Eukaryotic genomes are populated by repetitive sequences and transposable elements (TEs), mobile DNA sequences that are able to change their position and replicate within a host genome. Heterochromatin establishment at these sequences prevents ectopic recombination and TE mobilisation. In Drosophila ovaries, this depends on Piwi, which is guided by a bound small RNA to nascent TE transcripts where it regulates the formation of transcriptionally repressive chromatin states. As well as numerous chromatin modifying proteins, this mechanism depends on the Panx-induced co-transcriptional silencing (PICTS) complex, which contains Panx, Nxf2 and Nxt1. PICTS operates at the interface between piRNA-guided Piwi and heterochromatin effectors. While the N-terminal region of Panx is known to deliver the transcriptional silencing signal, the molecular mechanism by which PICTS induces heterochromatin formation at TE insertions is not fully resolved.

In the first part of this thesis, I identified a fourth component of PICTS, Cut-up (Ctp), which I showed is essential for co-transcriptional transposon repression. While initially identified as a component of cytoplasmic dynein, Ctp has been shown to function as a dimerisation hub protein, mediating the assembly and stabilisation of many dynein-independent protein complexes. I demonstrated that the primary function of Ctp within PICTS is to support the assembly of a higher-order complex through dimerisation of the Panx C-terminus. Next, I sought to further explore the molecular function of PICTS using biochemical and structural approaches. I established the expression and purification of full-length and mutant PICTS complexes and showed that PICTS exists as a constitutive homodimer that is able to associate with single-stranded RNA. While structural studies are still ongoing, together this work has contributed to a detailed understanding of the molecular assembly of PICTS.

Understanding transcriptional silencing and chromatin regulation more broadly relies on sophisticated tools to profile the genome-wide distribution of histone modifications. In the final part of this work, I established a modified CUT&RUN (Cleavage Under Targets & Release Using Nuclease) protocol that relies on chromatin reader domains fused directly to Micrococcal Nuclease (MNase) and allows rapid and specific antibody-free profiling of histone marks. Together, this work has contributed to a broader understanding of how small RNAs regulate chromatin structure.