Expression profiling by high throughput sequencing Genome binding/occupancy profiling by high throughput sequencing
Summary
During the S-phase, conflicts of replication forks with RNA Polymerase II (RNAPII) threaten genomic stability. While the PAF complex can resolve such conflicts during elongation, the particularly deleterious conflicts with stalling RNAPII are resolved by an as of yet unknown mechanism. Here we show that the MYCN oncoprotein forms a ternary complex with RNAPII and the nuclear RNA exosome, a 3’‑5’ exoribonuclease complex. Together with TFIIS, this complex restarts promoter‑proximal RNAPII, allows escape from co-directional transcription-replication conflicts and prevents double‑strand break accumulation. In cells lacking RNA exosome function, MYCN globally terminates transcription. MYCN-mediated termination is triggered by ATM‑dependent recruitment of BRCA1, which then stabilizes nuclear mRNA decapping complexes. Disruption of mRNA decapping activates ATR, indicative of head-on transcription-replication conflicts, and inhibits DNA replication. We propose that MYCN resolves transcription-replication conflicts via this two-step mechanism to sustain the rapid proliferation of neuroendocrine tumor cells.
Overall design
ChIPRX and 4sUseq experiments after ATM inhibition or after knock-down of EXOSC10, TCEA1, or DCP1A via Dox-inducible shRNAs in SH-EP-MYCNER cells, which allow MYCN activation via 4-OHT addition . Replicates indicated by R, all ChIPRX experiments normalized to mouse NIH3T3 spike-in, except for MYCN ChIPseq. No chase 4sU experiment normalized to 4sU-labelled mouse T-cell spike-in, all other 4sUseq experiments read normalized.
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