All eukaryotes organize their DNA together with histones and non-histone proteins into a highly complex nucleoprotein structure called chromatin, with the nucleosome as its monomeric subunit. Several interconnected mechanisms have evolved to regulate DNA accessibility, including nucleosome replacement of canonical histones with specialized histone variants. Deposition of histone variants can lead to profound chromatin structure alterations thereby influencing a multitude of biological processes ranging from transcriptional regulation to genome stability. At the focus of our research is the evolutionary highly conserved histone variant H2A.Z, which has been extensively studied and shown to play a role in gene expression, DNA repair, heterochromatin formation, chromosome segregation and mitosis. But the mechanism(s) of how H2A.Z controls these diverse biological processes is not understood. Using state-of-the-art biochemical, cell biological, genome-wide and bioinformatics approaches we are shedding light on H2A.Z biology by identifying its manifold binding proteins and their functional roles in gene regulation, cell cycle progression and organismal development.