The chromatin reader PHF6 at the crossroad of the replication stress and DNA damage responses in neuroblastoma through interaction with RRM2

Abstract

AbstractThe ‘plant homeodomain zinc finger protein 6’ (PHF6) is affected by germline mutations in patients with cognitive disabilities and somatic mutations in acute T-cell leukemia (T-ALL). We and others previously obtained evidence for a key role of PHF6 in hematopoietic precursor cell self-renewal capacity and lineage commitment during differentiation, while recent work revealed a role in non-homologous end joining and G2 checkpoint recovery. In this study, we identified the ribonucleotide reductase subunit M2 (RRM2), as a novel PHF6 interacting protein by immunoprecipitation coupled mass spectrometry. We confirmed the PHF6-RRM2 interaction in different normal and malignant cellular background and mapped the interaction interface towards the first PHD domain of PHF6 through NanoBRET™based deletion mapping. We also demonstrated that PHF6 knockdown in neuroblastoma cells causes increased replicative stress and DNA damage. Furthermore, we show binding of PHF6 to the DNA-damage associated H3K56ac histone mark. In view of our recent finding of RRM2 as a MYCN co-dependency and target for synergistic CHK1 inhibition in neuroblastoma, we performed CUT&RUN mapping of H3K56ac and PHF6 genome-wide binding sites in neuroblastoma cells and next to a significant overlap of the two, we also found evidence for a functional crosstalk with established core regulatory circuitry transcription factors. Our data suggest that PHF6 is a component of the earlier proposed ‘replitase’, implicated in active regulation of RRM2 function during replication and DNA repair.