Nucleosomes in which histone H3 is replaced by CENP-A direct kinetochore assembly. CENP-A nucleosomes extracted from human and Drosophila cells have been reported to have reduced heights relative to canonical octameric H3 nucleosomes, suggesting a unique tetrameric, hemisomal composition. We demonstrate that even octameric CENP-A nucleosomes assembled in vitro exhibit a reduced height, indicating that they are physically distinct from H3 nucleosomes, and negating the need to invoke the presence of hemisomes.
Using high-throughput sequencing, we have mapped sequence-directed nucleosome positioning in vitro on four plasmid DNAs containing DNA fragments derived from the genomes of sheep, drosophila, human and yeast. Chromatins were prepared by reconstitution using chicken, frog and yeast core histones. We also assembled yeast chromatin in which histone H3 was replaced by the centromere-specific histone variant, Cse4. The positions occupied by recombinant frog and native chicken histones were found to be very similar. In contrast, nucleosomes containing the canonical yeast octamer or, in particular, the Cse4 octamer were assembled at distinct populations of locations, a property that was more apparent on particular genomic DNA fragments. The factors that may contribute to this variation in nucleosome positioning and the implications of the behavior are discussed.
•Chromatins were formed on plasmids containing fragments of sheep, drosophila, human and yeast genomic DNAs with four types of histone octamer.•Nucleosome positioning was measured for each reconstitute.•Although similar, the binding profiles obtained with the yeast histone octamer, including one containing Cse4, were distinct from those obtained with chick or frog histones.•The difference in nucleosome positioning was best seen on AT-rich DNA substrates.
chromatin; nucleosome positioning; core histones; histone variants; Cse4
A new study takes an evolutionary approach to investigate to what extent nucleosome positioning is determined by underlying sequence or by trans-acting factors.
Evolution; genome-wide organization; nucleosome positioning; poly(dA:dT) sequences; yeast
Chromatin provides both a means to accommodate a large amount of genetic material in a small space and a means to package the same genetic material in different chromatin states. Transitions between chromatin states are enabled by chromatin-remodeling ATPases, which catalyze a diverse range of structural transformations. Biochemical evidence over the last two decades suggests that chromatin-remodeling activities may have emerged by adaptation of ancient DNA translocases to respond to specific features of chromatin. Here, we discuss such evidence and also relate mechanistic insights to our understanding of how chromatin-remodeling enzymes enable different in vivo processes.
The mechanisms by which histones are disassembled and reassembled into nucleosomes and chromatin structure during DNA replication, repair and transcription are poorly understood. A better understanding of the processes involved is, however, crucial if we are to understand whether and how histone variants and post-translationally modified histones are inherited in an epigenetic manner. To this end we have studied the interaction of histones H3–H4 with the human retinoblastoma-associated protein RbAp48 and their exchange with a second histone chaperone, anti-silencing function protein 1 (ASF1). Exchange of histones H3–H4 between these two histone chaperones plays a central role in the assembly of new nucleosomes and we show here that the H3–H4 complex has a surprising structural plasticity, which is important for this exchange.
One of the major problems facing distance determination by pulsed EPR, on spin-labelled proteins, has been the short relaxation time Tm. Solvent deuteration has previously been used to slow relaxation and so extend the range of distance measurement and sensitivity. We demonstrate here that deuteration of the underlying protein, as well as the solvent, extends the Tm to a considerable degree. Longer Tm gives greatly enhanced sensitivity, much extended distance measurement, more reliable distance distribution calculation and better baseline correction.
PELDOR; DEER; Relaxation; EPR; Tm; deuteration
Poly(ADP-ribosyl)ation plays a major role in DNA repair, where it regulates chromatin relaxation as one of the critical events in the repair process. However, the molecular mechanism by which poly(ADP-ribose) modulates chromatin remains poorly understood. Here we identify the poly(ADP-ribose)-regulated protein APLF as a DNA damage-specific histone chaperone. APLF preferentially binds to the histone H3/H4 tetramer via its C-terminal acidic motif, which is homologous to the motif conserved in the histone chaperones of the NAP1L family (NAP1L motif). We further demonstrate that APLF exhibits histone chaperone activities in a manner that is dependent on its acidic domain and that the NAP1L motif is critical for the repair capacity of APLF in vivo. Finally, we identify structural analogues of APLF in lower eukaryotes with the ability to bind histones and localize to the sites of DNA-damage-induced poly(ADP-ribosyl)ation. Collectively, these findings define the involvement of histone chaperones in poly(ADP-ribose)-regulated DNA repair reactions.
Global genome repair (GG-NER) removes DNA damage from non-transcribing DNA. In Saccharomyces cerevisiae, the RAD7 and RAD16 genes are specifically required for GG-NER. We reported that autonomously replicating sequence-binding factor 1 (A BF1) protein forms a stable complex with Rad7 and Rad16 proteins. ABF1 functions in transcription, replication, gene silencing and NER in yeast. We show that binding of ABF1 to its DNA recognition sequence found at multiple genomic locations promotes efficient GG-NER in yeast. Mutation of the I silencer ABF1 binding site at the HMLα locus causes loss of ABF1 binding, which results in a domain of reduced GG-NER efficiency on one side of the ABF1 binding site. During GG-NER, nucleosome positioning at this site is not altered, and this correlates with an inability of the GG-NER complex to reposition nucleosomes in vitro. We discuss how the GG-NER complex might facilitate GG-NER, whilst preventing unregulated gene transcription during this process.
ALC1, a novel PARP1-stimulated chromatin-remodelling enzyme promotes DNA repair.
The Snf2 family represents a functionally diverse class of ATPase sharing the ability to modify DNA structure. Here we use a magnetic trap and an Atomic Force Microscope to monitor the activity of a member of this class: the RSC complex. This enzyme causes transient shortenings in DNA length involving translocation of typically 400 bp within 2 seconds resulting in the formation of a loop whose size depends on both the force applied to the DNA and the ATP concentration. The majority of loops decrease in size within a time similar to that with which they are formed suggesting that the motor has the ability to translocate in different directions. Loop formation is also associated with the generation of negative DNA supercoiling. These observations support the idea that the ATPase motors of the Snf2 family proteins act as DNA translocases specialised to generate transient distortions in DNA structure.
The NuRD (nucleosome remodeling and deacetylase) complex serves as a crucial epigenetic regulator of cell differentiation, proliferation, and hematopoietic development by coupling the deacetylation and demethylation of histones, nucleosome mobilization, and the recruitment of transcription factors. The core nucleosome remodeling function of the mammalian NuRD complex is executed by the helicase-domain-containing ATPase CHD4 (Mi-2β) subunit, which also contains N-terminal plant homeodomain (PHD) and chromo domains. The mode of regulation of chromatin remodeling by CHD4 is not well understood, nor is the role of its PHD and chromo domains. Here, we use small-angle X-ray scattering, nucleosome binding ATPase and remodeling assays, limited proteolysis, cross-linking, and tandem mass spectrometry to propose a three-dimensional structural model describing the overall shape and domain interactions of CHD4 and discuss the relevance of these for regulating the remodeling of chromatin by the NuRD complex.
► The ATPase CHD4 mediates nucleosome remodeling by the NuRD complex. ► We present a three-dimensional small-angle X-ray scattering model of CHD4 and define its interdomain interactions. ► Cross-linking and limited proteolysis studies validate our model. ► Functional and binding assays suggest a regulatory role for the PHD and chromo domains.
CHD, chromo domain helicase DNA binding; NuRD, nucleosome remodeling and deacetylase; PHD, plant homeodomain; SAXS, small-angle X-ray scattering; LC–MS/MS, liquid chromatography–tandem mass spectrometry; DUF, domain of unknown function; TEV, tobacco etch virus; HRP, horseradish peroxidase; BSA, bovine serum albumin; Bistris, 2-[bis(2-hydroxyethyl)amino]-2-(hydroxymethyl)propane-1,3-diol; NuRD complex; chromatin remodeling; chromo domain helicase DNA-binding protein 4; histone; transcriptional regulation
Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodelling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best characterised chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome Assembly Proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein cross-linking we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone mediated lysine acetylation. This alternate mode of histone interaction also provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.
Nucleosome assembly; Histone Chaperone; Nap1; Vps75; Chromatin
The variant histone macroH2A helps maintain X inactivation and gene silencing. Previous work implied that nucleosomes containing macroH2A cannot be remodeled by ISWI and SWI/SNF chromatin remodeling enzymes. Using approaches that prevent misassembly of macroH2A nucleosomes, we find that macroH2A nucleosomes are excellent substrates for both enzyme families. Interestingly, SWI/SNF, which is involved in gene activation, preferentially binds H2A nucleosomes over macroH2A nucleosomes, but ACF, an ISWI complex implicated in gene repression, shows no preference. Thus, macroH2A may help regulate the balance between activating and repressive remodeling complexes.
Alteration of chromatin structure is key in the regulation of gene transcription. Some protein complexes remodel chromatin in an ATP-dependent manner to favor access to particular sequences. These chromatin remodeling factors form four families, whose archetypes are the yeast RSC (SWI/SNF) complex, the fly ISWI, the mouse CHD1 and the yeast INO80. All possess an ATPase subunit similar to the SF-II helicases which hydrolyze ATP to track along DNA. Translocation and the resulting torque in the DNA could drive chromatin remodeling. While the RSC complex exhibits ATP-dependent translocation and introduces negative supercoils into bare DNA, the ISWI complex was believed to be inactive on bare DNA. However new tethered particle motion assays and AFM images show that in absence of ATP, ISWI binds the DNA molecule wrapping it in an histone-like manner. In the presence of ATP, ISWI generated loops with negative supercoils.
The positioning of nucleosomes within the coding regions of eukaryotic genes is aligned with respect to transcriptional start sites. This organization is likely to influence many genetic processes, requiring access to the underlying DNA. Here we show that the combined action of Isw1 and Chd1 nucleosome spacing enzymes is required to maintain this organization. In the absence of these enzymes regular positioning of the majority of nucleosomes is lost. Exceptions include the region upstream of the promoter, the +1 nucleosome and a subset of locations distributed throughout coding regions where other factors are likely to be involved. These observations indicated that ATP-dependent remodeling enzymes are responsible for directing the positioning of the majority of nucleosomes within the Saccharomyces cerevisiae genome.
► The passage of RNA polymerase is intricately coupled with chromatin alterations. ► These include the action of histone chaperones, modifying and remodelling enzymes. ► The interplay between these events is complex involving parallel pathways and feedback loops. ► Overall the process acts to ensure disruption of chromatin during transcription is transient.
Eukaryotic cells package their genomes into a nucleoprotein form called chromatin. The basic unit of chromatin is the nucleosome, formed by the wrapping of ∼147 bp of DNA around an octameric complex of core histones. Advances in genomic technologies have enabled the locations of nucleosomes to be mapped across genomes [1,2]. This has revealed a striking organisation with respect to transcribed genes in a diverse range of eukaryotes. This consists of a nucleosome depleted region upstream of promoters, with an array of well spaced nucleosomes extending into coding regions . This observation reinforces the links between chromatin organisation and transcription. Central to this is the paradox that while chromatin is required by eukaryotes to restrict inappropriate access to DNA, this must be overcome in order for genetic information to be expressed. This conundrum is at its most flagrant when considering the need for nucleic acid polymerase's to transit 1000's of based pairs of DNA wrapped as arrays of nucleosomes.
We have mapped sequence-directed nucleosome positioning on genomic DNA molecules using high-throughput sequencing. Chromatins, prepared by reconstitution with either chicken or frog histones, were separately digested to mononucleosomes using either micrococcal nuclease (MNase) or caspase-activated DNase (CAD). Both enzymes preferentially cleave internucleosomal (linker) DNA, although they do so by markedly different mechanisms. MNase has hitherto been very widely used to map nucleosomes, although concerns have been raised over its potential to introduce bias. Having identified the locations and quantified the strength of both the chicken or frog histone octamer binding sites on each DNA, the results obtained with the two enzymes were compared using a variety of criteria. Both enzymes displayed sequence specificity in their preferred cleavage sites, although the nature of this selectivity was distinct for the two enzymes. In addition, nucleosomes produced by CAD nuclease are 8–10 bp longer than those produced with MNase, with the CAD cleavage sites tending to be 4–5 bp further out from the nucleosomal dyad than the corresponding MNase cleavage sites. Despite these notable differences in cleavage behaviour, the two nucleases identified essentially equivalent patterns of nucleosome positioning sites on each of the DNAs tested, an observation that was independent of the histone type. These results indicate that biases in nucleosome positioning data collected using MNase are, under our conditions, not significant.
► We measured nucleosome positioning using two distinct nucleases. ► CAD and MNase provided equivalent positioning profiles. ► The results were independent of DNA and histone type used to prepare chromatin. ► Our data are not consistent with the proposal that MNase provides biased nucleosome positioning measurements.
MNase, micrococcal nuclease; CAD, caspase-activated DNase; BLG, β-lactoglobulin; YRO, yeast replication origin; PDB, Protein Data Bank; caspase-activated DNase; nucleosome positioning; β-lactoglobulin; yeast replication origin; micrococcal nuclease
The large diversity in nucleosome-remodelling enzymes evokes great interest in unveiling common mechanistic themes in remodelling reactions. Here, the C-terminus of Chd1 contains a functionally important DNA-binding domain unexpectedly similar to the SANT and SLIDE domains in the ISWI ATPase.
The ATP-dependent chromatin-remodelling enzyme Chd1 is a 168-kDa protein consisting of a double chromodomain, Snf2-related ATPase domain, and a C-terminal DNA-binding domain. Here, we show the DNA-binding domain is required for Saccharomyces cerevisiae Chd1 to bind and remodel nucleosomes. The crystal structure of this domain reveals the presence of structural homology to SANT and SLIDE domains previously identified in ISWI remodelling enzymes. The presence of these domains in ISWI and Chd1 chromatin-remodelling enzymes may provide a means of efficiently harnessing the action of the Snf2-related ATPase domain for the purpose of nucleosome spacing and provide an explanation for partial redundancy between these proteins. Site directed mutagenesis was used to identify residues important for DNA binding and generate a model describing the interaction of this domain with DNA. Through inclusion of Chd1 sequences in homology searches SLIDE domains were identified in CHD6–9 proteins. Point mutations to conserved amino acids within the human CHD7 SLIDE domain have been identified in patients with CHARGE syndrome.
Chd1; DNA binding; nucleosomes; SANT; SLIDE
The SWI/SNF complex acts to constrain distribution of the centromeric histone variant Cse4
The SWI/SNF complex has an important role in regulating chromatin structure during transcriptional activation and DNA repair. Here, the SWI/SNF complex is also involved in the organisation of centromeric chromatin and prevention of the ectopic deposition of centromeric histone variants.
In order to gain insight into the function of the Saccharomyces cerevisiae SWI/SNF complex, we have identified DNA sequences to which it is bound genomewide. One surprising observation is that the complex is enriched at the centromeres of each chromosome. Deletion of the gene encoding the Snf2 subunit of the complex was found to cause partial redistribution of the centromeric histone variant Cse4 to sites on chromosome arms. Cultures of snf2Δ yeast were found to progress through mitosis slowly. This was dependent on the mitotic checkpoint protein Mad2. In the absence of Mad2, defects in chromosome segregation were observed. In the absence of Snf2, chromatin organisation at centromeres is less distinct. In particular, hypersensitive sites flanking the Cse4 containing nucleosomes are less pronounced. Furthermore, SWI/SNF complex was found to be especially effective in the dissociation of Cse4 containing chromatin in vitro. This suggests a role for Snf2 in the maintenance of point centromeres involving the removal of Cse4 from ectopic sites.
centromere; chromatin; Cse4; nucleosome; SWI/SNF
Histone chaperones physically interact with histones to direct proper assembly and disassembly of nucleosomes regulating diverse nuclear processes such as DNA replication, promoter remodeling, transcription elongation, DNA damage, and histone variant exchange. Currently, the best-characterized chaperone-histone interaction is that between the ubiquitous chaperone Asf1 and a dimer of H3 and H4. Nucleosome assembly proteins (Nap proteins) represent a distinct class of histone chaperone. Using pulsed electron double resonance (PELDOR) measurements and protein crosslinking, we show that two members of this class, Nap1 and Vps75, bind histones in the tetrameric conformation also observed when they are sequestered within the nucleosome. Furthermore, H3 and H4 trapped in their tetrameric state can be used as substrates in nucleosome assembly and chaperone-mediated lysine acetylation. This alternate mode of histone interaction provides a potential means of maintaining the integrity of the histone tetramer during cycles of nucleosome reassembly.
► Site-specific crosslinking of H3 shows Nap proteins bind a tetramer of H3 and H4 ► Tetrameric conformation confirmed by EPR measurements and in vivo crosslinking ► Nap1 can deposit a whole (H3-H4)2 tetramer onto DNA ► Vps75-Rtt109 preferentially acetylates tetrameric H3 and H4
Regulation of the Saccharomyces cerevisiae HO promoter has been shown to require the recruitment of chromatin-modifying and -remodeling enzymes. Despite this, relatively little is known about what changes to chromatin structure occur during the course of regulation at HO. Here, we used indirect end labeling in synchronized cultures to show that the chromatin structure is disrupted in a region that spans bp −600 to −1800 relative to the transcriptional start site. Across this region, there is a loss of canonical nucleosomes and a reduction in histone DNA cross-linking, as monitored by chromatin immunoprecipitation. The ATPase Snf2 is required for these alterations, but the histone acetyltransferase Gcn5 is not. This suggests that the SWI/SNF complex is directly involved in nucleosome removal at HO. We also present evidence indicating that the histone chaperone Asf1 assists in this. These observations suggest that SWI/SNF-related complexes in concert with histone chaperones act to remove histone octamers from DNA during the course of gene regulation.
The Saccharomyces cerevisiae Fun30 (Function unknown now 30) protein shares homology with an extended family of Snf2-related ATPases. Here we report the purification of Fun30 principally as a homodimer with a molecular mass of about 250 kDa. Biochemical characterization of this complex reveals that it has ATPase activity stimulated by both DNA and chromatin. Consistent with this, it also binds to both DNA and chromatin. The Fun30 complex also exhibits activity in ATP-dependent chromatin remodeling assays. Interestingly, its activity in histone dimer exchange is high relative to the ability to reposition nucleosomes. Fun30 also possesses a weakly conserved CUE motif suggesting that it may interact specifically with ubiquitinylated proteins. However, in vitro Fun30 was found to have no specificity in its interaction with ubiquitinylated histones.
Chromatin; Chromatin/Regulation; Chromatin/Remodeling; DNA/Protein Interaction; DNA/Transcription; Enzymes/ATPases; Gene/Regulation
The (H3-H4)2 histone tetramer forms the central core of nucleosomes and, as such, plays a prominent role in assembly, disassembly and positioning of nucleosomes. Despite its fundamental role in chromatin, the tetramer has received little structural investigation. Here, through the use of pulsed electron-electron double resonance spectroscopy coupled with site-directed spin labelling, we survey the structure of the tetramer in solution. We find that tetramer is structurally more heterogeneous on its own than when sequestered in the octamer or nucleosome. In particular, while the central region including the H3-H3′ interface retains a structure similar to that observed in nucleosomes, other regions such as the H3 αN helix display increased structural heterogeneity. Flexibility of the H3 αN helix in the free tetramer also illustrates the potential for post-translational modifications to alter the structure of this region and mediate interactions with histone chaperones. The approach described here promises to prove a powerful system for investigating the structure of additional assemblies of histones with other important factors in chromatin assembly/fluidity.
Lysine acetylation of histones defines the epigenetic status of human embryonic stem cells and orchestrates DNA replication, chromosome condensation, transcription, telomeric silencing, and DNA repair. A detailed mechanistic explanation of these phenomena is impeded by the limited availability of homogeneously acetylated histones. We report a general method for the production of homogeneously and site-specifically acetylated recombinant histones by genetically encoding acetyl-lysine. We reconstitute histone octamers, nucleosomes, and nucleosomal arrays bearing defined acetylated lysine residues. With these designer nucleosomes, we demonstrate that, in contrast to the prevailing dogma, acetylation of H3 K56 does not directly affect the compaction of chromatin and has modest effects on remodeling by SWI/SNF and RSC. Single-molecule FRET experiments reveal that H3 K56 acetylation increases DNA breathing 7-fold. Our results provide a molecular and mechanistic underpinning for cellular phenomena that have been linked with K56 acetylation.