Template-specific stimulation of RNA synthesis by phosphorylated non-histone chromatin proteins

Phosphorylated non-histone chromatin proteins are shown to stimulate the synthesis of RNA in a cell-free system using rat liver RNA polymerase and rat DNA as template. This stimulation is not observed when DNA's of other species are used as template, or when the phosphorylated proteins have been treated with alkaline phosphatase.     

Partial fractionation and physico-chemical properties of non-histone chromatin proteins]

The non-histone chromatin proteins (NHP) were isolated by a modified Wang technique. NHP were easily dissoluble in solutions of a physiological ionic strength within a wide pH range. NHP were subdivided into 18 zones by analytical polyacrylamide gel electrophoresis. NHP have molecular weights within the range 15,000 greater than 200,000. A part of NHP showed similar molecular weight but different values of molecular charge. NHP were separated by a gel filtration into 6 fractions. Two fractions were individual proteins. The great bulk of NHP has a molecular weight less than 40,000, and 6-6.5% of NHP-more than 100,000. The fractions different from each other in a specific UV-absorbtion, fluorescence and circular dichroism. The nhp fraction of a smaller molecular weight has a smaller content of alpha-helix (8%) and the greatest polarity of the environment of tryptophan residues; the molecules of this fraction may have a loose tertial structure. Other NHP have 15-24% of alpha-helix and possibly have compact globular sites.     

Association of newly synthesized 3H-arginine-labeled proteins with chromatin structures in fertilization

Zona-free hamster eggs have been fertilized in vitro with boar spermatozoa in a medium enriched by arginine-³H and the sites of localization of newly synthesized arginine-³H–labeled proteins have been investigated using fine-structure autoradiography. It was confirmed that such proteins are synthesized during fertilization and that they accumulate to a notable degree in decondensing sperm chromatin as well as in the chromatin of the female pronucleus and also of the second polar body. A similar process did evidently take place also in defective pronuclei, characterized by a core of a still condensed chromatin and by remaining nuclear membrane. In such male pronuclei the highest concentration of the label was seen just on the border of the condensed chromatin, on the expected site of nuclear protein exchange. It is supposed that, at least in this experimental system, any morphologically detectable sperm decondensation is accompanied immediately by a shift from sperm basic nuclear proteins to other nuclear proteins.     

Immunochemical study of chromatin non-histone proteins

Summary Rabbit antibodies against rat thymus and liver chromatin are obtained. Antithymus IgG are found to interact only with homologous chromatin, while antiliver IgG interact with both liver and thymus chromatin. After preincubation of antiliver IgG with thymus chromatin the antibodies interact with homologous chromatin only. Thus chromatin of both organs contains tissue-specific proteins. Antiliver and antithymus IgG are used to investigate a distribution of tissue-specific and tissue-non-specific immunogenic proteins in thymocyte nuclei. It is shown that these proteins are practically lacking in nuclear membranes, matrix, nuclear sap, nucleous chromatin and do not participate in attaching DNA to the nuclear matrix.Abbreviations NHP non-histone chromatin proteins - IgG immunoglobulins G - PMSF PhMeSO₃     

Lectin activity of chromatin non-histone proteins

Non-histone proteins from chromatin of sea urchin embryos were found to possess the ability to agglutinate erythrocytes.     

Interactions of non-histone proteins with immobilized components of chromatin

The specific interaction between non-histone proteins (NHP) of rat thymus and the dextran-immobilized components of chromatin has been investigated. DNA's from E. coli and rat thymus, total histone and reconstituted nucleohistone were used as the affinity sorbents. The distribution of protein fractions in the NHP groups dissociated from chromatin with different ionic strengths was studied by binding on the columns and by SDS-PAAG-electrophoresis. It is shown that NHP of chromatin include the protein components with selective affinity to nucleohistone. These results are discussed in connection with the different functions of NHP in chromatin.     

Distribution of tightly bound non-histone proteins in chromatin fractions obtained by DNAase II digestion

Digestion of pig liver chromatin with DNAse II afforded three different fractions which were characterized in terms of their DNA, RNA and tightly bound non-histone protein content, their DNA fragment size and their template activity. Two of these fractions are soluble after digestion with DNAase II and have been separated on the basis of their different solubility in MgCl2. A third fraction is not solubilized even after extensive digestion, although the size of its DNA is comparable to that of the enzyme solubilized fractions. The three fractions show qualitatively and quantitatively different distribution of tightly bound non-histone proteins, with specific protein components in each fraction; furthermore the non-solubilized fraction is greatly enriched in proteins tightly bound to DNA. From all the data obtained it can be suggested that the tightly bound proteins of the insoluble fraction may play, directly or indirectly, a role in maintaining an organized chromatin structure.     

Tightly-bound non-histone proteins in different nucleosome-like subpopulations from pig kidney chromatin

By differential sucrose gradient centrifugation of pig kidney chromatin in the presence or absence of Na-EDTA and under varying ionic strength conditions, three nucleosome-like subpopulations with different buoyant densities can be obtained. These particles, on the basis of their histones and HMG protein pattern, of the 5-methylcytosine level of their DNA and of the RNA polymerase activity associated with them, can be considered as originating from chromatin fractions differently involved in gene expression. Two-dimensional electrophoresis of the tightly-bound non-histone proteins shows a distinct pattern for each subpopulation, such protein components being notably present in restricted numbers but in high amounts in the subpopulation which was apparently derived from condensed heterochromatin.     

Role of non-histone proteins in chromatin solubility

Treatment of chromatin gel with low ionic strength solution of tRNA has produced the dioxyribonucleoprotein (dnptRNA) in which only part of non-histone proteins was removed without loss of any major histone fraction. The solubility of DNP in the presence of 0.15 M NaCl and 1 to 5 mM MgCl2 was considerably higher than that of initial untreated chromatin. It has been assumed that the solubility of chromatin depended primarily on some non-histone proteins and not on H1 histone.     

Changes in non-histone chromatin proteins during the storage of chromatin

We have described here the changes in stored chicken reticulocyte chromatin which take place among non-histone protein fractions based on SDS-polyacylamide gel electrophoresis and hybridization of globin cDNA with RNAs transcribed on native and reconstitited chromatin templates.     

Rat liver chromatin non-histone proteins and glucocorticoid binding

We have previously characterized a specific corticosterone binding protein in chromosomal non histone proteins (NHP) from rat liver. In this paper, we present evidence that a relationship exists between this protein and the cytoplasmic glucocorticoid receptor. The binding capacity of NHP is reduced by 40 p. cent when this fraction is isolated from adrenalectomized animals. Incubation of isolated nuclei with the glucocorticoid hormone receptor complex results in a decrease in the specific radioactivity of the cytoplasmic proteins and simultaneously in a rapid uptake of the isotope by the nucleus; radioactive hormone was extracted along with the NHP. Evidence is presented that the NHP component binding the hormone is closely related or identical to the cytoplasmic receptor-proteins. Progesterone and corticosterone compete similarly for the binding of dexamethasone to nuclear and cytoplasmic forms of the receptor. However the nuclear form of the receptor has a higher affinity for corticosterone (K_a : 6 × 10⁹ M⁻¹) than for dexamethasone (K_A : 10⁸ M⁻¹) in vitro.A mixture of rat liver NHP and cytosol was shown to bind specifically more corticosterone than when the two proteins were incubated separately with the hormone. The Scatchard analysis shows that the enhancement of binding is due to an interaction of nuclear and cytoplasmic proteins leading to the appearance of a stable protein-protein complex which has a high affinity for the hormone (K_a : 2 × 10⁸ M⁻¹). KCl prevented this interaction. Complex formation does not require the presence of the hormone. The experiments presented here favor the hypothesis of the existence of a regulatory protein in the nucleus. This protein associated with the binding protein to reveal or enhance the active form of the receptor.