Selective release of HMG nonhistone proteins during DNase digestion of Tetrahymena chromatin at different stages of the cell cycle.

The possible role of LG-1, a Tetrahymena specific HMG protein found in the macronuclear chromatin (Hamana, K. and Iwai, K. (1979) J. Biochem. 86, 789-794), was examined in relation to the chromatin structure. The chromatin isolated from cells synchronized at different stages of the cell cycle contained about one molecule of LG-1 per nucleosome. Limited digestion of the chromatin with DNase I or micrococcal nuclease selectively released LG-1 with the nucleosomal core histones and H1 remained insoluble, bound to the resistant DNA. Depending on the cell stages several types of chromatin structure were distinguished by their nuclease sensitivity. However, the chromatin at different stages exhibited the similar behavior of the LG-1 release with the nucleases as a function of the degree of chromatin solubilization. The results suggest that LG-1 proteins play a role in the chromatin organization which is rather independent of the cell stages.     

Characterization of the progesterone receptor solubilized by micrococcal nuclease and DNase I digestion

In order to investigate the functional organization of the progesterone receptor in chromatin we characterized the physical-chemical properties of the receptor bound chromatin fragments released by micrococcal nuclease and DNase I digestion. The crude nuclear fraction was isolated from T 47 D cells, previously exposed to 0.1 microM [3H]ORG 2058. The parameters determined in low and high salt concentrated buffers were: sedimentation coefficients (S) on a sucrose gradient, Stokes radii (Rs) by gel filtration on a Sephadex G-200 column and the binding abilities to a DNA-cellulose column. The molecular weights (Mr) and frictional ratios (f/fo) were calculated from the S and Rs values. Micrococcal nuclease digestion solubilized a receptor form sedimenting as a single peak at 4.4 S with a Rs = 7.78 nm and an estimated Mr = 144,000. About 53% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. 0.4 M KCl dissociated this receptor form into a smaller receptor sedimenting at 3.3 S with Rs = 5.53 nm and a calculated Mr = 76,000. A similar receptor form was extracted by 0.6 M KCl from the undigested crude nuclear fraction. DNase I digestion solubilized a receptor form sedimenting at 3.3 S with a Rs = 6.87 nm and a calculated Mr = 94,000. About 26% of the applied receptor bound to a DNA-cellulose column could be eluted by high salt concentrated buffer. Dissociation of this receptor form by 0.4 M KCl resulted in a receptor sedimenting at 2.8 S with a Rs = 6.53 nm and an estimated Mr = 76,000. These results suggest: The progesterone receptor in chromatin is associated with several molecules probably proteins which complexed it to DNA. Some of these molecules still associated with the progesterone receptor could be released by nucleases digestion. Micrococcal nuclease releases a larger portion of these molecules than those release by DNase I.     

Fractionation of nucleosomes by salt elution from micrococcal nuclease- digested nuclei

The solubilization of nucleosomes and histone H1 with increasing concentrations of NaCl has been investigated in rat liver nuclei that had been digested with micrococcal nuclease under conditions that did not substantially alter morphological properties with respect to differences in the extent of chromatin condensation. The pattern of nucleosome and H1 solubilization was gradual and noncoordinate and at least three different types of nucleosome packing interactions could be distinguished from the pattern. A class of nucleosomes containing 13-- 17% of the DNA and comprising the chromatin structures most available for micrococcal nuclease attack was eluted by 0.2 M NaCl. This fraction was solubilized with an acid-soluble protein of apparent molecular weight of 20,000 daltons and no histone H1. It differed from the nucleosomes released at higher NaCl concentrations in content of nonhistone chromosomal proteins. 40--60% of the nucleosomes were released by 0.3 M NaCl with 30% of the total nuclear histone H1 bound. The remaining nucleosomes and H1 were solublized by 0.4 M or 0.6 M NaCl. H1 was not nucleosome bound at these ionic strengths, and these fractions contained, respectively, 1.5 and 1.8 times more H1 per nucleosome than the population released by 0.3 M NaCl. These fractions contained the DNA least available for micrococcal nuclease attach. The strikingly different macromolecular composition, availability for nuclease digestion, and strength of the packing interactions of the nucleosomes released by 0.2 M NaCl suggest that this population is involved in a special function.     

Studies on histones and non-histone proteins from rats treated with dimethylnitrosamine.

A study has been made of the histone and non-histone chromosomal proteins of rat liver after treatment in vivo with dimethylnitrosamine (DMN) (2 mg/kg). DMN was found not to affect histone turnover, as measured by 3H-labelled amino-acids incorporation. A decrease was observed in specific activity of the histones with time after injection of [14C]DMN or [14C]-formate and this was attributable to demethylation of both abnormal and normal methylation sites in these proteins. In the case of the non-histone proteins, DMN was found to increase greatly the turnover of those non-histone proteins loosely associated with chromatin DNA and RNA; turnover of those non-histone proteins tightly bound to chromatin DNA and RNA was unaffected. Demethylation of both normal and abnormal methylation sites was found to take place from both non-histone protein fractions. In the case of the loosely bound non-histone proteins a lower rate of demethylation was observed after DMN treatment.     

Methylation and degradation of chromatin DNA in isolated rat liver cell nuclei]

Methylation of chromatin DNA in rat liver cell nuclei incubated in a medium with [3H]CH3-S-adenosyl methionine was studied. It was shown that under the given experimental conditions DNA methylation and chromatin degradation by endogenous nuclear nuclease (nucleases) with a formation of chromatin structural subunits occur simultaneously. An analysis of methylated chromatin DNA degradation products based on a number of approaches demonstrated a predominant methylation of extra-nucleosomal DNA. The data obtained suggest that chromatin of isolated nuclei contain sites with supermethylated DNA fragments incorporating not less than 400 nucleotide pairs. These sites possess an increased sensitivity to endogenous nuclease.     

Chromatin regions,released by endogenous nucleases,are enriched in immunogenic tissue-specific proteins

Localization of immunogenic tissue-specific proteins in chromatin regions, hypersensitive to endogenous nucleases, has been studied using rabbit antibodies against rat thymus chromatin. It is shown that the first 1–2,5% of the chromatin (calculating on DNA), released by Mg²⁺-, Mn²⁺-, and Ca²⁺/Mg²⁺-dependent nuclear endonucleases are drastically enriched in tissue-specific antigenic determinants. The released chromatin fractions are found to contain a heterogeneous set of nonhistone proteins and are deficient in histones. The cleavage of nuclear DNA by endogenous acidic nuclease, independent on bivalent ions, resulted in a significantly less enrichment of the released fractions with immunogenic proteins.     

Effect of irradiation and endogenous nucleases on rat liver chromatin

These assessment of the consequences of irradiation on chromatin is complicated by endogenous nucleases. Isolation and prolonged storage of rat liver nuclei in buffers containing divalent metal ions activates these enzymes and promotes the degradation of chromatin. Irradiation of rat liver nuclei to dose levels of 20,000 rad under conditions in which endogenous nucleases are inhibited and analysis of the irradiated chromatin by sucrose density gradient centrifugation gave no evidence for monosomes or oligosomes. When chromatin from irradiated nuclei was digested with micrococcal nuclease, the levels of monosomes and oligosomes were identical to those of micrococcal nuclease, the levels of monosomes and oligosomes are identical to suggest that irradiation results in neither a direct fragmentation of linkers nor the sensitization of linkers for subsequent cleavage by micrococcal nuclease. Histones isolated from monosomes of irradiated and unirradiated nuclei were intact, showing no fragmentation or loss of residues, as judged by sodium dodecyl sulfate-polyacrylamide electrophoresis.     

Chromatin structure. Nuclease digestion profiles reflect intermediate stages in the folding of the 30-nm fiber rather than the existence of subunit beads

Conditions have been found for the isolation of rat liver nuclei which maintain chromatin in its native state and suppresses endogenous nuclease activity. Chromatin prepared in this way can be dispersed into buffers containing various concentrations of monovalent or divalent cations so that the 30-nm fiber is either totally or partially decondensed. When probed with micrococcal nuclease, the digestion profiles show that although the 30-nm fiber can be cleaved periodically to generate superbead-like particles this only occurs under certain ionic conditions when the fiber is partially decondensed. It is likely that this cleavage pattern reflects the transient exposure of specific nuclease sensitive sites as the 30-nm fiber condenses, rather than the existence of a specific subunit of a beaded 30-nm fiber. The periodicity of these nuclease-sensitive sites appear to be related to the asymmetric distribution of histone H1 molecules along the length of the fiber.     

Fibroblast growth factor phosphorylation and receptors in rod outer segments.

Acidic and basic fibroblast growth factors (aFGF and bFGF) have been isolated and purified from rod outer segments (ROS). aFGF is tightly bound to ROS membranes and can be specifically released by ATP. We show that this mechanism is dependent on the phosphorylation of aFGF itself. Phorbol 12-myristate 13-acetate (PMA) enhances this phenomenon independently of rhodopsin phosphorylation. This demonstrates that aFGF release from ROS membranes is dependent on its phosphorylation by endogenous kinase C. In addition specific binding sites for exogenous FGFs have been identified on ROS and disc membranes. A single high affinity site with a Kd of 40 pM was present in intact ROS while an additional low affinity site with a Kd of 300-600 pM was present in leaky ROS or in disc membranes. Light or ATP modified neither these Kd nor the apparent number of sites. The presence of specific receptors for FGFs and the kinase C dependent release of endogenous membrane bound aFGF suggest an autocrine mechanism which may be involved in photoreceptor cell biology.     

Binding of androgen receptor to prostatic chromatin requires intact linker DNA.

Receptor-chromatin complexes were recovered from prostatic chromatin digested with micrococcal nuclease. The fragments of chromatin were separated on linear 7.6 to 76% (v/v) glycerol density gradients. With extensive digestion of DNA, receptor labeled with [1,2-3H]dihydrotestosterone was released from the chromatin. After 5% digestion of DNA to acid-soluble products, only a trace amount of labeled receptor was detected in the unbound form. In the latter instance, most of the labeled receptor was recovered from the gradients in association with five A260 peaks representing oligomeric and monomeric nucleosomes with a repeat length of 182 +/- 14 (mean +/- S.D.) base pairs. The concentration of receptors was highest in the A260 peaks, which contained large oligomers of nucleosomes, and lowest in fractions containing primarily monomer structures. Hence, the extent to which receptors remained bound to chromatin was dependent on the relative amount of intact, linker DNA present.     

Reassociation of histone H1 with nucleosomes.

The role of histone H1 in nucleosome heterogeneity and structure has been studied using a reconstitution procedure. Histone H1 and non-histone proteins are removed selectively from enzymatically fragmented chromatin by Dowex 50W-X2 treatment. The resulting "stripped" chromatin then is reassociated with purified histone H1 using step gradient dialysis. Material reconstituted in this manner was examined by gel electrophoresis, protein cross-linking, and chromatin fingerprinting. The results demonstrate that the histone H1 molecule efficiently binds to nucleosomes with fidelity in an apparent noncooperative manner. Polynucleosomes possess two specific binding sites for histone H1 per histone octamer; the first binding site is of higher affinity than the second. The 160-base pair nuclease digestion barrier and nucleosome electrophoretic class (MIII)n are established upon binding the 1st histone H1 molecule. Upon binding the 2nd histone H1 molecule, polynucleosomes assume a highly compact conformation. The experimental approach introduced here should permit determining whether nucleosomes possess independent specific binding sites for other chromosomal proteins, and should allow reconstitution of the other electrophoretic forms of nucleosomes which we have described previously.     

Binding of [3H]estradiol- and [3H]H1285-receptor complexes to rabbit uterine chromatin

In the present study we investigated the binding characteristics of estrogen and antiestrogen-receptor complexes to rabbit uterine chromatin. Activated or nonactivated estrogen receptors were partially purified by DEAE-cellulose chromatography using low (1 mM) or high (10 mM) concentrations of sodium molybdate. Activated [³H]estradiol-receptor complexes showed enhanced binding to chromatin acceptor sites unmasked by 1 M, 4 M and 6 M guanidine hydrochloride. We also examined the chromatin-binding characteristics of the estrogen receptors when bound by the high-affinity triphenylethylene antiestrogen, H1285. The acceptor site activity for the [³H]H1285-receptor complexes was markedly decreased at sites unmasked by 4 M and 6 M guanidine hydrochloride. Further, the nonactivated receptor complexes showed very low binding to deproteinized chromatin. The estrogen-receptor chromatin-acceptor sites were tissue specific and saturable. These chromatin acceptor sites differ in their affinity and capacity (number of binding sites per cell) for the estrogen- and antiestrogen-receptor complexes. Thus, we suggest that the differences in the physiological and physicochemical properties of estrogens and antiestrogens may be related to their differential interaction with uterine chromatin subfractions.     

ChIC and ChEC; genomic mapping of chromatin proteins

To map the genomic interaction sites of chromatin proteins, two related methods were developed and experimentally explored in Saccharomyces cerevisiae. The ChIC method (chromatin immunocleavage) consists of tethering a fusion protein (pA-MN) consisting of micrococcal nuclease (MN) and staphylococcal protein A to specifically bound antibodies. The nuclease is kept inactive during the tethering process (no Ca2+). The ChEC method (chromatin endogenous cleavage) consists of expressing fusion proteins in vivo, where MN is C-terminally fused to the proteins of interest. The specifically tethered nucleases are activated with Ca2+ ions to locally introduce double-stranded DNA breaks. We demonstrate that ChIC and ChEC map proteins with a 100-200 bp resolution and excellent specificity. One version of the method is applicable to formaldehyde-fixed nuclei, another to native cells with comparable results. Among various model experiments, these methods were used to address the conformation of yeast telomeres.     

Functional, structural, and immunological compartmentalisation of malaria invasive proteins

Conserved Plasmodium falciparum merozoite high activity binding peptides (HABPs) involved in red blood cell (RBC) invasion which are present in merozoite surface proteins (MSPs) involved in attachment, rolling over RBC, those derived from soluble proteins loosely bound to the membrane, and those present in microneme and rhoptry organelles have an alpha-helical structure and bind with high affinity to HLA-DR52 molecules. On the contrary, conserved HABPs belonging to molecules anchored to the membrane by a GPI tail, or a transmembranal region, or those molecules presenting PEXEL motifs have a strand, turn or unordered configuration and bind with high affinity to HLA-DR53 molecules. Such functional, cellular, structural, and immunological compartmentalisation has tremendous implications in subunit-based, multi-epitope, synthetic, anti-malarial vaccine development.     

Studies on the association of the high mobility group non-histone chromatin proteins with isolated nucleosomes.

Nucleosomes have been isolated from rabbit thymus by sucrose gradient centrifugation, and their high mobility group (HMG) protein content analysed by electrophoresis on polyacrylamide gels. The results suggest that proteins HMG 14 and HMG 17 are associated with the core particle of the nucleosome, and that there are two or more sub-populations of both HMG 1 and HMG 2 molecules. One sub-population appears to be fairly tightly bound to the nucleosome, while another is rapidly released from the chromatin by digestion with micrococcal nuclease. The latter fraction may participate in a higher order folding of the nucleosomes.     

Separation of bovine heart galactose lectin from endogenous glycoproteins co-purified with the lectin during affinity chromatography

During affinity chromatographic purification of bovine heart 14 kDa galactose-binding lectin (galectin 1) on lactose-Sepharose, several high molecular weight non-lectin glycoproteins were co-purified with the lectin. Glycoprotein binding to the affinity matrix was neither hydrophobic nor ionic, but galactose-dependent since lactose abolished binding. Purification of galectin from the co-purified glycoproteins by affinity electrophoresis in presence of the specific sugar lactose increased agglutination activity about 65-fold, indicating that a complex containing galectin molecules bound sugar specifically to endogenous glycoproteins with sugar binding sites still available had been retained on lactose-Sepharose.