Identification of nonhistone chromatin proteins in chromatin subunits (or mononucleosomes) devoid of histone H1.

Rat liver chromatin was digested by micrococcal nuclease. Chromatin subunits (or mononucleosomes) were isolated by sucrose density gradient and subsequently fractionated by 6% polyacrylamide gel electrophoresis into two major components. One component (MN1) of the mononucleosomes had a higher mobility, contained histones H2A, H2B, H3, H4, and shorter DNA fragments (140 base pairs) while the other (MN2) contained all five histones and longer DNA fragments (180 base pairs). Both submononucleosomes (MN1 and MN2) were found to contain nonhistone chromatin proteins (NHCP). By electrophoresis in 15% sodium dodecyl sulfate-polyacrylamide gel, 9 and 11 major fractions of NHCP were identified in the submononucleosomes MN1 and MN2, respectively. It was also observed that treatment of mononucleosomes with 0.6 M NaCl removes most of these NHCP and histone H1 except for two major NHCP which remain in the core particles.     

Chromosomal proteins of conifers

During imbibition and germination of jack pine seeds, the composition of the total extractable chromatin varied. Relative to DNA, the histone levels decreased as the nonhistone chromosomal proteins (NHCP) increased. New chromosomal proteins were synthesized after 2 days of imbibition as judged by recovery of ¹⁴C-amino acids from the major protein fractions. Phosphorylation of histones from ³²P-phosphoric acid was detected before the incorporation of ¹⁴C-amino acids. In the seed the synthesis and relative changes of chromatin coincided with a fall in total soluble protein and free arginine N. By contrast, adenylate energy charge, free glutamine N and in vitro template activity of chromatin increased during chromatin protein synthesis. When seeds had germinated for 4 days after the start of imbibition more radioactivity, derived from free ¹⁴C-amino acids, was recovered from the NHCP than from the histones. The percentage amino acid composition of most histone fractions remained stable, whereas the composition of NHCP changed more with time. The phosphorylation of NHCP was 8- to 41-fold greater than that of the histones. Phosphorylation of histone H4 was not detected at any stage of germination. Correlations between recovery of radioactivity (³²P and ¹⁴C) from chromosomal proteins and higher adenylate energy charge were positive.     

Histone H2A‐H2B binding by Pol α in the eukaryotic replisome contributes to the maintenance of repressive chromatin

The eukaryotic replisome disassembles parental chromatin at DNA replication forks, but then plays a poorly understood role in the re‐deposition of the displaced histone complexes onto nascent DNA. Here, we show that yeast DNA polymerase α contains a histone‐binding motif that is conserved in human Pol α and is specific for histones H2A and H2B. Mutation of this motif in budding yeast cells does not affect DNA synthesis, but instead abrogates gene silencing at telomeres and mating‐type loci. Similar phenotypes are produced not only by mutations that displace Pol α from the replisome, but also by mutation of the previously identified histone‐binding motif in the CMG helicase subunit Mcm2, the human orthologue of which was shown to bind to histones H3 and H4. We show that chromatin‐derived histone complexes can be bound simultaneously by Mcm2, Pol α and the histone chaperone FACT that is also a replisome component. These findings indicate that replisome assembly unites multiple histone‐binding activities, which jointly process parental histones to help preserve silent chromatin during the process of chromosome duplication.     

Interaction of short peptides with FITC-labeled wheat histones and their complexes with deoxyribooligonucleotides

Judging from fluorescence modulation (quenching), short peptides (Ala-Glu-Asp-Gly, Glu-Asp-Arg, Ala-Glu-Asp-Leu, Lys-Glu-Asp-Gly, Ala-Glu-Asp-Arg, and Lys-Glu-Asp-Trp) bind with FITC-labeled wheat histones H1, H2в, H3, and H4. This results from the interaction of the peptides with the N-terminal histone regions that contain respective and seemingly homologous peptide-binding motifs. Because homologous amino acid sequences in wheat core histones were not found, the peptides seem to bind with some core histone regions having specific conformational structure. Peptide binding with histones and histone-deoxyribooligonucleotide complexes depends on the nature of the histone and the primary structures of the peptides and oligonucleotides; thus, it is site specific. Histones H1 bind preferentially with single-stranded oligonucleotides by homologous sites in the C-terminal region of the protein. Unlike histone H1, the core histones bind pre-dominantly with double-stranded methylated oligonucleotides and methylated DNA. Stern-Volmer constants of interaction of histone H1 and core histones with double-stranded hemimethylated oligonucleotides are higher compared with that of binding with unmethylated ones. DNA or deoxyribooligonucleotides in a complex with histones can enhance or inhibit peptide binding. It is suggested that site-specific interactions of short biologically active peptides with histone tails can serve in chromatin as control epigenetic mechanisms of regulation of gene activity and cellular differentiation.     

Chromatin proteins associated with micrococcal nuclease-sensitive and nuclease-resistant chromatin fractions of Kirkman-Robbins hepatoma and hamster liver

Micrococcal nuclease-sensitive (SP) and nuclease-resistant (PP) chromatin fractions from Kirkman-Robbins hepatoma and hamster liver were obtained. The molecular distribution of three non-histone proteins (NHCP1, NHCP2 and NHCP3), histones, and chromatin-bound protease activity between SP and PP fractions of both tissues was compared. Differences, mainly of quantitative nature, among non-histone proteins of neoplastic and normal tissue were observed. Moreover, it was found that polypeptides with mol. wt 81 000 (NHCP1), 39 000 (NHCP2) and 21 000, 35 000, 37 000 (NHCP1), 70 000, 112 000, 141 000, 157 000 (NHCP2), 30 000–33 000 (NHCP3) were associated only with the nuclease-sensitive part of chromatin of hepatoma and normal tissue, respectively. A major difference in histone compostion of hamster hepatoma and liver concerns histones H2A and H1. Furthermore, an enrichment of high mobility group proteins as well as other soluble non-histone proteins in an acid extract of the SP fraction was observed. Apparently chromatin-bound protease activity can be found in both fractions of chromatin.     

Chromatin structure as probed by nucleases and proteases: Evidence for the central role of hitones H3 and H4

We have examined the role played by each histone in forming the structure of the ν-body. When DNAase I, DNAase II, trypsin, and chymotrypsin attack chromatin, characteristic discrete DNA and protein digest fragments are produced. Using this restriction of accessibility as diagnostic for chromatin structure, we have examined complexes of DNA with virtually all possible combinations of histones. The results strongly support our previous conclusion (Camerini-Otero, Sollner-Webb, and Felsenfeld, 1976) that the arginine-rich histones are unique in their ability to create, with DNA, a structure with many features of native chromatin. Acting together, slightly lysine-rich histones then modify this complex into one very similar to native chromatin. An analysis of the rate constants of staphylococcal nuclease digestion also confirms that the complex of H3, H4, and DNA is crucial to the structure of the ν-body.     

Affinity of chromatin constituents for isopeptidase

Isopeptidase is a novel eukaryotic enzyme that cleaves a structural chromatin protein, A24, stoichiometrically into H2A and ubiquitin. To understand the rapid turnover of ubiquitin in mitosis as wells as the high specific activity of the enzyme associated with metaphase chromosomes, attempts were made to determine chromatin constituents that show high affinity for this enzyme. Endogenous protease-free isopeptidase was prepared from calf thymus and applied to a Sepharose 4B affinity column on which histones, DNA, NHCP and ubiquitin were respectively immobilized. The enzyme proved to bind only histones. To further determine which of the histone fractions is involved, affinity columns with each histone fraction were also used. The enzyme showed affinity for all histone fractions. However, the strength of affinity varied in the order H2A>H³ H2B≥H4?H1, being inversely correlated with the ratio of basic/acidic amino acids in these molecules. These results suggest that the turnover of A24 in mitosis is controlled, at least in part, by the affinity of enzyme for histones, and also that such affinity is caused by a mechanism which cannot be explained simply by the electrostatic interaction between negatively charged enzyme molecules and positively charged histones.     

Triiodothyronine nuclear receptor. Role of histones and DNA in hormone binding

The triiodothyronine (T3) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T3 could restore, at least in part, the high affinity T3 binding. We now demonstrate that DNA alone increases the high affinity T3 binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T3 binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T3 binding sites but decrease the affinity for T3; addition of DNA restores the high affinity for T3 and stabilizes the T3-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T3 binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T3 binding site are of biological relevance in the regulation of T3 binding to its receptor remains to be determined.     

A Mechanism for Histone Chaperoning Activity of Nucleoplasmin: Thermodynamic and Structural Models

Nucleoplasmin (NP), a histone chaperone, acts as a reservoir for histones H2A-H2B in Xenopus laevis eggs and can displace sperm nuclear basic proteins and linker histones from the chromatin fiber of sperm and quiescent somatic nuclei. NP has been proposed to mediate the dynamic exchange of histones during the expression of certain genes and assists the assembly of nucleosomes by modulating the interaction between histones and DNA. Here, solution structural models of full-length NP and NP complexes with the functionally distinct nucleosomal core and linker histones are presented for the first time, providing a picture of the physical interactions between the nucleosomal and linker histones with NP core and tail domains. Small-angle X-ray scattering and isothermal titration calorimetry reveal that NP pentamer can accommodate five histones, either H2A-H2B dimers or H5, and that NP core and tail domains are intimately involved in the association with histones. The analysis of the binding events, employing a site-specific cooperative model, reveals a negative cooperativity-based regulatory mechanism for the linker histone/nucleosomal histone exchange. The two histone types bind with drastically different intrinsic affinity, and the strongest affinity is observed for the NP variant that mimicks the hyperphosphorylated active protein. The different “affinity windows” for H5 and H2A-H2B might allow NP to fulfill its histone chaperone role, simultaneously acting as a reservoir for the core histones and a chromatin decondensing factor. Our data are compatible with the previously proposed model where NP facilitates nucleosome assembly by removing the linker histones and depositing H2A-H2B dimers onto DNA.     

Human testis–specific Y-encoded protein-like protein 5 is a histone H3/H4-specific chaperone that facilitates histone deposition in vitro

DNA and core histones are hierarchically packaged into a complex organization called chromatin. The nucleosome assembly protein (NAP) family of histone chaperones is involved in the deposition of histone complexes H2A/H2B and H3/H4 onto DNA and prevents nonspecific aggregation of histones. Testis-specific Y-encoded protein (TSPY)–like protein 5 (TSPYL5) is a member of the TSPY-like protein family, which has been previously reported to interact with ubiquitin-specific protease USP7 and regulate cell proliferation and is thus implicated in various cancers, but its interaction with chromatin has not been investigated. In this study, we characterized the chromatin association of TSPYL5 and found that it preferentially binds histone H3/H4 via its C-terminal NAP-like domain both in vitro and ex vivo. We identified the critical residues involved in the TSPYL5–H3/H4 interaction and further quantified the binding affinity of TSPYL5 toward H3/H4 using biolayer interferometry. We then determined the binding stoichiometry of the TSPYL5–H3/H4 complex in vitro using a chemical cross-linking assay and size-exclusion chromatography coupled with multiangle laser light scattering. Our results indicate that a TSPYL5 dimer binds to either two histone H3/H4 dimers or a single tetramer. We further demonstrated that TSPYL5 has a specific affinity toward longer DNA fragments and that the same histone-binding residues are also critically involved in its DNA binding. Finally, employing histone deposition and supercoiling assays, we confirmed that TSPYL5 is a histone chaperone responsible for histone H3/H4 deposition and nucleosome assembly. We conclude that TSPYL5 is likely a new member of the NAP histone chaperone family.     

Induction of DNA synthesis by 2,4-dichlorophenoxyacetic acid during callus induction in Jerusalem artichoke tuber tissues

During the induction of DNA synthesis in Jerusalem artichoke (Helianthus tuberosus L.) tuber by 2,4-D, the 2-¹⁴C-2, 4-D from the agar medium rapidly incorporated into the ethanol soluble and insoluble fractions. Although the 2,4-D level in the ethanol soluble fraction decreased on transplantation of the tissue from the 2-¹⁴C-2,4-D medium to medium without the auxin, its level in the buffer-soluble and -insoluble macromolecular fractions increased. The purified, buffer-insoluble macromolecules were chromatin. The 2,4-D binding to chromatin particularly increased during DNA synthesis. The histone contents of chromatin decreased as DNA synthesis progressed. The polyacrylamide gel electrophoretic patterns of the histones showed a decrease in the moderately lysine-rich histone fraction as compared to other fractions. Thus, the decrease in the histone level caused by 2,4-D and the presence of the 2,4-D moderately lysine-rich histone complex may be closely related to the induction of DNA synthesis by 2,4-D in cells.     

Phosphorylation of casein by mitochondrial protein kinase(s)

Summary Chromatin fractions (DNA, histones and nonhistone chromosomal proteins NHCP) have been isolated from human peripheral B and T lymphocytes using different methods and analyzed in order to identify their lipid content.While DNA and histone fractions do not reveal the presence of lipids, a 2% of phospholipids is present in the NHCP fraction. The phospholipids associated with NHCP present a constant relative ratio among sphingomyelin, phosphatidyl-choline and phosphatidyl-ethanolamine both in B and T lymphocytes, whichever are the extraction procedures employed.These findings are related to the possible derepressive role of phospholipids on DNA-dependent RNA synthesis.     

Histone octamer trans-transfer: a signature mechanism of ATP-dependent chromatin remodelling unravelled in wheat nuclear extract

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.

Methods

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.

Conclusions

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.     

Triiodothyronine nuclear receptor: Role of histones and DNA in hormone binding

The triiodothyronine (T₃) nuclear receptor was previously shown to lose rapidly its high affinity hormone-binding property after a partial purification from the nuclear extract. It was then found that histones + DNA added to the incubation medium with labeled T₃ could restore, at least in part, the high affinity T₃ binding. We now demonstrate that DNA alone increases the high affinity T₃ binding site concentration moderately, and only at low ionic strength where it can bind to the receptor. Total histones and all histone fractions studied (total core histones, F2a, H2B, H3, H4, H1) specifically increase, at low concentrations, the level of T₃ binding; but higher concentrations of some individualized histones, particularly arginine-rich histones, have an inhibitory effect. DNA, or several other polynucleotides, in the presence of histones increase the stimulating histone effect and reverse the inhibitory effect into a true activation. Histones increase the number of T₃ binding sites but decrease the affinity for T₃; addition of DNA restores the high affinity for T₃ and stabilizes the T₃-receptor complexes. Thus, some of the histone molecules could play a role in the maintenance of the T₃ binding site, but multiple interactions between histones or with DNA seem necessary to impair the negative effect exerted by other parts of the histone molecules. Whether these positive and negative effects of histones on the T₃ binding site are of biological relevance in the regulation of T₃ binding to its receptor remains to be determined.     

Purification and characterization of a 400-kDa nonhistone chromatin protein that serves as an effective phosphate acceptor for casein kinase II from Ehrlich ascites tumor cells

A nonhistone chromatin protein (NHCP) has been purified to homogeneity from a 0.5 M NaCl extract of Ehrlich ascites tumor cell (EAT cell) nuclei as a phosphate acceptor for casein kinase II using ion-exchange column chromatographies and Sephacryl S300 gel filtration. The purified NHCP (approximate Mr = 400,000) was found to be a tetramer of an Mr = 98,000 polypeptide (pI = 6.9) and to have high contents of glycine (15%) and serine (11.6%). This protein (designated as 400-kDa NHCP) was highly phosphorylated by casein kinase II (Mr = 130,000), but not by histone kinase. Casein kinase II phosphorylated only seryl residues of the purified 400-kDa NHCP. The NHCP bound with DNA, but not with RNAs, and the DNA binding ability of the protein was reduced when it was phosphorylated by casein kinase II. Moreover, we found that (a) the 400-kDa NHCP is present in large quantities in malignant mouse cells, such as EAT, EL-4, and Meth-A cells, but only slightly in normal tissues and cells; (b) the protein level is rapidly increased when mouse lymphocytes are treated with recombinant interleukin 2 (T cell growth factor) or concanavalin A; and (c) the kinase responsible for the 400-kDa NHCP phosphorylation in the chromatin of various mouse cells is a casein kinase II. These experimental results suggest that the 400-kDa NHCP acts as an effective phosphate acceptor for casein kinase II at the chromatin level and that an increased phosphorylation of the protein by the kinase may be implicated in the progress of cell differentiation and proliferation.     

Structural transition in chromatin induced by ions in solution

Structural transition in chromatin was measured as a function of counter ions in solution (NaCl or MgCl(2)) and of histones bound on the DNA. The addition of counter ions to aqueous solutions of chromatin, partially dehistonized chromatin, and DNA caused a drastic reduction in viscosity and a significant increase in sedimentation coefficient. Transitions occurred primarily at about 2 x 10(-3) M NaCl and 1 x 10(-5) M MgCl(2) and are interpreted as a change in structure of chromatin induced by tight binding of cations (Na(+) or Mg(++)) to DNA, either free or bound by histones, and is an intrinsic property of DNA rather than of the type of histone bound. At a given ionic condition, removal of histone H1 from chromatin had only a minor effect on the hydrodynamic properties of chromatin while removal of other histones caused a drastic change in these properties. An increase in the sedimentation coefficient of DNA was observed also for protamine. DNA complexes wherein the bound protein contains only unordered coil rather than the alpha-helices found in histones.     

Ionic strength-dependent conformational transitions of chromatin. Circular dichroism and thermal denaturation studies

High-molecular-weight chicken erythrocyte chromatin was prepared by mild digestion of nuclei with micrococcal nuclease. Samples of chromatin containing both core (H3, H4, H2A, H2B) and lysine-rich (H1, H5) histone proteins (whole chromatin) or only core histone proteins (core chromatin) were examined by CD and thermal denaturation as a function of ionic strength between 0.75 and 7.0 × 10^?3M Na⁺. CD studies at 21°C revealed a conformational transition over this range of ionic strengths in core chromatin, which indicated a partial unfolding of a segment of the core particle DNA at the lowest ionic strength studied. This transition is prevented by the presence of the lysine-rich histones in whole chromatin. Thermal-denaturation profiles of both whole and core chromatins, recorded by hyperchromicity at 260 nm, reproducibly and systematically varied with the ionic strength of the medium. Both materials displayed three resolvable thermal transitions, which represented the total DNA hyperchromicity on denaturation. The fractions of the total DNA which melted in each of these transitions were extremely sensitive to ionic strength. These effects are considered to result from intra- and/or internucleosomal electrostatic repulsions in chromatin studied at very low ionic strengths. Comparison of the whole and core chromatin melting profiles indicated substantial stabilization of the core-particle DNA by binding sites between the H1/H5 histones and the 140-base-pair core particle.