active gene promoters and enhancers. Together, this study identifies the PAXIP1-PAGR1 complex as a novel regulator of cohesin occupancy on chromatin. Possibly, this role in cohesin regulation is also relevant for previously described functions of PAXIP1 in transcription, immune cell maturation and DNA repair.