Immunospecificity of nonhistone chromatin proteins tightly bound to DNA from chicken thrombocytes and erythrocytes

Erythroid cell-specific antisera capable of detecting tightly bound nonhistone chromatin protein-DNA complexes were obtained by injecting rabbits with dehistonized chicken reticulocyte chromatin. The antisera showed no crossreactivity with chromatin of thrombocytes which are regarded as cells genealogically closely related with erythrocytes. The lack of thrombocyte chromatin immunoactivity was not caused by conformational constrains. Tightly bound nonhistone protein-DNA complexes isolated from thrombocyte chromatin showed no immunological similarity with these of erythrocyte chromatin.     

Cell-specific antigens in chicken erythroid nuclei: species specificity.

Antisera raised to dehistonized chicken reticulocyte chromatin were tested for their cell and species specificity. Quantitative microcomplement fixation and immunohistochemical localization revealed the presence in chromatin of erythroid cell-specific nonhistone protein antigen(s). The antigenic specificity was shown to depend on the association of the antigenic protein(s) with deoxyribonucleic acid (DNA). Although the antisera were exceptionally cell specific, they cross-reacted with erythroid cells of other avian species. The extent of cross-reactivity was found to approximate the phylogenetic distances of the tested avian species. Erythroid cells from fish and amphibians were not reactive. Reconstitution experiments of partially purified chicken reticulocyte chromosomal nonhistone protein antigens with DNAs isolated from several vertebrate species showed that the species specificity of the antigenic complexes is determined principally by the species origin of the nonhistone proteins. Our results show that a cell-specific chromosomal nonhistone protein(s) has undergone evolutionary change and the relative immunological differences are consistent with the accepted phylogenetic distances of the species examined.     

Chicken erythrocyte chromatin and nuclear envelope antigens

Chromatin and inner layer nuclear envelope were isolated from chicken erythrocyte nuclei. Two antisera against dehistonized chromatin and nuclear envelope of chicken erythrocytes were obtained. Using the antiserum against dehistonized chromatin of erythrocytes we found: the presence of the antigens at approximate mol. wts of 56,000 and 77,000 tightly bound with DNA and characteristic of only erythrocyte chromatin; localized antigens at approximate mol. wts of 63,000, 68,000 and 92,000 tightly bound with DNA and common only for chromatin and nuclear envelope of chicken erythrocytes; heterogeneity of the antigens tightly bound with DNA. Using the antiserum against inner layer nuclear envelope we did not find antigens specific only for nuclear envelope and absent in erythrocyte chromatin. Some of the antigens were present in the control preparations of chicken liver chromatin and may be regarded as being species specific.     

Increased stability of the higher order structure of chicken erythrocyte chromatin: nanosecond anisotropy studies of intercalated ethidium

Internal motion of the DNA in chicken erythrocyte chromatin fibers was studied by measurement of the fluorescence anisotropy decay of ethidium intercalated in the linker region. A comparison of the decay curves of the dye in chicken erythrocyte chromatin with those of calf thymus chromatin [Ashikawa, I., Kinosita, K., Jr., Ikegami, A., Nishimura, Y., Tsuboi, M., Watanabe, K., Iso, K., & Nakano, T. (1983) Biochemistry 22, 6018-6026] revealed greater suppression of nucleosome movement in chicken erythrocyte chromatin. Furthermore, the transition of this chromatin to the compact (solenoidal) structure occurred at lower solvent concentrations of Na+ or Mg2+ than those for calf thymus chromatin. These results demonstrated increased stability of the higher order structure (the solenoid) of chicken erythrocyte chromatin, which may be related to the reduction of nuclear activity in the chicken erythrocyte cell. In addition to intact chicken erythrocyte chromatin, we studied the structural transitions of H1-depleted and H1,H5-depleted chromatins. The result indicated that histone H5 of this chromatin stabilizes the higher order structure in the presence of magnesium (or divalent) cation and did not induce the transition in the solution containing only sodium cation.     

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.     

Linker histone subtype composition and affinity for chromatin in situ in nucleated mature erythrocytes

The replacement linker histones H1(0) and H5 are present in frog and chicken erythrocytes, respectively, and their accumulation coincides with cessation of proliferation and compaction of chromatin. These cells have been analyzed for the affinity of linker histones for chromatin with cytochemical and biochemical methods. Our results show a stronger association between linker histones and chromatin in chicken erythrocyte nuclei than in frog erythrocyte nuclei. Analyses of linker histones from chicken erythrocytes using capillary electrophoresis showed H5 to be the subtype strongest associated with chromatin. The corresponding analyses of frog erythrocyte linker histones using reverse-phase high performance liquid chromatography showed that H1(0) dissociated from chromatin at somewhat higher ionic strength than the three additional subtypes present in frog blood but at lower ionic strength than chicken H5. Which of the two H1(0) variants in frog is expressed in erythrocytes has thus far been unknown. Amino acid sequencing showed that H1(0)-2 is the only H1(0) subtype present in frog erythrocytes and that it is 100% acetylated at its N termini. In conclusion, our results show differences between frog and chicken linker histone affinity for chromatin probably caused by the specific subtype composition present in each cell type. Our data also indicate a lack of correlation between linker histone affinity and chromatin condensation.     

Erythroid specific nuclear antigen and globin gene binding protein

Using the procedure of Bekhor and Mirell (Biochem, $\text{18}$, 609, 1979), we isolated a nonhistone protein fraction tightly bound to DNA which putatively has a role in globin gene regulation in chicken reticulocytes. This fraction was tested by gel electrophoresis and microcomplement fixation and appears by these criteria very similar to the chicken nuclear antigen previously identified in reticulocyte chromatin and structurally altered erythrocyte chromatin. This antigen is tissue and species specific (Pumo $\text{et}\text{al}$, Biochem. $\text{19}$, 2362, 1980).     

Disruption of the typical chromatin structure in a 2500 base-pair region at the 5' end of the actively transcribed ovalbumin gene.

We examined the chromatin organizations of approximately 3 kb of DNA in the 5'-end flanking region of the ovalbumin gene in chicken erythrocyte and oviduct cell nuclei. With specific DNA probes and an indirect end-labeling technique, we analysed the pattern of the DNA fragments obtained after micrococcal nuclease digestion and generated comparative maps of the nuclease cuts. This region of the chicken genome displays a "typical" chromatin arrangement in erythrocyte nuclei, with nucleosomes apparently positioned at random. In contrast, in oviduct nuclei, the same region has an "altered" chromatin structure, and lacks a typical nucleosomal array. The existence of specifically positioned proteins and of alterations in the DNA secondary structure in this region of the oviduct chromatin is suggested by comparison of the nuclease cleavage maps which reveals specific changes: disappearance of nuclease cuts present in "naked" and erythrocyte chromatin DNAs, and appearance of new cuts absent from these DNAs.     

Structural repeat units of Chinese hamster ovary chromatin. Evidence for variations in repeat unit DNA size in higher eukaryotes.

DNA lengths in the structural repeat units of Chinese hamster ovary (CHO) and chicken erythrocyte chromatin were compared by analyzing the sizes of DNA fragments produced after treatment of nuclei with staphylococcal nuclease. The repeat length of CHO chromatin (173 +- 4 BP) is about 20 base pairs (BP) smaller than that of chicken erythrocyte chromatin (194 +- 8 BP). Repeat lengths of rat liver and calf thymus chromatin were found to be about 10 BP shorter than that of chicken erythrocyte chromatin. Thus significant variations occur in repeat units of chromatin of higher eukaryotes. These variations occur in the lengths of "spacer" (or "internucleosomal") DNA segments, not in "core particle" (or "nucleosomal") DNA lengths. The concept of spacer regions and the possible influence of H1 histones is discussed.     

Determination of the RNA-content in samples of isolated chromatin by circular dichroism measurements

A method is presented to determine the RNA content in isolated chromatin. An increased elipticity at 260nm–290nm in the circular dichroism spectrum of chromatin is due to RNA released together with chromatin from the cell nuclei. Mixtures of chicken erythrocyte chromatin and pure 5s-RNA were used to reconstruct such spectra. From the obtained data an RNA content higher than 3–5% in samples of isolated chromatin can be determined.     

The superstructure of chromatin and its condensation mechanism

Comparison between the internucleosomal distance found by X-ray solution scattering for chicken erythrocyte (23 nm) and sea urchin (30 nm) chromatin indicates that this distance is proportional to the linker length. The diameter of the condensed sea urchin chromatin fibers is about 45 nm which is significantly larger than in chicken erythrocyte chromatin (35 nm). Trivalent cations (Gd, Tb, Cr) and the polyamines spermine and spermidine were found to induce compaction at much lower concentrations than the divalent cations but Gd, Tb and Cr induce aggregation before full compaction of the fibers. The influence of hydrogen bonding is illustrated by comparison of the effects of NaCl, ammonium chloride and alkylammonium chlorides on condensation. Solubility experiments indicate that there is a nearly linear dependence of the Mg^-- concentration at which precipitation occures on chromatin concentration and confirm the differences between cations observed by X-ray scattering.The chicken erythrocyte chromatin samples were further characterized by their reduced electric dichroism. The values found are consistent with the model derived from X-ray scattering and are compared with those reported in the literature.     

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.     

Conformation of control and acetylated HeLa stripped chromatin after reassociation with H1

The effect of histone H1 on the conformation and stability of control and acetylated HeLa high-molecular-weight chromatin that had been stripped of H1 and nonhistone proteins was compared by circular dichroism (CD) and thermal denaturation measurements. Two different preparations of H1, originating from rat thymus and chicken erythrocyte, were used in the reconstitution studies. The control and acetylated stripped chromatin had identical CD and thermal denaturation properties, as did their reconstitutes with rat thymus H1. Reconstitutes of the two chromatins with chicken erythrocyte H1 had similar CD properties, but thermal denaturation studies showed that the acetylated reconstitute was destabilized compared to the control reconstitute. Reconstitutes of both chromatins with chicken erythrocyte H1 had a more condensed and stabilized structure than the reconstitutes with rat thymus H1. Thus, acetylation caused a decrease in the stability of chromatin in the presence of erythrocyte H1, but more marked differences were detected in the structure of stripped chromatin after reassociation with different H1 preparations.     

Effect of formaldehyde on the circular dichroism of chicken erythrocyte chromatin.

Formaldehyde (HCHO) fixation of chicken erythrocyte chromatin produces a marked decrease in its positive circular dichroism (CD), above 260 nm, and the appearance of s small negative ellipticity around 295 nm. The ultraviolet spectrum of chromatin is unaffected, nor does HCHO produce any changes in the uv or CD spectra of chicken erythrocyte DNA. The extent of the circular dichroism transition from the native chromatin to the suppressed spectrum is dependent on the concentration of HCHO and salt concentration. The kinetics of the reactions are complex, implicating at least two reactive species. Studies of the reaction of HCHO with chromatin in ethylene glycol and CD measurements of aqueous chromatin solution with added glutaraldehyde preclude simple dehydration and general cross-linking effects as causes of the CD changes observed. The results are interpreted as indicating a conformational change of the DNA in chromatin caused by histone-DNA or histone-histone cross-linking.     

Monoclonal antibodies to tissue-specific chromatin proteins

Antisera raised in mice to chromatins from different tissues of the chicken reacted preferentially with the chromatin type that was used for immunization. This tissue specificity was also evident in the spectrum of monoclonal antibodies generated when mice were immunized with erythrocyte chromatin. Three erythroid-specific antigens and one antigen that was present in a number of chicken tissues were characterized in further detail. These antigens, which comprised less than 0.1% of the erythrocyte chromatin proteins, were nuclear localized although three were also detected in the cytoplasm. Two of the erythroid-specific antigens existed as multiple polypeptides in isolated chromatin. The multiple chromatin forms of one antigen were derived from a precursor protein that was selectively cleaved within 1 min after erythrocyte lysis. Analysis of this antigen in extracts from erythrocytes and reticulocytes indicated that the cleavage of the precursor protein was developmentally regulated in vivo.