Affinity purification of mammalian RNA polymerase I. Identification of an associated kinase.

Overlapping cDNA clones encoding the two largest subunits of rat RNA polymerase I, designated A194 and A127, were isolated from a Reuber hepatoma cDNA library. Analyses of the deduced amino acid sequences revealed that A194 and A127 are the homologues of yeast A190 and A135 and have homology to the beta' and beta subunits of Escherichia coli RNA polymerase I. Antibodies raised against the recombinant A194 and A127 proteins recognized single proteins of approximately 190 and 120 kDa on Western blots of total cellular proteins of mammalian origin. N1S1 cell lines expressing recombinant His-tagged A194 and FLAG-tagged A127 proteins were isolated. These proteins were incorporated into functional RNA polymerase I complexes, and active enzyme, containing FLAG-tagged A127, could be immunopurified to approximately 80% homogeneity in a single chromatographic step over an anti-FLAG affinity column. Immunoprecipitation of A194 from 32P metabolically labeled cells with anti-A194 antiserum demonstrated that this subunit is a phosphoprotein. Incubation of the FLAG affinity-purified RNA polymerase I complex with [gamma-32P]ATP resulted in autophosphorylation of the A194 subunit of RPI, indicating the presence of associated kinase(s). One of these kinases was demonstrated to be CK2, a serine/threonine protein kinase implicated in the regulation of cell growth and proliferation.

Fine resolution of histones by two-dimensional polyacrylamide gel electrophoresis: developmental implications.

A two-dimensional electrophoresis for fine separation of histones is described in detail. The method is relatively simple and gives very reproducible results. In the first dimension the histones are separated by their charge in acid-urea gels, while in the second dimension the separation is based on both the charge and the differential affinity of histones to Triton in acid-urea-Triton gels. In this electrophoretic system, the linker histones are resolved on the gel diagonal, while the core histones are separated above the diagonal. The electrophoresis is very sensitive to charge effect, and thus it is very well suited to resolving histone-modified forms. The application of the two-dimensional electrophoresis in Xenopus developmental studies is illustrated.

UV-laser crosslinking of proteins to DNA.

Photochemical crosslinking is now a powerful method for studying protein-nucleic acid interactions. UV light is a zero-length crosslinking agent that predominantly or exclusively crosslinks proteins to nucleic acids at their contact points. It can therefore provide strong evidence for close protein-nucleic acid interactions. However, to achieve an acceptable degree of crosslinking with conventional UV light sources, exposure times ranging from minutes to several hours are necessary. Such prolonged irradiation allows for the artifactual redistribution of proteins and precludes kinetic studies. The use of UV lasers overcomes these difficulties since the number of photons required for the crosslinking may be delivered in time intervals on the order of nano- or even picoseconds. We described detailed procedures for UV laser-induced protein-DNA crosslinking both in vivo and in vitro. Technical aspects, including the choice of UV laser for irradiation, the isolation of covalently crosslinked protein-DNA complexes, immunochemical techniques for both the identification and isolation of specific protein-DNA complexes and the identification of the crosslinked DNA sequences, are reviewed in detail. The application of UV laser crosslinking in kinetic studies is illustrated by the example of the TATA-binding protein (TBP) interaction with the adenovirus E4 promoter.

Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not.

Acetylation of histones bound to rat rRNA genes has been studied relative to their organization in chromatin, either as canonical nucleosomes, containing the inactive copies, or as anucleosomal nonrepeating structures, corresponding to the transcribed genes (Conconi, A., Widmer, R. M., Koller, T., and Sogo, J. M. (1989) Cell 57, 753-761). Nuclei from butyrate-treated rat tumor cells were irradiated with a UV laser to cross-link proteins to DNA, and the purified covalent complexes were immunofractionated by an antibody that specifically recognized the acetylated histones. Upon probing with sequences coding for mature rat 28 S RNA, DNA of the antibody-bound complexes was 5-20-fold enriched relative to the total rat DNA. Since the laser cross-links histones to DNA in both active and inactive genes, one cannot distinguish which one of them, or both, are bound to acetylated histones. Alternatively, purified mononucleosomes were immunofractionated, but DNA from the antibody-bound monosomes was not enriched in coding rDNA. Taken together, these results suggest that nucleosome-organized rRNA genes are bound to nonmodified histones and that the acetylated histones are associated with the active, anucleosomal gene copies.

Mapping of a sequence essential for the nuclear transport of the Xenopus ribosomal transcription factor xUBF using a simple coupled translation-transport and acid extraction approach.

The amino acid sequences directing the nuclear transport of the RNA polymerase I transcription factor xUBF have been studied by a novel combination of in oocyte-coupled translation-nuclear transport and selective HCl extraction. Synthetic mRNA was used to direct the translation of labeled xUBF and its mutants in microinjected oocytes. After manual dissection of nuclei and cytoplasm, labeled xUBF and mutants were isolated essentially pure by HCl extraction. Using deletion mutations, a sequence essential, but not necessarily sufficient, for nuclear transport was mapped to a 29-amino-acid segment lying between the most carboxy-terminal putative HMG-box DNA-binding domain, HMG-box 5, and the highly acidic carboxy-terminal domain. It was shown that deletion of only 5 amino acids from this segment eliminated xUBF transport, and it could be deduced that at least 11 of the 29 amino acids were essential for nuclear transport. The segment of xUBF necessary for nuclear transport contains a sequence conforming to the bipartite nuclear transport motif consensus, but this sequence in itself was insufficient for nuclear transport.

Binding of histones to Xenopus laevis ribosomal genes with different levels of expression.

The association of ribosomal RNA genes with histones as a function of their expression has been studied in Xenopus laevis erythrocytes, where the genes are silent, and tadpoles at stage 40, where these genes are actively transcribed. Isolated nuclei were either treated with formaldehyde or irradiated with an ultraviolet laser to cross-link proteins to DNA. The covalently linked protein-DNA complexes were purified by centrifugation through CsCl and immunoprecipitated with antibodies against H1, H2A and H4. DNA from the precipitated complexes was analysed for the presence of ribosomal DNA sequences by hybridization to specific probes. The actively transcribed ribosomal genes from X. laevis embryos are associated with H1, H2A and H4 as are the non-transcribed genes in the erythrocytes.

Crosslinking proteins to nucleic acids by ultraviolet laser irradiation.

Ultraviolet (UV) irradiation can initiate complex formation between proteins and DNA or RNA and so can be used to study such interactions. However, crosslink formation by standard UV light sources can take up to several hours. More recently, a beam of monochromatic UV light from a laser has been used to initiate crosslinking in nano- and picosecond time intervals. As noted in an earlier TIBS article 'the advantages of short pulse times and high-energy fluxes should make this a valuable technique in the future'. In this review we characterize laser-induced crosslinking and explore the applications of this method.

The enhancers and promoters of the Xenopus laevis ribosomal spacer are associated with histones upon active transcription of the ribosomal genes.

The presence of histones on the enhancer-promoter region of the X.laevis ribosomal spacer has been studied in embryos at stage 40, where the ribosomal genes are actively transcribed. Isolated tadpole nuclei were either fixed with formaldehyde or irradiated with UV laser to crosslink histones to DNA. The purified protein-DNA complexes were immunoprecipitated with antibodies to the histones H1, H2A and H4 and the DNA fragments carrying the respective histones were analyzed for the presence of spacer enhancer-promoter sequences by hybridization to specific DNA probe. The two independent crosslinking procedures revealed the presence of these DNA sequences in the precipitated DNA. The quantitative analysis of the UV laser-crosslinked complexes showed that histones H2A and H4 were associated with enhancer-promoter DNA in amounts similar to those found for bulk DNA, whilst the content of H1 was reduced.

The chromatin fiber: structure and conformational transitions as revealed by optical anisotropy studies.

Structure and conformational transitions of the chromatin fiber as revealed by optical anisotropy studies are reviewed. The data in the literature do not allow a definite interpretation; in fact some of them are contradictory. The major findings are reported here and an attempt is made to analyse them with respect to the internal dynamics and the various models suggested for the organization of the chromatin fiber.

Laser-induced crosslinking of histones to DNA in chromatin and core particles: implications in studying histone-DNA interactions.

UV laser irradiation has been used to covalently crosslink histones to DNA in nuclei, chromatin and core particles and the presence of the different histone species in the covalently linked material was detected immunochemically. When nuclei were irradiated and then trypsinized to cleave the N- and C- terminal histone tails, no histones have been found covalently linked to DNA. This finding shows that UV laser-induced crosslinking of histones to DNA is accomplished via the non-structured domains only. This unexpected way of crosslinking operated in chromatin, H1-depleted chromatin and core particles, i.e. independently of the chromatin structure. The efficiency of crosslinking, however, showed such a dependence: whilst the yield of crosslinks was similar in total and H1-depleted chromatin, in core particles the efficiency was 3-4 times lower for H2A, H2B and H4 and 10-12 times lower for H3. The decreased crosslinking efficiency, especially dramatic in the case of H3, is attributed to a reduced number of binding sites, and, respectively, is considered as a direct evidence for interaction of nonstructured tails of core histones with linker DNA.

Interactions of acetylated histones with DNA as revealed by UV laser induced histone-DNA crosslinking.

The interaction of acetylated histones with DNA in chromatin has been studied by UV laser-induced crosslinking histones to DNA. After irradiation of the nuclei, the covalently linked protein-DNA complexes were isolated and the presence of histones in them demonstrated immunochemically. When chromatin from irradiated nuclei was treated with clostripain, which selectively cleaved the N-terminal tails of core histones, no one of them was found covalently linked to DNA, thus showing that crosslinking proceeded solely via the N-terminal regions. However, the crosslinking ability of the laser was preserved both upon physiological acetylation of histones, known to be restricted to the N-terminal tails, and with chemically acetylated chromatin. This finding is direct evidence that the postsynthetic histone acetylation does not release the N-terminal tails from interaction with DNA.

Protein-DNA crosslinking in reconstituted nucleohistone, nuclei and whole cells by picosecond UV laser irradiation.

A picosecond UV laser was used to cross-link proteins to DNA in nuclei, whole cells and reconstituted nucleohistone. Irradiation of the nucleohistone resulted in crosslinking 15-20% of bound histones to DNA in a very short time (one or several picosecond pulses), the efficiency of crosslinking to single stranded DNA being higher than to double stranded DNA. All histones as well as high mobility group 1 proteins were identified in the covalently linked protein-DNA complexes upon irradiation of isolated nuclei and whole cells. A method is suggested for isolation of crosslinked material from cells and nuclei in amounts sufficient for further analysis. Experiments with reconstituted nucleohistones showed that upon irradiation at a constant dose the efficiency of crosslinking depended on the intensity of the light, thus suggesting a two-quantum process is involved in the reaction.

NaCl-induced chromatin condensation. Application of static light scattering at 90 degrees and stopped flow technique.

We have studied the NaCl-induced condensation of calf thymus chromatin by static light scattering of 90 degrees and shown that the increase in NaCl concentration up to 120-170 mM results in a large increase in scattering intensity of the total chromatin. Histones H1-depleted and trypsinized chromatin preparations do not reveal such a large increase in scattering intensity. The increase in the scattering intensity reflects the folding of the chromatin filaments, but not their aggregation. We have used this effect to monitor the kinetics of the chromatin condensation in response to a jump to higher NaCl concentrations by means of a stopped-flow technique. The results show that the condensation is a fast complex process consisting of at least two steps. The first step is only partially resolved by the stopped-flow apparatus. The second step has a time constant in the range of 20-50 ms, which does not depend on chromatin concentration.

Optical anisotropy of chromatin. Flow linear dichroism and electric dichroism studies.

The optical anisotropy of chromatin with different length of the linker DNA isolated from a variety of sources (Frend erythroleukemia cells, calf thymus, hen erythrocytes and sea urchin sperm) has been studied in a large range of mono- and bivalent cations concentrations by the use of flow linear dichroism (LD) and electric dichroism. We have found that all chromatins studied displayed negative LD values in the range of 0.25 mM EDTA - 2 mM NaCl and close positive values in the range of 2-100 mM NaCl. Mg2+ cations, in contrast to Na+ cations, induce optically isotropic chromatin fibers. All chromatin samples exhibit positive form effect amounting to 5-10% of LD amplitude observed at 260 nm. This form effect is determined by the anisotropic scattering of polarized light by single chromatin fibers. The conformational transition at 2 mM NaCl leads to the distortion of chromatin filament structure. The reversibility of this distortion depends on the length of the linker DNA - for chromatins with the linker DNA of 10-30 b.p. it is parially reversible, while for preparations with longer linker DNA it is irreversible. Relatively low electric field does not affect chromatin structure, while higher electric field (more than 7 kV/cm) distorts the structure of chromatin. Presented results explain the contradictory data obtained by electrooptical and hydrooptical methods.

Polyamine-DNA interactions. Condensation of chromatin and naked DNA.

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin by polyamines, such as spermine, spermidine and its analogs designated by formula NH3+(CH2)iNH2+(CH2)jNH3+, where i = 2,3 and j = 2,3, putrescine, cadaverine and MgCl2. It has been found that the different polyamines affect DNA and chromatin in a similar way. The level of compaction of the chromatin fibers induced by spermine, spermidine and the triamines NH3+(CH2)3NH2+(CH2)3NH3+ and NH3+(CH2)3NH2+(CH2)2NH3+ and MgCl2 is found to be identical. The triamine NH3+(CH2)3NH2+(CH2)2NH3+ and the diamines studied condense neither chromatin nor DNA. This drastic difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations might play essential roles in their interactions with DNA and chromatin. It is shown that a mixture of mono- and multivalent cations affect DNA and chromatin condensation competitively, but not synergistically, as claimed in a recent report by Sen and Crothers (Biochemistry 25, 1495-1503, 1986). We have also estimated the extent of negative charge neutralization produced by some of the polyamines on their binding to chromatin fibers. The stoichiometry of polyamine binding at which condensation of chromatin is completed is found to be two polyamine molecules per DNA turn. The extent of neutralization of the DNA phosphates by the histones in these compact fibers is estimated to be about 55%. The model of polyamine interaction with chromatin is discussed.

Accessibility of the globular domain of histones H1 and H5 to antibodies upon folding of chromatin.

Antibodies to the globular domain of histones H1 and H5 were purified by affinity chromatography and used to study the accessibility of this region of H1 and H5 in folded and unfolded rat liver and hen erythrocyte chromatin respectively. The different conformations of the chromatin filament were induced by varying the ionic strength from 1 mM to 80 mM NaCl and maintained by fixation with glutaraldehyde. Treatment with glutaraldehyde at a given salt concentration affected neither the orientation of nucleosomes relative to the fiber axis nor the compactness of chromatin. Solid-phase immunoassay and inhibition experiments showed no binding of the antibody against the globular domain of H1 to chromatin at the entire range of salt concentrations, while the antibody to the whole H1 molecule reacted with chromatin at low salt. On the other hand, the antibody to the globular region of H5 reacted with hen erythrocyte chromatin independently of the extent of chromatin condensation. These results indicate that the antigenic determinants of the globular domain of H5 are accessible to the antibody both in folded and unfolded chromatin, while those of the same region of H1 are masked, probably by interaction with DNA or proteins.

[Study of the process of chromatin condensation using light scattering and stop-flow technics]. / Izuchenie protsessa formirovaniia kompaktnykh khromatinovykh fibrill s pomoshch'iu metoda svetorasseianiia i tekhniki ostanovlennoi strui.

We have studied the condensation of calf thymus chromatin induced by NaCl by static light scattering at 90 degrees and showed that the increase of NaCl concentration up to 120-170 mM results in a large increase of scattering intensity of the total chromatin. H1-depleted and trypsinized chromatin preparations do not reveal such a large increase of scattering intensity. The increase of the scattering intensity reflects folding of the chromatin filaments, but not their aggregation. We have used this effect to monitor the kinetics of the chromatin condensation in response to a jump to higher NaCl concentrations by means of a stopped-flow technique. The results show that the condensation is a fast complex process consisting of at least two steps. The first step is only partially resolved by the stopped-flow apparatus. The second step has a time constant in the range of 20-50 ms and does not depend on chromatin concentration.