Bacteria frequently need to adapt to altered environmental conditions. Adaptation requires changes in gene expression, often mediated by global regulators of transcription. The nucleoid-associated protein H-NS is a key global regulator in Gram-negative bacteria and is believed to be a crucial player in bacterial chromatin organization via its DNA-bridging activity. H-NS activity in vivo is modulated by physico-chemical factors (osmolarity, pH, temperature) and interaction partners. Mechanistically, it is unclear how functional modulation of H-NS by such factors is achieved. Here, we show that a diverse spectrum of H-NS modulators alter the DNA-bridging activity of H-NS. Changes in monovalent and divalent ion concentrations drive an abrupt switch between a bridging and non-bridging DNA-binding mode. Similarly, synergistic and antagonistic co-regulators modulate the DNA-bridging efficiency. Structural studies suggest a conserved mechanism: H-NS switches between a ‘closed’ and an ‘open’, bridging competent, conformation driven by environmental cues and interaction partners.

In enterobacteria, AT-rich horizontally acquired genes, including virulence genes, are silenced through the actions of at least three nucleoid-associated proteins (NAPs): H-NS, StpA and Hha. These proteins form gene-silencing nucleoprotein filaments through direct DNA binding by H-NS and StpA homodimers or heterodimers. Both linear and bridged filaments, in which NAPs bind one or two DNA segments, respectively, have been observed. Hha can interact with H-NS or StpA filaments, but itself lacks a DNA-binding domain. Filaments composed of H-NS alone can inhibit transcription initiation and, in the bridged conformation, slow elongating RNA polymerase (RNAP) by promoting backtracking at pause sites. How the other NAPs modulate these effects of H-NS is unknown, despite evidence that they help regulate subsets of silenced genes in vivo (e.g. in pathogenicity islands). Here we report that Hha and StpA greatly enhance H-NS-stimulated pausing by RNAP at 20°C. StpA:H-NS or StpA-only filaments also stimulate pausing at 37°C, a temperature at which Hha:H-NS or H-NS-only filaments have much less effect. In addition, we report that both Hha and StpA greatly stimulate DNA–DNA bridging by H-NS filaments. Together, these observations indicate that Hha and StpA can affect H-NS-mediated gene regulation by stimulating bridging of H-NS/DNA filaments.

Many archaea express histones, which organize the genome and play a key role in gene regulation. The structure and function of archaeal histone–DNA complexes remain however largely unclear. Recent studies show formation of hypernucleosomes consisting of DNA wrapped around an ‘endless’ histone-protein core. However, if and how such a hypernucleosome structure assembles on a long DNA substrate and which interactions provide for its stability, remains unclear. Here, we describe micromanipulation studies of complexes of the histones HMfA and HMfB with DNA. Our experiments show hypernucleosome assembly which results from cooperative binding of histones to DNA, facilitated by weak stacking interactions between neighboring histone dimers. Furthermore, rotational force spectroscopy demonstrates that the HMfB–DNA complex has a left-handed chirality, but that torque can drive it in a right-handed conformation. The structure of the hypernucleosome thus depends on stacking interactions, torque, and force. In vivo, such modulation of the archaeal hypernucleosome structure may play an important role in transcription regulation in response to environmental changes.

Remodelling chromatin structure is important for regulating gene expression, DNA replication and repair and other fundamental nuclear processes. The basic chromatin unit is the nucleosome. SWR1 is a multi-subunit complex, whose chromatin remodelling activity is associated with regulation of gene expression in heterochromatin regions of chromosomes in plants and mammals, and with the cellular response to DNA damage. In yeast, the simplest eukaryotic organism, the SWR1 complex is responsible for the ATP-dependent nucleosome remodelling by exchanging its canonical H2A histone with Htz1 variant (also known as H2A.Z in mammalian cells). In spite of a large number of genetic, biochemical and structural studies on SWR1, its detailed histone exchange mechanism remains largely unknown. To investigate the mechanism of histone exchange by SWR1, we have developed a single-molecule FRET assay, which monitors the interaction between individual nucleosomes and yeast SWR1 complexes in real time. The data show distinct dynamic behaviours in the presence or absence of ATP, or in the presence of non-hydrolysable ATP analogue. We hypothesize that the observed dynamics are important for the removal of canonical H2A histones or deposition of the histone variant Htz1. We anticipate that our data will help elucidate the molecular mechanism of histone exchange by SWR1.

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