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The organization of chromatin into different types of compact versus open states provides a means to fine tune gene regulation. Recent studies have suggested a role for phase-separation in chromatin compaction, raising new possibilities for regulating chromatin compartments. This perspective discusses some specific molecular mechanisms that could leverage such phase-separation processes to control the functions and organization of chromatin.

In mammals, HP1-mediated heterochromatin forms positionally and mechanically stable genomic domains even though the component HP1 paralogs, HP1α, HP1β, and HP1γ, display rapid on-off dynamics. Here, we investigate whether phase-separation by HP1 proteins can explain these biological observations. Using bulk and single-molecule methods, we show that, within phase-separated HP1α-DNA condensates, HP1α acts as a dynamic liquid, while compacted DNA molecules are constrained in local territories. These condensates are resistant to large forces yet can be readily dissolved by HP1β. Finally, we find that differences in each HP1 paralog’s DNA compaction and phase-separation properties arise from their respective disordered regions. Our findings suggest a generalizable model for genome organization in which a pool of weakly bound proteins collectively capitalize on the polymer properties of DNA to produce self-organizing domains that are simultaneously resistant to large forces at the mesoscale and susceptible to competition at the molecular scale.

The CRISPR-Cas9 bacterial surveillance system has become a versatile tool for genome editing and gene regulation in eukaryotic cells, yet how CRISPR-Cas9 contends with the barriers presented by eukaryotic chromatin is poorly understood. Here we investigate how the smallest unit of chromatin, a nucleosome, constrains the activity of the CRISPR-Cas9 system. We find that nucleosomes assembled on native DNA sequences are permissive to Cas9 action. However, the accessibility of nucleosomal DNA to Cas9 is variable over several orders of magnitude depending on dynamic properties of the DNA sequence and the distance of the PAM site from the nucleosome dyad. We further find that chromatin remodeling enzymes stimulate Cas9 activity on nucleosomal templates. Our findings imply that the spontaneous breathing of nucleosomal DNA together with the action of chromatin remodelers allow Cas9 to effectively act on chromatin in vivo. CRISPR is a method of editing the genetic material inside living cells and has enabled dramatic advances in a broad variety of research fields in recent years. The method relies on a bacterial enzyme called Cas9 that can be programmed, via short guide molecules made from RNA, to target specific sites in the cell’s DNA. Once bound to its target, the Cas9 enzyme cuts the DNA molecule; this often leads to changes in the DNA sequence. In nature, bacteria use the CRISPR-Cas9 system to defend themselves against viruses. However, this system also works in other cell types and can be reprogrammed to target almost any site in the DNA. To date, the CRISPR-Cas9 system has been used in fungi, worms, flies, plants, mammals and other eukaryotes. Yet, unlike in bacteria, much of the DNA in eukaryotes is wrapped around proteins called histones to form units referred to as nucleosomes. This means eukaryotic DNA is often tightly packaged, which makes it less accessible to other proteins. Nevertheless, eukaryotic DNA will spontaneously detach and reattach to the histones – a phenomenon that is commonly known as DNA “breathing”. Also, protein machines known as chromatin remodelers can move, assemble and take apart the nucleosomes in eukaryotic cells. However, because there is much still to learn about how CRISPR-Cas9 works in eukaryotic cells, it is not clear how nucleosomes affect this system’s activity. Isaac et al. have now used a simplified biochemical system to test how nucleosomes and chromatin remodelers affect CRISP-Cas9 activity. The system comprised purified Cas9 enzymes, short guide RNA molecules and nucleosomes. The experiments revealed that the Cas9 enzyme was able to cut DNA on nucleosomes when the DNA sequence allowed more spontaneous breathing or when chromatin remodelers were present to destabilize or move the nucleosome out of the way. These results suggest that by taking the placement of the nucleosomes into account, researchers can better predict how effective the CRISPR-Cas9 system will be at targeting a specific DNA sequence in a eukaryotic cell. The findings also suggest ways to make genome editing with CRISPR-Cas9 even more efficient.

In eukaryotes, DNA is packed onto nucleosomes. For transcription factors and other proteins to gain access to DNA, nucleosomes must be moved out of the way, or “remodeled”—but not disassembled. Nucleosomes are composed of histone protein octamers, the cores of which have generally been considered to be fairly rigid. Sinha et al. used nuclear magnetic resonance and protein cross-linking to show that one of the enzyme complexes that remodel nucleosomes, SNF2h, is able to distort the histone octamer (see the Perspective by Flaus and Owen-Hughes). Nucleosome deformation was important for this remodeler to be able to slide nucleosomes out of the way. Science , this issue p. 10.1126/science.aaa3761 ; see also p. 245 The nucleosome histone octamer can be deformed by a nucleosome remodeling enzyme to slide nucleosomes out of the way. Adenosine 5′-triphosphate (ATP)–dependent chromatin remodeling enzymes play essential biological roles by mobilizing nucleosomal DNA. Yet, how DNA is mobilized despite the steric constraints placed by the histone octamer remains unknown. Using methyl transverse relaxation–optimized nuclear magnetic resonance spectroscopy on a 450-kilodalton complex, we show that the chromatin remodeler, SNF2h, distorts the histone octamer. Binding of SNF2h in an activated ATP state changes the dynamics of buried histone residues. Preventing octamer distortion by site-specific disulfide linkages inhibits nucleosome sliding by SNF2h while promoting octamer eviction by the SWI-SNF complex, RSC. Our findings indicate that the histone core of a nucleosome is more plastic than previously imagined and that octamer deformation plays different roles based on the type of chromatin remodeler. Octamer plasticity may contribute to chromatin regulation beyond ATP-dependent remodeling.

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 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.

Our understanding of the beads-on-a-string arrangement of nucleosomes has been built largely on high-resolution sequence-agnostic imaging methods and sequence-resolved bulk biochemical techniques. To bridge the divide between these approaches, we present the single-molecule adenine methylated oligonucleosome sequencing assay (SAMOSA). SAMOSA is a high-throughput single-molecule sequencing method that combines adenine methyltransferase footprinting and single-molecule real-time DNA sequencing to natively and nondestructively measure nucleosome positions on individual chromatin fibres. SAMOSA data allows unbiased classification of single-molecular 'states' of nucleosome occupancy on individual chromatin fibres. We leverage this to estimate nucleosome regularity and spacing on single chromatin fibres genome-wide, at predicted transcription factor binding motifs, and across human epigenomic domains. Our analyses suggest that chromatin is comprised of both regular and irregular single-molecular oligonucleosome patterns that differ subtly in their relative abundance across epigenomic domains. This irregularity is particularly striking in constitutive heterochromatin, which has typically been viewed as a conformationally static entity. Our proof-of-concept study provides a powerful new methodology for studying nucleosome organization at a previously intractable resolution and offers up new avenues for modeling and visualizing higher order chromatin structure.

Heterochromatin maintains genome integrity and function, and is organised into distinct nuclear domains. Some of these domains are proposed to form by phase separation through the accumulation of HP1ɑ. Mouse heterochromatin contains noncoding major satellite repeats (MSR), which are highly transcribed in mouse embryonic stem cells (ESCs). Here, we report that MSR transcripts can drive the formation of HP1ɑ droplets in vitro, and modulate heterochromatin into dynamic condensates in ESCs, contributing to the formation of large nuclear domains that are characteristic of pluripotent cells. Depleting MSR transcripts causes heterochromatin to transition into a more compact and static state. Unexpectedly, changing heterochromatin’s biophysical properties has severe consequences for ESCs, including chromosome instability and mitotic defects. These findings uncover an essential role for MSR transcripts in modulating the organisation and properties of heterochromatin to preserve genome stability. They also provide insights into the processes that could regulate phase separation and the functional consequences of disrupting the properties of heterochromatin condensates. Here the authors show satellite transcripts in mouse embryonic stem cells drive HP1α into droplets in vitro and also control HP1α organisation and association with chromatin in vivo. Depleting the satellite transcripts converts heterochromatin into a less dynamic and more static state and leads to chromosome instability.

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. Every human cell contains nearly two meters of DNA, which is carefully packaged to form a dense structure known as chromatin. The building block of chromatin is the nucleosome, a unit composed of a short section of DNA tightly wound up around a spool-like core of proteins called histones. The tight structure of the nucleosome prevents the cell from accessing and ‘reading’ the genes in the packaged DNA, effectively switching off these genes. So the exact placement of nucleosomes helps manage which genes are turned on. Changing the position of the nucleosomes can ‘free’ the DNA and make genes available to the cell. Enzymes called chromatin remodelers move nucleosomes around – for example, they can make the histone core slide on the DNA strand. However, it is still unclear how these enzymes recognize nucleosomes. Previous research indicates that many proteins bind to nucleosomes by using a surface on the histone proteins called the acidic patch. Could chromatin remodelers also work by interacting with this acidic patch? To address this further, Gamarra et al. investigate how a chromatin remodeler enzyme known as SNF2h interacts with a nucleosome. By default, SNF2h is inactive because two of its regions called AutoN and NegC act as brakes. The experiments show that the acidic patch helps to bypass this inactivation and switches on SNF2h. Gamarra et al. propose that, when SNF2h docks on to the nucleosome, the patch provides a landing pad for the AutoN and NegC modules; this interaction activates the enzyme, which can then start remodeling the nucleosome. However, another type of chromatin remodeler also uses the patch to interact with nucleosomes but it does not have the AutoN and NegC regions. This suggests that chromatin remodelers work with the acidic patch in different ways. Overall, the findings deepen our understanding of how DNA is packaged in cells, and how this process may go wrong and cause disease.