Heterochromatin is a conserved organizational structure of the genetic material that is indispensable for cell viability. Heterochromatin is associated with the formation of functionally conserved centromeric and telomeric structures. These structures are both essential for the integrity and proper segregation of the genetic material during mitosis and meiosis. Heterochromatin structure is also implicated in programming patterns of genes expression, in particular during cellular differentiation. Several evolutionary conserved molecular pathways have been implicated in the initiation, propagation and maintenance of heterochromatic domains during cell divisions. However, the formation of heterochromatin is an intricate process and despite much research many facets remain to be understood. The overall goal of this proposed research is to gain insight into the formation and maintenance of heterochromatin. The first aim of this study will propose experiments designed to analyze the contribution of the centromere CENP-B proteins to heterochromatin organization and gene expression in the genetically tractable model organism S. pombe. CENP-B are evolutionarily conserved sequence-specific DNA-binding proteins that associate not only with centromeric heterochromatin but also with the heterochromatin of silenced genes and the mating-type locus in yeast. Different studies point to an important role of the CENP-B proteins in heterochromatin formation at several locations in the genome, but their exact molecular role(s) is still debated. The second part of this study will aim to identify additional proteins involved in heterochromatin formation by means of a two-step genome-wide screen in the fission yeast. This screen will be based on properties of centromeric heterochromatin and is anticipated to increase our knowledge of the different pathways controlling heterochromatin formation and maintenance.