ELOF1 is a transcription-coupled DNA repair factor that directs RNA polymerase II ubiquitylation Yana van der Weegen1, Klaas de Lint2, Rob M.F. Wolthuis2 , and Martijn S. Luijsterburg1 1 Department of Human Genetics, Leiden University Medical Center, Leiden, The Netherlands 2 Cancer Center Amsterdam, Department of Clinical Genetics, Section Oncogenetics, Amsterdam University Medical Center, Amsterdam, the Netherlands Transcription-coupled repair (TCR) eliminates transcription-blocking DNA lesions. These lesions are recognised by the stalling of RNA Polymerase II (RNAPII) followed by the recruitment of CSB, the CSA-DDB1-CUL4-based E3 ubiquitin ligase complex (CRL4CSA), and UVSSA to target the TFIIH complex to lesion-stalled RNAPIIo to initiate repair. The full repertoire of factors required for TCR is currently unknown. We employed a genome-wide CRISPR screen to identify potential new genes involved in the cellular response to transcription-blocking DNA lesions. Among the top hits, we identified the previously uncharacterized RNAPII-associated factor ELOF1. With a strand-specific ChIP-seq (TCR-seq) approach we demonstrate that ELOF1 is required for the removal of UV induced DNA lesions from transcribed strands. In addition, we show that ELOF1-KO cells are just as defective in the recovery of RNA synthesis after UV irradiation as TCR-deficient CSB-KO cells, confirming that ELOF1 is a new core TCR gene. To study the role of ELOF1 in TCR complex assembly, we immunoprecipitated endogenous RNAPII in WT and ELOF1-KO cells. Our findings show that while ELOF1 is not required for recruitment of CSB, the CRL4CSA ligase, or UVSSA, ELOF1 is essential for the efficient association of the TFIIH complex with lesion-stalled RNAPIIo. The transfer of TFIIH onto RNAPII requires ubiquitylation of the largest subunit of RNAPII at a single lysine (K1268) by the CRL4CSA ubiquitin ligase complex. However, how the CRL4CSA ligase is specifically directed toward the K1268 site is unknown. We demonstrate that ELOF1 is the missing link that facilitates RNAPII ubiquitylation, thereby promoting the assembly of downstream repair factors. Importantly, RNAPII ubiquitylation requires the constitutive interaction of ELOF1 with RNAPII close to the K1268 ubiquitylation site. Our study provides a genetic framework of the transcription stress response and identifies ELOF1 as a new core TCR factor that facilitates RNAPII ubiquitylation, which gives us new insights into the molecular mechanism of TCR.