A novel role of small RNA pathways in regulating DNA replication in polyploid cells.

Small RNA-mediated gene regulation and DNA replication are fundamental genome-regulatory pathways essential for cellular life, organismal development, and disease prevention.

While small RNA pathways are known to sequence-specifically regulate DNA copy number in some unicellular animals, such connections have yet to be found in multicellular animals.

With this project, I will investigate and mechanistically characterize such a potential connection in the Drosophila animal model system.

Piwi-interacting RNAs (piRNAs), a class of animal small RNAs, safeguard the germline genome against repetitive DNA proliferation by guiding repressive heterochromatin formation at target loci.

In Drosophila melanogaster ovarian nurse cells, heterochromatic (peri)centromeric DNA repeats undergo selective local suppression of DNA replication thus lowering their relative copy number.

Interestingly, abundant piRNAs able to target to these 'underreplicated' regions have been found, yet the potential functional interactions between small RNA genome defense and DNA replication remain unexplored.

With this project, I will bridge this gap by investigating the role of satellite piRNAs in Drosophila melanogaster, focusing on the interplay between regulated DNA replication and small RNA genome defense.

Two interconnected hypotheses will be examined: (1) piRNA-directed heterochromatin formation regulates centromeric DNA copy number, and (2) piRNA-regulated heterochromatin sinks alter epigenetic inheritance.

To do so, I will apply advanced sequencing- and imaging-based methods for DNA copy number quantification and leverage the powerful Drosophila genetics to uncover underlying molecular mechanisms.In sum, my investigation of small RNA-based regulation of DNA replication holds the promise to reveal a new paradigm in animal genome regulation and to provide deeper insights into the mechanisms regulating genome stability and the role of piRNAs in maintaining it.