Conformational switching of Arp5 subunit differentially regulates INO80 chromatin remodeling.

The INO80 chromatin remodeler is a versatile enzyme capable of several functions, including spacing nucleosomes equal distances apart, precise positioning of nucleosomes based on DNA shape/sequence and exchanging histone dimers. Within INO80, the Arp5 subunit plays a central role in INO80 remodeling, evidenced by its interactions with the histone octamer, nucleosomal and extranucleosomal DNA, and its necessity in linking INO80's ATPase activity to nucleosome movement. Our investigation reveals that the grappler domain of Arp5 interacts with the acidic pocket of nucleosomes through two distinct mechanisms: an arginine anchor or a hydrophobic/acidic patch. These two modes of binding serve distinct functions within INO80 as shown in vivo by mutations in these regions resulting in varying phenotypes and in vitro by diverse effects on nucleosome mobilization. Our findings suggest that the hydrophobic/acidic patch of Arp5 is likely important for dimer exchange by INO80, while the arginine anchor is crucial for mobilizing nucleosomes.

Enhancer switching in cell lineage priming is linked to eRNA, Brg1's AT-hook, and SWI/SNF recruitment.

RNA transcribed from enhancers, i.e., eRNA, has been suggested to directly activate transcription by recruiting transcription factors and co-activators. Although there have been specific examples of eRNA functioning in this way, it is not clear how general this may be. We find that the AT-hook of SWI/SNF preferentially binds RNA and, as part of the esBAF complex, associates with eRNA transcribed from intronic and intergenic regions. Our data suggest that SWI/SNF is globally recruited in cis by eRNA to cell-type-specific enhancers, representative of two distinct stages that mimic early mammalian development, and not at enhancers that are shared between the two stages. In this manner, SWI/SNF facilitates recruitment and/or activation of MLL3/4, p300/CBP, and Mediator to stage-specific enhancers and super-enhancers that regulate the transcription of metabolic and cell lineage priming-related genes. These findings highlight a connection between ATP-dependent chromatin remodeling and eRNA in cell identity and typical- and super-enhancer activation.

The SWI/SNF ATP-dependent chromatin remodeling complex in cell lineage priming and early development.

ATP dependent chromatin remodelers have pivotal roles in transcription, DNA replication and repair, and maintaining genome integrity. SWI/SNF remodelers were first discovered in yeast genetic screens for factors involved in mating type switching or for using alternative energy sources therefore termed SWI/SNF complex (short for SWItch/Sucrose NonFermentable). The SWI/SNF complexes utilize energy from ATP hydrolysis to disrupt histone-DNA interactions and shift, eject, or reposition nucleosomes making the underlying DNA more accessible to specific transcription factors and other regulatory proteins. In development, SWI/SNF orchestrates the precise activation and repression of genes at different stages, safe guards the formation of specific cell lineages and tissues. Dysregulation of SWI/SNF have been implicated in diseases such as cancer, where they can drive uncontrolled cell proliferation and tumor metastasis. Additionally, SWI/SNF defects are associated with neurodevelopmental disorders, leading to disruption of neural development and function. This review offers insights into recent developments regarding the roles of the SWI/SNF complex in pluripotency and cell lineage primining and the approaches that have helped delineate its importance. Understanding these molecular mechanisms is crucial for unraveling the intricate processes governing embryonic stem cell biology and developmental transitions and may potentially apply to human diseases linked to mutations in the SWI/SNF complex.

New insights into the mechanism and DNA-sequence specificity of INO80 chromatin remodeling.

The INO80 complex stood out in a large family of ATP-dependent chromatin remodelers because of its ATPase domain binding and translocating on DNA at the edge of nucleosomes, rather than at two helical turns from the center of DNA that is wrapped around nucleosomes. This unique property of INO80 was thought to account for its singular role in nucleosome placement at gene promoters in a DNA-sequence dependent manner that is crucial for transcription regulation. Now, we uncover INO80 functions differently than previously thought with its ATPase domain translocating on DNA close to the center of nucleosomes, like other remodelers. Our discovery also reveals the physical properties of the first ~36 bp of DNA on the entry side of nucleosomes is the main determinant for the DNA specificity of INO80 rather than the properties of the extranucleosomal DNA. The DNA sequence sensitive step of INO80 is after DNA is displaced from the histone octamer on the entry side of nucleosomes and 20 bp of DNA are moved out the exit side. We find the ATPase domain and Arp5 subunit of INO80 are likely involved in INO80's DNA specificity and the mechanism of INO80 remodeling is substantially different than originally proposed.

The AT-hook is an evolutionarily conserved auto-regulatory domain of SWI/SNF required for cell lineage priming.

The SWI/SNF ATP-dependent chromatin remodeler is a master regulator of the epigenome, controlling pluripotency and differentiation. Towards the C-terminus of the catalytic subunit of SWI/SNF is a motif called the AT-hook that is evolutionary conserved. The AT-hook is present in many chromatin modifiers and generally thought to help anchor them to DNA. We observe however that the AT-hook regulates the intrinsic DNA-stimulated ATPase activity aside from promoting SWI/SNF recruitment to DNA or nucleosomes by increasing the reaction velocity a factor of 13 with no accompanying change in substrate affinity (KM). The changes in ATP hydrolysis causes an equivalent change in nucleosome movement, confirming they are tightly coupled. The catalytic subunit's AT-hook is required in vivo for SWI/SNF remodeling activity in yeast and mouse embryonic stem cells. The AT-hook in SWI/SNF is required for transcription regulation and activation of stage-specific enhancers critical in cell lineage priming. Similarly, growth assays suggest the AT-hook is required in yeast SWI/SNF for activation of genes involved in amino acid biosynthesis and metabolizing ethanol. Our findings highlight the importance of studying SWI/SNF attenuation versus eliminating the catalytic subunit or completely shutting down its enzymatic activity.

Dinucleosome specificity and allosteric switch of the ISW1a ATP-dependent chromatin remodeler in transcription regulation.

Over the last 3 decades ATP-dependent chromatin remodelers have been thought to recognize chromatin at the level of single nucleosomes rather than higher-order organization of more than one nucleosome. We show the yeast ISW1a remodeler has such higher-order structural specificity, as manifested by large allosteric changes that activate the nucleosome remodeling and spacing activities of ISW1a when bound to dinucleosomes. Although the ATPase domain of Isw1 docks at the SHL2 position when ISW1a is bound to either mono- or di-nucleosomes, there are major differences in the interactions of the catalytic subunit Isw1 with the acidic pocket of nucleosomes and the accessory subunit Ioc3 with nucleosomal DNA. By mutational analysis and uncoupling of ISW1a's dinucleosome specificity, we find that dinucleosome recognition is required by ISW1a for proper chromatin organization at promoters; as well as transcription regulation in combination with the histone acetyltransferase NuA4 and histone H2A.Z exchanger SWR1.

Author Correction: De novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens.

An amendment to this paper has been published and can be accessed via a link at the top of the paper.

De novo identification of essential protein domains from CRISPR-Cas9 tiling-sgRNA knockout screens.

High-throughput CRISPR-Cas9 knockout screens using a tiling-sgRNA design permit in situ evaluation of protein domain function. Here, to facilitate de novo identification of essential protein domains from such screens, we propose ProTiler, a computational method for the robust mapping of CRISPR knockout hyper-sensitive (CKHS) regions, which refer to the protein regions associated with a strong sgRNA dropout effect in the screens. Applied to a published CRISPR tiling screen dataset, ProTiler identifies 175 CKHS regions in 83 proteins. Of these CKHS regions, more than 80% overlap with annotated Pfam domains, including all of the 15 known drug targets in the dataset. ProTiler also reveals unannotated essential domains, including the N-terminus of the SWI/SNF subunit SMARCB1, which is validated experimentally. Surprisingly, the CKHS regions are negatively correlated with phosphorylation and acetylation sites, suggesting that protein domains and post-translational modification sites have distinct sensitivities to CRISPR-Cas9 mediated amino acids loss.

Histone Octamer Structure Is Altered Early in ISW2 ATP-Dependent Nucleosome Remodeling.

Nucleosomes are the fundamental building blocks of chromatin that regulate DNA access and are composed of histone octamers. ATP-dependent chromatin remodelers like ISW2 regulate chromatin access by translationally moving nucleosomes to different DNA regions. We find that histone octamers are more pliable than previously assumed and distorted by ISW2 early in remodeling before DNA enters nucleosomes and the ATPase motor moves processively on nucleosomal DNA. Uncoupling the ATPase activity of ISW2 from nucleosome movement with deletion of the SANT domain from the C terminus of the Isw2 catalytic subunit traps remodeling intermediates in which the histone octamer structure is changed. We find restricting histone movement by chemical crosslinking also traps remodeling intermediates resembling those seen early in ISW2 remodeling with loss of the SANT domain. Other evidence shows histone octamers are intrinsically prone to changing their conformation and can be distorted merely by H3-H4 tetramer disulfide crosslinking.

Regulation of ATP-dependent chromatin remodelers: accelerators/brakes, anchors and sensors.

All ATP-dependent chromatin remodelers have a DNA translocase domain that moves along double-stranded DNA when hydrolyzing ATP, which is the key action leading to DNA moving through nucleosomes. Recent structural and biochemical data from a variety of different chromatin remodelers have revealed that there are three basic ways in which these remodelers self-regulate their chromatin remodeling activity. In several instances, different domains within the catalytic subunit or accessory subunits through direct protein-protein interactions can modulate the ATPase and DNA translocation properties of the DNA translocase domain. These domains or subunits can stabilize conformations that either promote or interfere with the ability of the translocase domain to bind or retain DNA during translocation or alter the ability of the enzyme to hydrolyze ATP. Second, other domains or subunits are often necessary to anchor the remodeler to nucleosomes to couple DNA translocation and ATP hydrolysis to DNA movement around the histone octamer. These anchors provide a fixed point by which remodelers can generate sufficient torque to disrupt histone-DNA interactions and mobilize nucleosomes. The third type of self-regulation is in those chromatin remodelers that space nucleosomes or stop moving nucleosomes when a particular length of linker DNA has been reached. We refer to this third class as DNA sensors that can allosterically regulate nucleosome mobilization. In this review, we will show examples of these from primarily the INO80/SWR1, SWI/SNF and ISWI/CHD families of remodelers.

The Arp8 and Arp4 module acts as a DNA sensor controlling INO80 chromatin remodeling.

Nuclear actin and actin-related proteins (Arps) are key components of chromatin remodeling and modifying complexes. Although Arps are essential for the functions of chromatin remodelers, their specific roles and mechanisms are unclear. Here we define the nucleosome binding interfaces and functions of the evolutionarily conserved Arps in the yeast INO80 chromatin remodeling complex. We show that the N-terminus of Arp8, C-terminus of Arp4 and the HSA domain of Ino80 bind extranucleosomal DNA 37-51 base pairs from the edge of nucleosomes and function as a DNA-length sensor that regulates nucleosome sliding by INO80. Disruption of Arp8 and Arp4 binding to DNA uncouples ATP hydrolysis from nucleosome mobilization by disengaging Arp5 from the acidic patch on histone H2A-H2B and the Ino80-ATPase domain from the Super-helical Location (SHL) -6 of nucleosomes. Our data suggest a functional interplay between INO80's Arp8-Arp4-actin and Arp5 modules in sensing the DNA length separating nucleosomes and regulating nucleosome positioning.

Emerging roles of transcriptional enhancers in chromatin looping and promoter-proximal pausing of RNA polymerase II.

Initiation and regulation of transcription by RNA polymerase II (RNAPII) in eukaryotes rely on the transcriptional regulatory elements. Promoters and enhancers share similar architectures and functions, and the prevailing view is that they can initiate bidirectional transcription. We summarize functional roles of enhancer transcription and possible mechanisms in enhancer-promoter communication. We discuss the potential roles of enhancer RNAs (eRNAs) in early elongation and highlight that transcriptional enhancers might modulate the release of paused RNAPII via 3D chromatin looping. Emerging evidence suggests that transcriptional enhancers regulate the promoter-proximal pausing of RNAPII, a key rate-limiting step required for productive elongation.

Proteasomes beyond proteolysis: Roles in heterochromatin maintenance.

In addition to its proteolytic roles, the 26S proteasome is involved in regulating transcription and in promoting sites of active chromatin. In this report, Seo et al. provide evidence that the non-proteolytic 19S subunit of the 26S proteasome also regulates the spreading of inactive chromatin referred to as heterochromatin, suggesting further non-canonical roles of the proteasome in gene expression.

INO80 exchanges H2A.Z for H2A by translocating on DNA proximal to histone dimers.

ATP-dependent chromatin remodellers modulate nucleosome dynamics by mobilizing or disassembling nucleosomes, as well as altering nucleosome composition. These chromatin remodellers generally function by translocating along nucleosomal DNA at the H3-H4 interface of nucleosomes. Here we show that, unlike other remodellers, INO80 translocates along DNA at the H2A-H2B interface of nucleosomes and persistently displaces DNA from the surface of H2A-H2B. DNA translocation and DNA torsional strain created near the entry site of nucleosomes by INO80 promotes both the mobilization of nucleosomes and the selective exchange of H2A.Z-H2B dimers out of nucleosomes and replacement by H2A-H2B dimers without any additional histone chaperones. We find that INO80 translocates and mobilizes H2A.Z-containing nucleosomes more efficiently than those containing H2A, partially accounting for the preference of INO80 to replace H2A.Z with H2A. Our data suggest that INO80 has a mechanism for dimer exchange that is distinct from other chromatin remodellers including its paralogue SWR1.

Loss of Snf5 Induces Formation of an Aberrant SWI/SNF Complex.

The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.

Coordinated Action of Nap1 and RSC in Disassembly of Tandem Nucleosomes.

The SWI/SNF and RSC family of ATP-dependent chromatin remodelers disassembles nucleosomes by moving nucleosomes into the vicinity of adjoining nucleosomes. We found that the histone chaperone Nap1 efficiently promotes disassembly of adjacent nucleosomes with which RSC collides and not the disassembly of nucleosomes mobilized by RSC. Nap1 is specific to RSC, as it does not target SWI/SNF, its paralog in Saccharomyces cerevisiae Extensive mutational analysis of Nap1 has revealed that Nap1 affinity for histones H2A-H2B and H3-H4 and its ability to displace histones from DNA are required for Nap1 to enhance RSC-mediated disassembly. Other histone chaperones, such as Vps75, that also bind histones are not able to enhance RSC-mediated disassembly. Our study suggests a mechanism by which Nap1 is recruited to actively transcribed regions and assists in the passage of the transcription complex through chromatin, and it provides a novel mechanism for the coordinated action of RSC and Nap1.

Stepwise nucleosome translocation by RSC remodeling complexes.

The SWI/SNF-family remodelers regulate chromatin structure by coupling the free energy from ATP hydrolysis to the repositioning and restructuring of nucleosomes, but how the ATPase activity of these enzymes drives the motion of DNA across the nucleosome remains unclear. Here, we used single-molecule FRET to monitor the remodeling of mononucleosomes by the yeast SWI/SNF remodeler, RSC. We observed that RSC primarily translocates DNA around the nucleosome without substantial displacement of the H2A-H2B dimer. At the sites where DNA enters and exits the nucleosome, the DNA moves largely along or near its canonical wrapping path. The translocation of DNA occurs in a stepwise manner, and at both sites where DNA enters and exits the nucleosome, the step size distributions exhibit a peak at approximately 1-2 bp. These results suggest that the movement of DNA across the nucleosome is likely coupled directly to DNA translocation by the ATPase at its binding site inside the nucleosome.

INO80 Chromatin Remodeler Facilitates Release of RNA Polymerase II from Chromatin for Ubiquitin-Mediated Proteasomal Degradation.

Stalling of RNA Polymerase II (RNAPII) on chromatin during transcriptional stress results in polyubiquitination and degradation of the largest subunit of RNAPII, Rpb1, by the ubiquitin proteasome system (UPS). Here, we report that the ATP-dependent chromatin remodeling complex INO80 is required for turnover of chromatin-bound RNAPII in yeast. INO80 interacts physically and functionally with Cdc48/p97/VCP, a component of UPS required for degradation of RNAPII. Cells lacking INO80 are defective in Rpb1 degradation and accumulate tightly bound ubiquitinated Rpb1 on chromatin. INO80 forms a ternary complex with RNAPII and Cdc48 and targets Rpb1 primed for degradation. The function of INO80 in RNAPII turnover is required for cell growth and survival during genotoxic stress. Our results identify INO80 as a bona fide component of the proteolytic pathway for RNAPII degradation and suggest that INO80 nucleosome remodeling activity promotes the dissociation of ubiquitinated Rpb1 from chromatin to protect the integrity of the genome.

Histone Acetylation near the Nucleosome Dyad Axis Enhances Nucleosome Disassembly by RSC and SWI/SNF.

Signaling associated with transcription activation occurs through posttranslational modification of histones and is best exemplified by lysine acetylation. Lysines are acetylated in histone tails and the core domain/lateral surface of histone octamers. While acetylated lysines in histone tails are frequently recognized by other factors referred to as "readers," which promote transcription, the mechanistic role of the modifications in the lateral surface of the histone octamer remains unclear. By using X-ray crystallography, we found that acetylated lysines 115 and 122 in histone H3 are solvent accessible, but in biochemical assays they appear not to interact with the bromodomains of SWI/SNF and RSC to enhance recruitment or nucleosome mobilization, as previously shown for acetylated lysines in H3 histone tails. Instead, we found that acetylation of lysines 115 and 122 increases the predisposition of nucleosomes for disassembly by SWI/SNF and RSC up to 7-fold, independent of bromodomains, and only in conjunction with contiguous nucleosomes. Thus, in combination with SWI/SNF and RSC, acetylation of lateral surface lysines in the histone octamer serves as a crucial regulator of nucleosomal dynamics distinct from the histone code readers and writers.

Dynamic regulation of transcription factors by nucleosome remodeling.

The chromatin landscape and promoter architecture are dominated by the interplay of nucleosome and transcription factor (TF) binding to crucial DNA sequence elements. However, it remains unclear whether nucleosomes mobilized by chromatin remodelers can influence TFs that are already present on the DNA template. In this study, we investigated the interplay between nucleosome remodeling, by either yeast ISW1a or SWI/SNF, and a bound TF. We found that a TF serves as a major barrier to ISW1a remodeling, and acts as a boundary for nucleosome repositioning. In contrast, SWI/SNF was able to slide a nucleosome past a TF, with concurrent eviction of the TF from the DNA, and the TF did not significantly impact the nucleosome positioning. Our results provide direct evidence for a novel mechanism for both nucleosome positioning regulation by bound TFs and TF regulation via dynamic repositioning of nucleosomes.