The formation and stable maintenance of heterochromatin is essential for the functionality of the genome. Heterochromatin restrains the activity of parasitic mobile elements, keeps repetitive sequences recombinationally inert, and contributes to the functionality of euchromatin. A range of widely conserved proteins and pathways have been implicated in different aspects of heterochromatin regulation in eukaryotes. Hallmarks of constitutive heterochromatin are the presence of HP1 proteins and di- or tri-methylation of H3K9. Recent work carried out in the host laboratory uncovered the existence of a functional network of heterochromatin protein factors and small RNA pathways collaborating in ensuring gene and transposable element repression and proper development1. However, how they contribute to heterochromatin formation and organisation remains to be understood. I am addressing these questions by employing super-resolution nanoscopy and high-throughput genome-wide analyses. Towards this goal, I have adapted one- and two-colour direct-Stochastic Optical Reconstruction Microscopy (dSTORM) to C. elegansto analyse heterochromatin formation during early embryogenesis. In preliminary experiments, I show that H3K9me2 and H3K9me3 heterochromatin puncta appear early during embryo development and form larger nanoclusters by the 32-cell stage. The heterochromatin proteins LIN-13 and LET-418, which extensively co-localize with H3K9me2 and HPL-2 by ChIP-seq in adult mixed tissues1, are also organised into similar puncta. By combining in situ analyses by dSTORM with genome-wide chromatin immunoprecipitation experiments, my specific aims are: 1 - investigate the interdependencies and role of heterochromatin proteins in regulating heterochromatin establishment and structure during embryogenesis. 2 - identify sites of de novo heterochromatin formation by genome-wide chromatin profiling and determine whether and how heterochromatin is altered in mutant backgrounds. 1. McMurchy, A. N. et al.A team of heterochromatin factors collaborates with small RNA pathways to combat repetitive elements and germline stress. eLife6,
e21666 (2017).