The mechanism of enhancer RNA-induced RNA polymerase II pause release

• Applicant: Professor Claus-Dieter Kuhn, Ph.D.
• Subject Area: Biochemistry ; General Genetics and Functional Genome Biology
• Term: since 2016
• Project identifier: Deutsche Forschungsgemeinschaft (DFG) - Project number 314375425

Enhancers are regulatory DNA sequences that bind transcription factors and that are present all across metazoan genomes. The ability of enhancers to activate target gene transcription is primarily dependent on their spatial proximity. Thus, the 3D-genome arrangement is a measure of transcriptional activity. Beyond this chromatin-based model of enhancer activity, the last decade has seen the emergence of enhancer-derived long non-coding RNAs, so-called enhancer RNAs (eRNAs) as crucial for the induction of genes in stimulus-induced systems, such as neurons and macrophages. However, despite their importance, the mechanism of how eRNAs stimulate transcription remained unanswered. In neurons, for instance, eRNAs were hypothesized to activate transcription by abrogating Pol II (RNA polymerase II) pausing through dissociation of NELF (negative elongation factor). Thus, during the previous funding period we aimed to decipher whether eRNAs displace NELF from paused Pol II and whether this leads to transcription activation. Both questions could be answered in the affirmative - with the notable exception that eRNAs trigger NELF release from Pol II without actively displacing it. The success of the previous proposal was enabled by a wide range of biochemical techniques that ranged from RNA structure mapping to sophisticated in vitro assays combined with protein-RNA crosslinking and mass spectrometry. All together, these methodological advances allowed us to begin to decipher the mechanism of how eRNAs impinge on mammalian transcription for the first time. In building on this established framework this renewal is designed to answer three follow up questions. First, we aim to unravel the detailed mechanism of how multivalent interactions between eRNAs and NELF induce its dissociation and whether G quadruplex structures play an important role in this process. Second, gained knowledge on eRNA-induced Pol II pause release now allows us to study how the kinase P-TEFb controls the canonical Pol II pause release pathway by phosphorylating the pause-inducing protein complexes DSIF and NELF. We plan to decipher not only the role of DSIF and NELF phosphorylation during Pol II pause release, but to also determine any crosstalk between the eRNA-driven and P-TEFb-controlled pathways. Finally, we seek to confirm and expand our mechanistic insights into Pol II pause release gained in vitro in an estradiol-stimulated cell culture model. In summary, this proposal thrives on the successful establishment of a biochemical framework to study eRNA-driven Pol II pause release in the previous funding period. It utilizes this lead to unravel the precise molecular mechanism of eRNA-induced pause release, and it will reveal any crosstalk with the canonical pause release pathway controlled by P-TEFb.

• DFG Programme: Research Grants