Two new photoaffinity polyamines appear to alter the helical twist of DNA in nucleosome core particles

Two new photoaffinity derivatives of polyamines have been synthesized by the reaction of spermine or spermidine with methyl 4-azidobenzimidate. The new compounds were purified chromatographically and characterized by several methods including proton magnetic resonance spectroscopy. The spermine derivative is N1-ABA-spermine [(azidobenzamidino)spermine], and the spermidine derivative is a mixture of N1- and N8-ABA-spermidine. ABA-spermine stabilizes nucleosome core particles in thermal denaturation experiments, with similar but not identical effects when compared with the parent polyamine, spermine. In circular dichroism experiments, ABA-spermine was capable of producing a B----Z transition in poly(dG-m5dC) at a concentration of 30 microM, compared with 5 microM required to produce the same effect with spermine. On the other hand, ANB-spermine [(azidonitrobenzoyl)spermine; Morgan, J. E., Calkins, C. C., & Matthews, H. R. (1989) Biochemistry 28, 5095-5106] stabilized the B form of poly(dG-br5dC). ABA-spermine is a potent inhibitor of ornithine decarboxylase from Escherichia coli, giving 50% inhibition at 0.12 mM, while ANB-spermine is a modest inhibitor, comparable to spermine or spermidine. Under conditions of nitrogen-limited growth, yeast take up ABA-spermine and ABA-spermidine at approximately one-third to half the rate of spermidine or spermine. In contrast, ANB-spermine was not significantly taken up. The photoaffinity polyamines were used to photoaffinity label the DNA in nucleosome core particles, and the sites of labeling were determined by exonuclease protection. All photoaffinity reagents showed both nonspecific labeling and specific sites of higher occupancy. However, the positions of the sites varied: the ANB-spermine sites confirmed those previously reported (Morgan et al., 1989); the ABA-spermine and ABA-spermidine sites were spaced at 9.8 bp intervals from the 3' end of each DNA strand. This observation, together with the effect of spermine on the circular dichroism of DNA in nucleosome core particles, implies that polyamines alter the helical twist of DNA in nucleosome core particles. The ABA-polyamines are offered as general-purpose photoaffinity polyamine reagents.     

Elevated levels of polyamines alter chromatin in murine skin and tumors without global changes in nucleosome acetylation

Polyamines affect nucleosome oligomerization and DNA conformation in vitro, yet little information exists regarding the influence of naturally synthesized polyamines on mammalian chromatin. Capitalizing on the relative inefficiency of a moderate ionic strength extraction buffer to dissociate histones, we obtained evidence of altered chromatin in transgenic mice that overexpress ornithine decarboxylase (ODC), which catalyzes polyamine synthesis. Dissociation of histones from chromatin in ODC transgenic mouse skin, as well as in tumors that develop spontaneously in ODC/Ras bigenic mice, is dramatically reduced relative to normal littermate skin. This could reflect tighter tethering of nucleosomes to DNA or a more compacted chromatin structure due to elevated intracellular concentrations of polyamines since this effect is reversible upon treatment with alpha-difluoromethylornithine (DFMO), a specific inhibitor of ODC enzymatic activity. Impeded release of nonhistone chromatin proteins HP-1beta and nucleophosmin, but not Lamin B, HDAC-1, HMGB, HMGN2, or HMGA1, suggests that polyamines exert selective effects on specific chromatin protein complexes. Moreover, overall acetylation, as well as specific methylation, of nucleosomes in ODC mice is unaffected, implying that access by histone modifying enzymes is not generally restricted. The abnormal chromatin environment fostered by elevated levels of polyamines may be a necessary prerequisite for epithelial tumor growth and maintenance.     

Interaction of polyamines with chromatin and DNA: formation of compact structures

We have used flow linear dichroism (LD) and light scattering at 90 degrees to study the condensation of both DNA and calf thymus chromatin induced by spermine, triamines NH3+(CH2)iNH+(CH2)jNH3+, designated as much less than i, j much greater than: much less than 3, 4 much greater than (spermidine), much less than 3, 3 much greater than, much less than 2, 3 much greater than, much less than 2, 2 much greater than; the diamines putrescine and cadaverine and MgCl2. It is found that the different polyamines affected DNA and chromatin in a similar way. The degree of compaction of the chromatin fibers induced by spermine, triamines except much less than 2, 2 much greater than and Mg2+ has been found to be identical. The triamine much less than 2, 2 much greater than and the diamines studied do not condense either chromatin of DNA. Such a big difference in the action of the triamines indicates that not only the charge, but also the structure of the polycations are important for their interactions with DNA and chromatin. The stoichiometry of polyamine binding to chromatin at which condensation occurred is found to be 2 polyamine molecules per DNA helical turn. Polyamines are supposed to bind to the exposed sites of core DNA every 10 b.p. The extent of DNA phosphate neutralization by the histones is estimated to be about 55%. It has been shown that a mixture of mono- and multivalent cations affected DNA and chromatin condensation competitively and not synergistically, as claimed in a recent report by Sen and Crothers.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Local geometry and elasticity in compact chromatin structure

The hierarchical packaging of DNA into chromatin within a eukaryotic nucleus plays a pivotal role in both the accessibility of genomic information and the dynamics of replication. Our work addresses the role of nanoscale physical and geometric properties in determining the structure of chromatin at the mesoscale level. We study the packaging of DNA in chromatin fibers by optimization of regular helical morphologies, considering the elasticity of the linker DNA as well as steric packing of the nucleosomes and linkers. Our model predicts a broad range of preferred helix structures for a fixed linker length of DNA; changing the linker length alters the predicted ensemble. Specifically, we find that the twist registry of the nucleosomes, as set by the internucleosome repeat length, determines the preferred angle between the nucleosomes and the fiber axis. For moderate to long linker lengths, we find a number of energetically comparable configurations with different nucleosome-nucleosome interaction patterns, indicating a potential role for kinetic trapping in chromatin fiber formation. Our results highlight the key role played by DNA elasticity and local geometry in regulating the hierarchical packaging of the genome.     

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.     

Chromatin remodeling by ISW2 and SWI/SNF requires DNA translocation inside the nucleosome

Chromatin-remodeling complexes regulate access to nucleosomal DNA by mobilizing nucleosomes in an ATP-dependent manner. In this study, we find that chromatin remodeling by SWI/SNF and ISW2 involves DNA translocation inside nucleosomes two helical turns from the dyad axis at superhelical location-2. DNA translocation at this internal position does not require the propagation of a DNA twist from the site of translocation to the entry/exit sites for nucleosome movement. Nucleosomes are moved in 9- to 11- or approximately 50-base-pair increments by ISW2 or SWI/SNF, respectively, presumably through the formation of DNA loops on the nucleosome surface. Remodeling by ISW2 but not SWI/SNF requires DNA torsional strain near the site of translocation, which may work in conjunction with conformational changes of ISW2 to promote nucleosome movement on DNA. The difference in step size of nucleosome movement by SWI/SNF and ISW2 demonstrates how SWI/SNF may be more disruptive to nucleosome structure than ISW2.     

Changing Chromatin Fiber Conformation by Nucleosome Repositioning

Chromatin conformation is dynamic and heterogeneous with respect to nucleosome positions, which can be changed by chromatin remodeling complexes in the cell. These molecular machines hydrolyze ATP to translocate or evict nucleosomes, and establish loci with regularly and more irregularly spaced nucleosomes as well as nucleosome-depleted regions. The impact of nucleosome repositioning on the three-dimensional chromatin structure is only poorly understood. Here, we address this issue by using a coarse-grained computer model of arrays of 101 nucleosomes considering several chromatin fiber models with and without linker histones, respectively. We investigated the folding of the chain in dependence of the position of the central nucleosome by changing the length of the adjacent linker DNA in basepair steps. We found in our simulations that these translocations had a strong effect on the shape and properties of chromatin fibers: i), Fiber curvature and flexibility at the center were largely increased and long-range contacts between distant nucleosomes on the chain were promoted. ii), The highest destabilization of the fiber conformation occurred for a nucleosome shifted by two basepairs from regular spacing, whereas effects of linker DNA changes of ∼10 bp in phase with the helical twist of DNA were minimal. iii), A fiber conformation can stabilize a regular spacing of nucleosomes inasmuch as favorable stacking interactions between nucleosomes are facilitated. This can oppose nucleosome translocations and increase the energetic costs for chromatin remodeling. Our computational modeling framework makes it possible to describe the conformational heterogeneity of chromatin in terms of nucleosome positions, and thus advances theoretical models toward a better understanding of how genome compaction and access are regulated within the cell.     

Effect of histone acetylation on structure and in vitro transcription of chromatin.

n-Butyrate treatment of growing Hela cells produces a dramatic increase in the levels of histone acetylation. We have exploited this system to study the effect of histone acetylation on chromatin structure. Chromatin containing highly acetylated histones is more rapidly digested to acid-soluble material by DNase I, but not by micrococcal nuclease. The same pattern of nuclease sensitivity was exhibited by in vitro-assembled chromatin consisting of SV40 DNA Form I and the 2 M salt-extracted core histones from butyrate-treated cells. Using this very defined system, it was possible to demonstrate that acetylated nucleosomes do not have a greatly diminished stability. Stability was measured in terms of exhange of histone cores onto competing naked DNA or sliding of histone cores along ligated naked DNA. Finally, it was shown that acetylated nucleosomes are efficient inhibitors of in vitro RNA synthesis by the E. coli holoenzyme as well as by the mammalian polymerases A and B.     

Effects of histone acetylation and CpG methylation on the structure of nucleosomes

Nucleosomes are the fundamental packing units of the eukaryotic genome. A nucleosome core particle comprises an octameric histone core wrapped around by ~147bp DNA. Histones and DNA are targets for covalent modifications mediated by various chromatin modification enzymes. These modifications play crucial roles in various gene regulation activities. A group of common hypotheses for the mechanisms of gene regulation involves changes in the structure and structural dynamics of chromatin induced by chromatin modifications. We employed single molecule fluorescence methods to test these hypotheses by monitoring the structure and structural dynamics of nucleosomes before and after histone acetylation and DNA methylation, two of the best-conserved chromatin modifications throughout eukaryotes. Our studies revealed that these modifications induce changes in the structure and structural dynamics of nucleosomes that may contribute directly to the formation of open or repressive chromatin conformation.     

DNA-PK is activated by nucleosomes and phosphorylates H2AX within the nucleosomes in an acetylation-dependent manner

Eukaryotic DNA is organized into nucleosomes and higher order chromatin structure, which plays an important role in the regulation of many nuclear processes including DNA repair. Non-homologous end-joining, the major pathway for repairing DNA double-strand breaks (DSBs) in mammalian cells, is mediated by a set of proteins including DNA-dependent protein kinase (DNA-PK). DNA-PK is comprised of a large catalytic subunit, DNA-PKcs, and its regulatory subunit, Ku. Current models predict that Ku binds to the ends of broken DNA and DNA-PKcs is recruited to form the active kinase complex. Here we show that DNA-PK can be activated by nucleosomes through the ability of Ku to bind to the ends of nucleosomal DNA, and that the activated DNA-PK is capable of phosphorylating H2AX within the nucleosomes. Histone acetylation has little effect on the steps of Ku binding to nucleosomes and subsequent activation of DNA-PKcs. However, acetylation largely enhances the phosphorylation of H2AX by DNA-PK, and this acetylation effect is observed when H2AX exists in the context of nucleosomes but not in a free form. These results suggest that the phosphorylation of H2AX, known to be important for DSB repair, can be regulated by acetylation and may provide a mechanistic basis on which to understand the recent observations that histone acetylation critically functions in repairing DNA DSBs.     

High-mobility group and other nonhistone substrates for nuclear histoneN-acetyltransferase

A variety of nonhistone proteins and polyamines has been studied for their substrate activity for nuclear histone N-acetyltransferase. Nonhistone chromatin high-mobility group (HMG) proteins are found to be as good a substrate for the enzyme as histones. The enzyme also acetylates spermidine and spermine. However, protamine, bovine serum albumin, and ubiquitin are not substrates. Chymotryptic peptides of histone and HMGs retained about 64% of the substrate activity, but trypsin treatment reduced the substrate activity by more than 85%. Both N-acetyltransferase activities for HMGs and histones are copurified through salt extraction, polyethylene glycol fractionation, and chromatography on DEAE-cellulose, phosphocellulose columns, and a HPLC anionic-exchange column. The highly purified nuclear histone acetyltransferase shows similar optimal pH and ping-pong kinetics for both HMGs and histones. The Km for HMG is 0.25 mg/ml. HMGs are able to accept the acetyl group from isolated acetyl-enzyme intermediate. Denatured gel analysis shows that HMG 1 and HMG 2 are the major proteins acetylated. High salt concentrations, mononucleotides, and DNA, which inhibit histone substrate activity of the enzyme, also inhibit HMG substrate activity. These observations suggest that there is a major nuclear N-acetyltransferase which is responsible for the acetylation of both histones and HMGs and perhaps also of spermine and spermidine. Thus the regulation of the structure and function of chromatin through postsynthetic acetylation can be achieved by a single nuclear N-acetyltransferase.     

Epigenetic regulatory elements associate with specific histone modifications to prevent silencing of telomeric genes

In eukaryotic cells, transgene expression levels may be limited by an unfavourable chromatin structure at the integration site. Epigenetic regulators are DNA sequences which may protect transgenes from such position effect. We evaluated different epigenetic regulators for their ability to protect transgene expression at telomeres, which are commonly associated to low or inconsistent expression because of their repressive chromatin environment. Although to variable extents, matrix attachment regions (MARs), ubiquitous chromatin opening element (UCOE) and the chicken cHS4 insulator acted as barrier elements, protecting a telomeric-distal transgene from silencing. MARs also increased the probability of silent gene reactivation in time-course experiments. Additionally, all MARs improved the level of expression in non-silenced cells, unlike other elements. MARs were associated to histone marks usually linked to actively expressed genes, especially acetylation of histone H3 and H4, suggesting that they may prevent the spread of silencing chromatin by imposing acetylation marks on nearby nucleosomes. Alternatively, an UCOE was found to act by preventing deposition of repressive chromatin marks. We conclude that epigenetic DNA elements used to enhance and stabilize transgene expression all have specific epigenetic signature that might be at the basis of their mode of action.