Analysis of the dynamic equilibrium of histone oligomers in a solution. The nature of forces stabilizing the (H2A-H2B-H3-H4)2 octamer structure

Dynamic equilibrium analysis of the (H2A-H2B-H3-H4)2 histone octamer with lower oligomers was performed in 2 M NaCl. Calculated data on the relative content of histone oligomers upon changing protein concentration in solution are given. The red shift of lambda max for histone tyrosine fluorescence spectra is shown to be due to hydrogen bond formation by tyrosyl OH-groups. Analysis of free energy changes of histone oligomers upon association (delta G = -17,37 +/- 0,14 kcal/mole) as well as the effect of urea on histone octamer dissociation made it possible to conclude that virtually all tyrosyls in octamer form hydrogen bonds. Intermolecular hydrogen bonds formed by tyrosyls contribute substantially to octamer stabilization. The (H2A-H2B) dimer positive cooperativity in association with the (H3-H4)2 tetramer was found. This cooperativity is caused by interaction between association sites with a two order increase in an apparent constant of dimers with tetramer association. The histone octamer was determined to be of asymmetric structure due to unequivolency of the two binding sites for the (H2A-H2B) dimers.     

Spatial organization of the (H3-H4-H2A-H2B)2 histone octamer

Structure of the (H2A-H2B-H3-H4)2 histone octamer isolated from calf thymus chromatin at ionic strength 0.1 to 4.0 M NaCl, pH 7.6, was studied spectrofluorometrically. Sensitivity of lambda max tyrosine fluorescence position to structural changes of histone oligomers and to the processes of their association was shown. It were detect two ranges of cooperative changes in histone optical parameters at 0.6-1.4 M NaCl (transition I) and at 2.4-3.4 M NaCl (transition II): Transition I corresponds to the formation of equilibrium system (hexamer) + (dimer) in equilibrium octamer. Transition II corresponds to the structural changes of the histone octamer. Thus, fluorescence anisotropy increases, lambda max for fluorescence spectrum is shifted to the longer wavelengths, contributions of two components to fluorescence decay change, a fraction of fluorescence accessible to the quenching by I- decreases. Histone octamer formation is characterized by making specific contacts between the (H2A-H2B) dimer and (H3-H4)2 tetramer. These contacts are realized at gradual changing of ionic strengths (by dialysis). In the case of abrupt local changes of the environment the process is irreversibly shifted to formation of unspecific high molecular aggregates. The important function role for energetically degenerated states of histone oligomers, energy barriers between which can be overcome by changing total conditions of histone microenvironment in chromatin is discussed.     

Histones and histone phosphorylation during quail myogenesis in vitro

Cultured quail myoblasts were labelled with 32Pi and nuclear proteins extracted before and after myoblast fusion. Histones H1, H4 and the H1-H2B-H2A complex were all phosphorylated in proliferating prefusion cultures, while histone phosphorylation was absent in B1-arrested postfusion cultures except for minor phosphorylation of the H3-H2B-H2A complex. Postfused cultures were distinguished by the appearance of the histone-like protein whch migrated slightly faster than H1. Histone phosphorylation is therefore correlated with cell proliferation, while the appearance of the new histone-like protein is associated with G1 arrest and the absence of cell division.     

Yeast CAF-1 assembles histone (H3-H4)2 tetramers prior to DNA deposition

Following acetylation, newly synthesized H3-H4 is directly transferred from the histone chaperone anti-silencing factor 1 (Asf1) to chromatin assembly factor 1 (CAF-1), another histone chaperone that is critical for the deposition of H3-H4 onto replicating DNA. However, it is unknown how CAF-1 binds and delivers H3-H4 to the DNA. Here, we show that CAF-1 binds recombinant H3-H4 with 10- to 20-fold higher affinity than H2A-H2B in vitro, and H3K56Ac increases the binding affinity of CAF-1 toward H3-H4 2-fold. These results provide a quantitative thermodynamic explanation for the specific H3-H4 histone chaperone activity of CAF-1. Surprisingly, H3-H4 exists as a dimer rather than as a canonical tetramer at mid-to-low nanomolar concentrations. A single CAF-1 molecule binds a cross-linked (H3-H4)₂ tetramer, or two H3-H4 dimers that contain mutations at the (H3-H4)₂ tetramerization interface. These results suggest that CAF-1 binds to two H3-H4 dimers in a manner that promotes formation of a (H3-H4)₂ tetramer. Consistent with this idea, we confirm that CAF-1 synchronously binds two H3-H4 dimers derived from two different histone genes in vivo. Together, the data illustrate a clear mechanism for CAF-1-associated H3-H4 chaperone activity in the context of de novo nucleosome (re)assembly following DNA replication.     

Acetylation of histones of rat testis.

When [1-¹⁴C]acetate was injected into rats intratesticularly in the presence of cycloheximide to inhibit protein synthesis, the label was incorporated into histone fractions F2a1 and F3 and into non-histone chromosomal proteins of each of the following stages of spermatogenesis: spermatogonia-preleptotene spermatocytes, leptotene-zygotene-pachytene-diplotene primary spermatocytes, and spermatids. Acetylation of histones was particularly active in the spermatid stages. There was no significant incorporation of acetate into the lysine-rich histone fractions F1 and X₁.In early periods of in vivo incorporation of [³H]amino acids into histones the acetylated histone F2a1 fractions had higher specific activities than the main band of F2a1, but with the passage of time the label moved into the principal band to the extent that specific activities in the acetylated and principal bands were approximately equal at 6 days. However, at 24–36 days the specific activities were again higher in the acetylated bands than in the principal band of F2a1. These data support the conclusions of Candido, Louie, and Dixon, from experiments with trout testis, that acetylation of histone F2a1 may be important in the process of combination of this protein with DNA in chromatin at the spermatogonia-primary spermatocyte stage and also in the subsequent removal of this histone for replacement by protamines at the spermatid stage.[³H]Amino acids were incorporated into histone fractions X₁ and F1 at approximately equal rates, and there was no evidence that one of these fractions was a precursor of the other.Chromatin of the seminiferous epithelial cells of rat testis has a firmly bound acetylase which catalyzes the in vitro acetylation of histones F3 and F2a1 by acetyl CoA.     

A rapid method of preparing the (H3-H4-H2A-H2b)(2) histone octamer in large quantities]

A simple and fast method for isolation of large amounts of the histone octamer (H2A-H2B-H3-H4)2 is proposed. This method is based on chromatin adsorption by hydroxyapatite with subsequent extraction of the histone octamer with 50 mM sodium-phosphate buffer containing 4 M NaCl pH 8.0. It was shown that the properties of the histone octamer isolated by this extractive procedure are identical with those of the histone octamer obtained by elution on a Sephadex G-100 column. The histone tetramer (H3-H4)2 and dimer (H2A-H2B) were obtained after gel filtration on Sephadex G-100 in 50 mM sodium-acetate (pH 5.6).     

The presence of F3-F2a1 dimers and F1 oligomers in chromatin.

The oligomeric structure of histones in nuclei and chromatin has been studied by crosslinking nuclei and chromatin with 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide. Crosslinked histones were detected as new high molecular weight components on SDS gels, and the protomers of the crosslinked histones were identified by their characteristic ¹²⁵I-fingerprints. The results show that a considerable portion of histones F3 and F2a1 exist in nuclei and chromatin as an F3-F2a1 dimer. Evidence is presented that histone F1 probably exists in chromatin as large oligomers.     

Nonallelic histone gene clusters of individual sea urchins (Lytechinus pictus): Polarity and gene organization

We have analyzed the histone genes from the sea urchin Lytechinus pictus. Examination of native DNA from individuals reveals four major Eco RI restriction endonuclease histone gene DNA fragments which have been labeled A (6.0 kb), B (4.1 kb), C (3.1 kb) and D (1.2 kb). The fragments A, B and C have been cloned into E. coli plasmids (pLpA, pLpB and pLpC). These histone gene fragments display length and sequence heterogeneity in different individuals. The plasmid pLpA contains the coding regions for H1, H4, H2B and H3 histones, and we determined that the DNA fragment D is tandem to A in native DNA and that it contains the H2A gene. The plasmids pLpB and pLpC contain the histone genes H2A-H1-H4 and H2B-H3, respectively, and together contain the sequences for the five major histones. Restriction analysis of native L. pictus DNA reveals that B and C are tandem to each other but not intermingled with the A-D-type repeat units, and are thus in separate clusters with a repeat length of 7.2 kb. Since the two cluster types do not segregate, they are not alleles. Hybridization of histone mRNA to exonuclease III-digested linear DNA demonstrated an identical polarity of the histone genes in the A-D- and B-C-type repeat units. This result revealed that the L. pictus histone genes have a polarity which is the same as other sea urchin histone genes examined to date—that is, 3′ H1-H4-H2B-H3-H2A 5′. Restriction endonuclease cleavage patterns of the cloned segments indicate that considerable sequence heterogeneity exists between the two types of histone gene repeat units.     

Inhibition of histone H3-H4 chaperone pathways rescues C. elegans sterility by H2B loss

Oncohistone mutations are crucial drivers for tumorigenesis, but how a living organism governs the loss-of-function oncohistone remains unclear. We generated a histone H2B triple knockout (3KO) strain in Caenorhabditis elegans, which decreased the embryonic H2B, disrupted cell divisions, and caused animal sterility. By performing genetic suppressor screens, we uncovered that mutations defective in the histone H3-H4 chaperone UNC-85 restored H2B 3KO fertility by decreasing chromatin H3-H4 levels. RNA interference of other H3-H4 chaperones or H3 or H4 histones also rescued H2B 3KO sterility. We showed that blocking H3-H4 chaperones recovered cell division in C. elegans carrying the oncohistone H2B^E74K mutation that distorts the H2B-H4 interface and induces nucleosome instability. Our results indicate that reducing chromatin H3-H4 rescues the dysfunctional H2B in vivo and suggest that inhibiting H3-H4 chaperones may provide an effective therapeutic strategy for treating cancers resulting from loss-of-function H2B oncohistone.     

Studies on histone organization in the nucleosome using formaldehyde as a reversible cross-linking agent

A new procedure is described which allows selective reversal of formaldehyde cross-linking in both histone-histone and histone-DNA of nuclei isolated from calf thymus. All ten possible dimers of the four non-H1 histones, H3, H2B, H2A and H4, are observed, the major dimers being H3-H3, H3-H2A, H2B-H2A, H2a-H2A and two separate dimers of H2B-H4. Although oligomers of the non-H1 histones are formed by prolonged treatment with this reagent, 50% of the histones continue to remain resistant to cross-linking with each other. For those histones which cross-linking with each other. For those histones which cross-link, the site of cross-linking within the molecules is located in the "core" (trysin-resistant) regionand therfore indicates proximities for these molecules within the nucleosome. The core region also cross-links to DNA, indicating intimate interactions between this region in all the non-H1 histones with DNA.     

Fractionation of avian erythrocyte histones by hydrophobic interaction chromatography.

The interactions of H1 (H1A, H1B), H2A, H2B, H3, H4, and H5 with phenyl cross-linked agarose were studied. Procedures are described whereby all six histones can be bound, released, and fractionated by using appropriate salt concentrations or pH. The binding can be totally abolished by inclusion of hydrophobic disrupting agents. Control experiments with nonderivated cross-linked agarose ruled out a passive aggregation-disaggregation phenomenon governing the binding patterns. The absorption sequence based on the identification and quantitation of individual histones from either unfractionated (whole) histone or separate histone classes is as follows: H3 greater than or equal to H4 greater than H2B greater than or equal to H5 greater than or equal to H2A greater than H1A greater than or equal to H1B. The order differs only slightly from the reverse of the desorption sequence, H1B less than or equal to H1A less than or equal to H5 less than H2A less than or equal to H3. Preferential interaction of H2A-H2B, H3-H4, and H2A-H2B-H4 occur; these interactions can modify the original relative affinity of each individual component for the matrix. The variability in matrix affinity appears to involve simple stoichiometry of the histone components.     

纳米二氧化锆对人永生化角质形成细胞组蛋白H3修饰的影响

目的 阐明纳米二氧化锆暴露对人永生化角质形成细胞Ha Ca T组蛋白H3常见修饰位点的影响,探讨组蛋白H3修饰变化的潜在机制,为纳米材料的进一步安全应用提供理论基础。方法 在利用扫描电子显微镜、激光粒度仪、X射线衍射仪等技术对纳米二氧化锆进行详细表征的基础上,通过蛋白质免疫印迹及流式细胞术等方法评价纳米二氧化锆暴露对细胞生存率、细胞内蓄积量以及组蛋白H3修饰等的影响。结果 在分散介质中纳米二氧化锆明显团聚,比表面积减少,二次粒径增大,其短时间内（1 h）即诱导了组蛋白H3第10位丝氨酸的磷酸化、第9及14位赖氨酸的乙酰化、第4及27位赖氨酸的三甲基化修饰水平的升高。进一步分析发现,纳米二氧化锆的细胞内蓄积量及其引起的DNA损伤水平,与纳米二氧化锆诱导的组蛋白H3修饰水平均呈线性相关。结论 纳米二氧化锆暴露后诱导了Ha Ca T细胞组蛋白H3常见修饰位点的变化,其细胞内的蓄积是诱导组蛋白H3修饰变化的关键因素之一,且组蛋白H3修饰的调控机制可能涉及DNA损伤修复途径。     

A series of repetitive DNA sequences are associated with human core and H1 histone genes

Summary Repetitive DNA sequences, derived from the human β-globin gene cluster, were mapped within a series of human genomic DNA segments containing core (H2A, H2B, H3 and H4) and H1 histone genes. Cloned recombinant λCH4A phage with human histone gene inserts were analyzed by Southern blot analysis using the following³²P-labeled (nick translated) repetitive sequences as probes:Alu I,Kpn I and LTR-like. A cloned DNA designated RS002-5′C6 containing (i)a (TG)₁₆ simple repeat, (ii) an (ATTTT)n repeat and (iii)a 52 base pair alternating purine and pyrimidine sequence was also used as a radiolabelled hybridization probe. Analysis of 12 recombinant phage, containing 6 arrangements of core histone genes, indicated the presence ofAlu I,Kpn and RS002-5′C6 repetitive sequences. In contrast, analysis of 4 human genomic DNA segments, containing both core and H1 histone genes, indicated the presence of onlyAlu I family sequences. LTR-like sequences were not detected in association with any of the core or H1 histone genes examined. These results suggest that human histone and β-globin genes share certain aspects of sequence organization in flanking regions despite marked differences in their overall structure and pattern of expression.     

Synthesis and phosphorylation of chromatin-associated proteins in cAMP-induced "differentiated" neuroblastoma cells in culture.

Prostaglandin E₁ and a cAMP phosphodiesterase inhibitor 4-(3-butoxy-4-methoxybenzyl)-2-imidazolidinone, RO20-1724, were used to induce differentiation in mouse neuroblastoma cells in culture. The incorporation of amino acids and phosphate into nuclear proteins of control and drug-treated cells (1 h and 3 days after treatment) was examined using double radioisotopic techniques. A marked decrease in histone synthesis and H1-histone phosphorylation were observed in ‘differentiated’ neuroblastoma cells after 3 days of prostaglandin E₁ and RO20-1724 treatment, but only small differences were noted in the synthesis and phosphorylation of non-histone chromatin associated proteins after 3 days of drug treatment. Minimal changes were observed in the labeling of histone and non-histone nuclear proteins if the cells were treated for 1 h with prostaglandin E₁ and RO20-1724.     

Nucleosomes and subnucleosomes: heterogeneity and composition

Previous studies (Varshavsky, Bakayev and Georgiev, 1976a) have shown that chromatin subunits (mononucleosomes) and their oligomers in a mild staphylococcal nuclease digest of chromatin display a heterogeneous content of histone H1. We now report that a mild staphylococcal nuclease digest of either chromatin or nuclei from mouse Ehrlich tumor cells contains mononucleosomes of three discrete kinds. The smallest mononucleosome (MN₁) contains all histones except H1 and a DNA fragment 140 base pairs (bp) long. The intermediate mononucleosome (MN₂) contains all five histones and a DNA fragment 170 bp long. The third mononucleosome (MN₃) also contains all five histones, but its DNA fragment is longer and more heterogeneous in size (180–200 bp). Most of the MN₃ particles are rapidly converted by nuclease into mononucleosomes MN₁ and MN₂ There exists, however, a relatively nuclease-resistant subpopulation of the MN₃ mononucleosomes. These 200 bp MN₁ particles contain not only histones but also nonhistone proteins, and are significantly more resistant to nuclease than the bulk of MN₃ particles and the smaller mononucleosomes MN₁ and MN₂.There are eight major kinds of staphylococcal nuclease-produced soluble subnucleosomes (SN). The SN₁ is a set of naked double-stranded DNA fragments ～20 bp long. The SN₂ is a complex of a specific basic nonhistone protein (molecular weight ～16,000 daltons) and a DNA fragment ～27 bp long. The SN₃ contains histone H4, the above-mentioned specific nonhistone protein and a DNA fragment ～27 bp long. The SN₄ contains histones H2a, H2b, H4 and a DNA fragment ～45 bp long. The SN₅ contains histones H2a, H2b, H3 and a DNA fragment ～55 bp long. The SN₆ is a complex of histone H1 and a DNA fragment ～35 bp long. Subnucleosomes SN₇ and SN₈ each contain all the histones except H1, and DNA fragments ～100 and ～120 bp long, respectively.Nuclease digestion of isolated mono- or dinucleosomes does not produce some of the subnucleosomes. These and related findings indicate that the cleavage required to generate these subnucleosomes result from some aspect of chromatin structure which is lost upon digestion to mono- and dinucleosomes.     

The molecular size of glycans liberated by hydrazinolysis from semliki forest virus proteins

The glycans of well characterized, [6-³H]galactose-labelled glycopeptides, GC-4 from bovine IgG₁ as well as GP-V-2 and GP-V-5 from α₁-acid glycoprotein, were liberated by hydrazinolysis. Molecular weights close to the expected values were observed by gel filtration. Desialated glycans of Semliki Forest virus proteins were likewise liberated by hydrazinolysis and subjected to gel filtration. Metabolically labelled [1-³H]galactose-oligosaccharides of the mixed viral proteins revealed an apparent molecular weight of 1800. The bi-antennary glycan liberated from the reference glycopeptide GC-4 was of 1750 daltons. A mixture of [2-³H]mannose-labelled E₁-and E₂-proteins of the virus contained L-type glycans of 1800 daltons (formerly called A-type), and M-type glycans of 1200 daltons (formerly called B-type). A fraction of the E₃-glycans isolated by affinity chromatography on Concanavalin A-Sepharose showed an average molecular weight of 2150, a value intermediate between the three- and four-antennary glycans liberated from the reference glycopeptides GP-V-5 and GP-V-2. The rest of the E₃-glycans were of 1850 daltons, a value close to the bi-antennary GC-4 glycan. We suggest that the comparatively large size of the E₃-glycans and the exposed position of E₃-proteins on the viral surface may be interrelated.