Nucleosome composition regulates the histone H3 tail conformational ensemble and accessibility

Hexasomes and tetrasomes are intermediates in nucleosome assembly and disassembly. Their formation is promoted by histone chaperones, ATP-dependent remodelers, and RNA polymerase II. In addition, hexasomes are maintained in transcribed genes and could be an important regulatory factor. While nucleosome composition has been shown to affect the structure and accessibility of DNA, its influence on histone tails is largely unknown. Here, we investigate the conformational dynamics of the H3 tail in the hexasome and tetrasome. Using a combination of NMR spectroscopy, MD simulations, and trypsin proteolysis, we find that the conformational ensemble of the H3 tail is regulated by nucleosome composition. As has been found for the nucleosome, the H3 tails bind robustly to DNA within the hexasome and tetrasome, but upon loss of the H2A/H2B dimer, we determined that the adjacent H3 tail has an altered conformational ensemble, increase in dynamics, and increase in accessibility. Similar to observations of DNA dynamics, this is seen to be asymmetric in the hexasome. Our results indicate that nucleosome composition has the potential to regulate chromatin signaling and ultimately help shape the chromatin landscape.     

Structure of the CENP-A nucleosome and its implications for centromeric chromatin architecture

Centromeres are dictated by the epigenetic inheritance of the centromeric nucleosome containing the centromere-specific histone H3 variant, CENP-A. The structure of the CENP-A nucleosome has been considered to be the fundamental architecture of the centromeric chromatin. Controversy exists in the literature regarding the CENP-A nucleosome structures, with octasome, hemisome, compact octasome, hexasome, and tetrasome models being reported. Some of these CENP-A nucleosome models may correspond to transient intermediates for the assembly of the mature CENP-A nucleosome; however, their significances are still unclear. Therefore, the structure of the mature CENP-A nucleosome has been eagerly awaited. We reconstituted the human CENP-A nucleosome with its cognate centromeric DNA fragment, and determined its crystal structure. In this review, we describe the structure and the physical properties of the CENP-A nucleosome, and discuss their implications for centromeric chromatin architecture.     

盐离子作用下组蛋白变体H2A.Z增强核小体结构的稳定性

真核生物染色质的基本结构组成单元是核小体,基因组DNA被压缩在染色质中,核小体的存在通常会抑制转录、复制、修复和重组等发生在DNA模板上的生物学过程。组蛋白变体H2A.Z可以调控染色质结构进而影响基因的转录过程,但其详细的调控机制仍未研究清楚。为了比较含有组蛋白变体H2A.Z的核小体和常规核小体在盐离子作用下的稳定性差异,本文采用Förster共振能量转移的方法检测氯化钠、氯化钾、氯化锰、氯化钙、氯化镁等离子对核小体的解聚影响。实验对Widom 601 DNA序列进行双荧光Cy3和Cy5标记,通过荧光信号值的变化来反映核小体的解聚变化。Förster共振能量转移检测结果显示:在氯化钠、氯化钾、氯化锰、氯化钙和氯化镁作用下,含有组蛋白变体H2A.Z的核小体解聚速度相比于常规核小体要慢,且氯化钙、氯化锰和氯化镁的影响更明显。电泳分析结果表明,在75℃条件下含有组蛋白变体H2A.Z的核小体的解聚速率明显低于常规核小体。采用荧光热漂移检测(fluorescence thermal shift analysis , FTS)进一步分析含有组蛋白变体H2A.Z核小体的稳定性,发现两类核小体的荧光信号均呈现2个明显的增长期,含有组蛋白变体H2A.Z核小体的第1个荧光信号增速期所对应的温度明显高于常规核小体,表明核小体中H2A.Z/H2B二聚体的解聚变性温度要高于常规的H2A/H2B二聚体,含有组蛋白变体H2A.Z核小体的热稳定性高。研究结果均表明,含有组蛋白变体H2A.Z的核小体的结构比常规核小体的结构稳定。     

Nucleosome assembly protein 1 exchanges histone H2A-H2B dimers and assists nucleosome sliding

Eukaryotic chromatin is highly dynamic and turns over rapidly even in the absence of DNA replication. Here we show that the acidic histone chaperone nucleosome assembly protein 1 (NAP-1) from yeast reversibly removes and replaces histone protein dimer H2A-H2B or histone variant dimers from assembled nucleosomes, resulting in active histone exchange. Transient removal of H2A-H2B dimers facilitates nucleosome sliding along the DNA to a thermodynamically favorable position. Histone exchange as well as nucleosome sliding is independent of ATP and relies on the presence of the C-terminal acidic domain of yeast NAP-1, even though this region is not required for histone binding and chromatin assembly. Our results suggest a novel role for NAP-1 (and perhaps other acidic histone chaperones) in mediating chromatin fluidity by incorporating histone variants and assisting nucleosome sliding. NAP-1 may function either untargeted (if acting alone) or may be targeted to specific regions within the genome through interactions with additional factors.     

Effects of DNA Superhelical Stress on the Stability of H2B-Ubiquitylated Nucleosomes

On the nucleosome level, histone posttranslational modifications function mainly as the regulatory signals; in addition, some posttranslational modifications can enhance nucleosome stochastic folding, which is restricted in “canonic” nucleosomes. Recently, it has been shown in vitro that symmetric or asymmetric nucleosome ubiquitylation at H2BK34 (and H2BK120, to a lesser extent) can destabilize one of the nucleosomal H2A–H2B dimers and promote nucleosome conversion to a hexasome particle [Krajewski et al. (2018). Nucleic Acids Res., 46, 7631–7642]. Such lability of H2Bub nucleosomes raises a question of whether they could accommodate transient changes in DNA torsional tensions, which are generated by virtually any process that manipulates DNA strands. Using positively or negatively supercoiled DNA minicircles and homogeneously-modified H2Bub histones, we have found that DNA topology could strongly and selectively affect nucleosome stability depending on its ubiquitylation state (here the term “nucleosome stability” means the nucleosome property to maintain its structural integrity and dynamics characteristic to “canonic” nucleosomes). The results point to a role for H2B ubiquitylation in amplifying or mitigating the effects of a DNA torque on the nucleosome stability and dynamics.     

Histone H2A/H2B dimer exchange by ATP-dependent chromatin remodeling activities

ATP-dependent chromatin remodeling activities function to manipulate chromatin structure during gene regulation. One of the ways in which they do this is by altering the positions of nucleosomes along DNA. Here we provide support for the ability of these complexes to move nucleosomes into positions in which DNA is unraveled from one edge. This is expected to result in the loss of histone-DNA contacts that are important for retention of one H2A/H2B dimer within the nucleosome. Consistent with this we find that several chromatin remodeling complexes are capable of catalyzing the exchange of H2A/H2B dimers between chromatin fragments in an ATP-dependent reaction. This provides eukaryotes with additional means by which they may manipulate chromatin structure.     

Disruption of the nucleosomes at the replication fork.

The fate of parental nucleosomes during chromatin replication was studied in vitro using in vitro assembled chromatin containing the whole SV40 genome as well as salt-treated and native SV40 minichromosomes. In vitro assembled minichromosomes were able to replicate efficiently in vitro, when the DNA was preincubated with T-antigen, a cytosolic S100 extract and three deoxynucleoside triphosphates prior to chromatin assembly, indicating that the origin has to be free of nucleosomes for replication initiation. The chromatin structure of the newly synthesized daughter strands in replicating molecules was analysed by psoralen cross-linking of the DNA and by micrococcal nuclease digestion. A 5- and 10-fold excess of protein-free competitor DNA present during minichromosome replication traps the segregating histones. In opposition to published data this suggests that the parental histones remain only loosely or not attached to the DNA in the region of the replication fork. Replication in the putative absence of free histones shows that a subnucleosomal particle is randomly assembled on the daughter strands. The data are compatible with the formation of a H3/H4 tetramer complex under these conditions, supporting the notion that under physiological conditions nucleosome core assembly on the newly synthesized daughter strands occurs by the binding of H2A/H2B dimers to a H3/H4 tetramer complex.     

Assembly and properties of chromatin containing histone H1

The Xenopus oocyte supernatant (oocyte S-150) forms chromatin in a reaction that is affected by temperature and by the concentration of ATP and Mg. Under optimal conditions at 27 degrees C, relaxed DNA plasmids are efficiently assembled into supercoiled minichromosomes with the endogenous histones H3, H4, H2A and H2B. This assembly reaction is a gradual process that takes four to six hours for completion. Micrococcal nuclease digestions of the chromatin assembled under these conditions generate an extended series of DNA fragments that are, on average, multiples of 180 base-pairs. We have examined the effect of histone H1 in this system. Exogenous histone H1, when added at a molar ratio of H1 to nucleosome of 1:1 to 5:1, causes an increase in the micrococcal nuclease resistance of the chromatin without causing chromatin aggregation under these experimental conditions. Furthermore, the periodically arranged nucleosomes display longer internucleosome distances, and the average length of the nucleosome repeat is a function of the amount of histone H1 added, when this histone is present at the onset of the assembly process. In contrast, no major change in the length of the nucleosome repeat is observed when histone H1 is added at the end of the chromatin assembly process. Protein analyses of the purified minichromosomes show that histone H1 is incorporated in the chromatin that is assembled in the S-150 supplemented with histone H1. The amount of histone H1 bound to chromatin is a function of the total amount of histone H1 added. We define here the parameters that generate histone H1-containing chromatin with native nucleosome repeats from 160 to 220 base-pairs, and we discuss the implications of these studies.     

Structural Studies of Overlapping Dinucleosomes in Solution

An overlapping dinucleosome (OLDN) is a structure composed of one hexasome and one octasome and appears to be formed through nucleosome collision promoted by nucleosome remodeling factor(s). In this study, the solution structure of the OLDN was investigated through the integration of small-angle x-ray and neutron scattering (SAXS and SANS, respectively), computer modeling, and molecular dynamics simulations. Starting from the crystal structure, we generated a conformational ensemble based on normal mode analysis and searched for the conformations that reproduced the SAXS and SANS scattering curves well. We found that inclusion of histone tails, which are not observed in the crystal structure, greatly improved model quality. The obtained structural models suggest that OLDNs adopt a variety of conformations stabilized by histone tails situated at the interface between the hexasome and octasome, simultaneously binding to both the hexasomal and octasomal DNA. In addition, our models define a possible direction for the conformational changes or dynamics, which may provide important information that furthers our understanding of the role of chromatin dynamics in gene regulation.