When RNA Polymerase II (RNAPII) transcribes through a nucleosome, it must break all DNA- histone contacts. In vitro studies have shown that loss of an H2A/H2B dimer to form a hexasome can occur under high salt conditions that permit RNAPII transit through a nucleosome. However, whether these mechanisms operate in vivo is unknown. In this study, we show that transcriptional elongation in vivo drives hexasome formation, especially over the first (+1) nucleosome downstream of the transcriptional start site in Drosophila cells. We also identify DNA torsion as driving preferential loss of the distal dimer and show that the FACT histone chaperone is asymmetrically oriented over the +1 nucleosome and resists dimer loss. We obtained these new insights into nucleosome dynamics in vivo using Micrococcal Nuclease mapping using sequencing (MNase-seq) as a novel tool for structural epigenomics.
Having established the features of hexasomal intermediates in a cell line, we go on to show a similar phenomenon over expressed +1 positions in cell-free DNA (cfDNA) from human blood plasma DNA that was naturally digested during cell turnover (Snyder et al., Cell, 2016). Importantly, our subnucleosome particle analysis method requires sequence from only the first 300 bp of a transcription unit, ~2 orders of magnitude less than was used in the previous nucleosome spacing analysis, which when coupled to targeted DNA sequencing can greatly reduce the cost of cfDNA tissue-of-origin testing.