HUWE1 coordinates RNAPII-dependent ATM signaling at collisions with replication forks

Abstract

Oncogenic signaling deregulates RNA polymerase II (RNAPII) dependent transcription programs, which can lead to collisions of the transcription machinery with DNA replication complexes (transcription replication conflicts, TRCs). Although encounters of both machineries are thought to be frequent events at very long genes and may be readily resolved in healthy cells, perturbation of transcription or TRC-coordinating mechanisms can result in deleterious DNA damage. TRC-induced DNA damage is thought to underlie genomic instability in cancer cells. Both, transcription and replication, involve ubiquitin-dependent regulation of effector proteins. Previous studies highlighted the importance of the ubiquitin ligase HUWE1 in controlling RNAPII-dependent transcription through regulation of transcription factors, including MYC, TP53 and β-catenin. Furthermore, HUWE1 is involved in the control of DNA replication through interaction with the DNA sliding clamp PCNA, the replication factor CDC6 and it facilitates recovery from replication stress. In this study, I characterize the effect of the HUWE1 ubiquitin ligase function on conflicts between the transcription and DNA replication machineries and the influence of HUWE1 on DNA damage response (DDR) signaling and maintenance of genome integrity. Genetic inhibition of the ubiquitin ligase by mutation of the catalytic cysteine in the HUWE1-HECT domain shows that HUWE1 is required for the resolution of TRCs and suppression of DDR signaling via the ATM kinase. I identify the ATPase WRNIP1 is a novel interaction partner of HUWE1 and as critical effector protein to protect stalled replication forks at TRCs from collapse into severe double strand breaks. Catalytic activity of HUWE1 promotes the association of WRNIP1 with elongating RNAPII. Inactivation of HUWE1 or depletion of WRNIP1 results in elevated levels of TRCs without physical DNA breaks, but induces activation of ATM. Interestingly, the MRN complex, that is required for ATM recruitment to DNA double strand breaks, associates with elongating RNAPII in unperturbed cells. Consequently, elongating RNAPII is poised for ATM recruitment and activation at encounters with stalled replication forks or at TRCs. Upon induction of replication stress or catalytic mutation of HUWE1, WRNIP1 transfers from RNAPII to bind to the DNA replication machinery. This transfer likely enables ATM recruitment to RNAPII and protection of replication forks. Inhibition of RNAPII prior to induction of replication stress diminishes TRCs, impairs ATM signaling, abolishes DNA repair and results in severe growth defects. Taken together, the HUWE1 ubiquitin ligase and its effector WRNIP1 coordinate transcription and DNA replication to jointly resolve TRCs and redundantly stabilize replication forks. This coordination maintains genome integrity and facilitates cell proliferation. Further, this work suggests that contrary to the common perception of TRCs as a cause of genome instability, a conflict may provide a rescue mechanism in response to replication stress, since it enables RNAPII-dependent, preemptive ATM signaling to facilitate DNA repair.