Highlights d Chromatin undergoes liquid-liquid phase separation (LLPS) under physiologic conditions d Linker DNA length and patterning, histone H1, and acetylation modulate chromatin LLPS d Acetylated chromatin only phase separates upon binding multi-bromodomain proteins d LLPS could enable establishment and maintenance of distinct chromatin compartments

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.

Metazoan development requires robust proliferation of progenitor cells, whose identities are established by tightly controlled transcriptional networks 1 . As gene expression is globally inhibited during mitosis, the transcriptional programs defining cell identity must be restarted in each cell cycle 2 - 5 , yet how this is accomplished is poorly understood. Here, we identified a ubiquitin-dependent mechanism that integrates gene expression with cell division to preserve cell identity. We found that WDR5 and TBP, which bind active interphase promoters 6 , 7 , recruit the anaphase-promoting complex (APC/C) to specific transcription start sites (TSS) during mitosis. This allows APC/C to decorate histones with K11/K48-branched ubiquitin chains that recruit p97/VCP and the proteasome and ensure rapid expression of pluripotency genes in the next cell cycle. Mitotic exit and transcription re-initiation are thus controlled by the same regulator, APC/C, which provides a robust mechanism to maintain cell identity through cell division.

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 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.