Regulation of rDNA silencing via PAPAS

Project leader: Holger Bierhoff
Doctoral candidate: Hugo Costa

The nucleolus is the most prominent body in the cell nucleus. It contains ribosomal RNA genes (rDNA) and is responsible for ribosome production. Ribosome biogenesis starts with the transcription of rDNA into a long precursor rRNA (pre-rRNA) by RNA polymerase I (Pol I). This is a highly energy demanding process and therefore tightly regulated in response to cell growth and stress conditions.

In proliferating cells, pre-rRNA is heavily transcribed by Pol I. Under adverse conditions like growth factor deprivation, Pol I transcription is shut down and additional epigenetic mechanisms consolidate rDNA repression. Previous work showed that an essential component of this epigenetic regulation is the long non-coding RNA PAPAS (Promoter And Pre-rRNA AntiSense). Upon growth-factor deprivation, PAPAS is upregulated and recruits the histone methyltransferase Suv4-20h2 to the rDNA promoter to mediate histone H4 trimethylation at lysine 20 (H4K20me3). This histone mark leads to a condensed, transcription-refractory chromatin state. PAPAS-mediated epigenetic silencing is also induced upon different stress conditions and terminal cell differentiation.

Surprisingly, PAPAS is an rDNA antisense transcript synthesized by RNA polymerase II (Pol II). Given that it is still unknown how Pol II is recruited to rDNA, the main goal of our project is to unravel the underlying targeting mechanism. To this end, we will use the CRISPR technology to purify the PAPAS promoter at the 3´end of the pre-rRNA coding region. Co-purified Pol II transcription factors and coactivators will be identified by mass spectrometry. As a complimentary approach, we will tag and isolate rDNA-bound Pol II coactivators by proximity labeling. To gain further insights into the growth- and stress-dependent regulation of rDNA antisense transcription, we will monitor epigenetic changes at PAPAS promoter. Together, we will provide a detailed understanding of how Pol II can usurp rDNA to synthesize PAPAS and shut down Pol I transcription.