Polynucleosome Arrays Research Articles

The Dynamic Influence of Linker Histone Saturation within the Poly-Nucleosome Array

Linker histones bind to nucleosomes and modify chromatin structure and dynamics as a means of epigenetic regulation. Biophysical studies have shown that chromatin fibers can adopt a plethora of conformations with varying levels of compaction. Linker histone condensation, and its specific binding disposition, has been associated with directly tuning this ensemble of states. However, the atomistic dynamics and quantification of this mechanism remains poorly understood. Here, we present molecular dynamics simulations of octa-nucleosome arrays, based on a cryo-EM structure of the 30-nm chromatin fiber, with and without the globular domains of the H1 linker histone to determine how they influence fiber structures and dynamics. Results show that when bound, linker histones inhibit DNA flexibility and stabilize repeating tetra-nucleosomal units, giving rise to increased chromatin compaction. Furthermore, upon the removal of H1, there is a significant destabilization of this compact structure as the fiber adopts less strained and untwisted states. Interestingly, linker DNA sampling in the octa-nucleosome is exaggerated compared to its mono-nucleosome counterparts, suggesting that chromatin architecture plays a significant role in DNA strain even in the absence of linker histones. Moreover, H1-bound states are shown to have increased stiffness within tetra-nucleosomes, but not between them. This increased stiffness leads to stronger long-range correlations within the fiber, which may result in the propagation of epigenetic signals over longer spatial ranges. These simulations highlight the effects of linker histone binding on the internal dynamics and global structure of poly-nucleosome arrays, while providing physical insight into a mechanism of chromatin compaction.

Single-molecule and in silico dissection of the interaction between Polycomb repressive complex 2 and chromatin

Polycomb repressive complex 2 (PRC2) installs and spreads repressive histone methylation marks on eukaryotic chromosomes. Because of the key roles that PRC2 plays in development and disease, how this epigenetic machinery interacts with DNA and nucleosomes is of major interest. Nonetheless, the mechanism by which PRC2 engages with native-like chromatin remains incompletely understood. In this work, we employ single-molecule force spectroscopy and molecular dynamics simulations to dissect the behavior of PRC2 on polynucleosome arrays. Our results reveal an unexpectedly diverse repertoire of PRC2 binding configurations on chromatin. Besides reproducing known binding modes in which PRC2 interacts with bare DNA, mononucleosomes, and adjacent nucleosome pairs, our data also provide direct evidence that PRC2 can bridge pairs of distal nucleosomes. In particular, the "1-3" bridging mode, in which PRC2 engages two nucleosomes separated by one spacer nucleosome, is a preferred low-energy configuration. Moreover, we show that the distribution and stability of different PRC2-chromatin interaction modes are modulated by accessory subunits, oncogenic histone mutations, and the methylation state of chromatin. Overall, these findings have implications for the mechanism by which PRC2 spreads histone modifications and compacts chromatin. The experimental and computational platforms developed here provide a framework for understanding the molecular basis of epigenetic maintenance mediated by Polycomb-group proteins.

Murine leukemia virus p12 tethers the capsid-containing pre-integration complex to chromatin by binding directly to host nucleosomes in mitosis.

The murine leukaemia virus (MLV) Gag cleavage product, p12, is essential for both early and late steps in viral replication. The N-terminal domain of p12 binds directly to capsid (CA) and stabilises the mature viral core, whereas defects in the C-terminal domain (CTD) of p12 can be rescued by addition of heterologous chromatin binding sequences (CBSs). We and others hypothesised that p12 tethers the pre-integration complex (PIC) to host chromatin ready for integration. Using confocal microscopy, we have observed for the first time that CA localises to mitotic chromatin in infected cells in a p12-dependent manner. GST-tagged p12 alone, however, did not localise to chromatin and mass-spectrometry analysis of its interactions identified only proteins known to bind the p12 region of Gag. Surprisingly, the ability to interact with chromatin was conferred by a single amino acid change, M63I, in the p12 CTD. Interestingly, GST-p12_M63I showed increased phosphorylation in mitosis relative to interphase, which correlated with an increased interaction with mitotic chromatin. Mass-spectrometry analysis of GST-p12_M63I revealed nucleosomal histones as primary interactants. Direct binding of MLV p12_M63I peptides to histones was confirmed by biolayer-interferometry (BLI) assays using highly-avid recombinant poly-nucleosomal arrays. Excitingly, using this method, we also observed binding between MLV p12_WT and nucleosomes. Nucleosome binding was additionally detected with p12 orthologs from feline and gibbon ape leukemia viruses using both pull-down and BLI assays, indicating that this a common feature of gammaretroviral p12 proteins. Importantly, p12 peptides were able to block the binding of the prototypic foamy virus CBS to nucleosomes and vice versa, implying that their docking sites overlap and suggesting a conserved mode of chromatin tethering for different retroviral genera. We propose that p12 is acting in a similar capacity to CPSF6 in HIV-1 infection by facilitating initial chromatin targeting of CA-containing PICs prior to integration.

Understanding Chromatin Structure and Dynamics: A Molecular Dynamics Study

Chromatin fibers are a complex of DNA and proteins, which are the building blocks of chromosomes within the nucleus of eukaryotic cells. They consist of repeating units of the nucleosome core particle (NCP), which consists of ∼147 DNA base pairs densely wrapped around a histone octamer. The structure and dynamics of poly-NCP arrays therefore directly influences several processes, including gene expression, DNA replication, and DNA repair. Here, we report results from a series of fully atomistic molecular dynamics simulations of polynucleosomal arrays. Analyses focuses on the stability and conformational dynamics of the chromatin fiber, along with the importance of stacking interactions between adjacent nucleosomes and inter-NCP contacts formed by the of histone tails. These results build on previous experimental and simulation results of single NCP systems, and shed new light on the relationship between the structure, function, and dynamics of chromatin fibers.

SWI/SNF Has Intrinsic Nucleosome Disassembly Activity that Is Dependent on Adjacent Nucleosomes

The ATP-dependent chromatin remodeling complex SWI/SNF regulates transcription and has been implicated in promoter nucleosome eviction. Efficient nucleosome disassembly by SWI/SNF alone in biochemical assays, however, has not been directly observed. Employing a model system of dinucleosomes rather than mononucleosomes, we demonstrate that remodeling leads to ordered and efficient disassembly of one of the two nucleosomes. An H2A/H2B dimer is first rapidly displaced, and then, in a slower reaction, an entire histone octamer is lost. Nucleosome disassembly by SWI/SNF did not require additional factors such as chaperones or acceptors of histones. Observations in single molecules as well as bulk measurement suggest that a key intermediate in this process is one in which a nucleosome is moved toward the adjacent nucleosome. SWI/SNF recruited by the transcriptional activator Gal4-VP16 preferentially mobilizes the proximal nucleosome and destabilizes the adjacent nucleosome.

Molecular dynamics of DNA and nucleosomes in solution studied by fast-scanning atomic force microscopy

Nucleosome is a fundamental structural unit of chromatin, and the exposure from or occlusion into chromatin of genomic DNA is closely related to the regulation of gene expression. In this study, we analyzed the molecular dynamics of poly-nucleosomal arrays in solution by fast-scanning atomic force microscopy (AFM) to obtain a visual glimpse of nucleosome dynamics on chromatin fiber at single molecule level. The influence of the high-speed scanning probe on nucleosome dynamics can be neglected since bending elastic energy of DNA molecule showed similar probability distributions at different scan rates. In the sequential images of poly-nucleosomal arrays, the sliding of the nucleosome core particle and the dissociation of histone particle were visualized. The sliding showed limited fluctuation within ∼50 nm along the DNA strand. The histone dissociation occurs by at least two distinct ways: a dissociation of histone octamer or sequential dissociations of tetramers. These observations help us to develop the molecular mechanisms of nucleosome dynamics and also demonstrate the ability of fast-scanning AFM for the analysis of dynamic protein–DNA interaction in sub-seconds time scale.

Repression by Groucho/TLE/Grg Proteins: Genomic Site Recruitment Generates Compacted Chromatin In Vitro and Impairs Activator Binding In Vivo

Groucho-related (Gro/TLE/Grg) corepressors meditate embryonic segmentation, dorsal-ventral patterning, neurogenesis, and Notch and Wnt signaling. Although Gro/TLE/Grgs disrupt activator complexes and recruit histone deacetylases (HDAC), activator complexes can be disrupted in various ways, HDAC recruitment does not account for full corepressor activity, and a direct role for Gro/TLE/Grg binding and altering chromatin structure has not been explored. Using diverse chromatin substrates in vitro, we show that Grg3 creates higher-order, condensed complexes of polynucleosome arrays. Surprisingly, such complexes are in an open, exposed configuration. We find that chromatin binding enables Grg3 recruitment by a transcription factor and the creation of a closed, poorly accessible domain spanning three to four nucleosomes. Targeted recruitment of Grg3 blankets a similar-sized region in vivo, impairing activator recruitment and repressing transcription. These activities of a Groucho protein represent a newly discovered mechanism which differs from that of other classes of corepressors.

Human SWI/SNF Generates Abundant, Structurally Altered Dinucleosomes on Polynucleosomal Templates

Human SWI/SNF (hSWI/SNF) is an evolutionarily conserved ATP-dependent chromatin remodeling complex required for transcriptional regulation and cell cycle control. The regulatory functions of hSWI/SNF are correlated with its ability to create a stable, altered form of chromatin that constrains fewer negative supercoils than normal. Our current studies indicate that this change in supercoiling is due to the conversion of up to one-half of the nucleosomes on polynucleosomal arrays into asymmetric structures, termed "altosomes," each composed of two histone octamers and bearing an asymmetrically located region of nuclease-accessible DNA. Altosomes can be formed on chromatin containing the abundant mammalian linker histone H1 and have a unique micrococcal nuclease digestion footprint that allows their position and abundance on any DNA sequence to be measured. Over time, altosomes spontaneously revert to structurally normal but improperly positioned nucleosomes, suggesting a novel mechanism for transcriptional attenuation as well as transcriptional memory following hSWI/SNF action.

The pituitary-specific transcription factor, Pit-1, can direct changes in the chromatin structure of the prolactin promoter.

The chromatin structure of a promoter is an important determinant of its transcriptional activity. Many promoters are assembled into repressive polynucleosomal arrays that are subsequently remodeled to allow for the activation of gene expression. This study addresses the contribution of a single transcription factor, Pit-1, in orchestrating the chromatin structure of the prolactin gene. Utilizing an in vivo reconstitution system, we found that Pit-1 can bind to multiple sites in the chromatin-assembled 5'-flanking region of the prolactin gene and activate transcription from the chromatin-assembled template. Interestingly, Pit-1 was able to substantially alter micrococcal nuclease digestion of the prolactin 5'-flanking region, and the results are consistent with presence of a translationally positioned nucleosome on the prolactin promoter. Changes in micrococcal nuclease digestion were also observed with a truncated Pit-1 mutant containing only the DNA-binding domain. As the truncation mutant was unable to activate transcription from the chromatin-assembled template, the ability of Pit-1 to alter chromatin structure of the prolactin gene is not dependent on transcriptional activation. We propose that Pit-1 likely plays a role in altering chromatin to facilitate recruitment and subsequent transcriptional activation by additional factors.

ATP-dependent Remodeling by SWI/SNF and ISWI Proteins Stimulates V(D)J Cleavage of 5 S Arrays

Control of V(D)J recombination is critical for the generation of a fully developed immune repertoire. The molecular mechanisms underlying the regulation of antigen receptor gene assembly are beginning to be revealed. Here we studied the influence of chromatin modifications on V(D)J cleavage of a polynucleosomal substrate, in which V(D)J cleavage is greatly reduced compared with naked DNA. ATP-dependent remodeling by human SWI/SNF (hSWI/SNF) in the presence of HMG1 led to a substantial increase of cleavage by the recombination activation gene (RAG) proteins. Either BRG1, the ATPase subunit of hSWI/SNF, or SNF2h, the ATPase of human ISWI complexes, was capable of stimulating V(D)J cleavage of the array, although these remodelers act by different mechanisms. No effect of histone hyperacetylation was detectable in this system. As is observed on naked DNA, in the presence of core RAG1, the full-length RAG2 protein proved to be more active than core RAG2 on these polynucleosomal arrays, reinforcing the importance of the RAG2 C-terminal domain for efficient recombination. Comparison of 5 S array cleavage by the RAG proteins or by the restriction enzyme HhaI after remodeling by hSWI/SNF suggested that RAG proteins and HhaI might have different requirements for maximal accessibility of the substrate.

Identification of a 34 amino acid stretch within the C-terminus of histone H1 as the DNA-condensing domain by site-directed mutagenesis.

The C-terminus of histone H1 is necessary for the folding of polynucleosomal arrays into higher-order structure(s) and contains octapeptide repeats each having DNA binding S/TPKK motifs. These repeat motifs were earlier shown to mimic the DNA/chromatin-condensing properties of the C-terminus of histone H1 (Khadake, J. R., and Rao, M. R. S. (1995) Biochemistry 36, 1041-1051). In the present study, we have generated a series of C-terminal mutants of rat histone H1d and studied their DNA-condensation properties. The single proline to alanine mutation in the S/TPKK motifs either singly or in combination resulted in only a 20% decrease in the DNA-condensation property of histone H1. Deletion of all the three S/TPKK motifs resulted in a 45% decrease in DNA condensation. When the three octapeptide repeats encompassing the S/TPKK motifs were deleted, there was again a 45% decrease in DNA condensation. On the other hand, when the entire 34 amino acid stretch (residue 145-178) was deleted, there was nearly a 90% decrease in DNA condensation brought about by histone H1d. Interestingly, deletion of the 10 amino acid spacer between the octapeptide repeats (residues 161-170) also reduced the DNA condensation by 70%. Deletion of the region (residues 115-141) immediately before the 34 amino acid stretch and after the globular domain and the region (residues 184-218) immediately after the 34 amino acid stretch had only a marginal effect on DNA condensation. The importance of the 34 amino acid stretch, including the 10 amino acid spacer, was also demonstrated with the recombinant histone H1d C-terminus. We have also determined the induced alpha-helicity of histone H1 and its various mutants in the presence of 60% trifluoroethanol, and the experimentally determined induced helical contents agree with the theoretical predictions of secondary structural elements in the C-terminus of histone H1d. Thus, we have identified a 34 amino acid stretch in the C-terminus of histone H1d as the DNA-condensing domain.

Population Analysis of Subsaturated 172-12 Nucleosomal Arrays by Atomic Force Microscopy Detects Nonrandom Behavior That Is Favored by Histone Acetylation and Short Repeat Length

Concatameric 5 S rDNA templates reconstituted in vitro into nucleosomal arrays provide very popular chromatin models for many kinds of studies. Here, atomic force microscopy is used to determine the population distributions for one such nucleosomal array, the 172-12, reconstituted to various subsaturated levels with nonacetylated or hyperacetylated HeLa histones. This array is a model for short linker length genomes and transcriptionally active and newly replicated chromatins. The analysis shows that as input histone levels increase, template occupation increases progressively as discrete population distributions. The distributions are random at low (n(av) < 4) and high (n(av) > 8) loadings but display specific nonrandom features, such as a deficit of molecules with one nucleosome more or less than the peak species in the distribution and enhanced distribution breadths, in the mid-range (n(av) = 4-8). Thus, the mid-range of occupation on polynucleosomal arrays may be a special range for chromatin structure and/or assembly. The mid-range nonrandom features are enhanced in distributions from short repeat (172-12) arrays, particularly for unacetylated chromatin, and in distributions from hyperacetylated chromatin, particularly for long repeat (208-12) arrays. Thus, short repeat length and acetylation can affect basic chromatin properties, like population tendencies, in very similar ways and therefore may cause similar changes in chromatin structure. Some possible effects are suggested. The data also indicate that it is thermodynamically more difficult for hyperacetylated nucleosomes to assemble onto the 172-12 templates, a result having implications for in vivo chromatin assembly.

The SWI/SNF complex creates loop domains in DNA and polynucleosome arrays and can disrupt DNA-histone contacts within these domains.

To understand the mechanisms by which the chromatin-remodeling SWI/SNF complex interacts with DNA and alters nucleosome organization, we have imaged the SWI/SNF complex with both naked DNA and nucleosomal arrays by using energy-filtered microscopy. By making ATP-independent contacts with DNA at multiple sites on its surface, SWI/SNF creates loops, bringing otherwise-distant sites into close proximity. In the presence of ATP, SWI/SNF action leads to the disruption of nucleosomes within domains that appear to be topologically constrained by the complex. The data indicate that the action of one SWI/SNF complex on an array of nucleosomes can lead to the formation of a region where multiple nucleosomes are disrupted. Importantly, nucleosome disruption by SWI/SNF results in a loss of DNA content from the nucleosomes. This indicates a mechanism by which SWI/SNF unwraps part of the nucleosomal DNA.

Nucleosome Positioning Properties of the Albumin Transcriptional Enhancer

Considering the importance of nucleosome position with regard to how regulatory factors recognize their binding sites in chromatin, we have investigated the inherent nucleosome positioning properties of a transcriptional enhancer of the albumin gene. In the liver, where the albumin gene is highly expressed, the enhancer exists in an array of precisely positioned, nucleosome-like particles with transcription factors bound. In the absence of specific binding factors, such as in non-liver tissues or in polynucleosome arrays assembled in vitro, nucleosomes are randomly positioned over the enhancer. Herein we investigate the intrinsic nucleosome positioning properties of the central enhancer sequence assembled into mononucleosome core particles in vitro. We find that the enhancer DNA prefers three translational positions, each of which utilizes different rotational settings on the nucleosome core. We conclude that DNA binding factors that position nucleosomes may do so by stabilizing one configuration out of several that can be adopted by the underlying DNA, and that the potential exists for different positions to be stabilized at different stages of development.

Interactions of nuclear proteins with DNA, during sperm differentiation in the ram.

Ram spermatid nuclei and caput epididymal sperm nuclei were prepared and treated with DTT under conditions avoiding proteolysis. Whole-mount preparations for the electron microscope were made in the presence or absence of the detergent Joy. The chromatin of the less mature, non-round spermatid nuclei displayed a nucleosomal organization that gradually disappeared at the time the histones leave the nuclei (elongating spermatids). Digestion with micrococcal nuclease suggests that polynucleosome arrays are scarcer and more accessible to nuclease in the elongating than in the round nuclei, with increasing amounts of DNA becoming devoid of nucleosomes. In the protamine-containing nuclei (elongated spermatids), only smooth filaments were observed, which formed thick fibers by parallel aggregation. The change from a nucleosomal organization to bundles of smooth filaments appeared to result from a complex process involving the transitory presence of conspicuous "knobby fibers" that suggest a periodicity in the organization of the spermatidal proteins along the DNA molecules. X-ray diffraction patterns obtained with protamine-containing spermatid nuclei and with sperm nuclei confirm that the DNA is arranged in smoothly bent bundles of parallel molecules. No higher-order reflections that might correspond to nucleosome structures were detected in the 30-200 A region.

Chromatin structure of the potential Z-forming sequence (dT-dG) n · (dC-dA) n: Evidence for an “alternating- B” conformation

The sequence (dT-dG) n · (dC-dA) n is the most abundant purine-pyrimidine dinucleotide repeat in eukaryotic genomes. This sequence and certain others that contain alternating purine-pyrimidine residues have been shown to adopt the left-handed, Z-DNA conformation in vitro when subjected to negative torsional stress or elevated ionic strengths. We have asked whether (dT-dG) n · (dC-dA) n tracts exist in topologically constrained Z-form structures in vivo by examining the chromatin organization of these sequences in cultured mouse cell nuclei. We find that these elements are quantitatively packaged into typical core particles which are embedded in canonical polynucleosomal arrays. In addition, these sequences neither flank nor reside within regions of chromatin that are preferentially sensitive to S 1 nuclease. These characteristics suggest that these tracts do not exist predominantly in the Z-form in vivo. Furthermore, employing techniques that permit prominent hybridization to DNA fragments as short as 18 bases, we provide evidence that in vivo, most (dT-dG) n · (dC-dA) n elements instead adopt an “alternating- B” conformation on the nucleosomal surface.

Subunit structure of chromatin and the organization of eukaryotic highly repetitive DNA: Nucleosomal proteins associated with a highly repetitive mammalian DNA

Component alpha DNA is a homogeneous, highly repetitive fraction that comprises nearly a quarter of the African green monkey (Cercopithecus aethiops) genome. By restriction enzyme analysis, it has a repeat periodicity of 176 +/- 4 nucleotide base pairs, corresponding closely with the length of DNA contained within a nucleosome. The sequence is organized into large blocks of constitutive heterochromatin. A method is described here for the isolation of intact polynucleosomal arrays containing only component alpha sequences. Isolated monkey nuclei are treated with EcoRI, which releases only component alpha nucleosomal arrays; the arrays are then fractionated and purified by sedimentation in sucrose gradients. The method permits a compositional analysis of the proteins associated with a constitutively repressed, heterochromatic sequence. The major differences in the proteins associated with component alpha nucleosomes that distinguish them from the bulk DNA nucleosomes are a decrease in the content of the H1 histones in the component alpha nucleosomes and a concomitant increase in the amount of certain nonhistone proteins. The specific observations are: (i) In the component alpha nucleosomes, 65-70% of the proteins were nonhistone proteins; this contrasts with the value, 40%, for nonhistone proteins associated with nucleosomes containing bulk DNA. (ii) The amount of H1 histone in chromatin containing predominantly bulk DNA was about 13.7%. However, the H1 histone was depleted and possibly absent in component alpha oligonucleosomes. (iii) Coincident with the decrease in the H1 histones and in the same molecular weight range (24,000-43,000), there appeared five minor nonhistone proteins. The minor, low-molecular-weight, nonhistone proteins were not detected in chromatin containing bulk DNA but they represented nearly 12% of the protein in component alpha nucleosomes. The resistance to salt extraction (0.6-2.0 M NaCl) indicates that the low-molecular-weight nonhistone proteins are tenaciously bound to the component alpha nucleosomes. In addition, a class of high-molecular-weight (>100,000) nonhistone proteins was enriched 5- or 6-fold in component alpha oligonucleosomes. The relative amounts of the nucleosome core histones were not changed.