Compaction Of Chromatin Fiber Research Articles

Structural basis for linker histone H5-nucleosome binding and chromatin fiber compaction.

The hierarchical packaging of chromatin fibers plays a critical role in gene regulation. The 30-nm chromatin fibers, a central-level structure bridging nucleosomal arrays to higher-order organizations, function as the first level of transcriptional dormant chromatin. The dynamics of 30-nm chromatin fiber play a crucial role in biological processes related to DNA. Here, we report a 3.6-angstrom resolution cryogenic electron microscopy structure of H5-bound dodecanucleosome, i.e., the chromatin fiber reconstituted in the presence of linker histone H5, which shows a two-start left-handed double helical structure twisted by tetranucleosomal units. An atomic structural model of the H5-bound chromatin fiber, including an intact chromatosome, is built, which provides structural details of the full-length linker histone H5, including its N-terminal domain and an HMG-motif-like C-terminal domain. The chromatosome structure shows that H5 binds the nucleosome off-dyad through a three-contact mode in the chromatin fiber. More importantly, the H5-chromatin structure provides a fine molecular basis for the intra-tetranucleosomal and inter-tetranucleosomal interactions. In addition, we systematically validated the physiological functions and structural characteristics of the tetranucleosomal unit through a series of genetic and genomic studies in Saccharomyces cerevisiae and in vitro biophysical experiments. Furthermore, our structure reveals that multiple structural asymmetries of histone tails confer a polarity to the chromatin fiber. These findings provide structural and mechanistic insights into how a nucleosomal array folds into a higher-order chromatin fiber with a polarity in vitro and in vivo.

Local chromatin fiber folding represses transcription and loop extrusion in quiescent cells.

A longstanding hypothesis is that chromatin fiber folding mediated by interactions between nearby nucleosomes represses transcription. However, it has been difficult to determine the relationship between local chromatin fiber compaction and transcription in cells. Further, global changes in fiber diameters have not been observed, even between interphase and mitotic chromosomes. We show that an increase in the range of local inter-nucleosomal contacts in quiescent yeast drives the compaction of chromatin fibers genome-wide. Unlike actively dividing cells, inter-nucleosomal interactions in quiescent cells require a basic patch in the histone H4 tail. This quiescence-specific fiber folding globally represses transcription and inhibits chromatin loop extrusion by condensin. These results reveal that global changes in chromatin fiber compaction can occur during cell state transitions, and establish physiological roles for local chromatin fiber folding in regulating transcription and chromatin domain formation.

Surface structures consisting of chromatin fibers in isolated barley (Hordeum vulgare) chromosomes revealed by helium ion microscopy.

The chromosome compaction of chromatin fibers results in the formation of the nucleosome, which consists of a DNA unit coiled around a core of histone molecules associated with linker histone. The compaction of chromatin fibers has been a topic of controversy since the discovery of chromosomes in the 19th century. Although chromatin fibers were first identified using electron microscopy, the chromatin fibers on the surface of chromosome structures in plants remain unclear due to shrinking and breaking caused by prior chromosome isolation or preparation with alcohol and acid fixation, and critical point drying occurred into dehydration and denatured chromosomal proteins. This study aimed to develop a high-quality procedure for the isolation and preparation of plant chromosomes, maintaining the native chromosome structure, to elucidate the organization of chromatin fibers on the surface of plant chromosomes by electron microscopy. A simple technique to isolate intact barley (Hordeum vulgare) chromosomes with a high yield was developed, allowing chromosomes to be observed with a high-resolution scanning ion microscopy and helium ion microscopy (HIM) imaging technology, based on a scanning helium ion beam. HIM images from the surface chromatin fibers were analyzed to determine the size and alignment of the chromatin fibers. The unit size of the chromatin fibers was 11.6 ± 3.5 nm and was closely aligned to the chromatin network model. Our findings indicate that compacting the surface structure of barley via a chromatin network and observation via HIM are powerful tools for investigating the structure of chromatin.

Compaction and self-association of megabase-sized chromatin are induced by anionic protein crowding.

Highly compacted chromatin, a complex of DNA with cationic histone proteins, is found in the nucleus of eukaryotic cells in an environment with a high concentration of macromolecular species, many of which possess a negative charge. In the majority of previous studies, however, these crowding conditions were experimentally modelled using neutral synthetic macromolecules such as polyethylene glycol (PEG). Despite the importance of the crowding agent charge in the condensation process of chromatin, to the best of our knowledge, the behavior of chromatin under conditions of anionic protein crowding has not been studied. Here, compaction of nearly megabase-long chromatin in the presence of the anionic globular protein BSA was investigated by single-molecule fluorescent microscopy (FM). We demonstrate different effects of anionic macromolecular crowders (MMCs) on DNA and chromatin, compared to neutral MMCs. While DNA molecules undergo gradual compaction into a globular form in the presence of ca. 20% w/v of BSA, chromatin fibres complete coil to globule transition at a much lower concentration of BSA (ca. 5% w/v). Furthermore, at higher concentrations of BSA in solution (>5% w/v), chromatin fibres self-associate and form large spherical or fibrillar supramolecular microstructures characterized by a high colloidal stability and dynamic intermolecular fluctuations. Formation of such self-organized colloids from chromatin is universal and characteristic of chromatin fibres of various lengths. Our results highlight the hitherto underappreciated effect of anionic MMC environment on chromatin higher-order structures that may play an important role in self-organization of chromatin in vivo.

Identification of a functional hotspot on ubiquitin required for stimulation of methyltransferase activity on chromatin

Ubiquitylation of histone H2B at lysine 120 (H2B-Ub) plays a critical role in transcriptional elongation, chromatin conformation, as well as the regulation of specific histone H3 methylations. Herein, we report a strategy for the site-specific chemical attachment of ubiquitin to preassembled nucleosomes. This allowed expedited structure-activity studies into how H2B-Ub regulates H3K79 methylation by the methyltransferase human Dot1. Through an alanine scan of the ubiquitin surface, we identified a functional hotspot on ubiquitin that is required for the stimulation of human Dot1 in vitro. Importantly, this result was validated in chromatin from isolated nuclei by using a synthetic biology strategy that allowed selective incorporation of the hotspot-deficient ubiquitin mutant into H2B. The ubiquitin hotspot additionally impacted the regulation of ySet1-mediated H3K4 methylation but was not required for H2B-Ub-induced impairment of chromatin fiber compaction. These data demonstrate the utility of applying chemical ligation technologies to preassembled chromatin and delineate the multifunctionality of ubiquitin as a histone posttranslational modification.

One, Two, Three: Polycomb Proteins Hit All Dimensions of Gene Regulation.

Polycomb group (PcG) proteins contribute to the formation and maintenance of a specific repressive chromatin state that prevents the expression of genes in a particular space and time. Polycomb repressive complexes (PRCs) consist of several PcG proteins with specific regulatory or catalytic properties. PRCs are recruited to thousands of target genes, and various recruitment factors, including DNA-binding proteins and non-coding RNAs, are involved in the targeting. PcG proteins contribute to a multitude of biological processes by altering chromatin features at different scales. PcG proteins mediate both biochemical modifications of histone tails and biophysical modifications (e.g., chromatin fiber compaction and three-dimensional (3D) chromatin conformation). Here, we review the role of PcG proteins in nuclear architecture, describing their impact on the structure of the chromatin fiber, on chromatin interactions, and on the spatial organization of the genome in nuclei. Although little is known about the role of plant PcG proteins in nuclear organization, much is known in the animal field, and we highlight similarities and differences in the roles of PcG proteins in 3D gene regulation in plants and animals.

Thermodynamic model of heterochromatin formation through epigenetic regulation

Gene regulation in eukaryotes requires the segregation of silenced genomic regions into densely packed heterochromatin, leaving the active genes in euchromatin regions more accessible. We introduce a model that connects the presence of epigenetically inherited histone marks, methylation at histone 3 lysine-9, to the physical compaction of chromatin fibers via the binding of heterochromatin protein 1 (HP1). Our model demonstrates some of the key physical features that are necessary to explain experimental observations. In particular, we demonstrate that strong cooperative interactions among the HP1 proteins are necessary to see the phase segregation of heterochromatin and euchromatin regions. We also explore how the cell can use the concentration of HP1 to control condensation and under what circumstances there is a threshold of methylation over which the fibers will compact. Finally, we consider how different potential in vivo fiber structures as well as the flexibility of the histone 3 tail can affect the bridging of HP1. Many of the observations that we make about the HP1 system are guided by general thermodynamics principles and thus could play a role in other DNA organizational processes such as the binding of linker histones.

Polymorphism of Histone Tail Interactions in Nucleosome

Histone tails protruding from nucleosome core are known to play important role in gene expression regulation. They affect nucleosome dynamics and remodeling, chromatin compaction, transcription initiation and elongation and serve as targets for multiple posttranslational modifications, known as histone-code. The disordered nature of histone tails makes their structural characterization elusive. To address this question for the first time we perform extensive microsecond molecular dynamics simulations of nucleosome including linker DNA in explicit solvent in order to analyze the evolution of histone tails’ conformation and their interaction patterns with nucleosome core and linker DNA. We show that histone tails readily bind to nucleosomal DNA and become kinetically trapped in a certain number of distinct conformational states at microsecond timescale. Certain bound states of histone tails are associated with secondary structure formation, rearrangement of DNA conformation and protein DNA-interactions in the nucleosome core as well as neutralization of linker DNA. The implications of our findings for nucleosome dynamics, chromatin fiber compaction and histone code functioning are discussed.

Data on force-dependent structural changes of chromatin fibers measured with magnetic tweezers.

The compaction of chromatin fibers regulates the accessibility of embedded DNA, highly associated with transcriptional activities [1]. Single molecule force spectroscopy has revealed the great details of the structural changes of chromatin fibers in the presence of external exerted force [2–7]. However, most of the studies focus on a specific force regime [2,3,8,9]. The data here show force-extension (FE) traces of chromatin fibers as measured with magnetic tweezers, covering the force regime from 0 pN to 27 pN. Those traces provide information for further studies at varied force regimes.

Electrostatic effect of H1-histone protein binding on nucleosome repeat length

Within a simple biophysical model we describe the effect of electrostatic binding of H1 histone proteins on the nucleosome repeat length in chromatin. The length of wrapped DNA optimizes its binding energy to the histone core and the elastic energy penalty of DNA wrapping. The magnitude of the effect predicted from our model is in agreement with the systematic experimental data on the linear variation of nucleosome repeat lengths with H1/nucleosome ratio (Woodcock C L et al 2006 Chromos. Res. 14 17–25). We compare our model to the data for different cell types and organisms, with a widely varying ratio of bound H1 histones per nucleosome. We underline the importance of this non-specific histone-DNA charge-balance mechanism in regulating the positioning of nucleosomes and the degree of compaction of chromatin fibers in eukaryotic cells.

CENP-A Arrays Are More Condensed than Canonical Arrays at Low Ionic Strength

The centromeric histone H3 variant centromeric protein A (CENP-A), whose sequence is the least conserved among all histone variants, is responsible for specifying the location of the centromere. Here, we present a comprehensive study of CENP-A nucleosome arrays by cryo-electron tomography. We see that CENP-A arrays have different biophysical properties than canonical ones under low ionic conditions, as they are more condensed with a 20% smaller average nearest-neighbor distance and a 30% higher nucleosome density. We find that CENP-A nucleosomes have a predominantly crossed DNA entry/exit site that is narrowed on average by 8°, and they have a propensity to stack face to face. We therefore propose that CENP-A induces geometric constraints at the nucleosome DNA entry/exit site to bring neighboring nucleosomes into close proximity. This specific property of CENP-A may be responsible for generating a fundamental process that contributes to increased chromatin fiber compaction that is propagated under physiological conditions to form centromeric chromatin.

Condensin I Interacts with the PARP-1-XRCC1 Complex and Functions in DNA Single-Strand Break Repair

Condensins are essential protein complexes critical for mitotic chromosome organization. Little is known about the function of condensins during interphase, particularly in mammalian cells. Here we report the interphase-specific interaction between condensin I and the DNA nick-sensor poly(ADP-ribose) polymerase 1 (PARP-1). We show that the association between condensin I, PARP-1, and the base excision repair (BER) factor XRCC1 increases dramatically upon single-strand break damage (SSB) induction. Damage-specific association of condensin I with the BER factors flap endonuclease 1 (FEN-1) and DNA polymerase delta/epsilon was also observed, suggesting that condensin I is recruited to interact with BER factors at damage sites. Consistent with this, DNA damage rapidly stimulates the chromatin association of PARP-1, condensin I, and XRCC1. Furthermore, depletion of condensin in vivo compromises SSB but not double-strand break (DSB) repair. Our results identify a SSB-specific response of condensin I through PARP-1 and demonstrate a role for condensin in SSB repair.

The structure of C-banded human metaphase chromosomes as observed by atomic force microscopy

The ultrastructure of C-banded human metaphase chromosomes was studied by the combined use of light microscopy and atomic force microscopy (AFM). Light microscopy of the C-banded chromosomes showed that the centromeric regions of all chromosomes except the Y chromosome were positively stained. AFM further revealed that the C-positive region was higher than the C-negative region. The area of the C-positive region was specific depending on each chromosome; it ranged from the centromere to the proximal end of the long arm in chromosome 1, while it was restricted to the centromere in chromosomes 2 and 3. At higher magnification, chromatin fibers about 50 nm thick were clearly shown in the entire length of the chromosomes. In the C-positive region, these chromatin fibers were densely packed, while chromatin fibers were loosely packed with gentle twisting in the C-negative region. These AFM findings suggest that certain factors related to the chromatin fiber compaction remain in the C-positive region even after successive C-banging treatment.

DNA methylation-dependent chromatin fiber compaction in vivo and in vitro: requirement for linker histone.

Dynamic alterations in chromatin structure mediated by postsynthetic histone modifications and DNA methylation constitute a major regulatory mechanism in DNA functioning. DNA methylation has been implicated in transcriptional silencing, in part by inducing chromatin condensation. To understand the methylation-dependent chromatin structure, we performed atomic force microscope (AFM) studies of fibers isolated from cultured cells containing normal or elevated levels of m5C. Chromatin fibers were reconstituted on control or methylated DNA templates in the presence or absence of linker histone. Visual inspection of AFM images, combined with quantitative analysis of fiber structural parameters, suggested that DNA methylation induced fiber compaction only in the presence of linker histones. This conclusion was further substantiated by biochemical results.

Hybrid trypsinized nucleosomal arrays: identification of multiple functional roles of the H2A/H2B and H3/H4 N-termini in chromatin fiber compaction.

A defined 12-mer nucleosomal array in solution exists in a complex equilibrium between an unfolded 29S conformation, a 40S folding intermediate, an extensively folded 55S conformation, and soluble oligomeric states formed from cooperative intermolecular association of individual 12-mer arrays. Proteolytic removal of all of the core histone N-terminal tail domains previously has been shown to abolish both salt-dependent nucleosomal array folding and oligomerization. To elucidate the individual contributions of the H2A/H2B and H3/H4 tail domains to nucleosomal array condensation, "hybrid" trypsinized nucleosomal arrays have been assembled from tandemly repeated 5S rDNA and either trypsinized H3/H4 tetramers and intact H2A/H2B dimers or trypsinized H2A/H2B dimers and intact H3/H4 tetramers. Neither of the hybrid trypsinized arrays formed either the 40S or the 55S folded conformations in 2 mM MgCl2. In >/=4 mM MgCl2, however, both fully trypsinized arrays and each hybrid trypsinized array formed the 40S folding intermediate, but not the 55S conformation. In contrast to folding, each hybrid trypsinized nucleosomal array oligomerized completely in MgCl2. These studies have identified three mechanistically distinct functions performed by the core histone N-termini during salt-dependent condensation of nucleosomal arrays. The complexity of tail domain function in chromatin is discussed in the context of a competitive interaction model in which the core histone N-termini provide direct mechanistic links between the structure and function of the chromatin fiber.

ATP-Dependent Positive Supercoiling of DNA by 13S Condensin: A Biochemical Implication for Chromosome Condensation

13S condensin is a five-subunit protein complex that plays a central role in mitotic chromosome condensation in Xenopus egg extracts. Two core subunits of this complex, XCAP-C and XCAP-E, belong to an emerging family of putative ATPases, the SMC family. We report here that 13S condensin has a DNA-stimulated ATPase activity and exhibits a high affinity for structured DNAs such as cruciform DNA. 13S condensin is able to introduce positive supercoils into a closed circular DNA in the presence of bacterial or eukaryotic topoisomerase I. The supercoiling reaction is ATP-dependent. We propose that 13S condensin wraps DNA in a right-handed direction by utilizing the energy of ATP hydrolysis. This reaction may represent a key mechanism underlying the compaction of chromatin fibers during mitosis.