ISWI Remodelers Research Articles

ISWI catalyzes nucleosome sliding in condensed nucleosome arrays.

How chromatin enzymes work in condensed chromatin and how they maintain diffusional mobility inside remains unexplored. Here we investigated these challenges using the Drosophila ISWI remodeling ATPase, which slides nucleosomes along DNA. Folding of chromatin fibers did not affect sliding in vitro. Catalytic rates were also comparable in- and outside of chromatin condensates. ISWI cross-links and thereby stiffens condensates, except when ATP hydrolysis is possible. Active hydrolysis is also required for ISWI's mobility in condensates. Energy from ATP hydrolysis therefore fuels ISWI's diffusion through chromatin and prevents ISWI from cross-linking chromatin. Molecular dynamics simulations of a 'monkey-bar' model in which ISWI grabs onto neighboring nucleosomes, then withdraws from one before rebinding another in an ATP hydrolysis-dependent manner, qualitatively agree with our data. We speculate that monkey-bar mechanisms could be shared with other chromatin factors and that changes in chromatin dynamics caused by mutations in remodelers could contribute to pathologies.

A basic motif anchoring ISWI to nucleosome acidic patch regulates nucleosome spacing.

Recent studies have implicated the nucleosome acidic patch in the activity of ATP-dependent chromatin remodeling machines. We employed a photocrosslinking-based nucleosome profiling technology - ‘photoscanning’- to identify a conserved basic motif within the catalytic subunit of ISWI remodelers, SNF2h, which engages this nucleosomal epitope. This region of SNF2h is essential for chromatin remodeling activity in a reconstituted biochemical system and in cells. Our studies suggest that the basic motif in SNF2h plays a critical role in anchoring the remodeler to the nucleosomal surface. We also examine the functional consequences of several cancer-associated histone mutations that map to the nucleosome acidic patch. Kinetic studies employing physiologically relevant heterotypic nucleosomal substrates (‘Janus’ nucleosomes) indicate that these cancer mutations can disrupt regularly spaced chromatin structure by inducing ISWI-mediated unidirectional nucleosome sliding. These results imply a potential mechanistic link between oncogenic histones and alterations to the chromatin landscape.

The DNA repair protein SHPRH is a nucleosome-stimulated ATPase and a nucleosome-E3 ubiquitin ligase

BackgroundMaintenance of genome integrity during DNA replication is crucial to the perpetuation of all organisms. In eukaryotes, the bypass of DNA lesions by the replication machinery prevents prolonged stalling of the replication fork, which could otherwise lead to greater damages such as gross chromosomal rearrangements. Bypassing DNA lesions and subsequent repair are accomplished by the activation of DNA damage tolerance pathways such as the template switching (TS) pathway. In yeast, the RAD5 (Radiation-sensitive 5) protein plays a crucial role in initiating the TS pathway by catalyzing the polyubiquitination of PCNA (Proliferation Cell Nuclear Antigen). Likewise, one of the mammalian RAD5-homologs, SHPRH (SNF2, histone linker, PHD, RING, helicase) mediates PCNA polyubiquitination. To date, the study of SHPRH enzymatic functions has been limited to this modification. It is therefore unclear how SHPRH carries out its function in DNA repair. Moreover, how this protein regulates gene transcription at the enzymatic level is also unknown.ResultsGiven that SHPRH harbors domains found in chromatin remodeling proteins, we investigated its biochemical properties in the presence of nucleosomal substrates. We find that SHPRH binds equally well to double-stranded (ds) DNA and to nucleosome core particles, however, like ISWI and CHD-family remodelers, SHPRH shows a strong preference for nucleosomes presenting extranucleosomal DNA. Moreover, nucleosomes but not dsDNA strongly stimulate the ATPase activity of SHPRH. Intriguingly, unlike typically observed with SNF2-family enzymes, ATPase activity does not translate into conventional nucleosome remodeling, under standard assay conditions. To test whether SHPRH can act as a ubiquitin E3 ligase for nucleosomes, we performed a screen using 26 E2-conjugating enzymes. We uncover that SHPRH is a potent nucleosome E3-ubiquitin-ligase that can function with at least 7 different E2s. Mass spectrometry analyses of products generated in the presence of the UBE2D1-conjugating enzyme reveal that SHPRH can catalyze the formation of polyubiquitin linkages that are either branched or associated with the recruitment of DNA repair factors, as well as linkages involved in proteasomal degradation.ConclusionsWe propose that, in addition to polyubiquitinating PCNA, SHPRH promotes DNA repair or transcriptional regulation in part through chromatin ubiquitination. Our study sets a biochemical framework for studying other RAD5- and RAD16-related protein functions through the ubiquitination of nucleosomes.

Asymmetry between the two acidic patches dictates the direction of nucleosome sliding by the ISWI chromatin remodeler.

The acidic patch is a functionally important epitope on each face of the nucleosome that affects chromatin remodeling. Although related by 2-fold symmetry of the nucleosome, each acidic patch is uniquely positioned relative to a bound remodeler. An open question is whether remodelers are distinctly responsive to each acidic patch. Previously we reported a method for homogeneously producing asymmetric nucleosomes with distinct H2A/H2B dimers (Levendosky et al., 2016). Here, we use this methodology to show that the Chd1 remodeler from Saccharomyces cerevisiae and ISWI remodelers from human and Drosophila have distinct spatial requirements for the acidic patch. Unlike Chd1, which is equally affected by entry- and exit-side mutations, ISWI remodelers strongly depend on the entry-side acidic patch. Remarkably, asymmetry in the two acidic patches stimulates ISWI to slide mononucleosomes off DNA ends, overriding the remodeler's preference to shift the histone core toward longer flanking DNA.

Structural Architecture of the Nucleosome Remodeler ISWI Determined from Cross-Linking, Mass Spectrometry, SAXS, and Modeling

Chromatin remodeling factors assume critical roles by regulating access to nucleosomal DNA. To determine the architecture of the Drosophila ISWI remodeling enzyme, we developed an integrative structural approach that combines protein cross-linking, mass spectrometry, small-angle X-ray scattering, and computational modeling. The resulting structural model shows the ATPase module in a resting state with both ATPase lobes twisted against each other, providing support for a conformation that was recently trapped by crystallography. The autoinhibiting NegC region does not protrude from the ATPase module as suggested previously. The regulatory NTR domain is located near both ATPase lobes. The full-length enzyme is flexible and can adopt a compact structure in solution with the C-terminal HSS domain packing against the ATPase module. Our data imply a series of conformational changes upon activation of the enzyme and illustrate how the NTR, NegC, and HSS domains contribute to regulation of the ATPase module.

The Chd1 chromatin remodeler can sense both entry and exit sides of the nucleosome.

Chromatin remodelers are essential for establishing and maintaining the placement of nucleosomes along genomic DNA. Yet how chromatin remodelers recognize and respond to distinct chromatin environments surrounding nucleosomes is poorly understood. Here, we use Lac repressor as a tool to probe how a DNA-bound factor influences action of the Chd1 remodeler. We show that Chd1 preferentially shifts nucleosomes away from Lac repressor, demonstrating that a DNA-bound factor defines a barrier for nucleosome positioning. Rather than an absolute block in sliding, the barrier effect was achieved by altered rates of nucleosome sliding that biased redistribution of nucleosomes away from the bound Lac repressor site. Remarkably, in addition to slower sliding toward the LacO site, the presence of Lac repressor also stimulated sliding in the opposite direction. These experiments therefore demonstrate that Chd1 responds to the presence of a bound protein on both entry and exit sides of the nucleosome. This sensitivity to both sides of the nucleosome allows for a faster and sharper response than would be possible by responding to only the entry side, and we speculate that dual entry/exit sensitivity is also important for regularly spaced nucleosome arrays generated by Chd1 and the related ISWI remodelers.

Mobilization of hyperacetylated mononucleosomes by purified yeast ISW2 in vitro

Catalytic activity of ISWI chromatin remodelers, which regulate nucleosome positioning on the DNA, depends on interactions of the putative acidic patch in ISWI helicase domain with the N-termini of nucleosomal H4 – such, that removal of H4 termini abolishes ISWI remodeling. Acetylation of H4 termini is also known to disrupt H4 interactions with acidic protein surfaces, and thus, histone acetylation could potentially impede ISWI functions. Since active chromatin in vivo is hyperacetylated, it is important to clarify if ISWI activities can function on the in vivo hyperacetylated nucleosomes. We evaluated if purified yeast ISW2 can act on mononucleosomes in which all four core histones are highly acetylated. Mononucleosomes were assembled using purified histones from mammalian CV1 cells grown in the presence of deacetylase inhibitor Trichostatin A (TSA). The CV1 cell line is characterized by fast kinetic of accumulation of highly acetylated histone isoforms in response to TSA treatment. However, such ‘native’ histone hyperacetylation had no apparent effects on the nucleosome remodeling propensities, suggesting that histone hyperacetylation does not necessarily block ISWI functions and that ISWI enzymes can function on active chromatin as well.

Nucleosome spacing generated by ISWI and CHD1 remodelers is constant regardless of nucleosome density.

Arrays of regularly spaced nucleosomes are a hallmark of chromatin, but it remains unclear how they are generated. Recent genome-wide studies, in vitro and in vivo, showed constant nucleosome spacing even if the histone concentration was experimentally reduced. This counters the long-held assumption that nucleosome density determines spacing and calls for factors keeping spacing constant regardless of nucleosome density. We call this a clamping activity. Here, we show in a purified system that ISWI- and CHD1-type nucleosome remodelers have a clamping activity such that they not only generate regularly spaced nucleosome arrays but also generate constant spacing regardless of nucleosome density. This points to a functionally attractive nucleosome interaction that could be mediated either directly by nucleosome-nucleosome contacts or indirectly through the remodelers. Mutant Drosophila melanogaster ISWI without the Hand-Sant-Slide (HSS) domain had no detectable spacing activity even though it is known to remodel and slide nucleosomes. This suggests that the role of ISWI remodelers in generating constant spacing is not just to mediate nucleosome sliding; they actively contribute to the attractive interaction. Additional factors are necessary to set physiological spacing in absolute terms.

Regulatory motifs on ISWI chromatin remodelers: molecular mechanisms and kinetic proofreading

Recently, kinetic proofreading scenarios have been proposed for the regulation of chromatin remodeling, first on purely theoretical grounds (Blossey and Schiessel 2008 HFSP J. 2 167–70) and deduced from experiments on the ISWI/ACF system (Narlikar 2010 Curr. Opin. Chem. Biol. 14 660). In the kinetic proofreading scenario of chromatin remodeling, the combination of the recognition of a histone tail state and ATP-hydrolysis in the remodeler motor act together to select (i.e. proofread) a nucleosomal substrate. ISWI remodelers have recently been shown to have an additional level of regulation as they contain auto-inhibitory motifs which need to be inactivated through an interaction with the nucleosome. In this paper we show that the auto-regulatory effect enhances substrate recognition in kinetic proofreading. We further report some suggestive additional insights into the molecular mechanism underlying ISWI-autoregulation.

ISWI chromatin remodeling: one primary actor or a coordinated effort?

The ISWI family of ATP-dependent chromatin remodelers regulates transcription of coding and noncoding RNA by mobilizing nucleosomes and controlling the length of linker DNA separating nucleosomes (spacing). Nucleosome movement is tightly coupled to the DNA translocation activity of the helicase domain in the catalytic subunit. There may be other domains besides the helicase domain needed to move DNA in and out of nucleosomes. The C terminus of the ISWI catalytic subunit with the conserved HAND, SANT, and SLIDE domains may be involved in nucleosome spacing. There are several models of how the C terminus may facilitate in ISWI remodeling such as regulating the activity of the helicase domain and causing the helicase domain to translocate more efficiently on DNA or to enhance its selectivity for nucleosomes. Another possibility is that domains like SLIDE promote linker DNA entering into nucleosomes in a coordinated manner with the helicase domain.

Dynamic Regulation of Transcription Factors by Nucleosome Remodeling

In eukaryotic cells, transcription activation and repression are regulated by transcription factors (TFs) and nucleosomes. Although it has been demonstrated that a TF and a nucleosome can directly compete for binding to the same region of DNA, it is unclear whether and how such a competition may continue after a TF has been bound and a nucleosome has been assembled. In this study, we have addressed an aspect of this question by investigating whether a nucleosome under remodeling can displace a bound TF. We determined the locations of both a nucleosome and a TF on DNA by mechanically unzipping the DNA molecule. We found that under the action of ISWI remodeling, a nucleosome was not repositioned past a TF. In contrast, under the action of SWI/SNF remodeling, a nucleosome was repositioned past a TF with concurrent eviction of the TF from the DNA. Our results demonstrate a novel mechanism for how TFs may be regulated via dynamic repositioning of nucleosomes.

Regulation of ATP‐dependent Chromatin Remodeling

The objective of our research is to understand how ATP‐dependent chromatin remodeling machines (Remodelers) are regulated. Remodelers contain a catalytic ATPase subunit that conducts DNA translocation, used to pump DNA around the nucleosome. Here, we define and characterize new domains/regions present on the ATPases of the RSC and ISWI remodelers, and show how they regulate ATPase, translocase and nucleosome remodeling activities.The catalytic ATPase of the RSC remodeler is bound by a heterodimer of actin‐related proteins (ARPs). Here, we present a crystal structure of the ARP module at high resolution. In order to understand how ARPs regulate DNA translocation, we applied genetic, biophysical and biochemical approaches. Here, we reveal a new domain that functions as a negative regulator, restricting ATPase and DNA translocation activities, which is antagonized by the ARP/HSA module. For ISWI remodelers, we reveal an autoinhibition mechanism for ISWI regulation involving an N‐terminal domain, and also define a novel region in the C‐terminus of the ISWI enzyme important for the coupling of energy‐consumption with DNA translocation. Together, these discoveries emphasize the importance of the regulation of ATPase, translocase and remodeling activities by an elaborate network of domains. This work is supported by NIH GM60415 and HHMI.

ISWI and CHD chromatin remodelers bind to promoters but act in gene bodies

ATP-dependent nucleosome remodelers influence genetic processes by altering nucleosome occupancy, positioning, and composition. In vitro, Saccharomyces cerevisiae ISWI and CHD remodelers require ∼30-85 bp of extranucleosomal DNA to reposition nucleosomes, but linker DNA in S. cerevisiae averages <20 bp. To address this discrepancy between in vitro and in vivo observations, we have mapped the genomic distributions of the yeast Isw1, Isw2, and Chd1 remodelers at base-pair resolution on native chromatin. Although these remodelers act in gene bodies, we find that they are also highly enriched at nucleosome-depleted regions (NDRs), where they bind to extended regions of DNA adjacent to particular transcription factors. Surprisingly, catalytically inactive remodelers show similar binding patterns. We find that remodeler occupancy at NDRs and gene bodies is associated with nucleosome turnover and transcriptional elongation rate, suggesting that remodelers act on regions of transient nucleosome unwrapping or depletion within gene bodies subsequent to transcriptional elongation.

ISWI and CHD Chromatin Remodelers Bind Promoters but Act in Gene Bodies

ATP-dependent nucleosome remodelers influence genetic processes by altering nucleosome occupancy, positioning, and composition. In vitro, Saccharomyces cerevisiae ISWI and CHD remodelers require ∼30–85 bp of extranucleosomal DNA to reposition nucleosomes, but linker DNA in S. cerevisiae averages <20 bp. To address this discrepancy between in vitro and in vivo observations, we have mapped the genomic distributions of the yeast Isw1, Isw2, and Chd1 remodelers at base-pair resolution on native chromatin. Although these remodelers act in gene bodies, we find that they are also highly enriched at nucleosome-depleted regions (NDRs), where they bind to extended regions of DNA adjacent to particular transcription factors. Surprisingly, catalytically inactive remodelers show similar binding patterns. We find that remodeler occupancy at NDRs and gene bodies is associated with nucleosome turnover and transcriptional elongation rate, suggesting that remodelers act on regions of transient nucleosome unwrapping or depletion within gene bodies subsequent to transcriptional elongation.

Single-Molecule Studies of Nucleosome Translocation by the ACF Chromatin Remodeling Complex

The packaging of eukaryotic genomes as chromatin restricts access to the DNA for critical processes such as replication, transcription, recombination, and repair. Thus, eukaryotic cells depend on a dynamic balance between genome compaction and access facilitated in part by chromatin remodeling enzymes (remodelers). Remodelers read epigenetic marks such as histone modifications and use the energy of ATP hydrolysis to assemble, disassemble, reposition, and modify the composition of nucleosomes. Single-molecule techniques enable characterization of transient intermediates formed during the remodeling reaction and can therefore uncover previously unobtainable insights into the mechanisms of chromatin remodeling.In this study, we developed a single-molecule fluorescence resonance energy transfer assay to study the dynamics of chromatin remodeling by human ACF, a prototypical member of the ISWI family of remodelers. ISWI remodelers are endowed with the ability to create regularly-spaced nucleosome arrays characteristic of transcriptionally silent heterochromatin.With the addition of ACF and ATP, nucleosomes exhibit gradual translocation along the DNA interrupted by kinetic pauses after approximately seven or three base pairs (bp) of translocation, thereby dividing the remodeling process into alternating translocation phases and pause phases. Moreover, we found that ACF is a highly processive and bidirectional nucleosome translocase capable of sliding a nucleosome back-and-forth for an average of 200 bp before dissociating.The nucleosome spacing activity of ISWI remodelers is regulated by two substrate features: (1) length of linker DNA, and (2) histone H4 N-terminal tail. Shortening the linker DNA or altering the H4 tail decrease the catalytic activity of ISWI remodelers with little effect on binding affinity. We discovered that the catalytic defects associated with these substrate modifications are attributed solely to changes in the pause phases and not the translocation phases, providing new insights into how nucleosomal features regulate chromatin remodelers.

ISWI Remodelers Slide Nucleosomes with Coordinated Multi-Base-Pair Entry Steps and Single-Base-Pair Exit Steps

ISWI-family enzymes remodel chromatin by sliding nucleosomes along DNA, but the nucleosome translocation mechanism remains unclear. Here we use single-molecule FRET to probe nucleosome translocation by ISWI-family remodelers. Distinct ISWI-family members translocate nucleosomes with a similar stepping pattern maintained by the catalytic subunit of the enzyme. Nucleosome remodeling begins with a 7 bp step of DNA translocation followed by 3 bp subsequent steps toward the exit side of nucleosomes. These multi-bp, compound steps are comprised of 1 bp substeps. DNA movement on the entry side of the nucleosome occurs only after 7 bp of exit-side translocation, and each entry-side step draws in a 3 bp equivalent of DNA that allows three additional base pairs to be moved to the exit side. Our results suggest a remodeling mechanism with well-defined coordination at different nucleosomal sites featuring DNA translocation toward the exit side in 1 bp steps preceding multi-bp steps of DNA movement on the entry side.

Functional Antagonism between Sas3 and Gcn5 Acetyltransferases and ISWI Chromatin Remodelers

Chromatin-modifying enzymes and ATP-dependent remodeling complexes have been intensely studied individually, yet how these activities are coordinated to ensure essential cell functions such as transcription, replication, and repair of damage is not well understood. In this study, we show that the critical loss of Sas3 and Gcn5 acetyltransferases in yeast can be functionally rescued by inactivation of ISWI remodelers. This genetic interaction depends on the ATPase activities of Isw1 and Isw2, suggesting that it involves chromatin remodeling activities driven by the enzymes. Genetic dissection of the Isw1 complexes reveals that the antagonistic effects are mediated specifically by the Isw1a complex. Loss of Sas3 and Gcn5 correlates with defective RNA polymerase II (RNAPII) occupancy at actively transcribed genes, as well as a significant loss of H3K14 acetylation. Inactivation of the Isw1a complex in the acetyltransferase mutants restores RNAPII recruitment at active genes, indicating that transcriptional regulation may be the mechanism underlying suppression. Dosage studies and further genetic dissection reveal that the Isw1b complex may act in suppression through down-regulation of Isw1a. These studies highlight the importance of balanced chromatin modifying and remodeling activities for optimal transcription and cell growth.

Human ISWI chromatin-remodeling complexes sample nucleosomes via transient binding reactions and become immobilized at active sites

Chromatin remodeling complexes can translocate nucleosomes along the DNA in an ATP-dependent manner. Here, we studied autofluorescent protein constructs of the human ISWI family members Snf2H, Snf2L, the catalytically inactive Snf2L+13 splice variant, and the accessory Acf1 subunit in living human and mouse cells by fluorescence microscopy/spectroscopy. Except for Snf2L, which was not detected in the U2OS cell line, the endogenous ISWI proteins were abundant at nuclear concentrations between 0.14 and 0.83 μM. A protein interaction analysis showed the association of multimeric Snf2H and Acf1 into a heterotetramer or higher-order ACF complex. During the G1/2 cell cycle phase, Snf2H and Snf2L displayed average residence times <150 ms in the chromatin-bound state. The comparison of active and inactive Snf2H/Snf2L indicated that an immobilized fraction potentially involved in active chromatin remodeling comprised only 1-3%. This fraction was largely increased at replication foci in S phase or at DNA repair sites. To rationalize these findings we propose that ISWI remodelers operate via a "continuous sampling" mechanism: The propensity of nucleosomes to be translocated is continuously tested in transient binding reactions. Most of these encounters are unproductive and efficient remodeling requires an increased binding affinity to chromatin. Due to the relatively high intranuclear remodeler concentrations cellular response times for repositioning a given nucleosome were calculated to be in the range of tens of seconds to minutes.

ISWI remodeling complexes in Xenopus egg extracts: identification as major chromosomal components that are regulated by INCENP-aurora B.

We previously characterized major components of mitotic chromosomes assembled in Xenopus laevis egg extracts and collectively referred to them as Xenopus chromosome-associated polypeptides (XCAPs). They included five subunits of the condensin complex essential for chromosome condensation. In an effort to identify novel proteins involved in this process, we have isolated XCAP-F and found it to be the Xenopus ortholog of ISWI, a chromatin remodeling ATPase. ISWI exists in two major complexes in Xenopus egg extracts. The first complex contains ACF1 and two low-molecular-weight subunits, most likely corresponding to Xenopus CHRAC. The second complex is a novel one that contains the Xenopus ortholog of the human Williams syndrome transcription factor (WSTF). In the absence of the ISWI complexes, the deposition of histones onto DNA is apparently normal, but the spacing of nucleosomes is greatly disturbed. Despite the poor spacing of nucleosomes, ISWI depletion has little effect on DNA replication, chromosome condensation or sister chromatid cohesion in the cell-free extracts. The association of ISWI with chromatin is cell cycle regulated and is under the control of the INCENP-aurora B kinase complex that phosphorylates histone H3 during mitosis. Apparently contradictory to the generally accepted model, we find that neither chromosome condensation nor chromosomal targeting of condensin is compromised when H3 phosphorylation is drastically reduced by depletion of INCENP-aurora B.