RNA polymerase II-mediated rDNA transcription mediates rDNA copy number expansion in Drosophila.

Ribosomal DNA (rDNA), which encodes ribosomal RNA, is an essential but unstable genomic element due to its tandemly repeated nature. rDNA's repetitive nature causes spontaneous intrachromatid recombination, leading to copy number (CN) reduction, which must be counteracted by a mechanism that recovers CN to sustain cells' viability. Akin to telomere maintenance, rDNA maintenance is particularly important in cell types that proliferate for an extended time period, most notably in the germline that passes the genome through generations. In Drosophila, the process of rDNA CN recovery, known as 'rDNA magnification', has been studied extensively. rDNA magnification is mediated by unequal sister chromatid exchange (USCE), which generates a sister chromatid that gains the rDNA CN by stealing copies from its sister. However, much remains elusive regarding how germ cells sense rDNA CN to decide when to initiate magnification, and how germ cells balance between the need to generate DNA double-strand breaks (DSBs) to trigger USCE vs. avoiding harmful DSBs. Recently, we identified an rDNA-binding Zinc-finger protein Indra as a factor required for rDNA magnification, however, the underlying mechanism of action remains unknown. Here we show that Indra is a negative regulator of rDNA magnification, balancing the need of rDNA magnification and repression of dangerous DSBs. Mechanistically, we show that Indra is a repressor of RNA polymerase II (Pol II)-dependent transcription of rDNA: Under low rDNA CN conditions, Indra protein amount is downregulated, leading to Pol II-mediated transcription of rDNA. This results in the expression of rDNA-specific retrotransposon, R2, which we have shown to facilitate rDNA magnification via generation of DBSs at rDNA. We propose that differential use of Pol I and Pol II plays a critical role in regulating rDNA CN expansion only when it is necessary. Author summary: Ribosomal DNA (rDNA) exists as tandemly-repeated copies in eukaryotic genome, making it unstable due to spontaneous intrachromatid recombination that causes copy number loss. The germline, which is the sole cell type that transmits genome from generation to generation, must expand rDNA copy number to counteract spontaneous copy number loss. It has been shown that the process of rDNA copy number expansion, called rDNA magnification, involves rDNA binding protein Indra and retrotransposon R2. However, the underlying molecular mechanism of rDNA magnification and how Indra and R2 may functionally intersect remained unknown. This study shows that Indra is a transcriptional repressor of the intergenic spacer (IGS) sequence of rDNA, orchestrating R2 expression and rDNA copy number expression. Low rDNA copy number downregulates Indra, leading to expression of IGS, which in turn leads to expression of R2. It was found that IGS expression is mediated by RNA polymerase II, which is specifically recruited to nucleolus upon rDNA copy number reduction. Together, the results lead to a model how rDNA copy number reduction triggers the process of rDNA copy number expansion. [ABSTRACT FROM AUTHOR]