Crystallographic and NMR approaches have provided a wealth of structural information about protein domains. However, often these domains are found as components of larger multi domain polypeptides or complexes. Orienting domains within such contexts can provide powerful new insight into their function. The combination of site specific spin labelling and Pulsed Electron Double Resonance (PELDOR) provide a means of obtaining structural measurements that can be used to generate models describing how such domains are oriented. Here we describe a pipeline for modelling the location of thio-reactive nitroxyl spin locations to engineered sties on the histone chaperone Vps75. We then use a combination of experimentally determined measurements and symmetry constraints to model the orientation in which homodimers of Vps75 associate to form homotetramers using the XPLOR-NIH platform. This provides a working example of how PELDOR measurements can be used to generate a structural model.
Cys, cysteine; Glu, glutamic acid; Tyr, tyrosine; Vps75, vacuolar protein sorting protein 75; PELDOR; DEER; XPLOR-NIH; MTSSLwizard; Chromatin; Vps75
•Echo decay curves were measured on segmentally deuterated, octameric proteins.•The echo dephasing time (Tm) was extracted from each data set.•Spin relaxation was correlated to the spatial distribution of protons.•Temperature and concentration dependence of Tm and T1 was measured.•The effect of deuteration on relaxation and dipolar coupling were discussed.
Pulsed electron–electron double resonance (PELDOR) coupled with site-directed spin labeling is a powerful technique for the elucidation of protein or nucleic acid, macromolecular structure and interactions. The intrinsic high sensitivity of electron paramagnetic resonance enables measurement on small quantities of bio-macromolecules, however short relaxation times impose a limit on the sensitivity and size of distances that can be measured using this technique. The persistence of the electron spin-echo, in the PELDOR experiment, is one of the most crucial limitations to distance measurement. At a temperature of around 50 K one of the predominant factors affecting persistence of an echo, and as such, the sensitivity and measurable distance between spin labels, is the electron spin echo dephasing time (Tm). It has become normal practice to use deuterated solvents to extend Tm and recently it has been demonstrated that deuteration of the underlying protein significantly extends Tm. Here we examine the spatial effect of segmental deuteration of the underlying protein, and also explore the concentration and temperature dependence of highly deuterated systems.
EPR; Relaxation; Tm; Spin-label; PELDOR; DEER; Deuteration
Chromatin facilitates the housing of eukaryotic DNA within the nucleus and restricts access to the underlying sequences. Thus, the regulation of chromatin structure provides an excellent platform for regulating processes that require information stored within genomic DNA. Snf2 proteins are a family of helicase-like proteins that direct energy derived from ATP hydrolysis into the mechanical remodelling of chromatin structure. Here, we highlight some of the recent discoveries regarding this family of proteins and show Snf2 proteins have roles in many aspects of genetic metabolism. Recent developments include new insights into the mechanism for nucleosome spacing and histone dimer exchange; together with growing evidence for the involvement of Snf2 proteins in DNA repair.
The PICH protein localises to ultra-fine anaphase DNA bridges (UFBs) in mitosis alongside a complex of DNA repair proteins, including BLM. However, very little is known about the function of PICH or how it is recruited selectively to UFBs. Using a combination of microfluidics, fluorescence microscopy and optical tweezers, we have defined the properties of PICH in an in vitro model of an anaphase bridge. We show that PICH binds with a remarkably high affinity to dsDNA, resulting in ATP-dependent protein translocation and extension of the DNA. Most strikingly, the affinity of PICH for binding dsDNA increases with tension-induced DNA stretching, which mimics the effect of the mitotic spindle on a UFB. PICH binding also appears to diminish force-induced DNA melting. We propose a new model in which PICH recognizes, binds to and stabilizes DNA under tension during anaphase, thereby facilitating the resolution of entangled sister chromatids prior to cytokinesis.
Chromatin remodelling is the ATP-dependent change in nucleosome organisation driven by Snf2 family ATPases. The biochemistry of this process depends on the behaviours of ATP dependent motor proteins and their dynamic nucleosome substrates, which brings significant technical and conceptual challenges. Steady progress has been made in characterizing the polypeptides that these enzymes are comprised of. Divergence in the sequences of different subfamilies of Snf2 related proteins suggests that the motors are adapted for different functions. Recently structural insights have suggested that the Snf2 ATPase acts as a context-sensitive DNA translocase. This may have arisen as a means to enable efficient access to DNA in the high density of the eukaryotic nucleus. How the enzymes engage nucleosomes and how the network of non-covalent interactions within the nucleosome respond to the force applied remains unclear, and it remains prudent to recognise the potential for both DNA distortions and dynamics within the underlying histone octamer structure.
NAP-1 fold histone chaperones play an important role in escorting histones to and from sites of nucleosome assembly and disassembly. The two NAP-1 fold histone chaperones in budding yeast, Vps75 and Nap1, have previously been crystalized in a characteristic homodimeric conformation. In this study, a combination of small angle X-ray scattering, multi angle light scattering and pulsed electron–electron double resonance approaches were used to show that both Vps75 and Nap1 adopt ring-shaped tetrameric conformations in solution. This suggests that the formation of homotetramers is a common feature of NAP-1 fold histone chaperones. The tetramerisation of NAP-1 fold histone chaperones may act to shield acidic surfaces in the absence of histone cargo thus providing a ‘self-chaperoning’ type mechanism.
Post-translational modifications of proteins have emerged as a major mechanism for regulating gene expression. However, our understanding of how histone modifications directly affect chromatin function remains limited. In this study, we investigate acetylation of histone H3 at lysine 64 (H3K64ac), a previously uncharacterized acetylation on the lateral surface of the histone octamer. We show that H3K64ac regulates nucleosome stability and facilitates nucleosome eviction and hence gene expression in vivo. In line with this, we demonstrate that H3K64ac is enriched in vivo at the transcriptional start sites of active genes and it defines transcriptionally active chromatin. Moreover, we find that the p300 co-activator acetylates H3K64, and consistent with a transcriptional activation function, H3K64ac opposes its repressive counterpart H3K64me3. Our findings reveal an important role for a histone modification within the nucleosome core as a regulator of chromatin function and they demonstrate that lateral surface modifications can define functionally opposing chromatin states.
DNA is a very long molecule, so it needs to be packaged carefully to fit into the nucleus of a cell. To achieve this, the DNA is wrapped around proteins called histones to form a structure termed a nucleosome, which is the building block of a more compacted substance called chromatin. However, to express the genes in the DNA it is necessary to open up parts of the chromatin to give various enzymes access to the DNA.
Cells often chemically modify histones by adding acetyl or methyl groups, and these modifications are known to influence what proteins can bind to the nucleosomes, which ultimately influences what genes are expressed in the cell at a given time. It has been suspected for some time that histone modifications can also influence gene expression more directly, but there has been little evidence for this idea.
Now Di Cerbo et al. have studied what happens when acetyl or methyl groups are added to a specific site within a histone called H3K64, which is close to where the DNA wraps around this histone. These experiments showed that this site tends to be acetylated when a nearby gene is active, and to be unmodified or methylated when this gene is not active. It appears that the addition of the acetyl group makes this region of the chromatin less stable: this, in turn, makes it easier for the chromatin to be unpacked, thus giving access to the enzymes that transcribe the DNA and allowing transcription to take place. The work of Di Cerbo et al. shows that methylation and acetylation at the same site within a histone can define two opposing states of chromatin and DNA: an active state and a repressive state.
histone; chromatin; acetylation; Human; Mouse
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