Long range chromatin interaction in gene silencing

Heterochromatin is a highly compacted form of chromatin that silences transcription sequence independently. Importantly, heterochromatin structure is epigenetically inherited and crucial for proper development and genome integrity. This means that changes in heterochromatin can influence cell fate across generations, altering pathways of the integrity of cell differentiation. This has obvious biomedical impact: for instance, the mis-regulation of telomeric silencing in humans has been implicated in aberrant recombination events that are associated with 3-5% of otherwise unexplained mental retardations. The largest heterochromatic domains are found at telomeres and centromeres, and in many eukaryotes these regions form dark-staining, condensed foci within the interphase nucleus. As the dark-staining foci of heterochromatin imply, chromatin within silent domains is highly compact and is thought to assume higher-order folded structures. In addition to the compaction, heterochromatic domains seem to interact, as interactions in trans between silent domains, as well as intra-chromosome looping, have been reported for yeast, flies and mammalian cells. Although heterochromatin is not cytologically visible in Saccharomyces cerevisiae, this organism is a powerful model for studying the basic features of chromatin mediated repression. Sites of chromatin-mediated repression are nucleated at the mating-type loci HMRa and HMLa and at telomeres. The Silent Information Regulatory proteins Sir2, Sir3 and Sir4 form a heterotrimeric complex which spreads up to 4kb along the chromatin fiber, repressing nearby promoters. Intriguingly, the association of this Sir complex with chromatin is not sufficient to repress transcription. This argues against a simple model in which Sir complex binding alone sterically blocks the access of the transcription machinery. Trans interactions between silent chromatin domains have been detected in budding yeast, the most striking example being the clustering of the 32 telomeres in 5-8 foci at the nuclear periphery. These regions are highly competent for silencing, whereas silencing is prevented outside these foci. Looping of silent chromatin was also observed in vivo in yeast. This suggests that higher-order structural changes in chromatin are likely to be needed to establish Sir-mediated repression although the nature of this higher-order structure remains unknown. In the proposed project, we aim to explore the importance of such trans interactions on gene silencing in S. cerevisiae. Using a recently developed in vitro system for the reconstitution of Sir proteins on nucleosomes, we will first define the molecular players required for interactions in trans between silent chromatin domains in vitro. We will then address their importance for the formation of higher order chromatin structure and heterochromatic silencing in vivo.