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At least 145 records · Page 8Linked to original sources
PubMed2001

Detection of nucleosome particles in serum and plasma from patients with systemic lupus erythematosus using monoclonal antibody 4H7.

OBJECTIVE: To develop a monoclonal antibody reagent that would react with nucleosomes but not directly with constituent double stranded DNA (dsDNA) or with histones. METHODS: Mice were immunized with highly purified chicken mononucleosomes and hybridomas employed to produce Mab that did not react with dsDNA or histones but still showed reactivity with nucleosomes. RESULTS: Murine monoclonal IgG antibody 4H7, generated from a mouse immunized with highly purified chicken erythrocyte nucleosomes, showed no direct ELISA reactivity with either dsDNA or isolated histones or with Sm and RNP antigens or combinations of any of these components. Mab 4H7 did show strong ELISA reactivity for chicken erythrocyte and calf thymus nucleosomes as well as for human leukocyte nucleosomes. The Mab did show strong ELISA reactions with peptides 1-25 of histone H2B and 1-21 of H3, which correspond to sequences known to be located at the surface of nucleosomes. We then measured relative serum levels of 4H7 reactive nucleosome antigen in 140 patients with systemic lupus erythematosus (SLE) in parallel with 50 non-SLE patients with other types of connective tissue disease and 92 healthy subjects. Occasional low levels of serum nucleosomal antigen were seen in 4 of 92 controls, but many patients with SLE (66/140) showed marked elevations of serum nucleosomal antigen. No difference was observed when serum or plasma samples were studied. A marked correlation (R = 0.401, p < 0.0001) was noted when disease activity score (SLEDAI) was plotted against optical density value measured with 4H7 in ELISA. Further, the levels of circulating nucleosomes were raised in SLE patients with very active central nervous system and renal involvement. CONCLUSION: Presence of nucleosome related antigen in sera from patients with SLE may provide insight into the sequence of disease related antigenic stimuli in active SLE.

Adult↗
PubMed1999

Termination of human T cell tolerance to histones by presentation of histones and polyomavirus T antigen provided that T antigen is complexed with nucleosomes.

OBJECTIVE: To investigate whether polyomavirus T antigen linked to histones through nucleosome-T antigen complexes has the potential to terminate histone-specific T cell anergy. METHODS: Blood mononuclear cells from healthy individuals were used as the source to establish T cell lines initiated and maintained by T antigen, histones, nucleosome-T antigen complexes, or nucleosomes. Proliferative responses of these lines to T antigen, histones, and nucleosomes were determined. RESULTS: Whereas T cell lines could be established using T antigen or T antigen-nucleosome complexes, histones or nucleosomes did not have this potential. However, T cell lines selected by T antigen-nucleosome complexes responded subsequently to histones and nucleosomes. Identical results were obtained with murine and human nucleosomes, provided that they were complexed with T antigen. CONCLUSION: T antigen-specific T cells possess the potential to proliferate when interacting with an antigen-presenting cell that presents T antigen. In the presence of T antigens complexed with nucleosomes, T antigen-specific T cells offer bystander help that may terminate histone-specific T cell anergy. These T cells may progress into functional, autoimmune T cells if histones are properly presented.

Animals↗
PubMed2000

Nucleosomes are major T and B cell autoantigens in systemic lupus erythematosus.

OBJECTIVE: Double-stranded DNA (dsDNA) is a well-known target of autoantibodies in systemic lupus erythematosus (SLE). The majority of these autoantibodies are of the IgG isotype and show affinity maturation, both of which are known hallmarks of T cell help. T cell responses to autoantigens, including DNA, have been reported only incidentally in SLE patients. Nevertheless, in murine SLE, naked DNA and complexed DNA (nucleosomes) are known to be recognized by T cells. This study aimed to characterize the antinucleosome response and its clinical impact on human SLE. METHODS: Nucleosomes were prepared from chicken erythrocytes. Sera from SLE and control patients were investigated by enzyme-linked immunosorbent assay (ELISA) for nucleosome-specific antibody responses. Peripheral blood mononuclear cells (PBMC) from SLE and control patients were analyzed by a kinetic T cell proliferation assay. PBMC were subsequently analyzed for nucleosome-specific T cell proliferation. RESULTS: Of 136 SLE patients, 56% were seropositive for antinucleosome antibodies. In contrast, only 3% of 309 control patients (with rheumatoid arthritis, mixed connective tissue disease, undifferentiated connective tissue disease, Lyme borreliosis, scleroderma, Sjögren's syndrome, ulcerative colitis, hepatitis B virus infection, or human immunodeficiency virus infection) were seropositive. Thus, the antinucleosome ELISA had a sensitivity of 56%, a specificity of 97%, and a diagnostic confidence of 90% when applied to SLE. It was therefore superior to an anti-DNA ELISA that demonstrated a 69% diagnostic confidence in the same population. Antinucleosome reactivity in SLE patients correlated significantly with disease activity (P < 0.0001), nephritis (P < 0.002), and psychosis (P < 0.02). When proliferation assays were applied, 14 of 26 SLE patients (54%) were positive for nucleosome-specific T cells that proliferated in response to their cognate antigen. A suppressed response was elicited in 3 SLE patients (12%); in these patients, the PBMC response to nucleosomes was lower than the proliferation of PBMC in the presence of culture medium only. PBMC from the remaining 9 SLE patients (35%) were nonresponsive to nucleosomes in either way. Responding, nonresponding, and suppressed populations differed from each other significantly (P < 0.0001). None of the PBMC from 7 healthy donors and 10 control patients could be stimulated with nucleosomal antigens. CONCLUSION: We present evidence that nucleosomes are major autoantigens in human SLE and that antinucleosomal antibodies are highly specific for the disease. The antinucleosome ELISA has been shown to be superior to the anti-dsDNA ELISA and may thus be a significantly better tool for diagnosing SLE. Nucleosome-specific T cells in SLE patients may help B cells class switch to IgG and undergo affinity maturation.

Adult↗
PubMed1997

Antigen specificity of anti-nuclear antibodies complexed to nucleosomes determines glomerular basement membrane binding in vivo.

Monoclonal anti-nuclear antibodies which are complexed to nucleosomes are able to bind to the glomerular basement membrane (GBM) in vivo, whereas purified antibodies do not bind. The positively charged histone moieties in the nucleosome are-responsible for the binding to anionic determinants in the GBM. We tested the hypothesis that the specificity of the autoantibodies complexed to the nucleosome influences the glomerular binding of the antibody-nucleosome complex. We induced the formation of these immune complexes in vivo, by intraperitoneal inoculation of hybridomas producing monoclonal anti-nuclear antibodies (four anti-histone, three anti-double stranded (ds)DNA and three anti-nucleosome antibodies) into nude BALB/c mice. In ascites and plasma from the mice inoculated with these hybridomas, nucleosome/autoantibody complexes were detected in comparable amounts. Immunofluorescence of kidney sections revealed that about 60% of the mice inoculated with anti-nucleosome or anti-dsDNA hybridomas had immunoglobulin deposits in the GBM, whereas only 15% of the mice with anti-histone hybridomas showed these deposits (p < or = 0.04). In the Matrigel-ELISA (used as a GBM surrogate) ascites from anti-nucleosome or anti-DNA hybridomas displayed significantly higher titers (p < or = 0.002) than ascites from anti-histone hybridomas. In conclusion, nucleosome/immunoglobulin complexes comprising anti-nucleosome or anti-dsDNA auto-antibodies do bind more frequently to the GBM in vivo than nucleosome/immunoglobulin complexes containing anti-histone antibodies. It therefore appears that the specificity of the antibody bound to the nucleosome is a critical determinant for the nephritogenic potential of the nucleosome-autoantibody complex.

Animals↗
PubMed1995

Electrostatic mechanism of nucleosome spacing.

Native bulk chromatin is characterized by regular arrays of nucleosomes with defined internucleosomal distances. The nucleosome repeat length is not a constant but varies between species and cell-types, during differentiation and during gene activation. Previous studies have highlighted the importance of linker histones as a major determinant of nucleosome repeat length in vivo. We used a physiological reconstitution system derived from Drosophila embryos to study nucleosome spacing. In these extracts, histone H1 incorporation increases the apparent linker length in a gradual way. Manipulation of the chromatin assembly conditions in vitro allowed us to define additional parameters that modulate nucleosomal distances, such as protein phosphorylation events and the precise ionic conditions during the reconstitution. Interestingly, moderate changes in the concentrations of mono-, di-, and multivalent cations affect the precise distances between nucleosome cores remarkably. These changes in the ionic environment are unlikely to affect the association of linker proteins but are known to influence the folding of the nucleosomal fiber by modulation of electrostatic forces. Our results suggest electrostatic interactions in chromatin units as major determinants of nucleosome spacing. Nucleosome spacing and the folding of the nucleosomal fiber can therefore be explained by common principles, most notably the neutralization of charges in linker DNA.

Animals↗
PubMed2001

Specific interactions of the telomeric protein Rap1p with nucleosomal binding sites.

The telomeres of Saccharomyces cerevisiae are structurally and functionally well characterized. Their telomeric DNA is packaged by the protein Rap1p (repressor activator protein 1). Rap1p is a multifunctional, sequence-specific, DNA-binding protein which, besides participating in the regulation of telomeres structure and length, is also involved in transcriptional regulation of genes essential for cell growth and in silencing. Whereas the long tracts of telomeric DNA repeats of higher eukaryotes are mostly organized in closely spaced canonical nucleosomal arrays, it has been proposed that the 300 base-pairs of S. cerevisiae telomeric DNA are organized in a large non-nucleosomal structure that has been called the telosome. Recently, nucleosomes have been found also in Tetrahymena thermophila telomeres, suggesting that, in general, telomere structural differences between lower and higher eukaryotes could be quantitative, rather than qualitative. Using an in vitro model system, we have addressed the question of whether Rap1p can form a stable ternary complex with nucleosomes containing telomeric binding sites, or competes with nucleosome core formation. The approach we have taken is to place a single Rap1p-binding site at different positions within a nucleosome core and then test the binding of Rap1p and its DNA-binding domain (Rap1p-DBD). We show here that both proteins are able to specifically recognize their nucleosomal binding site, but that binding is dependent on the location of the site within the nucleosome core structure. These results show that a ternary complex between a nucleosome and Rap1p is stable and could be a possible intermediate between telomeric nucleosomes and telosomes in the dynamics of S. cerevisiae telomere organization.

Binding Sites↗
PubMed2003

Nucleosome transactions on the Hypocrea jecorina (Trichoderma reesei) cellulase promoter cbh2 associated with cellulase induction.

The 5' regulatory region of the cbh2 gene of Hypocrea jecorina contains the cbh2 activating element (CAE) which is essential for induction of cbh2 gene expression by sophorose and cellulose. The CAE consists of two motifs, a CCAAT box on the template strand and a GTAATA box on the coding strand, which cooperate during induction. Northern analyses of cbh2 gene expression has revealed an absolute dependence on induction, but no direct effect of Cre1-mediated carbon catabolite repression. Investigation of the chromatin structure in the wild-type strain showed that, under repressing conditions, there is a nucleosome free region (nfr) around the CAE, which is flanked by strictly positioned nucleosomes. Induction results in a loss of positioning of nucleosomes -1 and -2 downstream of the CAE, thus making the TATA box accessible. Simultaneous mutation of both motifs of the CAE, or of the CCAAT-box alone, also leads to shifting of nucleosome -1, which normally covers the TATA-box under repressing conditions, whereas mutation of the GTAATA element results in a narrowing of the nfr, indicating that the proteins that bind to both motifs in the CAE interact with chromatin, although in different ways. A cellulase-negative mutant strain, which has previously been shown to be altered in protein binding to the CAE, still displayed the induction-specific changes in nucleosome structure, indicating that none of the proteins that directly interact with CAE are affected, and that nucleosome rearrangement and induction of cbh2 expression are uncoupled. Interestingly, the carbon catabolite repressor Cre1 is essential for strict nucleosome positioning in the 5' regulatory sequences of cbh2 under all of the conditions tested, and induction can occur in a promoter that lacks positioned nucleosomes. These data suggest that Cre1, the Hap2/3/5 complex and the GTAATA-binding protein are all involved in nucleosome assembly on the cbh2 promoter, and that the latter two respond to inducing conditions by repositioning nucleosome -1.

Base Sequence↗
PubMed1991

In vitro assembly of a positioned nucleosome near the hypersensitive region in simian virus 40 chromatin.

Previous studies have identified a nucleosome near a potential late boundary for the nuclease-hypersensitive region in simian virus 40 chromatin. We have performed in vitro reconstitution analysis to determine whether the underlying DNA sequence encodes for the assembly of this nucleosome and applied hydroxyl radical and DNase I footprinting techniques to examine the structure of the reconstituted nucleosome. Both methods revealed the formation of a precisely positioned nucleosome in vitro, on a fragment spanning the strong in vivo nucleosome location site determined previously in the viral chromatin. The center of the positioned nucleosome maps between nucleotide 384 and 387 on simian virus 40 DNA. The corresponding nucleosome core includes the major-late transcription site (12 base-pairs within the core), the MspI site, and a segment shown previously to adopt a bent structure in the absence of proteins. The hydroxyl radical produces a strikingly well-defined cleavage pattern over the bent DNA incorporated in nucleosomes. The dominant periodicity of DNA in this nucleosome is 10.26 base-pairs per turn. The distribution of the .OH cut sites in the positioned nucleosome provides strong support for models in which the minor grooves of the A/T-rich tracts are oriented toward the histone core while the minor grooves of the G/C-rich sequences are facing outward.

Base Sequence↗
PubMed1979

A phase relationship associates tRNA structural gene sequences with nucleosome cores.

DNA (760 bp) isolated from nucleosome tetramers of staphylococcal nuclease-digested chicken embryo chromatin was highly enriched for tRNA genes and subsequently cloned in E. coli chi 1776. The location of genes coding for chicken embryo tRNALys, tRNAPhe and tRNAiMet within the cloned nucleosome tetramer DNA was determined using restriction endonucleases for which single cleavage sites could be predicted from the respective tRNA base sequence. All our tRNA genes reside nonrandomly at four locations on nucleosome tetramer DNA. The spacing between the tRNA gene locations is approximately 190 bp, similar to the DNA repeat length of chicken embryo chromatin. The four tRNA gene locations were also defined in noncloned nucleosome tetramer DNA highly enriched for tRNA genes. The majority of genes coding for tRNALys, tRNAPhe and tRNAiMet, respectively, are located in equal proportion 40-45, 230, 420 and 610 bp distant from the 5' end of the tRNA-identical strand. Thus the tRNA structural gene sequences all appear to begin about 20 bp "inside" the nucleosome core. As observed with nucleosomal DNA not enriched for tRNA genes, the phase relationship between tRNA genes and nucleosome location is maintained over a distance of 4-6 subsequent nucleosomes. A cloned molecule of nucleosomal DNA containing both a tRNALys gene and a tRNAiMet gene in the same polarity reveals that a phase adjustment might be necessary for the nucleosomes between these two tRNA genes in chicken embryo chromatin.

Animals↗
PubMed2003

Mechanism of nucleosome disruption and octamer transfer by the chicken SWI/SNF-like complex.

We had recently characterized SLC, a SWI/SNF-like chromatin remodelling activity, from chicken liver. The SLC efficiently disrupts nucleosomes, transfers histone octamers from nucleosomal substrates onto acceptor DNA, and slides histone octamers along DNA. Here, we present evidence that SLC is indeed a SWI/SNF homologue, and it disrupts nucleosomes by inducing extensive dynamic helical distortions in the nucleosomal DNA. Both the nucleosome disruption and octamer transfer functions are indifferent to nucleosomal histone tails. We further show that the nucleosome disruption precedes the octamer transfer and that the latter requires continuous presence of ATP. Based on these observations, we propose that a disrupted nucleosome is not a spontaneous substrate for octamer transfer; rather the nucleosome disruption and the octamer transfer are two temporally successive, ATP-dependent events during nucleosome remodelling by SLC in vitro.

Adenosine Triphosphate↗
PubMed1983

Binding of daunomycin to calf thymus nucleosomes.

We report equilibrium, hydrodynamic, and electric dichroism studies of the complex of daunomycin with H1-depleted 175 base pair nucleosomes, along with some comparative data for ethidium. In contrast to ethidium, daunomycin binding to nucleosomes is strongly reduced relative to the affinity for free DNA. The salt concentration dependence of the binding constant indicates that approximately one Na+ ion is released from both nucleosomes and free DNA upon daunomycin binding. The early melting transition of nucleosomes is preferentially stabilized by low levels of both drugs, but more markedly by ethidium. Ethidium also stabilizes the second nucleosome melting transition, but daunomycin barely does so. Dichroism and rotational relaxation time measurements indicate that daunomycin unfolds nucleosomes in a manner analogous to the influence of ethidium, although about twice as much daunomycin as ethidium is required complete the unfolding process. The data support an unfold structure in which the nucleosome elongates along the DNA superhelical axis. Levels of daunomycin greater than about 0.15 per DNA base pair promote nucleosome aggregation. To relate our results to the activity of daunomycin as an antitumor agent, we propose that the drug, because of its special intercalation geometry, strongly prefers free DNA regions over the bent helices found in nucleosomes and chromatin. The result of this preference should be an increased local concentration of the drug in the genetically active regions of nuclear DNA in which nucleosomal structure is less prevalent. Presumably the abundance of such regions in tumor cells makes them especially sensitive to daunomycin.

Animals↗
PubMed1980

Unfolding of nucleosomes by ethidium binding.

We report spectroscopic, hydrodynamic, and biochemical studies on the complex of ethidium bromide with 140 base pair nucleosomal core particles. Fluorescence titration indicates a greater intrinsic affinity of ethidium for nucleosomes than for DNA, and fluorescence depolarization measurements imply increased immobilization of ethidium bound to nucleosomes, but with more extensive dye-dye energy transfer compared to DNA-bound dye. Ethidium intercalated into DNA in nucleosomes has a limiting reduced linear dichroism of -0.45 at 320 nm and -0.25 at 530 nm. Both the energy transfer and dichroism results are consistent with clustering of the nucleosome-bound dye molecules. Electric dichroism measurements and ultracentrifugation studies reveal that structural distortion of the nucleosome accompanies ethidium binding, occurring in the range of r (ethidium residues per base pair) values from 0.02 to 0.06. The distortion transition is characterized by an increase in the negative limiting reduced dichroism from 0.29 to 0.45 at 265 nm, an increase in the field-induced viscosity-limited rotational orientation time from 0.8 to 3 mus, and a decrease in sedimentation coefficient from 10.5 to 8.2 S. The complex was modeled hydrodynamically as a cylinder of 335-A length and 67-A diameter, containing 1.4 superhelical turns of DNA. Dimethylsuberimidate cross-linked nucleosomes, or native nucleosomes in the presence of Mg2+, bind ethidium weakly and are not distorted. The periodicity of cutting sites produced by DNase II digestion of nucleosomes remains constant as ethidium is added, but the bandwidth increases. A thermodynamic model is proposed to interpret the binding isotherm, based on enhancement of drug binding affinity due to release of superhelical stress in the nucleosome-ethidium complex.

Base Composition↗
PubMed2002

Interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments containing isolated ligand binding sites.

We have examined the interaction of Hoechst 33258 and echinomycin with nucleosomal DNA fragments which contain isolated ligand binding sites. A 145 base pair fragment was prepared on the basis of the sequence of tyrT DNA, which contained no CpG or (A/T)(4) binding sites for these ligands. Isolated binding sites were introduced into this fragment at discrete locations where the minor groove is known to face toward or away from the protein core when reconstituted onto nucleosome core particles. The interaction of ligands with target sites on these nucleosomal DNA fragments was assessed by DNase I footprinting. We find that Hoechst 33258 can bind to single nucleosomal sites which face both toward and away from the protein core, without affecting the nucleosome structure. Hoechst binding is also observed on nucleosomal fragments which contain two or more drug binding sites, though in these cases the footprints are accompanied by the presence of new cleavage products in positions which suggest that the ligand has caused a proportion of the DNA molecules to adopt a new rotational positioning on the protein surface. Hoechst 33258 does not affect nucleosome reconstitution with any of these fragments. In contrast, the bifunctional intercalating antibiotic echinomycin is not able to bind to single nucleosomal CpG sites. Echinomycin footprints are observed on nucleosomal fragments containing two or more CpG sites, but there are no changes in the cleavage patterns in the remainder of the fragment. Echinomycin abolishes nucleosome reconstitution when included in the reconstitution mixture.

Base Sequence↗
PubMed1999

Evidence for nonrandom behavior in 208-12 subsaturated nucleosomal array populations analyzed by AFM.

Atomic force microscopy was used to determine the population distributions in reconstituted, subsaturated 208-12 nucleosomal arrays. The features found in these distributions vary with the average nucleosome loading per template molecule (n(av)): at n(av) < 4, the distributions show a single peak whose breadth is equal to that expected for a random loading process; at n(av) = 4-8, the distributions are broader than random distributions and are complex; i.e., they contain multiple peaks and/or shoulders. Moreover, the peaks/shoulders typically occur at two nucleosome intervals, i.e., 2, 4, 6 or 3, 5, 7 nucleosomes. This two-nucleosome periodicity is statistically significant. The precise cause for such discrete features within the distributions is unknown, but at least these features would seem to indicate some pairwise preference in nucleosome occupation at these loading levels. In these intermediate-level (n(av) = 4-8) distributions, the major peak contains a larger fraction of the total templates than a random nucleosome loading process would produce. This feature indicates that at these intermediate population levels there is some tendency for correlated nucleosome loading among the templates. Hyperacetylated nucleosomal arrays show only subtle differences in their population distributions compared to nonacetylated arrays and demonstrate the above features. AFM allows one to study unfixed chromatin arrays; we find that nucleosomes on the 208-12 template demonstrate significant lability when they are not glutaraldehyde-fixed.

Acetylation↗
PubMed2000

DNA sequence-dependent contributions of core histone tails to nucleosome stability: differential effects of acetylation and proteolytic tail removal.

Modulation of nucleosome stability in chromatin plays an important role in eukaryotic gene expression. The core histone N-terminal tail domains are believed to modulate the stability of wrapping nucleosomal DNA and the stability of the chromatin filament. We analyzed the contribution of the tail domains to the stability of nucleosomes containing selected DNA sequences that are intrinsically straight, curved, flexible, or inflexible. We find that the presence of the histone tail domains stabilizes nucleosomes containing DNA sequences that are intrinsically straight or curved. However, the tails do not significantly contribute to the free energy of nucleosome formation with flexible DNA. Interestingly, hyperacetylation of the core histone tail domains does not recapitulate the effect of tail removal by limited proteolysis with regard to nucleosome stability. We find that acetylation of the tails has the same minor effect on nucleosome stability for all the selected DNA sequences. A comparison of histone partitioning between long donor chromatin, acceptor DNA, and free histones in solution shows that the core histone tails mediate internucleosomal interactions within an H1-depleted chromatin fiber amounting to an average free energy of about 1 kcal/mol. Thus, such interactions would be significant with regard to the free energies of sequence-dependent nucleosome positioning. Last, we analyzed the contribution of the H2A/H2B dimers to nucleosome stability. We find that the intact nucleosome is stabilized by 900 cal/mol by the presence of the dimers regardless of sequence. The biological implications of these observations are discussed.

Acetylation↗
PubMed1999

Histone-DNA contacts in structure/function relationships of nucleosomes as revealed by crosslinking.

We describe studies of histone-DNA contacts in the nucleosome using the method of covalent zero length protein-DNA crosslinking. These studies show that in intact nuclei isolated from different sources the linear sequential arrangement of histone-DNA contacts in the nucleosomal core is essentially the same. However, the relative strength of certain contacts varies and correlates with the level of chromatin activity and condensation. These altered contacts are located in the sharply bent regions of the nucleosomal DNA and are supposed to be sensitive to the structural changes that may occur during nucleosome functions. Studies of the mechanism of these alterations revealed that the difference in strength of these contacts is attributed to the different conformational state of the nucleosomal core and is caused by stretching of the nucleosomal DNA upon chromatin decondensation during its activation. Histone-terminal domains may be involved in this process through posttranslational modifications affecting chromatin condensation. The described localization of the histone H2A C-terminal domain in the nucleosome by crosslinking demonstrates the ability of this methodology to determine the location of histone-terminal domains and thereby elucidate their role in nucleosome function. Results of the described experiments suggest that chromatin decondensation may alter the nucleosomal DNA conformation and affect the histone-DNA contacts resulting in a structural transition that may play a role in rendering the nucleosome competent for transcription and/or replication.

Animals↗
PubMed2004

Sin mutations alter inherent nucleosome mobility.

Previous studies have identified sin mutations that alleviate the requirement for the yeast SWI/SNF chromatin remodelling complex, which include point changes in the yeast genes encoding core histones. Here we characterise the biochemical properties of nucleosomes bearing these mutations. We find that sin mutant nucleosomes have a high inherent thermal mobility. As the SWI/SNF complex can alter nucleosome positioning, the higher mobility of sin mutant nucleosomes provides a means by which sin mutations may substitute for SWI/SNF function. The location of sin mutations also provides a new opportunity for insights into the mechanism for nucleosome mobilisation. We find that both mutations altering histone DNA contacts at the nucleosome dyad and mutations in the dimer-tetramer interface influence nucleosome mobility. Furthermore, incorporation of H2A.Z into nucleosomes, which also alters dimer-tetramer interactions, affects nucleosome mobility. Thus, variation of histone sequence or subtype provides a means by which eukaryotes may regulate access to chromatin through alterations to nucleosome mobility.

Calcium Chloride↗
PubMed1996

The affinity of nuclear factor 1 for its DNA site is drastically reduced by nucleosome organization irrespective of its rotational or translational position.

A DNA-bending sequence has been used for in vitro reconstitution of nucleosomes in order to direct a nuclear factor 1 (NF-1) binding site into different nucleosome positions. By this strategy nucleosomes were obtained that had one of two rotational positions of the NF-1 binding site, one oriented toward the periphery and the other toward the histone octamer, translationally positioned 50 and 45 base pairs, respectively, from the nucleosome dyad. The affinity of partially purified NF-1 for these nucleosomal targets was compared with its affinity for free DNA by dimethylsulfate methylation protection and DNase I footprinting assays. The binding affinity of NF-1 to all nucleosomal targets was reduced 100-300-fold compared with its affinity for free DNA. The two rotational settings of the NF-1 site showed the same binding affinity for NF-1 as did other nucleosome constructs in which the NF-1 binding site was translationally positioned from 10 to 40 base pairs from the nucleosome dyad. We conclude that the nucleosomal inhibition of NF-1 binding is an inherent characteristic of NF-1 since another transcription factor, the glucocorticoid receptor, is able to bind to its DNA site in a nucleosome.

Base Sequence↗