Significant compaction of H4 histone tail upon charge neutralization by acetylation and its mimics,possible effects on chromatin structure

The intrinsically disordered, positively charged H4 histone tail is important for chromatin structure and function. We have explored conformational ensembles of human H4 tail in solution, with varying levels of charge neutralization via acetylation or amino-acid substitutions such as $K\to Q$. We have employed an explicit water model shown recently to be well suited for simulations of intrinsically disordered proteins.Upon progressive neutralization of the H4, its radius of gyration decreases linearly with the tail charge q, the trend is explained using a simple polymer model. While the wild type state ($q=+8$) is essentially a random coil, hyper-acetylated H4 ($q=+3$) is virtually as compact and stable as a globular protein of the same number of amino-acids.Conformational ensembles of acetylated H4 match the corresponding $K\to X$ substitutions only approximately: based on the ensemble similarity, we propose $K\to M$ as a possible alternative to the commonly used $K\to Q$.Possible effects of the H4 tail compaction on chromatin structure are discussed within a qualitative model in which the chromatin is highly heterogeneous, easily inter-converting between various structural forms. We predict that upon progressive charge neutralization of the H4 tail, the least compact sub-states of chromatin de-condense first, followed by de-condensation of more compact structures, e.g. those that harbor a high fraction of stacked di-nucleosomes. The predicted hierarchy of DNA accessibility increase upon progressive acetylation of H4 might be utilized by the cell for selective DNA accessibility control.     

Activation of tachykinin,neurokinin 3 receptors affects chromatin structure and gene expression by means of histone acetylation

The tachykinin, neurokinin 3 receptor (NK3R) is a g-protein coupled receptor that is broadly distributed in the nervous system and exerts its diverse physiological actions through multiple signaling pathways. Despite the role of the receptor system in a range of biological functions, the effects of NK3R activation on chromatin dynamics and gene expression have received limited attention. The present work determined the effects of senktide, a selective NK3R agonist, on chromatin organization, acetylation, and gene expression, using qRT-PCR, in a hypothalamic cell line (CLU 209) that expresses the NK3R. Senktide (1 nM, 10 nM) caused a relaxation of chromatin, an increase in global acetylation of histone H3 and H4, and an increase in the expression of a common set of genes involved in cell signaling, cell growth, and synaptic plasticity. Pretreatment with histone acetyltransferase (HAT) inhibitor (garcinol and 2-methylene y-butylactone), that inhibits p300, p300/CREB binding protein (CBP) associated factor (PCAF), and GCN 5, prevented the senktide-induced increase in expression of most, but not all, of the genes upregulated in response to 1 nM and 10 nM senktide. Treatment with 100 nM had the opposite effect: a reduction in chromatin relaxation and decreased acetylation. The expression of four genes was significantly decreased and the HAT inhibitor had a limited effect in blocking the upregulation of genes in response to 100 nM senktide. Activation of the NK3R appears to recruit multiple pathways, including acetylation, and possibly histone deactylases, histone methylases, or DNA methylases to affect chromatin structure and gene expression.     

基于NGS的染色质测序在肿瘤研究中的应用

染色质是真核生物细胞核内由核酸和蛋白质组成的复合结构,有着精密且复杂的三维结构。染色质除基本的DNA序列外,内部还存在着不同化学修饰,DNA-蛋白质相互作用,DNA-DNA相互作用和DNA-RNA相互作用,以上这些若发生改变都可能在肿瘤发生发展过程中起到至关重要的作用。通过不同的染色质测序方法,可以解析出这些改变,并进一步加深研究者对肿瘤形成机制的理解,最终应用于肿瘤的治疗。本文对常见的染色质测序技术部分原理和应用进行综述。     

MeCP2 preferentially binds to methylated linker DNA in the absence of the terminal tail of histone H3 and independently of histone acetylation

Methyl CpG binding protein 2 (MeCP2) is a basic protein that contains a DNA methyl binding domain. The mechanism by which the highly positive charge of MeCP2 and its ability to bind methylated DNA contribute to the specificity of its binding to chromatin has long remained elusive. In this paper, we show that MeCP2 binds to nucleosomes in a very similar way to linker histones both in vitro and in vivo. However, its binding specificity strongly depends on DNA methylation. We also observed that as with linker histones, this binding is independent of the core histone H3 N-terminal tail and is not affected by histone acetylation.     

Reversible acetylation of chromatin: Implication in regulation of gene expression,disease and therapeutics

The eukaryotic genome is a highly dynamic nucleoprotein complex that is comprised of DNA, histones, nonhistone proteins and RNA, and is termed as chromatin. The dynamicity of the chromatin is responsible for the regulation of all the DNA-templated phenomena in the cell. Several factors, including the nonhistone chromatin components, ATP-dependent remodeling factors and the chromatin-modifying enzymes, mediate the combinatorial post-translational modifications that control the chromatin fluidity and, thereby, the cellular functions. Among these modifications, reversible acetylation plays a central role in the highly orchestrated network. The enzymes responsible for the reversible acetylation, the histone acetyltransferases (HATs) and histone deacetylases (HDACs), not only act on histone substrates but also on nonhistone proteins. Dysfunction of the HATs/HDACs is associated with various diseases like cancer, diabetes, asthma, cardiac hypertrophy, retroviral pathogenesis and neurodegenerative disorders. Therefore, modulation of these enzymes is being considered as an important therapeutic strategy. Although substantial progress has been made in the area of HDAC inhibitors, we have focused this review on the HATs and their small-molecule modulators in the context of disease and therapeutics. Recent discoveries from different groups have established the involvement of HAT function in various diseases. Furthermore, several new classes of HAT modulators have been identified and their biological activities have also been reported. The scaffold of these small molecules can be used for the design and synthesis of better and efficient modulators with superior therapeutic efficacy.     

Nucleosome Interactions and Stability in an Ordered Nucleosome Array Model System

Although it is well established that the majority of eukaryotic DNA is sequestered as nucleosomes, the higher-order structure resulting from nucleosome interactions as well as the dynamics of nucleosome stability are not as well understood. To characterize the structural and functional contribution of individual nucleosomal sites, we have developed a chromatin model system containing up to four nucleosomes, where the array composition, saturation, and length can be varied via the ordered ligation of distinct mononucleosomes. Using this system we find that the ligated tetranucleosomal arrays undergo intra-array compaction. However, this compaction is less extensive than for longer arrays and is histone H4 tail-independent, suggesting that well ordered stretches of four or fewer nucleosomes do not fully compact to the 30-nm fiber. Like longer arrays, the tetranucleosomal arrays exhibit cooperative self-association to form species composed of many copies of the array. This propensity for self-association decreases when the fraction of nucleosomes lacking H4 tails is systematically increased. However, even tetranucleosomal arrays with only two octamers possessing H4 tails recapitulate most of the inter-array self-association. Varying array length shows that systems as short as dinucleosomes demonstrate significant self-association, confirming that relatively few determinants are required for inter-array interactions and suggesting that in vivo multiple interactions of short runs of nucleosomes might contribute to complex fiber-fiber interactions. Additionally, we find that the stability of nucleosomes toward octamer loss increases with array length and saturation, suggesting that in vivo stretches of ordered, saturated nucleosomes could serve to protect these regions from histone ejection.     

Insights into role of bromodomain, testis-specific (Brdt) in acetylated histone H4-dependent chromatin remodeling in mammalian spermiogenesis

Mammalian spermiogenesis is of considerable biological interest especially due to the unique chromatin remodeling events that take place during spermatid maturation. Here, we have studied the expression of chromatin remodeling factors in different spermatogenic stages and narrowed it down to bromodomain, testis-specific (Brdt) as a key molecule participating in chromatin remodeling during rat spermiogenesis. Our immunocytochemistry experiments reveal that Brdt colocalizes with acetylated H4 in elongating spermatids. Remodeling assays showed an acetylation-dependent but ATP-independent chromatin reorganization property of Brdt in haploid round spermatids. Furthermore, Brdt interacts with Smarce1, a member of the SWI/SNF family. We have studied the genomic organization of smarce1 and identified that it has two splice variants expressed during spermatogenesis. The N terminus of Brdt is involved in the recognition of Smarce1 as well as in the reorganization of hyperacetylated round spermatid chromatin. Interestingly, the interaction between Smarce1 and Brdt increases dramatically upon histone hyperacetylation both in vitro and in vivo. Thus, our results indicate this interaction to be a vital step in the chromatin remodeling process during mammalian spermiogenesis.     

Histone H4 acetylation dynamics determined by stable isotope labeling with amino acids in cell culture and mass spectrometry

This paper describes an integrated approach that couples stable isotope labeling with amino acids in cell culture to acetic acid-urea polyacrylamide gel electrophoresis (AU-PAGE) and matrix-assisted laser desorption/ionization time-of-flight mass spectrometry (MALDI-TOF MS) for the quantitation and dynamics of histone H4 acetylation. The 697 acute lymphoblastic cell lines were grown in regular medium and in medium in which lysine was substituted with deuterium-labeled lysine. Histone deacetylase (HDAC) activity was inhibited by addition of the HDAC inhibitor depsipeptide to the culture medium for different exposure times. Histones were extracted from cells pooled from unlabeled, untreated cells and from labeled, treated cells, followed by AU-PAGE separation. Gel bands corresponding to different acetylation states of H4 were excised, in-gel digested with trypsin, and analyzed by MALDI-TOF MS. Detailed information was obtained for both the change of histone H4 acetylation specific to the N terminus and the global transformation of H4 from one acetylation state to another following treatment with the HDAC inhibitor depsipeptide. The kinetics of H4 acetylation was also assessed. This study provides a quantitative basis for developing potential therapies by using epigenetic regulation and the developed methodology can be applied to quantitation of change for other histone modifications induced by external stimuli.     

Histone H3 lysine 14 acetylation is required for activation of a DNA damage checkpoint in fission yeast

Histone lysine acetylation has emerged as a key regulator of genome organization. However, with a few exceptions, the contribution of each acetylated lysine to cellular functions is not well understood because of the limited specificity of most histone acetyltransferases and histone deacetylases. Here we show that the Mst2 complex in Schizosaccharomyces pombe is a highly specific H3 lysine 14 (H3K14) acetyltransferase that functions together with Gcn5 to regulate global levels of H3K14 acetylation (H3K14ac). By analyzing the effect of H3K14ac loss through both enzymatic inactivation and histone mutations, we found that H3K14ac is critical for DNA damage checkpoint activation by directly regulating the compaction of chromatin and by recruiting chromatin remodeling protein complex RSC.     

A Distinct Switch in Interactions of the Histone H4 Tail Domain upon Salt-dependent Folding of Nucleosome Arrays

The core histone tail domains mediate inter-nucleosomal interactions that direct folding and condensation of nucleosome arrays into higher-order chromatin structures. The histone H4 tail domain facilitates inter-array interactions by contacting both the H2A/H2B acidic patch and DNA of neighboring nucleosomes (1, 2). Likewise, H4 tail-H2A contacts stabilize array folding (3). However, whether the H4 tail domains stabilize array folding via inter-nucleosomal interactions with the DNA of neighboring nucleosomes remains unclear. We utilized defined oligonucleosome arrays containing a single specialized nucleosome with a photo-inducible cross-linker in the N terminus of the H4 tail to characterize these interactions. We observed that the H4 tail participates exclusively in intra-array interactions with DNA in unfolded arrays. These interactions are diminished during array folding, yet no inter-nucleosome, intra-array H4 tail-DNA contacts are observed in condensed chromatin. However, we document contacts between the N terminus of the H4 tail and H2A. Installation of acetylation mimics known to disrupt H4-H2A surface interactions did not increase observance of H4-DNA inter-nucleosomal interactions. These results suggest the multiple functions of the H4 tail require targeted distinct interactions within condensed chromatin.     

体外装配核小体过程组蛋白浓度依赖的动力学模型

为探索组蛋白浓度对核小体体外装配的影响,本研究表达纯化了4种组蛋白,通过控制实验反应体系中组蛋白的浓度,利用盐透析法在体外装配了核小体,检测分析了组蛋白浓度与核小体组装效率的关系。以此实验数据为基础,提出了核小体组装过程组蛋白浓度依赖性的动力学模型。实验结果发现,反应体系中组蛋白浓度与核小体生成量呈典型的线性关系。依据动力学理论模型,进行线性回归拟合,回归系数达到0.963;经计算601 DNA序列组装核小体的反应速率常数k为1.49×10^-5mL·h·μg^-1。CS1序列验证动力学模型的线性回归相关系数为0.989,反应速率常数为1.52×10^-5mL·h·μg^-1。该实验方法及动力学模型中反应速率常数k可用于评价相同长度的DNA序列组装核小体的能力、组蛋白与其突变体以及组蛋白变体之间形成核小体结构能力的差异。该动力学模型的建立为理解核小体装配、核小体定位、染色质结构等相关问题提供了理论指导。     

On the function of the internal cavity of histone deacetylase protein 8: R37 is a crucial residue for catalysis

Biochemical studies reveal that a conserved arginine residue (R37) at the centre of the 14 Å internal cavity of histone deacetylase (HDAC) 8 is important for catalysis and acetate affinity. Computational studies indicate that R37 forms multiple hydrogen bonding interactions with the backbone carbonyl oxygen atoms of two conserved glycine residues, G303 and G305, resulting in a ‘closed’ form of the channel. One possible rationale for these data is that water or product (acetate) transit through the catalytically crucial internal channel of HDAC8 is regulated by a gating interaction between G139 and G303 tethered in position by the conserved R37.     

Surface effects in the acetylation of granular potato starch

The occurrence of surface effects in the acetylation of granular potato starch with acetic anhydride to degrees of substitution 0.04-0.2 was studied by two different approaches. The first approach involved the fractionation of granular starch acetates into five different size classes and analysis of their acetate content. Alternatively, two narrow size fractions of potato starch acetate granules were surface-peeled by chemical gelatinization in 5M CaCl(2), and the remaining cores were analyzed for acetyl content at different peeling levels. It was established that true surface peeling occurs in this medium and that the ester linkages are stable under the conditions applied. Both approaches led to the conclusion that the acetylation of potato starch granules is accompanied by a pronounced surface effect. The surface peeling method allows determination of the extent of substitution as a function of the radial position in the starch granule.     

Elevated levels of polyamines alter chromatin in murine skin and tumors without global changes in nucleosome acetylation

Polyamines affect nucleosome oligomerization and DNA conformation in vitro, yet little information exists regarding the influence of naturally synthesized polyamines on mammalian chromatin. Capitalizing on the relative inefficiency of a moderate ionic strength extraction buffer to dissociate histones, we obtained evidence of altered chromatin in transgenic mice that overexpress ornithine decarboxylase (ODC), which catalyzes polyamine synthesis. Dissociation of histones from chromatin in ODC transgenic mouse skin, as well as in tumors that develop spontaneously in ODC/Ras bigenic mice, is dramatically reduced relative to normal littermate skin. This could reflect tighter tethering of nucleosomes to DNA or a more compacted chromatin structure due to elevated intracellular concentrations of polyamines since this effect is reversible upon treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC enzymatic activity. Impeded release of nonhistone chromatin proteins HP-1beta and nucleophosmin, but not Lamin B, HDAC-1, HMGB, HMGN2, or HMGA1, suggests that polyamines exert selective effects on specific chromatin protein complexes. Moreover, overall acetylation, as well as specific methylation, of nucleosomes in ODC mice is unaffected, implying that access by histone modifying enzymes is not generally restricted. The abnormal chromatin environment fostered by elevated levels of polyamines may be a necessary prerequisite for epithelial tumor growth and maintenance.