Comprehensive structural analysis of mutant nucleosomes containing lysine to glutamine (KQ) substitutions in the H3 and H4 histone-fold domains

Post-translational modifications (PTMs) of histones play important roles in regulating the structure and function of chromatin in eukaryotes. Although histone PTMs were considered to mainly occur at the N-terminal tails of histones, recent studies have revealed that PTMs also exist in the histone-fold domains, which are commonly shared among the core histones H2A, H2B, H3, and H4. The lysine residue is a major target for histone PTM, and the lysine to glutamine (KQ) substitution is known to mimic the acetylated states of specific histone lysine residues in vivo. Human histones H3 and H4 contain 11 lysine residues in their histone-fold domains (five for H3 and six for H4), and eight of these lysine residues are known to be targets for acetylation. In the present study, we prepared 11 mutant nucleosomes, in which each of the lysine residues of the H3 and H4 histone-fold domains was replaced by glutamine: H3 K56Q, H3 K64Q, H3 K79Q, H3 K115Q, H3 K122Q, H4 K31Q, H4 K44Q, H4 K59Q, H4 K77Q, H4 K79Q, and H4 K91Q. The crystal structures of these mutant nucleosomes were determined at 2.4-3.5 ? resolutions. Some of these amino acid substitutions altered the local protein-DNA interactions and the interactions between amino acid residues within the nucleosome. Interestingly, the C-terminal region of H2A was significantly disordered in the nucleosome containing H4 K44Q. These results provide an important structural basis for understanding how histone modifications and mutations affect chromatin structure and function.     

NF-Y associates with H3-H4 tetramers and octamers by multiple mechanisms

NF-Y is a CCAAT-binding trimer with two histonic subunits, NF-YB and NF-YC, resembling H2A-H2B. We previously showed that the short conserved domains of NF-Y efficiently bind to the major histocompatibility complex class II Ea Y box in DNA nucleosomized with purified chicken histones. Using wild-type NF-Y and recombinant histones, we find that NF-Y associates with H3-H4 early during nucleosome assembly, under conditions in which binding to naked DNA is not observed. In such assays, the NF-YB-NF-YC dimer forms complexes with H3-H4, for whose formation the CCAAT box is not required. We investigated whether they represent octamer-like structures, using DNase I, micrococcal nuclease, and exonuclease III, and found a highly positioned nucleosome on Ea, whose boundaries were mapped; addition of NF-YB-NF-YC does not lead to the formation of octameric structures, but changes in the digestion patterns are observed. NF-YA can bind to such preformed DNA complexes in a CCAAT-dependent way. In the absence of DNA, NF-YB-NF-YC subunits bind to H3-H4, but not to H2A-H2B, through the NF-YB histone fold. These results indicate that (i) the NF-Y histone fold dimer can efficiently associate DNA during nucleosome formation; (ii) it has an intrinsic affinity for H3-H4 but does not form octamers; and (iii) the interactions between NF-YA, NF-YB-NF-YC, and H3-H4 or nucleosomes are not mutually exclusive. Thus, NF-Y can intervene at different steps during nucleosome formation, and this scenario might be paradigmatic for other histone fold proteins involved in gene regulation.     

The mechanics behind DNA sequence-dependent properties of the nucleosome

Chromatin organization and composition impart sophisticated regulatory features critical to eukaryotic genomic function. Although DNA sequence-dependent histone octamer binding is important for nucleosome activity, many aspects of this phenomenon have remained elusive. We studied nucleosome structure and stability with diverse DNA sequences, including Widom 601 derivatives with the highest known octamer affinities, to establish a simple model behind the mechanics of sequence dependency. This uncovers the unique but unexpected role of TA dinucleotides and a propensity for G|C-rich sequence elements to conform energetically favourably at most locations around the histone octamer, which rationalizes G|C% as the most predictive factor for nucleosome occupancy in vivo. In addition, our findings reveal dominant constraints on double helix conformation by H3-H4 relative to H2A-H2B binding and DNA sequence context-dependency underlying nucleosome structure, positioning and stability. This provides a basis for improved prediction of nucleosomal properties and the design of tailored DNA constructs for chromatin investigations.     

Roles for Gcn5 in promoting nucleosome assembly and maintaining genome integrity

The coordinated process of DNA replication and nucleosome assembly, termed replication-coupled (RC) nucleosome assembly, is important for the maintenance of genome integrity. Loss of genome integrity is linked to aging and cancer. RC nucleosome assembly involves deposition of histone H3-H4 by the histone chaperones CAF-1, Rtt106 and Asf1 onto newly-replicated DNA. Coordinated actions of these three histone chaperones are regulated by modifications on the histone proteins. One such modification is histone H3 lysine 56 acetylation (H3K56Ac), a mark of newly-synthesized histone H3 that regulates the interaction between H3-H4 and the histone chaperones CAF-1 and Rtt106 following DNA replication and DNA repair. Recently, we have shown that the lysine acetyltransferase Gcn5 and H3 N-terminal tail lysine acetylation also regulates the interaction between H3-H4 and CAF-1 to promote the deposition of newly-synthesized histones. Genetic studies indicate that Gcn5 and Rtt109, the H3K56Ac lysine acetyltransferase, function in parallel to maintain genome stability. Utilizing synthetic genetic array analysis, we set out to identify additional genes that function in parallel with Gcn5 in response to DNA damage. We summarize here the role of Gcn5 in nucleosome assembly and suggest that Gcn5 impacts genome integrity via multiple mechanisms, including nucleosome assembly.     

水稻H3.2型组蛋白基因RH3.2A的克隆与盐胁迫下的表达分析

组蛋白H3与其他类型的组蛋白分子H2A,H2B,H4共同构成了真核生物核小体的八聚体核心。研究发现组蛋白H3的多种翻译修饰,如甲基化、乙酰化、磷酸化等在调控基因转录过程种发挥了重要的作用。本研究从盐胁迫处理的水稻幼苗组织中分离了一个新的水稻组蛋白H3基因RH3．2A,编码具有136个氨基酸残基的多肽,与多种植物的组蛋白H3蛋白具有高度的氨基酸一致性。多序列比较发现,除了基因结构差异之外,还有3个位置的氨基酸残基（32、88、91）在H3．1与H3．2型组蛋白H3中存在差异。研究了RH3．2A基因在高盐和ABA胁迫下的表达,结果发现在水稻根部RH3．2A基因受高盐的强烈诱导,而在叶片RH3．2A基因的表达则不受高盐诱导,此外RH3．2A基因也受外源ABA的诱导,结合启动子分析的结果,我们认为RH3．2A基因可能参与了依赖于ABA的高盐胁迫应答反应。文章讨论了植物组蛋白H3基因在高盐胁迫应答反应中可能的作用。     

Evidence for an early prokaryotic origin of histones H2A and H4 prior to the emergence of eukaryotes.

Histones have been identified recently in many prokaryotes. These histones, unlike their eukaryotic homologs, are of a single uniform type that is thought to resemble the archetypal ancestor of the eukaryotic histone family. In this paper we report the finding, the cloning and the phylogenetic analysis of the sequence of a prokaryotic histone from the hyperthermophile Methanopyrus kandleri . Unlike previously described prokaryotic histones, the Methanopyrus sequence has a novel structure consisting of two tandemly repeated histone fold motifs in a single polypeptide. Sequence analyses indicate that the N-terminal repeat is most closely related to eukaryotic H2A and H4 histones, whereas the C-terminal repeat resembles that found in prokaryotic histones. These results imply an early divergence within the histone gene family prior to the emergence of eukaryotes and may represent an evolutionary step leading to eukaryotic histones.     

Structure of the Drosophila nucleosome core particle highlights evolutionary constraints on the H2A-H2B histone dimer

We determined the 2.45 A crystal structure of the nucleosome core particle from Drosophila melanogaster and compared it to that of Xenopus laevis bound to the identical 147 base-pair DNA fragment derived from human alpha-satellite DNA. Differences between the two structures primarily reflect 16 amino acid substitutions between species, 15 of which are in histones H2A and H2B. Four of these involve histone tail residues, resulting in subtly altered protein-DNA interactions that exemplify the structural plasticity of these tails. Of the 12 substitutions occurring within the histone core regions, five involve small, solvent-exposed residues not involved in intraparticle interactions. The remaining seven involve buried hydrophobic residues, and appear to have coevolved so as to preserve the volume of side chains within the H2A hydrophobic core and H2A-H2B dimer interface. Thus, apart from variations in the histone tails, amino acid substitutions that differentiate Drosophila from Xenopus histones occur in mutually compensatory combinations. This highlights the tight evolutionary constraints exerted on histones since the vertebrate and invertebrate lineages diverged.     

Analysis of Primary Structural Determinants That Distinguish the Centromere-Specific Function of Histone Variant Cse4p from Histone H3

Cse4p is a variant of histone H3 that has an essential role in chromosome segregation and centromere chromatin structure in budding yeast. Cse4p has a unique 135-amino-acid N terminus and a C-terminal histone-fold domain that is more than 60% identical to histone H3 and the mammalian centromere protein CENP-A. Cse4p and CENP-A have biochemical properties similar to H3 and probably replace H3 in centromere-specific nucleosomes in yeasts and mammals, respectively. In order to identify regions of Cse4p that distinguish it from H3 and confer centromere function, a systematic site-directed mutational analysis was performed. Nested deletions of the Cse4p N terminus showed that this region of the protein contains at least one essential domain. The C-terminal histone-fold domain of Cse4p was analyzed by changing Cse4p amino acids that differ between Cse4p and H3 to the analogous H3 residues. Extensive substitution of contiguous Cse4p residues with H3 counterparts resulted in cell lethality. However, all large lethal substitution alleles could be subdivided into smaller viable alleles, many of which caused elevated rates of mitotic chromosome loss. The results indicate that residues critical for wild-type Cse4p function and high-fidelity chromosome transmission are distributed across the entire histone-fold domain. Our findings are discussed in the context of the known structure of H3 within the nucleosome and compared with previous results reported for CENP-A.     

Replication-coupled chromatin assembly generates a neuronal bilateral asymmetry in C. elegans

Although replication-coupled chromatin assembly is known to be important for the maintenance of patterns of gene expression through sequential cell divisions, the role of replication-coupled chromatin assembly in controlling cell differentiation during animal development remains largely unexplored. 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. A mutation in 1 of 24 C. elegans histone H3 genes specifically eliminates this aspect of neuronal asymmetry by causing 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.     

Dramatic diversity of ciliate histone H4 genes revealed by comparisons of patterns of substitutions and paralog divergences among eukaryotes

The accumulation of divergent histone H4 amino acid sequences within and between ciliate lineages challenges traditional views of the evolution of this essential eukaryotic protein. We analyzed histone H4 sequences from 13 species of ciliates and compared these data with sequences from well-sampled eukaryotic clades. Ciliate histone H4s differ from one another at as many as 46% of their amino acids, in contrast with the highly conserved character of this protein in most other eukaryotes. Equally striking, we find paralogs of histone H4 within ciliate genomes that differ by up to 25% of their amino acids, whereas paralogs in other eukaryotes share identical or nearly identical amino acid sequences. Moreover, the most divergent H4 proteins within ciliates are found in the lineages with highly processed macronuclear genomes. Our analyses demonstrate that the dual nature of ciliate genomes-the presence of a "germline" micronucleus and a "somatic" macronucleus within each cell-allowed the dramatic variation in ciliate histone genes by altering functional constraints or enabling adaptive evolution of the histone H4 protein, or both.     

Histones of Neurospora crassa.

Neurospora crassa chromatin isolated by a rapid method minimizing proteolytic degradation contains approximately one weight of acid-extractable basic protein per weight of DNA. This basic protein consists of five major polypeptide species which are similar in size to the histone proteins of higher eukaryotes and are present in approximately the same molar ratios. These five polypeptides have been purified by polyacrylamide gel electrophoresis in the presence of sodium dodecyl sulfate. Their electrophoretic mobilities in polyacrylamide gels and their amino acid compositions indicate that they are histones homologous, although not identical, to the H1, H2A, H2B, H3, and H4 histones of mammals. The first 3 residues in the amino acid sequence of Neurospora H3 histone are identical to the first 3 residues in calf and pea H3; Neurospora H1, H2A, and H4 histones have blocked NH2 termini, like their mammalian counterparts. The finding of recognizable H1, H2A, H2B, H3, and H4 histones in Neurospora extends the range of eukaryotes now shown to contain a full complement of these strongly conserved chromosomal proteins, and supports the view that histones became involved in chromosome structure at a very early point in the evolution of eukaryotes.     

技术与方法体外组装含有组蛋白变体H2A.Z及H3.3的核小体

核小体是构成真核生物染色质的基本结构单位,组蛋白变体H2A.Z及H3.3对染色质结构及基因转录过程发挥着重要的调控作用。体内研究核小体及染色质结构受到诸多因素限制,体外重构含有H2A.Z及H3.3的核小体结构是研究与组蛋白变体相关基因表达调控的重要方法之一。实验表达纯化了6种组蛋白,在复性的过程中装配了含有H2A.Z和H3.3的组蛋白八聚体。基于DNA序列10bp周期性及序列模体设计了3条易于形成核小体的DNA序列,通过PCR大量扩增的方法,回收了标记Cy3荧光分子的目的DNA序列。采用盐透析法体外组装了含有H2A.Z和H3.3的核小体结构,利用荧光标记、EB染色及考马斯亮蓝染色检测了含有组蛋白变体的核小体形成效率及形成过程的吉布斯自由能变化。结果发现,设计的3条DNA序列可以有效地组装形成含有组蛋白电梯的核小体结构,而且随着组蛋白八聚体与DNA比例的增加,核小体的形成效率显著提高;采用Cy3荧光标记可以灵敏且定量地计算组装过程的吉布斯自由能。该方法的建立对研究组蛋白变体相关的结构生物学及转录调控等具有一定的意义。     

Histone tails and the H3 alphaN helix regulate nucleosome mobility and stability

Nucleosomes fulfill the apparently conflicting roles of compacting DNA within eukaryotic genomes while permitting access to regulatory factors. Central to this is their ability to stably associate with DNA while retaining the ability to undergo rearrangements that increase access to the underlying DNA. Here, we have studied different aspects of nucleosome dynamics including nucleosome sliding, histone dimer exchange, and DNA wrapping within nucleosomes. We find that alterations to histone proteins, especially the histone tails and vicinity of the histone H3 alphaN helix, can affect these processes differently, suggesting that they are mechanistically distinct. This raises the possibility that modifications to histone proteins may provide a means of fine-tuning specific aspects of the dynamic properties of nucleosomes to the context in which they are located.     

ASF1 binds to a heterodimer of histones H3 and H4: a two-step mechanism for the assembly of the H3-H4 heterotetramer on DNA

The first step in the formation of the nucleosome is commonly assumed to be the deposition of a histone H3-H4 heterotetramer onto DNA. Antisilencing function 1 (ASF1) is a major histone H3-H4 chaperone that deposits histones H3 and H4 onto DNA. With a goal of understanding the mechanism of deposition of histones H3 and H4 onto DNA, we have determined the stoichiometry of the Asf1-H3-H4 complex. We have established that a single molecule of Asf1 binds to an H3-H4 heterodimer using gel filtration, amino acid, reversed-phase chromatography, and analytical ultracentrifugation analyses. We demonstrate that Asf1 blocks formation of the H3-H4 heterotetramer by a mechanism that likely involves occlusion of the H3-H3 dimerization interface.