Chromatin fibers are dynamic macromolecular assemblages that are intimately involved in nuclear function. This review focuses on recent advances centered on the molecular mechanisms and determinants of chromatin fiber dynamics in solution. Major points of emphasis are the functions of the core histone tail domains, linker histones, and a new class of proteins that assemble supramolecular chromatin structures. The discussion of important structural issues is set against a background of possible functional significance.

The compaction level of arrays of nucleosomes may be understood in terms of the balance between the selfrepulsion of DNA (principally linker DNA) and countering factors including the ionic strength and composition of the medium, the highly basic N termini of the core histones, and linker histones. However, the structural principles that come into play during the transition from a loose chain of nucleosomes to a compact 30-nm chromatin fiber have been difficult to establish, and the arrangement of nucleosomes and linker DNA in condensed chromatin fibers has never been fully resolved. Based on images of the solution conformation of native chromatin and fully defined chromatin arrays obtained by electron cryomicroscopy, we report a linker histone-dependent architectural motif beyond the level of the nucleosome core particle that takes the form of a stem-like organization of the entering and exiting linker DNA segments. DNA completes Ϸ1.7 turns on the histone octamer in the presence and absence of linker histone. When linker histone is present, the two linker DNA segments become juxtaposed Ϸ8 nm from the nucleosome center and remain apposed for 3-5 nm before diverging. We propose that this stem motif directs the arrangement of nucleosomes and linker DNA within the chromatin fiber, establishing a unique threedimensional zigzag folding pattern that is conserved during compaction. Such an arrangement with peripherally arranged nucleosomes and internal linker DNA segments is fully consistent with observations in intact nuclei and also allows dramatic changes in compaction level to occur without a concomitant change in topology.

We have examined the effects of core histone acetylation on the transcriptional activity and higher-order folding of defined 12-mer nucleosomal arrays. Purified HeLa core histone octamers containing an average of 2, 6, or 12 acetates per octamer (8, 23, or 46% maximal site occupancy, respectively) were assembled onto a DNA template consisting of 12 tandem repeats of a 208-bp Lytechinus 5S rRNA gene fragment. Reconstituted nucleosomal arrays were transcribed in a Xenopus oocyte nuclear extract and analyzed by analytical hydrodynamic and electrophoretic approaches to determine the extent of array compaction. Results indicated that in buffer containing 5 mM free Mg 2؉ and 50 mM KCl, high levels of acetylation (12 acetates/octamer) completely inhibited higher-order folding and concurrently led to a 15-fold enhancement of transcription by RNA polymerase III. The molecular mechanisms underlying the acetylation effects on chromatin condensation were investigated by analyzing the ability of differentially acetylated nucleosomal arrays to fold and oligomerize. In MgCl 2 -containing buffer the folding of 12-mer nucleosomal arrays containing an average of two or six acetates per histone octamer was indistinguishable, while a level of 12 acetates per octamer completely disrupted the ability of nucleosomal arrays to form higher-order folded structures at all ionic conditions tested. In contrast, there was a linear relationship between the extent of histone octamer acetylation and the extent of disruption of Mg 2؉ -dependent oligomerization. These results have yielded new insight into the molecular basis of acetylation effects on both transcription and higher-order compaction of nucleosomal arrays.The packaging of eukaryotic DNA into chromatin presents a major obstacle to the transcriptional machinery (reviewed in references 61 and 67). Acetylation of the core histone N termini is a post-translational modification of chromatin that has been widely correlated with enhanced transcriptional activity in vivo (3,34,55,57). Understanding of the connection between histone acetylation and transcriptional regulation has been further strengthened by the recent demonstrations that transcriptional coactivators possess histone acetyltransferase activity (11) and that transcriptional repressors associate with histone deacetylases (52). Despite this strong correlative evidence, the mechanism(s) through which histone acetylation influences transcription remains speculative. At the nucleosome level, the decreased access of transcription factors to regulatory DNA elements in vitro due to wrapping of the DNA around the histone octamer in some cases can be relieved by acetylation of the core histone N termini (38, 63; reviewed in reference 44). Beyond the level of the nucleosome, histone acetylation may function by disrupting higher-order folding of nucleosomal arrays. Studies of selectively trypsinized nucleosomal arrays have established that the core histone N termini perform multiple essential functions during nucleosomal array condensation (...

Regularly spaced nucleosomal arrays equilibrate between unfolded and highly folded conformations in <2 mM MgCl2, and self-associate above 2 mM MgCl2 [Schwarz, P. M., & Hansen, J. C. (1994) J. Biol. Chem. 269, 16284-16289]. Here we use analytical and differential sedimentation techniques to characterize the molecular mechanism and determinants of oligonucleosome self-association. Divalent cations induce self-association of intact nucleosomal arrays by binding to oligonucleosomal DNA and neutralizing its negative charge. Neither linker histones nor H2A/H2B dimers are required for Mg2+ - dependent self-association. However, divalent cations are unable to induce self-association of trypsinized nucleosomal arrays lacking their N- and C-terminal core histone tail domains. This suggests that the H3/H4 tail domains directly mediate oligonucleosome self-association through a non-Coulombic-based mechanism. Self-association occurs independently of whether the oligonucleosome monomers are folded or unfolded. The first step in the self-association pathway is strongly cooperative and produces a soluble association intermediate that sediments approximately 10 times faster than the oligonucleosome monomers. The size of the oligonucleosome polymers increases rapidly as a consequence of small increases in the divalent cation concentration, eventually producing polymeric species that sediment at >> 10 000 S. Importantly, all steps in the self-association pathway are freely reversible upon removal of the divalent cations. Taken together, these data indicate that short oligonucleosome fragments composed of only core histone octamers and DNA possess all of the structural features required to achieve chromosome-level DNA compaction. These findings provide a molecular basis for explaining many of the recently uncovered structural features of interphase and metaphase chromosomal fibers.

The release of GA (mitochondrial glutaminase) from neurons following acute ischaemia or during chronic neurodegenerative diseases may contribute to the propagation of glutamate excitotoxicity. Thus an inhibitor that selectively inactivates the released GA may limit the accumulation of excess glutamate and minimize the loss of neurological function that accompanies brain injury. The present study examines the mechanism of inactivation of rat KGA (kidney GA isoform) by the small-molecule inhibitor BPTES [bis-2-(5-phenylacetamido-1,2,4-thiadiazol-2-yl)ethyl sulfide]. BPTES is a potent inhibitor of KGA, but not of the liver GA isoform, glutamate dehydrogenase or gamma-glutamyl transpeptidase. Kinetic studies indicate that, with respect to glutamine, BPTES has a K(i) of approx. 3 microM. Moreover, these studies suggest that BPTES inhibits the allosteric activation caused by phosphate binding and promotes the formation of an inactive complex. Gel-filtration chromatography and sedimentation-velocity analysis were used to examine the effect of BPTES on the phosphate-dependent oligomerization of KGA. This established that BPTES prevents the formation of large phosphate-induced oligomers and instead promotes the formation of a single oligomeric species with distinct physical properties. Sedimentation-equilibrium studies determined that the oligomer produced by BPTES is a stable tetramer. Taken together, the present work indicates that BPTES is a unique and potent inhibitor of rat KGA and elucidates a novel mechanism of inactivation.

To investigate the role of human apolipoprotein E (apoE) on A␤ deposition in vivo, we crossed PDAPP mice lacking mouse Apoe to targeted replacement mice expressing human apoE (PDAPP/TRE2, PDAPP/TRE3, or PDAPP/TRE4). We then measured the levels of apoE protein and A␤ peptides in plasma, CSF, and brain homogenates in these mice at different ages. We also quantified the amount of brain A␤ and amyloid burden in 18-month-old mice. In young PDAPP/TRE4 mice that were analyzed at an age before brain A␤ deposition, we observed a significant decrease in the levels of apoE in CSF and brain when compared with age-matched mice expressing either human E2 or E3. The brain levels of A␤42 in PDAPP/TRE4 mice were substantially elevated even at this very early time point. In older PDAPP/TRE4 mice, the levels of insoluble apoE protein increased in parallel to the dramatic rise in brain A␤ burden, and the majority of apoE was associated with A␤. In TRE4 only mice, we also observed a significant decrease in the level of apoE in brain homogenates. Since the relative level of apoE mRNA was equivalent in PDAPP/TRE and TRE only mice, it appears that post-translational mechanisms influence the levels of apoE protein in brain (E4 Ͻ E3 Ͻ Ͻ E2), resulting in early and dramatic apoE isoform-dependent effects on brain A␤ levels (E4 Ͼ Ͼ E3 Ͼ E2) that increase with age. Therapeutic strategies aimed at increasing the soluble levels of apoE protein, regardless of isoform, may effectively prevent and (or) treat Alzheimer's disease.

Using the method of salt dialysis, we have reconstituted histone octamers onto DNA templates consisting of 12 tandem repeats, each containing a fragment of the sea urchin 5S rRNA gene [Simpson, R.T., Thoma, F., & Brubaker, J.M. (1985) Cell 42, 799-808]. In these templates, each sea urchin repeat contains a sequence for preferred nucleosome positioning. Sedimentation velocity and sedimentation equilibrium studies in the analytical ultracentrifuge indicate that at molar histone/DNA ratios of 1.0-1.1 extremely homogeneous preparations of fully loaded oligonucleosomes (12 nucleosomes/template) can be regularly obtained. Digestion of the oligonucleosomes with micrococcal nuclease, followed by restriction mapping of purified nucleosome-bound DNA sequences, yields a complicated but consistent pattern of nucleosome positioning. Roughly 50% of the nucleosomes appear to be phased at positions 1-146 of each repeat, while the remainder of the nucleosomes occupy a number of other minor discrete positions along the template that differ by multiples of 10 bp. From sedimentation velocity studies of the oligonucleosomes in 0-0.2 M NaCl, we observe a reversible increase in mean sedimentation coefficient by almost 30%, accompanied by development of heterogeneity in sedimentation. These results, in combination with theoretical predictions, indicate that linear stretches of chromatin in the absence of lysine-rich histones exist in solution in a salt-dependent equilibrium between an extended (low salt) conformation and one or more folded (high salt) structures. In addition, by 100 mM NaCl, salt-dependent dissociation of histone octamers from these linear oligonucleosomes is observed.

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.