ATP Hydrolysis Coordinates the Activities of Two Motors in a Dimeric Chromatin Remodeling Enzyme.

ATP-dependent chromatin remodelers are essential enzymes that restructure eukaryotic genomes to enable all DNA-based processes. The diversity and complexity of these processes arethe complexity of the enzymes that carry them out, making remodelers a challenging class of molecular motors to study by conventional methods. Here we use a single molecule biophysical assay to overcome some of these challenges, enabling a detailed mechanistic dissection of a paradigmatic remodeler reaction, that of sliding a nucleosome towards the longer DNA linker. We focus on how two motors of a dimeric remodeler coordinate to accomplish such directional sliding. We find that ATP hydrolysis by both motors promotes coordination, suggesting a role for ATP in resolving the competition for directional commitment. Furthermore, we show an artificially constitutive dimer is no more or less coordinated, but is more processive, suggesting a cell could modulate a remodeler's oligomeric state to modulate local chromatin dynamics.

Cryo-EM structures of remodeler-nucleosome intermediates suggest allosteric control through the nucleosome.

The SNF2h remodeler slides nucleosomes most efficiently as a dimer, yet how the two protomers avoid a tug-of-war is unclear. Furthermore, SNF2h couples histone octamer deformation to nucleosome sliding, but the underlying structural basis remains unknown. Here we present cryo-EM structures of SNF2h-nucleosome complexes with ADP-BeFx that capture two potential reaction intermediates. In one structure, histone residues near the dyad and in the H2A-H2B acidic patch, distal to the active SNF2h protomer, appear disordered. The disordered acidic patch is expected to inhibit the second SNF2h protomer, while disorder near the dyad is expected to promote DNA translocation. The other structure doesn't show octamer deformation, but surprisingly shows a 2 bp translocation. FRET studies indicate that ADP-BeFx predisposes SNF2h-nucleosome complexes for an elemental translocation step. We propose a model for allosteric control through the nucleosome, where one SNF2h protomer promotes asymmetric octamer deformation to inhibit the second protomer, while stimulating directional DNA translocation.

Ion counting demonstrates a high electrostatic field generated by the nucleosome.

In eukaryotes, a first step towards the nuclear DNA compaction process is the formation of a nucleosome, which is comprised of negatively charged DNA wrapped around a positively charged histone protein octamer. Often, it is assumed that the complexation of the DNA into the nucleosome completely attenuates the DNA charge and hence the electrostatic field generated by the molecule. In contrast, theoretical and computational studies suggest that the nucleosome retains a strong, negative electrostatic field. Despite their fundamental implications for chromatin organization and function, these opposing views of nucleosome electrostatics have not been experimentally tested. Herein, we directly measure nucleosome electrostatics and find that while nucleosome formation reduces the complex charge by half, the nucleosome nevertheless maintains a strong negative electrostatic field. Our studies highlight the importance of considering the polyelectrolyte nature of the nucleosome and its impact on processes ranging from factor binding to DNA compaction.

The nucleosomal acidic patch relieves auto-inhibition by the ISWI remodeler SNF2h.

ISWI family chromatin remodeling motors use sophisticated autoinhibition mechanisms to control nucleosome sliding. Yet how the different autoinhibitory domains are regulated is not well understood. Here we show that an acidic patch formed by histones H2A and H2B of the nucleosome relieves the autoinhibition imposed by the AutoN and the NegC regions of the human ISWI remodeler SNF2h. Further, by single molecule FRET we show that the acidic patch helps control the distance travelled per translocation event. We propose a model in which the acidic patch activates SNF2h by providing a landing pad for the NegC and AutoN auto-inhibitory domains. Interestingly, the INO80 complex is also strongly dependent on the acidic patch for nucleosome sliding, indicating that this substrate feature can regulate remodeling enzymes with substantially different mechanisms. We therefore hypothesize that regulating access to the acidic patch of the nucleosome plays a key role in coordinating the activities of different remodelers in the cell.

The Yeast INO80 Complex Operates as a Tunable DNA Length-Sensitive Switch to Regulate Nucleosome Sliding.

The yeast INO80 chromatin remodeling complex plays essential roles in regulating DNA damage repair, replication, and promoter architecture. INO80's role in these processes is likely related to its ability to slide nucleosomes, but the underlying mechanism is poorly understood. Here we use ensemble and single-molecule enzymology to study INO80-catalyzed nucleosome sliding. We find that the rate of nucleosome sliding by INO80 increases ∼100-fold when the flanking DNA length is increased from 40 to 60 bp. Furthermore, once sliding is initiated, INO80 moves the nucleosome rapidly at least 20 bp without pausing to re-assess flanking DNA length, and it can change the direction of nucleosome sliding without dissociation. Finally, we show that the Nhp10 module of INO80 plays an auto-inhibitory role, tuning INO80's switch-like response to flanking DNA. Our results indicate that INO80 is a highly processive remodeling motor that is tightly regulated by both substrate cues and non-catalytic subunits.

Mechanisms of ATP-Dependent Chromatin Remodeling Motors.

Chromatin remodeling motors play essential roles in all DNA-based processes. These motors catalyze diverse outcomes ranging from sliding the smallest units of chromatin, known as nucleosomes, to completely disassembling chromatin. The broad range of actions carried out by these motors on the complex template presented by chromatin raises many stimulating mechanistic questions. Other well-studied nucleic acid motors provide examples of the depth of mechanistic understanding that is achievable from detailed biophysical studies. We use these studies as a guiding framework to discuss the current state of knowledge of chromatin remodeling mechanisms and highlight exciting open questions that would continue to benefit from biophysical analyses.

The everyday routines of families of children with autism: examining the impact of sensory processing difficulties on the family.

The purpose of this qualitative study was to explore the lived experience of how sensory-related behaviors of children with autism affected family routines. In-depth semi-structured interviews were conducted with four primary caregivers regarding the meaning and impact of their child's sensory-related behaviors on family routines that occurred inside and outside the home. Findings indicated that sensory behaviors are one factor that limited family participation in work, family and leisure activities; and that parents employed specific strategies to manage individual and family routines in light of the child's sensory-related behaviors. This information has important implications for professionals who work with families of children with autism to decrease caregiver stress and to increase life satisfaction for the child and family.