Role of CLRC complex in telomeric assembly of shelterin proteins

Heterochromatin represents a specific structural organization of chromatin that is essential for cellular development and genome stability. In S. pombe, all members of the Cul4-Rik1Raf1/2 E3 ubiquitin ligase complex are critical for heterochromatin integrity. This key complex assembles with Clr4 (the sole H3K9 methyltransferase) to form the CLRC, which is required for silencing of the three main heterochromatic regions (centromere, telomeres and mating locus). Although much effort has been invested in understanding the function of this key complex, the ubiquitylation substrate(s) of CLRC remain unknown. To overcome the challenge of transient interactions between substrate and ubiquitin ligase in vivo, we have established the Ubiquitin Ligase Trapping method in S. pombe (Mark KG et al., Mol. Cell 2014). This approach is based on fusing a ubiquitin associated domain (UBA) to the E3 ligase by a flexible linker in order to trap the ubiquitylated substrate allowing the analysis of E3-associated proteins by mass spectrometry. We have been able to reproducibly detect chromatin related proteins associated with CRLC and to validate their interactions. Interestingly, among these candidates we identified the telomeric shelterin complex as a new interactor of CLRC.