Multimodal multiscale analysis reveals flexible DNA contacts of bacterial nucleoprotein filaments

Gene silencing in bacteria is mediated by chromatin proteins, of which Escherichia coli H-NS is a paradigmatic example. H-NS forms nucleoprotein filaments with either one or two DNA duplexes. However, the overall structures, arrangements of DNA-binding domains (DBDs), positions of DBD–DNA contacts, and determinants of genomic distribution for these linear and bridged filaments are uncertain. To connect H-NS structures that silence genes in vivo with features elucidated in vitro, we developed a multimodal multiscale analysis that combines a new method for chromatin reconstitution and mapping (Nitro-seq), ChIP-seq, tethered-nuclease mapping of DBD–DNA contacts (TEN-map), ·OH footprinting, molecular dynamics, and bioinformatics. We find that DNA sequence principally governs H-NS-filament location with indistinguishable sequence specificity in bridged or linear forms with or without the H-NS modifiers StpA and Hha and that DBD–DNA contacts vary in orientation and position with ~10-bp average spacing. Our results support a hemi-sequestration model of linear-to-bridged filament switching. ChIP-seq on H-NS and RNA polymerase in Escherichia coli K-12 genome with or without rifampicin treatment to inhibit active transcription. There are 12 total ChIP-seq samples including input (no-IP) control. 2 replicates were performed per IP. Nitro-seq, a new method chromatin reconsitution and mapping, on H-NS, StpA, a mixture of Hha and H-NS, and Fis binding to E. coli K-12 purified genomic DNA. The mixture of protein and DNA was applied to a nitrocellulose filter to enrich for protein::DNA complexes. The DNA bound by protein was prepared for sequencing by eluting the DNA from the nitrocellulose filter and then preparing a sequencing library. There are a total of 25 Nitro-seq samples, including 1 input DNA control. Each sample was performed in triplicate, execpt for the 5 nM Fis sample which was done in duplicate.