Stress-Induced Transcriptional Memory Accelerates Promoter-Proximal Pause-Release and Decelerates Termination over Mitotic Divisions

Anniina Vihervaara; Dig Bijay Mahat; Samu V. Himanen; Malin A.H. Blom; John T. Lis; Lea Sistonen

Hallmarks of cancer cells include high stress-tolerance and active proliferation even in the 393! presence of sustained stress (Hanahan and Weinberg, 2011) . In accordance, human K562 394! erythroleukemia cells proliferated through multiple heat shocks that challenge the proteome 395! integrity ( Figure S3 ). The persistence of protein-damaging stress in the daughters of repeatedly 396! stressed K562 cells was reflected in their transcriptional profile; two mitotic divisions after nine 397! heat exposures, transcription of certain pro-survival genes was elevated, whereas expression of 398! genes that are involved in maintaining protein production was decreased (Figure 3 ). The declined 399! synthesis of protein production machinery was accompanied with reduced processing of 400! transcripts at the 3'-ends of active genes ( Figure 6 ). In cells with decreased protein synthesis, the 401! decelerated transcription termination likely serves to reduce the mRNA load, as less pre-mRNA 402! is released from the chromatin, and fewer Pol II molecules become available for new rounds of 403! heat-induced transcription (Figure 7) . Moreover, increased association of uncleaved transcripts at 404! the 3'-ends of genes could provide a reservoir of pre-mRNAs that are rapidly processed to mature 405! mRNAs once proteotoxicity is relieved and the cell restores its protein synthesis. This regulatory 406! step at the 3'-end of the gene would be particularly important to instantly restore production of 407! ! ! 16! mRNAs from long genes, where transcription, proceeding on average 2.5 kb/minute (Jonkers et 408! al., 2014) , can take hours to complete after the Pol II pause-release. Intriguingly, the mechanisms 409! that primed gene activation and refined transcription over mitotic divisions were mediated via 410! regulation of Pol II, but they were not detected to involve altered activity of HSF1 (Figure 4 ; 411! Figure 7 ). Taken together, in cells exposed to either single or multiple heat shocks, the re-wiring 412! EtOH-precipitation, the RNA was air-dried, base hydrolyzed with 0.1 N NaOH for 5 minutes on 476! ice, and the NaOH was neutralized with Tris-HCl (pH 6.8). Unincorporated nucleotides were 477! removed using P-30 columns (Bio-Rad), and the biotinylated nascent transcripts were isolated in 478! a total of three rounds of streptavidin-coated magnetic bead (M-280, Invitrogen) purifications.