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PubMed1998

Recruitment of phosphorylated chromatin assembly factor 1 to chromatin after UV irradiation of human cells.

The subcellular distribution and posttranslational modification of human chromatin assembly factor 1 (CAF-1) have been investigated after UV irradiation of HeLa cells. In an asynchronous cell population only a subfraction of the two large CAF-1 subunits, p150 and p60, were found to exist in a chromatin-associated fraction. This fraction is most abundant during S phase in nonirradiated cells and is much reduced in G2 cells. After UV irradiation, the chromatin-associated form of CAF-1 dramatically increased in all cells irrespective of their position in the cell cycle. Such chromatin recruitment resembles that seen for PCNA, a DNA replication and repair factor. The chromatin-associated fraction of p60 was predominantly hypophosphorylated in nonirradiated G2 cells. UV irradiation resulted in the rapid recruitment to chromatin of phosphorylated forms of the p60 subunit. Furthermore, the amount of the p60 and p150 subunits of CAF-1 associated with chromatin was a function of the dose of UV irradiation. Consistent with these in vivo observations, we found that the amount of CAF-1 required to stimulate nucleosome assembly during the repair of UV photoproducts in vitro depended upon both the number of lesions and the phosphorylation state of CAF-1. The recruitment of CAF-1 to chromatin in response to UV irradiation of human cells described here supports a physiological role for CAF-1 in linking chromatin assembly to DNA repair.

Animals↗
PubMed2006

Transcriptional coactivator PC4, a chromatin-associated protein, induces chromatin condensation.

Human transcriptional coactivator PC4 is a highly abundant multifunctional protein which plays diverse important roles in cellular processes, including transcription, replication, and repair. It is also a unique activator of p53 function. Here we report that PC4 is a bona fide component of chromatin with distinct chromatin organization ability. PC4 is predominantly associated with the chromatin throughout the stages of cell cycle and is broadly distributed on the mitotic chromosome arms in a punctate manner except for the centromere. It selectively interacts with core histones H3 and H2B; this interaction is essential for PC4-mediated chromatin condensation, as demonstrated by micrococcal nuclease (MNase) accessibility assays, circular dichroism spectroscopy, and atomic force microscopy (AFM). The AFM images show that PC4 compacts the 100-kb reconstituted chromatin distinctly compared to the results seen with the linker histone H1. Silencing of PC4 expression in HeLa cells results in chromatin decompaction, as evidenced by the increase in MNase accessibility. Knocking down of PC4 up-regulates several genes, leading to the G2/M checkpoint arrest of cell cycle, which suggests its physiological role as a chromatin-compacting protein. These results establish PC4 as a new member of chromatin-associated protein family, which plays an important role in chromatin organization.

Cell Cycle↗
PubMed2001

Relationship between chromatin compactness and dye uptake for in situ chromatin stained with DAPI.

BACKGROUND: This study investigated the relationship between chromatin compactness, which is directly related to chromatin condensation, and DAPI uptake. Materials and Methods For the structural characterization of in situ chromatin, we used fluorescence microscopy and differential scanning calorimetry on calf thymocytes. The compactness of nuclear chromatin was altered by permeabilizing native cells with NP40 detergent. A time-dependent analysis of detergent effects was performed by acquiring nuclear images at different time intervals after permeabilization. In order to compare nuclei of different sizes, we implemented a geometrical correction in the calculation of the integrated fluorescence intensity. For a quantitative evaluation of chromatin condensation we introduced two new parameters, "average chromatin packing ratio" and "average dye spatial density." RESULTS: This approach allowed us to estimate the effects of NP40 detergent at the level of in situ chromatin. Detergent effects could be modulated by changing the ionic composition of buffer. Moreover, changes of chromatin condensation induced by detergent were inversely related to modifications of nuclear volume. CONCLUSIONS: The combination of complementary information obtained by fluorescence microscopy, supported by a proper geometrical correction, and differential calorimetry allowed us to interpret the patterns of fluorescence intensities inside the nucleus in terms of chromatin structure.

Animals↗
PubMed1988

Reconstitution of chromatin higher-order structure from histone H5 and depleted chromatin.

Reconstitution of the 30 nm filament of chromatin from pure histone H5 and chromatin depleted of H1 and H5 has been studied using small-angle neutron-scattering. We find that depleted, or stripped, chromatin is saturated by H5 at the same stoichiometry as that of linker histone in native chromatin. The structure and condensation behavior of fully reconstituted chromatin is indistinguishable from that of native chromatin. Both native and reconstituted chromatin condense continuously as a function of salt concentration, to reach a limiting structure that has a mass per unit length of 6.4 nucleosomes per 11 nm. Stripped chromatin at all ionic strengths appears to be a 10 nm filament, or a random coil of nucleosomes. In contrast, both native and reconstituted chromatin have a quite different structure, showing that H5 imposes a spatial correlation between neighboring nucleosomes even at low ionic strength. Our data also suggest that five to seven contiguous nucleosomes must have H5 bound in order to be able to form a higher-order structure.

Animals↗
PubMed1982

Fractionation of chromatin, released by nuclease digestion, on ECTHAM-cellulose. Separation of active and inactive chromatin.

Chromatin released by two nucleases under various ionic conditions has been fractionated by chromatography on ECTHAM-cellulose. Mg2+ -soluble chromatin, which according to Gottesfeld and Partington is enriched in transcribed DNA sequences (Gottesfeld, J.M. and Partington, G.A., (1977) Cell 12, 953-962) and produced by DNAase II digestion at intermediate ionic strength, comprises material eluting from ECTHAM-cellulose at 80-100 mM Cl-, pH 6.8-7.0, whereas bulk, Mg2+ -insoluble chromatin comprises more tightly binding material. Free hnRNP particles elute at 30 mM Cl-, pH 6.8. Oligonucleosomes, which according to Dimitriadis and Tata are enriched in transcribed sequences (Dimitriadis, G.J. and Tata, J.R. (1980) Biochem. J. 187, 467-477) and produced by micrococcal nuclease digestion at physiological ionic strength, also elute predominantly at 80-100 mM Cl-, pH 6.8-7.0. When liver nuclei are digested with micrococcal nuclease at low ionic strength, the most rapidly released chromatin is enriched in nascent RNA and hnRNP particles, and binds weakly to ECTHAM-cellulose. More slowly solubilised chromatin, containing fewer hnRNP particles, binds much more strongly to ECTHAM-cellulose. In confirmation of results with mechanically sheared chromatin, the affinity of particular chromatin fractions is not dependent on the size of chromatin particles, rather it reflects the differing composition, and in particular the non-histone protein and hnRNP content, which, we propose, determines the conformation adopted by different chromatin fractions in the cation conditions used for elution from ECTHAM-cellulose.

Animals↗
PubMed1986

Tubulin-chromatin interactions: evidence for tubulin-binding sites on chromatin and isolated oligonucleosomes.

The interaction of tubulin with chromatin has been studied using a radiolabeled tubulin binding assay and velocity sedimentation analysis on isokinetic sucrose gradients. Soluble chromatin was prepared by mild micrococcal nuclease digestion of rat liver nuclei and tubulin was purified from rat brain by temperature-dependent assembly-disassembly and phosphocellulose chromatography. The tubulin-binding assay is based on the ability of chromatin to precipitate quantitatively at physiological ionic strength allowing separation of free tubulin from chromatin-bound tubulin. The binding of tubulin to unfractionated soluble chromatin was rapid, reversible and saturable. Saturation of binding sites was obtained using tubulin concentrations ranging from 0.5 to 400 micrograms/ml, in the presence of a high concentration (2.5 mg/ml) of another acidic protein, bovine serum albumin. The Scatchard and Hill plots showed that tubulin bound to a single class of non-interacting sites and yielded values of (0.5-0.6) X 10(7) M-1 for an apparent Ka and a maximal binding capacity of 0.8 nmol tubulin/mg DNA, i.e. about 1 molecule of tubulin/10 nucleosomes. Similar binding parameters were obtained when binding experiments were performed with insoluble chromatin in 0.15 M NaCl. Velocity sedimentation analysis of tubulin-chromatin complexes revealed that tubulin bound to all classes of chromatin oligomers, irrespective of the length of the nucleosomal chain. Tubulin-trinucleosome complexes formed from isolated trinucleosome in the presence of an excess of tubulin were separated from free reactants. It was found that 10-15% of the starting oligonucleosomal species reacted with tubulin, in a stoichiometry of about 0.8 molecule of tubulin/nucleosome. Given the characteristics of the binding and the expected cellular free tubulin concentration, the tubulin-chromatin interaction could possibly take place in vivo, when the nuclear membrane breaks down during the first steps of mitosis.

Animals↗
PubMed1976

Chromatin-bound protease: degradation of chromosomal proteins under chromatin dissociation conditions.

A chromatin-bound protease, active in 2 M NaCl-5 M urea or 5 M urea alone, was demonstrated in rat liver, kidney, testes, brain, rabbit bone marrow, chicken reticulocyte, and Ehrlich ascites chromatin. Chicken erythrocyte chromatin did not possess any detectable proteolytic activity in salt and urea. The proteolytic activity of rat liver chromatin in salt and urea was found to be independent of the methods of chromatin preparation. The protease can be inhibited by the serine specific reagents phenylmethanesulfonyl fluoride and diisopropyl fluorophosphate and the alkylating reagent, carbobenzoxyphenylalanine chloromethyl ketone, in the presence of organic solvents at 1 mM concentration. The inhibitions of chromatin-bound protease in rat liver by these compounds are irreversible. On the other hand, carbobenzoxyphenylalanine and p-nitrophenyl acetate were shown to be reversible inhibitors of rat liver chromatin-bound protease. The application of these inhibitors during the dissociation of chromatin by salt and urea may be useful to researchers interested in purifying various chromosomal proteins or to those researchers doing reconstitution studies with labile chromatins.

Animals↗
PubMed1990

Pentraxin-chromatin interactions: serum amyloid P component specifically displaces H1-type histones and solubilizes native long chromatin.

Pure serum amyloid P component (SAP) and native long chromatin, mixed together at wt/wt ratios between 1:1 and 1:2 in the presence of physiological concentrations of NaCl and calcium, both remained in solution, whereas each alone precipitates rapidly under these conditions. This solubilization accompanies the binding of SAP to chromatin and the displacement of H1-type histones, which are essential for condensation and higher order folding of chromatin. Such binding of SAP to chromatin is remarkable since displacement of H1 and H5 by salt alone requires approximately 0.5 M NaCl. SAP also bound to nucleosome core particles forming soluble complexes with an apparent stoichiometry of 1:2, a result that is compatible with attachment of SAP at the nucleosome dyad, the site of H1 in intact chromatin. SAP thus undergoes a specific, avid interaction with chromatin that promotes its solubilization and may thereby contribute to the physiological handling of chromatin released from cells in vivo. In contrast, C-reactive protein (CRP) did not bind significantly to either chromatin or to core particles at physiological ionic strength. Incubation of chromatin with either normal serum, or acute phase human serum containing raised levels of CRP, did not induce complement activation regardless of the presence of added SAP or CRP, nor was any cleavage of DNA observed.

Animals↗
PubMed2005

Valproic acid alters chromatin structure by regulation of chromatin modulation proteins.

Histone acetylation and deacetylation are crucial in the regulation of gene expression. Dynamic changes in gene expression may affect chromatin structure and, consequently, the interaction of chromatin with regulatory factors. In this study, the effects of the antiseizure drug valproic acid (VPA) on the expression of genes that regulate the structure of chromatin and the access of macromolecules to the DNA were investigated. Exposure of breast cancer cells to VPA resulted in rapid dose-dependent hyperacetylation of the histones H3 and H4. VPA further induced a depletion of several members of the structural maintenance of chromatin (SMC) proteins, SMC-associated proteins, DNA methyltransferase, and heterochromatin proteins. Down-regulation of these proteins was associated with chromatin decondensation. The observed alterations of chromatin structure correlated with enhanced sensitivity of DNA to nucleases and increased interaction of DNA with intercalating agents. VPA-induced chromatin decondensation led to a sequence-specific potentiation of DNA-damaging agents in cell culture and xenograft models. Modulation of heterochromatin maintenance proteins was not a direct, but a downstream, effect of histone acetylation. The effects on the chromatin structure were reversible upon drug withdrawal, but obligatory for the potentiation of DNA-damaging agents. In addition to their antitumor activity as single agents, the chromatin decondensation induced by histone deacetylase inhibitors may enhance the efficacy of cytotoxic agents that act by targeting DNA. The proposed mechanism of action suggests an effect of drug sequencing on the antitumor activity of these drugs.

Acetylation↗
PubMed1984

[Intranuclear localization of hepatoma chromatin antigens not detected in liver chromatin].

Localization of malignant cell antigens which are not detected in the liver chromatin was investigated by antibodies to chromatin of Zajdela ascite hepatoma and solid hepatoma 27. Antibodies to chromatin of Zajdela ascite hepatoma do not interact with nuclear matrix of both hepatoma and liver cells. Zajdela ascite hepatoma and solid hepatoma 27 chromatin regions hypersensitive to DNase I and endogenous Mg2+-dependent nuclease are enriched with immunogenic proteins. Antibodies to hepatoma chromatins pretreated with liver chromatin show that hepatoma chromatin antigens which are not detected in liver chromatin are localized in chromatin regions hypersensitive to nucleases but are absent (or scanty) in actively transcribed regions.

Animals↗
PubMedDate not supplied

[Electron microscopic morphometric study of activated chromatin of lymphocytes. I. Variation of chromatin condensations during early action of phytohemagglutinin].

We made a quantitative electron microscopic investigation of rat blood lymphocyte chromatin on cell sections in the early period of action of phytohaemoagglutinin (PHA). According to the dynamics of binding of acridine orange to chromatin, there were chosen time lapse after the beginning of stimulation of cells. In one hour of PHA action when the binding of acridine orange is intensified two-fold the total area of profiles of condensed chromatin was not changed, however, the area of membrane-free chromatin profiles was simultaneously diminished by 36%. At that period, the packing of DNP-fibers of condensed chromatin loosened. After 1.5 hr, parallel to a noticeable decrease of binding of acridine orange, there were observed tendencies for restoration of area of chromatin profiles and partial diminishing of relative distances between DNP-fibers. In 6 hrs, the binding of acridine orange was sharply intensified, which was accompanied by a decrease by 26% of the total area of chromatin profiles at the expense of diminishing of perimembranous chromatin. At the same time, the low-electron--density regions of condensed chromatin were enlarged, and the packing of DNP-fibers was strongly loosened.

Acridine Orange↗
PubMed2005

Two distinct mechanisms of chromatin interaction by the Isw2 chromatin remodeling complex in vivo.

We have previously shown that Saccharomyces cerevisiae Isw2 complex slides nucleosomes to remodel chromatin in vivo. Our data suggested a model in which Isw2 complex binds the histone octamer and DNA separately to generate the force necessary for nucleosome movement. Here we find that the histone H4 "basic patch" is the only portion of any amino-terminal histone tail required for both target-specific association of Isw2 complex with chromatin and chromatin remodeling in vivo, whereas it is dispensable for basal levels of chromatin binding. Similarly, we find that nonremodeled chromatin structure and integrity of Isw2 complex are required only for target-specific association of Isw2 with chromatin. These data demonstrate fundamental differences between the target-specific and basal modes of chromatin binding by Isw2 complex in vivo and suggest that only the former involves contributions from DNA, histone H4, and sequence-specific DNA binding proteins. We propose a model for target recognition and chromatin remodeling by Isw2 complex in vivo.

Adenosine Triphosphatases↗
PubMed1984

Immunochemical study of chromatin non-histone proteins. II. Localization of immunogenic tissue-specific proteins in nuclease-hypersensitive sites of chromatin.

Localization of immunogenic tissue-specific and tissue-non-specific non-histone proteins in thymocyte chromatin has been investigated using antibodies against rat thymus and liver chromatin. After chromatin digestion by DNAase II and subsequent fractionation with 2 mM MgCl2, the Mg2+-soluble fraction interacts with both types of antibodies 5-6 times more effectively than Mg2+-insoluble chromatin. The experiments on chromatin digestion with DNAase I indicate that tissue-specific and tissue-non-specific proteins, reacting with antibodies, are released only upon hydrolysis of the first 1-3% of DNA. Further digestion with DNAase I causes no additional solubilization of these proteins. The chromatin fraction enriched in immunogenic proteins is also released upon autolytic digestion of chromatin with endogenous nuclease. The data obtained suggest that by their function tissue-specific and tissue-non-specific antigenic determinants belong to the same class of non-histone proteins localized in the chromatin sites hypersensitive to nucleases. The possible role of these proteins in regulation of the transcription is discussed.

Animals↗
PubMed1981

Different chromatin structures in Physarum polycephalum: a special form of transcriptionally active chromatin devoid of nucleosomal particles.

Nonnucleolar chromatin from interphase nuclei of Physarum polycephalum plasmodia occurs in two different structural configurations as seen in electron microscopic spread preparations. While the majority of the chromatin is devoid of nascent ribonucleoprotein (RNP) fibrils and compacted into nucleosomal particles, a minor proportion (10-20%) is organized differently and reveals a smooth contour. It is this form of smooth chromatin which is rich in transcription units (mean length: 1.36 +/- 0.21 micrometer). Only occasionally are solitary nascent RNP fibrils observed which are associated with beaded strands of chromatin. In transcribed smooth chromatin nucleosomal particles are not only absent from the transcription units but also from their nontranscribed flanking regions, indicating that this special structural aspect is not merely a direct consequence of the transcriptional process. The existence of ca. 10-20% of Physarum chromatin in the smoothly contoured form is discussed in relation to reports of a preferential digestibility of a similar proportion of Physarum chromatin by DNAse I (Jalouzot et al., 1980) and to the altered configuration of "peak A" chromatin subunits after micrococcal nuclease digestion (Johnson et al., 1978 a, b).

Chromatin↗
PubMed1979

Effect of salts and chromatin concentrations on the buoyant density of chromatin in metrizamide gradient.

Using isopycnic centrifugation in metrizamide gradient, effect of ions and chromatin concentration on the buoyant density of chromatin was quantitatively examined. An elevation followed by gradual decline and secondary increase of the density occurred in accordance with increase in MgCl2 or NaCl concentration. Maximum density was observed at a concentration of these salts known to result in the condensation of chromatin. Release of protein occurred during the phase of density decline. The second increase in density is mainly due to the density increment of DNA in the chromatin. The density was dependent upon the concentration of chromatin in a band formed in the metrizamide gradient, while the density of free DNA and protein was not so greatly affected by their concentration. The density of chromatin in the presence of 0.14 M NaCl was less affected by the chromatin concentration than that in the absence of salt. Calculation of results indicates that grade of hydration of chromatin at concentrations lower than 400 microgram/ml in 1 mM Tris-HCl (pH 8.0) is higher than that expected from its DNA and protein components.

Animals↗
PubMed2005

Gal4-VP16 directs ATP-independent chromatin reorganization in a yeast chromatin assembly system.

Major insights into the regulation of chromatin organization have stemmed from biochemical studies using Gal4-VP16, a chimeric transcriptional activator in which the DNA binding domain of Gal4p is fused to the activation domain of viral protein VP16. Unexpectedly, given previous intensive efforts to understand how Gal4-VP16 functions in the context of chromatin, we have uncovered a new mode of chromatin reorganization that is dependent on Gal4-VP16. This reorganization is performed by an activity in a crude DEAE (CD) fraction from budding yeast which also supports ATP-dependent assembly of physiologically spaced nucleosome arrays. Biochemical analysis reveals that the activity tightly associates with chromatin and reorganizes nucleosome arrays by a mechanism which is insensitive to ATP depletion after nucleosome assembly. It generates a chromatin organization in which a nucleosome is stably positioned immediately adjacent to Gal4p binding sites in the template DNA. Individual deletion of genes previously implicated in chromatin assembly and remodeling, namely, the histone chaperones NAP1, ASF1, and CAC1 and the SNF2-like DEAD/H ATPases SNF2, ISW1, ISW2, CHD1, SWR1, YFR038w, and SPT20, does not significantly perturb reorganization. Therefore, Gal4-VP16-directed chromatin reorganization in yeast can occur by an ATP-independent mechanism that does not require SAGA, SWI/SNF, Isw1, or Isw2 chromatin remodeling complexes.

Adenosine Triphosphate↗
PubMed1997

Structure of active chromatin: isolation and characterization of transcriptionally active chromatin from rat liver.

Rat liver nuclei were isolated in low-ionic-strength buffer in the absence of bi- and multi-valent cations. Digestion of these nuclei by endogenous nuclease, micrococcal nuclease and DNase I revealed that a minor chromatin fraction was preferentially digested into poly- and oligo-nucleosomes. Southern blot hybridization with various active gene probes confirmed that these chromatin fragments represent coding and 5' upstream regions of transcriptionally active chromatin. Active chromatin fragments were released selectively into the medium, with inactive chromatin remaining inside the nuclei, under the above ionic conditions. The inclusion of bivalent cations during the digestion of nuclei reversed the solubility behaviour of active chromatin. Rearrangement and exchange of histone H1 between chromatin fragments was prevented by using low-salt conditions in all steps in the absence of bivalent cations. All histones, including H1, were present in stoichiometric amounts in this active chromatin fraction. Active nucleosomes showed a lower electrophoretic mobility than bulk nucleosomes in an acrylamide/agarose composite gel in the absence of Mg2+, but were selectively bound to the gel in the presence of this ion.

Animals↗