Linker histones stabilize the intrinsic salt-dependent folding of nucleosomal arrays: mechanistic ramifications for higher-order chromatin folding.

Defined nucleosomal arrays reconstituted from core histone octamers and twelve 208 bp tandem repeats of Lytechinus 5S rDNA (208-12 nucleosomal arrays) possess the ability to form an unstable folded species in MgCl2 whose extent of compaction equals that of canonical higher-order 30 nm diameter chromatin structures [Schwarz, P. M., and Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. To address the mechanistic functions of linker histones in chromatin condensation, purified histone H5 has been assembled with 208-12 nucleosomal arrays in 50 mM NaCl. Novel purification procedures subsequently were developed that yielded preparations of 208-12 chromatin model systems in which a majority of the sample contained both one histone octamer per 5S rDNA repeat and one molecule of histone H5 per histone octamer. The integrity of the purified 208-12 chromatin has been extensively characterized under low-salt conditions using analytical ultracentrifugation, quantitative agarose gel electrophoresis, electron cryomicroscopy, and nuclease digestion. Results indicate that histone H5 binding to 208-12 nucleosomal arrays constrains the entering and exiting linker DNA in a way that produces structures that are indistinguishable from native chicken erythrocyte chromatin. Folding experiments performed in NaC1 and MgC12 have shown that H5 binding markedly stabilizes both the intermediate and extensively folded states of nucleosomal arrays without fundamentally altering the intrinsic nucleosomal array folding pathway. These results provide new insight into the mechanism of chromatin folding by demonstrating for the first time that distinctly different macromolecular determinants are required for formation and stabilization of higher-order chromatin structures.

The Epstein-Barr virus-encoded nuclear antigen EBNA-5 accumulates in PML-containing bodies.

EBNA-5 is one of the Epstein-Barr virus (EBV)-encoded nuclear proteins required for immortalization of human B lymphocytes. In the nuclei of EBV-transformed lymphoblastoid cell lines EBNA-5 is preferentially targetted to distinct nuclear foci. Previously we have shown (W.Q. Jiang, L. Szekely, V. Wendel-Hansen, N. Ringertz, G. Klein, and A. Rosen, Exp. Cell Res. 197:314-318, 1991) that the same foci also contained the retinoblastoma (Rb) protein. Using a similar double immunofluorescence technique, we now show that these foci colocalize with nuclear bodies positive for PML, the promyelocytic leukemia-associated protein. Artificial spreading of the chromatin by exposure to the forces of fluid surface tension disrupts this colocalization gradually, suggesting that the bodies consist of at least two subcomponents. Heat shock or metabolic stress induced by high cell density leads to the release of EBNA-5 from the PML-positive nuclear bodies and induces it to translocate to the nucleoli. In addition to their presence in nuclear bodies, both proteins are occasionally present in nuclear aggregates and doughnut-like structures in which PML is concentrated in an outer shell. Nuclear bodies with prominent PML staining are seen in resting B lymphocytes. This staining pattern does not change upon EBV infection. In freshly infected cells EBNA-5 antigens are first distributed throughout the nucleoplasm. After a few days intensely staining foci develop. These foci coincide with PML-positive nuclear bodies. At a later stage and in established lymphoblastoid cell lines EBNA-5 is almost exclusively present in the PML-positive nuclear foci. The colocalization is restricted to EBV-infected human lymphoblasts. The data presented indicate that the distinct EBNA-5 foci are not newly formed structures but the result of translocation of the viral protein to a specialized domain present already in the nuclei of uninfected cells.

Modulation of chromatin folding by histone acetylation.

A homogeneous oligonucleosome complex was prepared by reconstitution of highly hyperacetylated histone octamers onto a linear DNA template consisting of 12 tandemly arranged 208-base pair fragments of the 5 S rRNA gene from the sea urchin Lytechinus variegatus. The ionic strength-dependent folding of this oligonucleosome assembly was monitored by sedimentation velocity and electron microscopy. Both types of analysis indicate that under ionic conditions resembling those found in the physiological range and in the absence of histone H1, the acetylated oligonucleosome complexes remain in an extended conformation in contrast to their nonacetylated counterparts. The implications of this finding in the context of a multistate model of chromatin folding (Hansen, J. C., and Ausio, J. (1992) TIBS 197, 187-191) as well as its biological relevance are discussed.

Quantitative analysis of macromolecular conformational changes using agarose gel electrophoresis: application to chromatin folding.

Quantitative analysis of chromatin electrophoretic mobility (mu) in agarose gels provides a measure of three structural parameters: average surface electrical charge density, which is proportional to the gel-free mu (mu 0), effective radius (Re), and particle deformability [Fletcher, T. M., Krishnan, U., Serwer, P., & Hansen, J. C. (1994) Biochemistry 33, 2226-2233]. To determine whether the intramolecular conformational changes associated with salt-dependent chromatin folding influence these electrophoretic parameters, defined oligonucleosomes were reconstituted from monodisperse tandemly repeated 5S DNA and varying amounts of histone octamers. These oligonucleosomes were subjected to both quantitative agarose gel electrophoresis and analytical velocity ultracentrifugation in buffers containing 0-2 mM MgCl2. Ionic conditions that caused a 40% increase in the oligonucleosome sedimentation coefficient (s20,w) also caused both a 30% decrease in Re and a 60% decrease in the magnitude of the mu 0. Furthermore, the Mg(2+)-dependent changes in s20,w, Re, and mu 0 each exhibited the same nonlinear dependence on the degree of nucleosome saturation of the DNA. These data demonstrate that quantitative agarose gel electrophoresis can be used to detect and characterize the process of chromatin folding. In addition, they suggest that this approach can be used for characterization of the conformational dynamics of many other types of macromolecular assemblies, including those systems that are not yet amenable for study by more traditional quantitative biophysical techniques.