MNase-seq analysis of the Methnocaldococcus jannaschii nucleoid reveals a general size-variable hypernucleosome architecture

In eukaryotes, histone paralogues form obligate heterodimers such as H3/H4 and H2A/H2B that assemble into octameric nucleosome particles. Archaeal histones are dimeric and assemble on DNA into ‘hypernucleosome’ particles of varying sizes with each dimer wrapping 30 bp of DNA. These are likely composed of canonical and variant histone paralogues, but the function of these variants is poorly understood. Here, we characterise the structure and function of the histone paralogue MJ1647 from Methanocaldococcus jannaschii that has a unique C-terminal extension that enables MJ1647 homo-tetramerisation. The accompanying paper solves the crystal structure of MJ1647, but this part of the study specifically uses MNase-seq methodology to explore the hypernucleosome protection landscape of the in vivo M. jannaschii nucleoid. M. jannaschii cells were grown to log phase and nucleoids rendered accessible by detergent permeabilisation in an isotonic buffer. Two nucleoid samples were digested with different concentrations of MNase and nuclease-resistant and fragmented DNA species over a wide size range (typically 30 – 600bp) were converted to Illumina NGS libraries and sequenced in paired-end mode. A deproteinised gDNA sample was also digested with MNase to provide a control dataset to reveal any MNase-cleavage bias at the DNA sequence level. The mid-points between paired reads are then mapped to the reference genome, with peaks in mid-point frequency in nucleoid datasets taken to imply the presence of positioned MNase-resistant DNA protein complexes which we term: “chromatin particle positions”. Sequence data is then separated according to paired read end-to-end distances/insert size to directly reflect the extent of MNase protection of DNA by a specific DNA:protein complex and defining a “chromatin particle size”. In eukaryote experiments, sequence read pairs with an end-to-end distance of approximately 150bp effectively map positioned histone octamer nucleosomes (which are wrapped by 147bp of DNA). Here, the read pair mid-point positions effectively define hypernucleosomal dyad sequences of widely varying lengths (archaeal DNA:histone “slinkies” or “curly-fries”).