Nucleosome phase

Cross-linkers, Chromatin condensation. Histone tails, Nucleosome phasing... 

At Oak Ridge, the focus was to develop specific-sequence DNA to improve the diffraction quality of NCP crystals. The positioning of the DNA on the histone core has to be precise so that all the NCPs are identical. A project was undertaken to understand the DNA sequence effects on nucleosome phasing [25]. Second, a DNA palindrome was developed to extend the two-fold symmetry of the histone core to the DNA. The objective was to eliminate the two-fold disorder caused by the indeterminacy of packing of an asymmetric particle into the crystal lattice. A palindrome based on one-half of the primary candidate sequence was constructed and methods were developed to produce the palindrome fragment in large quantities for reconstitution of NCPs. 

Heterogeneous reaction (Section 6 1) A reaction involving two or more substances present in different phases Hydro genation of alkenes is a heterogeneous reaction that takes place on the surface of an insoluble metal catalyst Heterolytic cleavage (Section 4 16) Dissociation of a two electron covalent bond in such a way that both electrons are retained by one of the initially bonded atoms Hexose (Section 25 4) A carbohydrate with six carbon atoms High density lipoprotein (HDL) (Section 26 11) A protein that carries cholesterol from the tissues to the liver where it is metabolized HDL is often called good cholesterol Histones (Section 28 9) Proteins that are associated with DNA in nucleosomes... 

The assembly of nucleosomes is mediated by one of several chromatin assembly factors facilitated by histone chaperones, proteins such as the anionic nuclear protein nucleoplasmin. As the nucleosome is assembled, histones are released from the histone chaperones. Nucleosomes appear to exhibit preference for certain regions on specific DNA molecules, but the basis for this nonrandom distribution, termed phasing, is not completely... 

Simon RH, Eelsenfeld G (1979) A new procedure for purifying histone pairs H2A + H2B and H3 + H4 from chromatin using hydroxylapatite. Nucleic Acids Res 6 689-696 Simpson RT (1978) Structure of the chromatosome, a chromatin particle containing 160 base pairs of DNA and all the histones. Biochemistry 17 5524-5531 Simpson RT, Stafford DW (1983) Structural features of a phased nucleosome core particle. Proc Natl Acad Sci U S A 80 51-55... 

The crystallization and structural determination of the histone octamer was first reported in 1984 [34], However, the overall dimensions of the 3.3 A structure [15] did not appear to fit within the known X-ray structures of the nucleosome core particle [12,13], In an elegant analysis [16], re-examination of the original phasing of the histone octamer data revealed misplacement of the heavy atom site by 2.7 A. The structure was resolved, after which it was possible to build molecular models of the individual histones into the 3.1 A resolution electron density map of the histone core of the nucleosome [17]. Figure 2 shows the first atomic resolution model of the core histone octamer. Several additional publications followed in which the histone octamer structure formed the basis for constructing models of the NCP [17-21],... 

Simpson, R.T. and Stafford, D.W. (1983) Structural features of a phased nucleosome core particle. Proc. Natl. Acad. Sci. USA 80, 51-55. 

Duplicating the GFP-H2B experiments with both GFP-H3 and GFP-H4 vectors, very little H3 and H4 exchange outside of S phase was observed. Fluorescence of GFP-H3 and GFP H4 in HeLa cells in G1 recovered rapidly. The extremely rapid recovery rate is similar to that of a diffuse soluble protein, indicating that at this stage of the cell cycle, GFP-tagged H3 and -H4 are not incorporated into chromatin. FRAP experiments of transfected cells in S or G2 show that there is very little recovery of GFP-H3 or -H4 fluorescence. The fluorescence imaging indicates that once the H3 and H4 proteins are incorporated into the chromatin, they are essentially immobile for the remainder of the cell cycle. Unlike histones H2A and H2B, which associate as dimers in the nucleosome histone octamer, there is very little exchange of the components of the H3/H4 tetramer. 

Nucleosome-nucleosome interaction potentials can be calibrated by comparison with the characteristics of liquid crystals of mononucleosomes at high concentrations. Under suitable conditions, nucleosome core particles form a hexagonal-columnar phase with a distance of 11.55 1 nm between the columns and a mean distance of 7.16 0.65 nm between the particles in one column [44,46]. These distances may be assumed to correspond to the positions of the minima of an attractive internucleosomal potential. The depth of the interaction potential (i.e., the binding energy per nucleosome) was estimated in the stretching experiments of Cui and Bustamante [66] to 2.6-3.4 kT. A slightly lower potential minimum of 1.25 kT is obtained by a comparison of the stability of the nucleosome liquid crystal phase with simulations [50]. 

Leforestier, A. and Livolant, F. (1997) Liquid crystalline ordering of nucleosome core particles under macromolecular crowding conditions evidence for a discotic columnar hexagonal phase. Biophys. J. 73, 1771-1776. 

Mangenot, S., Leforestier, A., Durand, D., and Livolant, F. (2003) X-ray diffraction characterization of the dense phases formed by nucleosome core particles. Biophys. J. 84, 2570-2584. 

MCF-7 cells exposed to 50 nM TSA were in G1 phase (>70%). However, cells treated with 1 J,M TSA arrested cells predominantly in G2/M phase, suggesting a dose-dependent fashion. With either dose, cells accumulated least in the S phase. In addition to accumulation of hyperacetylated nucleosome core histones, TSA enhanced p21 expression. Therefore, flow cytometry (FACS) offers an efficient tool for analyzing the action of HDACIs in cell proliferation. This method is particularly useful for application to clinical samples, where cell numbers may be small. The effects of HDACIs on the cell cycle are dose-dependent. 

---