Synthesis of a eukaryotic chromosome reveals a role for N6-methyladenine in nucleosome organization

Abstract

SummaryBiochemical studies of chromatin have typically used either artificial DNA templates with unnaturally high affinity for histones, or small genomic DNA fragments deprived of their cognate physical environment. It has thus been difficult to dissect chromatin structure and function within fully native DNA substrates. Here, we circumvent these limitations by exploiting the minimalist genome of the eukaryoteOxytricha trifallax, whose notably small ~3kb chromosomes mainly encode single genes. Guided by high-resolution epigenomic maps of nucleosome organization, transcription, and DNA N6-methyladenine (m6dA) locations, we reconstruct full-lengthOxytrichachromosomesin vitroand use these synthetic facsimiles to dissect the influence of m6dA and histone post-translational modifications on nucleosome organization. We show that m6dA directly disfavors nucleosomes in a quantitative manner, leading to local decreases in nucleosome occupancy that are synergistic with histone acetylation. The effect of m6dA can be partially reversed by the action of an ATP-dependent chromatin remodeler. Furthermore, erasing m6dA marks fromOxytrichachromosomes leads to proportional increases in nucleosome occupancy across the genome. This work showcasesOxytrichachromosomes as powerful yet practical models for studying eukaryotic chromatin and transcription in the context of biologically relevant DNA substrates.HighlightsDe novosynthesis of complete, epigenetically definedOxytrichachromosomesEpigenomic profiles of chromatin organization inOxytricha’sminiature chromosomesm6dA directly disfavors nucleosome occupancy in natural and synthetic chromosomesHistone acetylation and chromatin remodelers temper the impact of m6dA on chromatin