Harvey, Z. H.; Stevens, K. M.; Warnecke, T.; Berger, F. H.

Since eukaryogenesis, chromatin has evolved to regulate gene expression. The histone variant H2A.Z is an ancestral, essential feature of eukaryotic chromatin linked to transcription. Curiously, it is a transcriptional activator in some species and a repressor in others, suggesting species-specific differences shape its function. Here we engineer H2A.Z sequence variants from diverse eukaryotes into a single host, Schizosaccharomyces pombe, to understand H2A.Zs transcriptional impact. We show that H2A.Z sequences representing more than a billion years of evolution encode functional variation attributable to as little as single amino acid substitutions in the unstructured loop 2 (L2) region of H2A.Zs core domain. We show that L2 binds elongation factor Spt6, with L2 sequence influencing its binding to Spt6 and whether it promotes or restricts transcription elongation. In this way, the H2A.Z-Spt6 interaction clarifies H2A.Zs fundamental regulatory function, providing a direct physical and functional linkage between transcription and its chromatinized template across eukaryotes.