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1.  Kinetics of Core Histones in Living Human Cells 
The Journal of Cell Biology  2001;153(7):1341-1354.
Histones H2A and H2B form part of the same nucleosomal structure as H3 and H4. Stable HeLa cell lines expressing histones H2B, H3, and H4 tagged with green fluorescent protein (GFP) were established; the tagged molecules were assembled into nucleosomes. Although H2B-GFP was distributed like DNA, H3-GFP and H4-GFP were concentrated in euchromatin during interphase and in R-bands in mitotic chromosomes. These differences probably result from an unregulated production of tagged histones and differences in exchange. In both single cells and heterokaryons, photobleaching revealed that H2B-GFP exchanged more rapidly than H3-GFP and H4-GFP. About 3% of H2B exchanged within minutes, whereas ∼40% did so slowly (t1/2 ∼ 130 min). The rapidly exchanging fraction disappeared in 5,6-dichloro-1-β-d-ribofuranosylbenzimidazole and so may represent H2B in transcriptionally active chromatin. The slowly exchanging fraction was probably associated with chromatin domains surrounding active units. H3-GFP and H4-GFP were assembled into chromatin when DNA was replicated, and then >80% remained bound permanently. These results reveal that the inner core of the nucleosome is very stable, whereas H2B on the surface of active nucleosomes exchanges continually.
PMCID: PMC2150718  PMID: 11425866
cell fusion; FRAP; histone actetylation; nucleosome; transcription
2.  Global Replication-Independent Histone H4 Exchange in Budding Yeast▿  
Eukaryotic Cell  2006;5(10):1780-1787.
The eukaryotic genome is packaged together with histone proteins into chromatin following DNA replication. Recent studies have shown that histones can also be assembled into chromatin independently of DNA replication and that this dynamic exchange of histones may be biased toward sites undergoing transcription. Here we show that epitope-tagged histone H4 can be incorporated into nucleosomes throughout the budding yeast (Saccharomyces cerevisiae) genome regardless of the phase of the cell cycle, the transcriptional status, or silencing of the region. Direct comparisons reveal that the amount of histone incorporation that occurs in G1-arrested cells is similar to that occurring in cells undergoing DNA replication. Additionally, we show that this histone incorporation is not dependent on the histone H3/H4 chaperones CAF-1, Asf1, and Hir1 individually. This study demonstrates that DNA replication and transcription are not necessary prerequisites for histone exchange in budding yeast, indicating that chromatin is more dynamic than previously thought.
PMCID: PMC1595336  PMID: 16936140
3.  Chromatin is an ancient innovation conserved between Archaea and Eukarya 
eLife  2012;1:e00078.
The eukaryotic nucleosome is the fundamental unit of chromatin, comprising a protein octamer that wraps ∼147 bp of DNA and has essential roles in DNA compaction, replication and gene expression. Nucleosomes and chromatin have historically been considered to be unique to eukaryotes, yet studies of select archaea have identified homologs of histone proteins that assemble into tetrameric nucleosomes. Here we report the first archaeal genome-wide nucleosome occupancy map, as observed in the halophile Haloferax volcanii. Nucleosome occupancy was compared with gene expression by compiling a comprehensive transcriptome of Hfx. volcanii. We found that archaeal transcripts possess hallmarks of eukaryotic chromatin structure: nucleosome-depleted regions at transcriptional start sites and conserved −1 and +1 promoter nucleosomes. Our observations demonstrate that histones and chromatin architecture evolved before the divergence of Archaea and Eukarya, suggesting that the fundamental role of chromatin in the regulation of gene expression is ancient.
eLife digest
Single-celled microorganisms called archaea are one of the three domains of cellular life, along with bacteria and eukaryotes. Archaea are similar to bacteria in that they do not have nuclei, but genetically they have more in common with eukaryotes. Archaea are found in a wide range of habitats including the human colon, marshlands, the ocean and extreme environments such as hot springs and salt lakes.
It has been known since the 1990s that the DNA of archaea is wrapped around histones to form complexes that closely resemble the nucleosomes found in eukaryotes, albeit with four rather than eight histone subunits. Nucleosomes are the fundamental units of chromatin, the highly-ordered and compact structure that all the DNA in a cell is packed into. Now we know exactly how many nucleosomes are present in a given cell for some eukaryotes, notably yeast, and to a good approximation we know the position of each nucleosome during a variety of metabolic states and physiological conditions. We can also quantify the nucleosome occupancy, which is measure of the length of time that the nucleosomes spend in contact with the DNA: this is a critical piece of information because it determines the level of access that other proteins, including those that regulate gene expression, have to the DNA. These advances have been driven in large part by advances in technology, notably high-density microarrays for genome wide-studies of nucleosome occupancy, and massively parallel sequencing for direct nucleosome sequencing.
Ammar et al. have used these techniques to explore how the DNA of Haloferax volcanii, a species of archaea that thrives in the hyper-salty waters of the Dead Sea, is organized on a genome-wide basis. Despite some clear differences between the genomes of archaea and eukaryotes—for example, genomic DNA is typically circular in archaea and linear in eukaryotes—they found that the genome of Hfx. volcanii is organized into chromatin in a way that is remarkably similar to that seen in all eukaryotic genomes studied to date. This is surprising given that the chromatin in eukaryotes is confined to the nucleus, whereas there are no such constraints in archaea. In particular, Ammar et al. found that those regions of the DNA near the ends of genes that mark where the transcription of the DNA into RNA should begin and end contain have lower nucleosome occupancy than other regions. Moreover, the overall level of occupancy in Hfx. volcanii was twice that of eukaryotes, which is what one would expect given that nucleosomes in archaea contain half as many histone subunits as nucleosomes in eukaryotes. Ammar et al. also confirmed that that the degree of nucleosome occupancy is correlated with gene expression.
These two findings—the similarities between the chromatin in archaea and eukaryotes, and the correlation between nucleosome occupancy and gene expression in archaea—raise an interesting evolutionary possibility: the initial function of nucleosomes and chromatin formation might have been for the regulation of gene expression rather than the packaging of DNA. This is consistent with two decades of research that has shown that there is an extraordinary and complex relationship between the structure of chromatin and the process of gene expression. It is possible, therefore, that as the early eukaryotes evolved, nucleosomes and chromatin started to package DNA into compact structures that, among other things, helped to prevent DNA damage, and that this subsequently enabled the early eukaryotes to flourish.
PMCID: PMC3510453  PMID: 23240084
Haloferax volcanii; Nucleosome; Chromatin; Transcriptome; RNA-seq; Archaea; Other
4.  Sequence-dependent histone variant positioning signatures 
BMC Genomics  2010;11(Suppl 4):S3.
Nucleosome, the fundamental unit of chromatin, is formed by wrapping nearly 147bp of DNA around an octamer of histone proteins. This histone core has many variants that are different from each other by their biochemical compositions as well as biological functions. Although the deposition of histone variants onto chromatin has been implicated in many important biological processes, such as transcription and replication, the mechanisms of how they are deposited on target sites are still obscure.
By analyzing genomic sequences of nucleosomes bearing different histone variants from human, including H2A.Z, H3.3 and both (H3.3/H2A.Z, so-called double variant histones), we found that genomic sequence contributes in part to determining target sites for different histone variants. Moreover, dinucleotides CA/TG are remarkably important in distinguishing target sites of H2A.Z-only nucleosomes with those of H3.3-containing (both H3.3-only and double variant) nucleosomes.
There exists a DNA-related mechanism regulating the deposition of different histone variants onto chromatin and biological outcomes thereof. This provides additional insights into epigenetic regulatory mechanisms of many important cellular processes.
PMCID: PMC3005914  PMID: 21143812
5.  The Histone Database: an integrated resource for histones and histone fold-containing proteins 
Eukaryotic chromatin is composed of DNA and protein components—core histones—that act to compactly pack the DNA into nucleosomes, the fundamental building blocks of chromatin. These nucleosomes are connected to adjacent nucleosomes by linker histones. Nucleosomes are highly dynamic and, through various core histone post-translational modifications and incorporation of diverse histone variants, can serve as epigenetic marks to control processes such as gene expression and recombination. The Histone Sequence Database is a curated collection of sequences and structures of histones and non-histone proteins containing histone folds, assembled from major public databases. Here, we report a substantial increase in the number of sequences and taxonomic coverage for histone and histone fold-containing proteins available in the database. Additionally, the database now contains an expanded dataset that includes archaeal histone sequences. The database also provides comprehensive multiple sequence alignments for each of the four core histones (H2A, H2B, H3 and H4), the linker histones (H1/H5) and the archaeal histones. The database also includes current information on solved histone fold-containing structures. The Histone Sequence Database is an inclusive resource for the analysis of chromatin structure and function focused on histones and histone fold-containing proteins.
Database URL: The Histone Sequence Database is freely available and can be accessed at
PMCID: PMC3199919  PMID: 22025671
6.  A Computational Model for Histone Mark Propagation Reproduces the Distribution of Heterochromatin in Different Human Cell Types 
PLoS ONE  2013;8(9):e73818.
Chromatin is a highly compact and dynamic nuclear structure that consists of DNA and associated proteins. The main organizational unit is the nucleosome, which consists of a histone octamer with DNA wrapped around it. Histone proteins are implicated in the regulation of eukaryote genes and they carry numerous reversible post-translational modifications that control DNA-protein interactions and the recruitment of chromatin binding proteins. Heterochromatin, the transcriptionally inactive part of the genome, is densely packed and contains histone H3 that is methylated at Lys 9 (H3K9me). The propagation of H3K9me in nucleosomes along the DNA in chromatin is antagonizing by methylation of H3 Lysine 4 (H3K4me) and acetylations of several lysines, which is related to euchromatin and active genes. We show that the related histone modifications form antagonized domains on a coarse scale. These histone marks are assumed to be initiated within distinct nucleation sites in the DNA and to propagate bi-directionally. We propose a simple computer model that simulates the distribution of heterochromatin in human chromosomes. The simulations are in agreement with previously reported experimental observations from two different human cell lines. We reproduced different types of barriers between heterochromatin and euchromatin providing a unified model for their function. The effect of changes in the nucleation site distribution and of propagation rates were studied. The former occurs mainly with the aim of (de-)activation of single genes or gene groups and the latter has the power of controlling the transcriptional programs of entire chromosomes. Generally, the regulatory program of gene transcription is controlled by the distribution of nucleation sites along the DNA string.
PMCID: PMC3777982  PMID: 24069233
7.  -In silico functional characterization of a double histone fold domain from the Heliothis zea virus 1 
BMC Bioinformatics  2005;6(Suppl 4):S15.
Histones are short proteins involved in chromatin packaging; in eukaryotes, two H2a-H2b and H3-H4 histone dimers form the nucleosomal core, which acts as the fundamental DNA-packaging element. The double histone fold is a rare globular protein fold in which two consecutive regions characterized by the typical structure of histones assemble together, thus originating a histone pseudodimer. This fold is included in a few prokaryotic histones and in the regulatory region of guanine nucleotide exchange factors of the Sos family. For the prokaryotic histones, there is no direct structural counterpart in the nucleosomal core particle, while the pseudodimer from Sos proteins is very similar to the dimer formed by histones H2a and H2b
The absence of a H3-H4-like histone pseudodimer in the available structural databases prompted us to search for proteins that could assume such fold. The application of several secondary structure prediction and fold recognition methods allowed to show that the viral protein gi|22788712 is compatible with the structure of a H3-H4-like histone pseudodimer. Further in silico analyses revealed that this protein module could retain the ability of mediating protein-DNA interactions, and could consequently act as a DNA-binding domain.
Our results suggest a possible functional role in viral pathogenicity for this novel double histone fold domain; thus, the computational analyses here reported will be helpful in directing future biochemical studies on gi|22788712 protein.
PMCID: PMC1866393  PMID: 16351741
8.  Stability of the conservative mode of nucleosome assembly. 
Nucleic Acids Research  1983;11(9):2717-2732.
The conservative assembly of nucleosome histone octamer cores has been confirmed by electrophoretic analysis of density labeled histones following equilibrium buoyant density centrifugation. After normal replication, crosslinked octamers are shown not to contain a mixture of new and old core histones. Moreover, when DNA synthesis is inhibited by ara-C nucleosome cores are still assembled exclusively from nascent histone. Similarly, after release from cycloheximide inhibition newly synthesized core histone is conservatively deposited. Thus, a conservative mechanism of histone octamer assembly occurs when nascent histone is present in the normal stoichiometry to nascent DNA and when chromatin is assembled in nascent histone or nascent DNA excess.
PMCID: PMC325919  PMID: 6856473
9.  Nucleosome remodeling by hMSH2-hMSH6 
Molecular cell  2009;36(6):1086-1094.
DNA nucleotide mismatches and lesion arise on chromosomes that are a complex assortment of protein and DNA (chromatin). The fundamental unit of chromatin is a nucleosome that contains ~146 bp DNA wrapped around an H2A, H2B, H3, and H4 histone octamer. We demonstrate that the mismatch recognition heterodimer hMSH2-hMSH6 disassembles a nucleosome. Disassembly requires a mismatch that provokes the formation of hMSH2-hMSH6 hydrolysis-independent sliding clamps, which translocate along the DNA to the nucleosome. The rate of disassembly is enhanced by actual or mimicked acetylation of histone H3 within the nucleosome entry-exit and dyad axis that occurs during replication and repair in vivo and reduces DNA-octamer affinity in vitro. Our results support a passive mechanism for chromatin remodeling where hMSH2-hMSH6 sliding clamps trap localized fluctuations in nucleosome positioning and/or wrapping that ultimately leads to disassembly, and highlights unanticipated strengths of the Molecular Switch Model for mismatch repair (MMR).
PMCID: PMC3010363  PMID: 20064472
10.  The Inheritance of Histone Modifications Depends upon the Location in the Chromosome in Saccharomyces cerevisiae 
PLoS ONE  2011;6(12):e28980.
Histone modifications are important epigenetic features of chromatin that must be replicated faithfully. However, the molecular mechanisms required to duplicate and maintain histone modification patterns in chromatin remain to be determined. Here, we show that the introduction of histone modifications into newly deposited nucleosomes depends upon their location in the chromosome. In Saccharomyces cerevisiae, newly deposited nucleosomes consisting of newly synthesized histone H3-H4 tetramers are distributed throughout the entire chromosome. Methylation of lysine 4 on histone H3 (H3-K4), a hallmark of euchromatin, is introduced into these newly deposited nucleosomes, regardless of whether the neighboring preexisting nucleosomes harbor the K4 mutation in histone H3. Furthermore, if the heterochromatin-binding protein Sir3 is unavailable during DNA replication, histone H3-K4 methylation is introduced onto newly deposited nucleosomes in telomeric heterochromatin. Thus, a conservative distribution model most accurately explains the inheritance of histone modifications because the location of histones within euchromatin or heterochromatin determines which histone modifications are introduced.
PMCID: PMC3244422  PMID: 22216151
11.  Nuclear receptors and chromatin remodeling machinery 
Molecular and cellular endocrinology  2007;265-266:162-167.
Eukaryotic genetic information is stored within the association of DNA and histone proteins resulting in a dynamic polymer called chromatin. The fundamental structural unit of chromatin is the nucleosome which consists of ~146 bp of DNA wrapped around an octamer of histones containing two copies each of four core histones, H2A, H2B, H3 and H4. It is this DNA/protein fiber that transcription factors and other agents of chromatin metabolism must access and regulate. We have developed model systems to study the mechanisms by which steroid receptors control physiological activities by regulating gene expression within a higher order chromatin organization. Our studies have focused on the glucocorticoid receptor and its ability to remodel chromatin which is mediated by the BRG1 complex. Using novel cell systems, we demonstrate that GR-mediated transactivation from chromatin templates requires BRG1 remodeling activity and that other ATP-dependent remodeling proteins cannot substitute for this activity.
PMCID: PMC3582388  PMID: 17240047
GR; Chromatin; Epigenetics; SWI/SNF; BRG1; Transcriptional activation
12.  Acetylation of Histone H3 Lysine 56 Regulates Replication-Coupled Nucleosome Assembly 
Cell  2008;134(2):244-255.
Chromatin assembly factor 1 (CAF-1) and Rtt106 participate in the deposition of newly synthesized histones onto replicating DNA to form nucleosomes. This process is critical for the maintenance of genome stability and inheritance of functionally specialized chromatin structures in proliferating cells. However, the molecular functions of the acetylation of newly synthesized histones in this DNA replication-coupled nucleosome assembly pathway remain enigmatic. Here we show that histone H3 acetylated at lysine 56 (H3K56Ac) is incorporated onto replicating DNA and, by increasing the binding affinity of CAF-1 and Rtt106 for histone H3, H3K56Ac enhances the ability of these histone chaperones to assemble DNA into nucleosomes. Genetic analysis indicates that H3K56Ac acts in a nonredundant manner with the acetylation of the N-terminal residues of H3 and H4 in nucleosome assembly. These results reveal a mechanism by which H3K56Ac regulates replication-coupled nucleosome assembly mediated by CAF-1 and Rtt106.
PMCID: PMC2597342  PMID: 18662540
13.  The CHD1 motor protein is required for deposition of histone variant H3.3 into chromatin in vivo 
Science (New York, N.Y.)  2007;317(5841):1087-1090.
Deposition of core histones into chromatin in vivo transpires through an active, ATP-dependent mechanism catalyzed by molecular motor proteins, such as CHD1.
The organization of chromatin affects all aspects of nuclear DNA metabolism in eukaryotes. H3.3 is an evolutionarily conserved histone variant and a key substrate for replication-independent chromatin assembly. Elimination of maternal chromatin remodelling factor CHD1 in Drosophila embryos abolishes incorporation of H3.3 into the male pronucleus, renders the paternal genome unable to participate in zygotic mitoses and leads to the development of haploid embryos. Furthermore, CHD1 but not ISWI interacts with HIRA in cytoplasmic extracts. Our findings establish CHD1 as a major factor in replacement histone metabolism in the nucleus and reveal a critical role for CHD1 in the earliest developmental instances of genome-scale, replication-independent nucleosome assembly. Furthermore, our results point to the general requirement of ATP-utilizing motor proteins for histone deposition in vivo.
PMCID: PMC3014568  PMID: 17717186
14.  The Role of FACT in Making and Breaking Nucleosomes 
Biochimica et Biophysica Acta  2011;1819(3-4):247-255.
FACT is a roughly 180 kDa heterodimeric protein complex important for managing the properties of chromatin in eukaryotic cells. Chromatin is a repressive barrier that plays an important role in protecting genomic DNA and regulating access to it. This barrier must be temporarily removed during transcription, replication, and repair, but it also must be rapidly restored to the original state afterwards. Further, the properties of chromatin are dynamic and must be adjusted as conditions dictate. FACT was identified as a factor that destabilizes nucleosomes in vitro, but it has now also been implicated as a central factor in the deposition of histones to form nucleosomes, as an exchange factor that swaps the histones within existing nucleosomes for variant forms, and as a tether that prevents histones from being displaced by the passage of RNA polymerases during transcription. FACT therefore plays central roles in building, maintaining, adjusting, and overcoming the chromatin barrier. This review summarizes recent results that have begun to reveal how FACT can promote what appear to be contradictory goals, using a simple set of binding activities to both enhance and diminish the stability of nucleosomes.
PMCID: PMC3229669  PMID: 21807128
15.  Structural plasticity of histones H3-H4 facilitates their allosteric exchange between RbAp48 and ASF1 
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.
PMCID: PMC3538076  PMID: 23178455
16.  The evolutionary history of histone H3 suggests a deep eukaryotic root of chromatin modifying mechanisms 
The phenotype of an organism is an outcome of both its genotype, encoding the primary sequence of proteins, and the developmental orchestration of gene expression. The substrate of gene expression in eukaryotes is the chromatin, whose fundamental units are nucleosomes composed of DNA wrapped around each two of the core histone types H2A, H2B, H3 and H4. Key regulatory steps involved in the determination of chromatin conformations are posttranslational modifications (PTM) at histone tails as well as the assembly of histone variants into nucleosomal arrays. Although the mechanistic background is fragmentary understood, it appears that the chromatin signature of metazoan cell types is inheritable over generations. Even less understood is the conservation of epigenetic mechanisms among eukaryotes and their origins.
In the light of recent progress in understanding the tree of eukaryotic life we discovered the origin of histone H3 by phylogenetic analyses of variants from all supergroups, which allowed the reconstruction of ancestral states. We found that H3 variants evolved frequently but independently within related species of almost all eukaryotic supergroups. Interestingly, we found all core histone types encoded in the genome of a basal dinoflagellate and H3 variants in two other species, although is was reported that dinoflagellate chromatin is not organized into nucleosomes.
Most probably one or more animal/nuclearid H3.3-like variants gave rise to H3 variants of all opisthokonts (animals, choanozoa, fungi, nuclearids, Amoebozoa). H3.2 and H3.1 as well as H3.1t are derivatives of H3.3, whereas H3.2 evolved already in early branching animals, such as Trichoplax. H3.1 and H3.1t are probably restricted to mammals.
We deduced a model for protoH3 of the last eukaryotic common ancestor (LECA) confirming a remarkable degree of sequence conservation in comparison to canonical human H3.1. We found evidence that multiple PTMs are conserved even in putatively early branching eukaryotic taxa (Euglenozoa/Excavata).
At least a basal repertoire of chromatin modifying mechanisms appears to share old common ancestry and may thus be inherent to all eukaryotes. We speculate that epigenetic principles responsive to environmental triggers may have had influenced phenotypic variation and concomitantly may potentially have had impact on eukaryotic diversification.
PMCID: PMC2939574  PMID: 20738881
17.  Chromatin Remodeling in Arabidopsis Root Growth 
Plant Signaling & Behavior  2007;2(3):160-162.
The basic structural unit of chromatin is the nucleosome, which consists of 146 bp of DNA wrapped around the histone octamer constituted by two molecules each of histones H2A, H2B, H3 and H4. Nucleosome assembly/disassembly/reassembly processes occur primarily during DNA replication and also during transcription, DNA repair and recombination. Several chromatin-remodeling factors had been previously shown to have pleiotropic roles in different processes of plant growth and development. We have recently demonstrated that the Arabidopsis NRP1 and NRP2 genes encode H2A/H2B chaperones and are required for the maintenance of post-embryonic root growth. The nrp1-1nrp2-1 double mutant plants specifically showed a short-root phenotype in normal growth conditions. They were also hypersensitive to DNA damage and showed release of transcriptional gene silencing. We propose that NRP1 and NRP2 act as histone H2A/H2B chaperones in nucleosome assembly, playing critical roles for a correct genome transcription in the maintenance of root growth.
PMCID: PMC2634044  PMID: 19704743
Arabidopsis thaliana; histone chaperone; chromatin; transcription; root growth
18.  Drosophila Yemanuclein and HIRA Cooperate for De Novo Assembly of H3.3-Containing Nucleosomes in the Male Pronucleus 
PLoS Genetics  2013;9(2):e1003285.
The differentiation of post-meiotic spermatids in animals is characterized by a unique reorganization of their nuclear architecture and chromatin composition. In many species, the formation of sperm nuclei involves the massive replacement of nucleosomes with protamines, followed by a phase of extreme nuclear compaction. At fertilization, the reconstitution of a nucleosome-based paternal chromatin after the removal of protamines requires the deposition of maternally provided histones before the first round of DNA replication. This process exclusively uses the histone H3 variant H3.3 and constitutes a unique case of genome-wide replication-independent (RI) de novo chromatin assembly. We had previously shown that the histone H3.3 chaperone HIRA plays a central role for paternal chromatin assembly in Drosophila. Although several conserved HIRA-interacting proteins have been identified from yeast to human, their conservation in Drosophila, as well as their actual implication in this highly peculiar RI nucleosome assembly process, is an open question. Here, we show that Yemanuclein (YEM), the Drosophila member of the Hpc2/Ubinuclein family, is essential for histone deposition in the male pronucleus. yem loss of function alleles affect male pronucleus formation in a way remarkably similar to Hira mutants and abolish RI paternal chromatin assembly. In addition, we demonstrate that HIRA and YEM proteins interact and are mutually dependent for their targeting to the decondensing male pronucleus. Finally, we show that the alternative ATRX/XNP-dependent H3.3 deposition pathway is not involved in paternal chromatin assembly, thus underlining the specific implication of the HIRA/YEM complex for this essential step of zygote formation.
Author Summary
Chromosome organization relies on a basic functional unit called the nucleosome, in which DNA is wrapped around a core of histone proteins. However, during male gamete formation, the majority of histones are replaced by sperm-specific proteins that are adapted to sexual reproduction but incompatible with the formation of the first zygotic nucleus. These proteins must therefore be replaced by histones upon fertilization, in a replication-independent chromatin assembly process that requires the histone deposition factor HIRA. In this study, we identified the protein Yemanuclein (YEM) as a new partner of HIRA at fertilization. We show that, in eggs laid by yem mutant females, the male pronucleus fails to assemble its nucleosomes, resulting in the loss of paternal chromosomes at the first zygotic division. In addition, we found that YEM and HIRA are mutually dependent to perform chromatin assembly at fertilization, demonstrating that they tightly cooperate in vivo. Finally, we demonstrate that the replication-independent chromatin assembly factor ATRX/XNP is not involved in the assembly of paternal nucleosomes. In conclusion, our results shed new light into critical mechanisms controlling paternal chromosome formation at fertilization.
PMCID: PMC3567178  PMID: 23408912
19.  Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract 
Annals of Botany  2011;108(7):1235-1246.
Background and Scope
In eukaryotes, chromatin remodelling complexes are shown to be responsible for nucleosome mobility, leading to increased accessibility of DNA for DNA binding proteins. Although the existence of such complexes in plants has been surmised mainly at the genetic level from bioinformatics studies and analysis of mutants, the biochemical existence of such complexes has remained unexplored.
Histone H1-depleted donor chromatin was prepared by micrococcal nuclease digestion of wheat nuclei and fractionation by exclusion chromatography. Nuclear extract was partially purified by cellulose phosphate ion exchange chromatography. Histone octamer trans-transfer activity was analysed using the synthetic nucleosome positioning sequence in the absence and presence of ATP and its analogues. ATPase activity was measured as 32Pi released using liquid scintillation counting.
Key Results
ATP-dependent histone octamer trans-transfer activity, partially purified from wheat nuclei using cellulose phosphate, showed ATP-dependent octamer displacement in trans from the H1-depleted native donor chromatin of wheat to the labelled synthetic nucleosome positioning sequence. It also showed nucleosome-dependent ATPase activity. Substitution of ATP by ATP analogues, namely ATPγS, AMP-PNP and ADP abolished the octamer trans-transfer, indicating the requirement of ATP hydrolysis for this activity.
ATP-dependent histone octamer transfer in trans is a recognized activity of chromatin remodelling complexes required for chromatin structure dynamics in non-plant species. Our results suggested that wheat nuclei also possess a typical chromatin remodelling activity, similar to that in other eukaryotes. This is the first report on chromatin remodelling activity in vitro from plants.
PMCID: PMC3197459  PMID: 21896571
ATPase; chromatin remodelling; histone; octamer-transfer; SWI/SNF-like complex; wheat; Triticum aestivum
20.  Histone Transfer Among Chaperones 
Biochemical Society transactions  2012;40(2):357-363.
The eukaryotic processes of nucleosome assembly and disassembly govern chromatin dynamics, in which histones exchange in a highly regulated manner to promote genome accessibility for all DNA-dependent processes. This regulation is partly carried out by histone chaperones, which serve multifaceted roles in coordinating the interactions of histone proteins with modification enzymes, nucleosome remodelers, other histone chaperones, and nucleosomal DNA. The molecular details of the processes by which histone chaperones promote delivery of histones among their many functional partners are still largely undefined, but promise to offer insights into epigenome maintenance. Here we review recent findings on the histone chaperone interactions that guide the assembly of histones H3 and H4 into chromatin. This evidence supports the concepts of histone post-translational modifications and specific histone chaperone interactions as guiding principles for histone H3/H4 transactions during chromatin assembly.
PMCID: PMC3494481  PMID: 22435812
21.  CAF-1-Mediated Chromatin Assembly Generates a Bilateral Asymmetry in C. elegans Neuroanatomy 
Cell  2011;147(7):1525-1536.
Chromatin assembly is a fundamental cellular process, but its role during animal development remains largely elusive. Here we report that the CAF-1 protein complex, an evolutionarily conserved histone chaperone that deposits histone H3-H4 proteins onto replicating DNA, is required to generate a bilateral asymmetry in the C. elegans nervous system. We describe our findings that a mutation in one of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry. This histone H3 mutation causes a defect in the formation of a histone H3-H4 tetramer and the consequent inhibition of CAF-1-mediated nucleosome formation. Our results reveal that replication-coupled nucleosome assembly is necessary to generate a bilateral asymmetry in C. elegans neuroanatomy and suggest that left-right asymmetric epigenetic regulation can establish bilateral asymmetry in the nervous system.
PMCID: PMC3290763  PMID: 22177093
22.  Prothymosin α is a component of a linker histone chaperone 
FEBS letters  2010;584(13):2833-2836.
Linker histone H1 binds with high affinity to naked and nucleosomal DNA in vitro but is rapidly exchanged between chromatin sites in vivo suggesting the involvement of one or more linker histone chaperones. Using permeabilized cells, we demonstrate that the small acidic protein prothymosin α (ProTα) can facilitate H1 displacement from and deposition onto the native chromatin template. Depletion of ProTα levels in vivo by siRNA-mediated mRNA degradation resulted in a decreased rate of exchange of linker histones as assayed by photobleaching techniques. These results indicate that ProTα is a component of a linker histone chaperone.
PMCID: PMC2891112  PMID: 20434447
Prothymosin α; Histone H1; linker histone; histone chaperone; chromatin
23.  Drosophila NAP-1 is a core histone chaperone that functions in ATP-facilitated assembly of regularly spaced nucleosomal arrays. 
Molecular and Cellular Biology  1996;16(6):3112-3124.
We describe the cloning and analysis of Drosophila nucleosome assembly protein 1 (dNAP-1), a core histone-binding protein that functions with other chromatin assembly activities in a Drosophila chromatin assembly factor 1-containing fraction (dCAF-1 fraction) in the ATP-facilitated assembly of regularly spaced nucleosomal arrays from purified core histones and DNA. Purified, recombinant dNAP-1 acts cooperatively with a factor(s) in the dCAF-1 fraction in the efficient and DNA replication-independent assembly of chromatin. In the presence of histone H1, the repeat length of the chromatin is similar to that of native chromatin from Drosophila embryos. By coimmunoprecipitation analysis, dNAP-1 was found to be associated with histones H2A and H2B in a crude whole-embryo extract, which suggests that dNAP-1 is bound to the histones in vivo. Studies of the localization of dNAP-1 in the Drosophila embryo revealed that the factor is present in the nucleus during S phase and is predominantly cytoplasmic during G2 phase. These data suggest that NAP-1 acts as a core histone shuttle which delivers the histones from the cytoplasm to the chromatin assembly machinery in the nucleus. Thus, NAP-1 appears to be one component of a multifactor chromatin assembly machinery that mediates the ATP-facilitated assembly of regularly spaced nucleosomal arrays.
PMCID: PMC231306  PMID: 8649423
24.  Identification of a Rapidly Formed Non-nucleosomal Histone-DNA Intermediate That Is Converted into Chromatin by ACF 
Molecular cell  2011;43(4):638-648.
Chromatin assembly involves the combined action of histone chaperones and ATP-dependent motor proteins. Here we investigate the mechanism of nucleosome assembly with a purified chromatin assembly system containing the histone chaperone NAP1 and the ATP-dependent motor protein ACF. These studies revealed the rapid formation of a stable non-nucleosomal histone-DNA intermediate that is converted into canonical nucleosomes by ACF. The histone-DNA intermediate does not supercoil DNA like a canonical nucleosome, but has a nucleosome-like appearance by atomic force microscopy. This intermediate contains all four core histones, lacks NAP1, and is formed by the initial deposition of histones H3-H4. Conversion of the intermediate into histone H1-containing chromatin results in increased resistance to micrococcal nuclease digestion. These findings suggest that the histone-DNA intermediate corresponds to nascent nucleosome-like structures, such as those observed at DNA replication forks. Related complexes might be formed during other chromatin-directed processes such as transcription, DNA repair, and histone exchange.
PMCID: PMC3160715  PMID: 21855802
25.  Roles of chromatin insulator proteins in higher-order chromatin organization and transcription regulation 
Nucleus (Austin, Tex.)  2011;2(5):358-369.
Eukaryotic chromosomes are condensed into several hierarchical levels of complexity: DNA is wrapped around core histones to form nucleosomes, nucleosomes form a higher-order structure called chromatin, and chromatin is subsequently compartmentalized in part by the combination of multiple specific or unspecific long-range contacts. The conformation of chromatin at these three levels greatly influences DNA metabolism and transcription. One class of chromatin regulatory proteins called insulator factors may organize chromatin both locally, by setting up barriers between heterochromatin and euchromatin, and globally by establishing platforms for long-range interactions. Here, we review recent data revealing a global role of insulator proteins in the regulation of transcription through the formation of clusters of long-range interactions that impact different levels of chromatin organization.
PMCID: PMC3796873  PMID: 21983085

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