Archaeal nucleosomes

Archaea contain histones that have primary sequences in common with eukaryal nucleosome core histones and a three-dimensional structure that is essentially only the histone fold. Here we report the results of experiments that document that archaeal histones compact DNA in vivo into structures similar to the structure formed by the histone (H3+H4)2 tetramer at the center of the eukaryal nucleosome. After formaldehyde cross-linking in vivo, these archaeal nucleosomes have been isolated from Methanobacterium thermoautotrophicum and Methanothermus fervidus, visualized by electron microscopy on plasmid and genomic DNAs, and shown by immunogold labeling, SDS/PAGE, and immunoblotting to contain archaeal histones, cross-linked into tetramers. Archaeal nucleosomes protect approximately 60 bp of DNA and multiples of approximately 60 bp from micrococcal nuclease digestion, and immunoprecipitation has demonstrated that most, but not all, M. fervidus genomic DNA sequences are associated in vivo with archaeal histones.     

Histone stoichiometry and DNA circularization in archaeal nucleosomes

Recombinant (r)HMfB (archaealhistone B fromMethanothermusfervidus) formed complexes with increasing stability with DNA molecules increasing in length from 52 to 100 bp, but not with a 39 bp molecule. By using125I-labeled rHMfB-YY (an rHMfB variant with I31Y and M35Y replacements) and32P-labeled 100 bp DNA, these complexes, designated archaeal nucleosomes, have been shown to contain an archaeal histone tetramer. Consistent with DNA bending and wrapping, addition of DNA ligase to archaeal nucleosomes assembled with 88 and 128 bp DNAs resulted in covalently-closed monomeric circular DNAs which, following histone removal, were positively supercoiled based on their electrophoretic mobilities in the presence of ethidium bromide before and after relaxation by calf thymus topoisomerase I. Ligase addition to mixtures of rHMfB with 53 or 30 bp DNA molecules also resulted in circular DNAs but these were circular dimers and trimers. These short DNA molecules apparently had to be ligated into longer linear multimers for assembly into archaeal nucleosomes and ligation into circles. rHMfB assembled into archaeal nucleosomes at lower histone to DNA ratios with the supercoiled, circular ligation product than with the original 88 bp linear version of this molecule. Archaeal histones are most similar to the globular histone fold region of eukaryal histone H4, and the results reported are consistent with archaeal nucleosomes resembling the structure formed by eukaryal histone (H3+H4)2tetramers.     

Histones associated with non-nucleosomal rat ribosomal genes are acetylated while those bound to nucleosome-organized gene copies are not

Acetylation of histones bound to rat rRNA genes has been studied relative to their organization in chromatin, either as canonical nucleosomes, containing the inactive copies, or as anucleosomal nonrepeating structures, corresponding to the transcribed genes (Conconi, A., Widmer, R. M., Koller, T., and Sogo, J. M. (1989) Cell 57, 753-761). Nuclei from butyrate-treated rat tumor cells were irradiated with a UV laser to cross-link proteins to DNA, and the purified covalent complexes were immunofractionated by an antibody that specifically recognized the acetylated histones. Upon probing with sequences coding for mature rat 28 S RNA, DNA of the antibody-bound complexes was 5-20-fold enriched relative to the total rat DNA. Since the laser cross-links histones to DNA in both active and inactive genes, one cannot distinguish which one of them, or both, are bound to acetylated histones. Alternatively, purified mononucleosomes were immunofractionated, but DNA from the antibody-bound monosomes was not enriched in coding rDNA. Taken together, these results suggest that nucleosome-organized rRNA genes are bound to nonmodified histones and that the acetylated histones are associated with the active, anucleosomal gene copies.     

Circular dichroism as a probe of DNA structure inside reconstituted nucleohistones

Reconstituted nucleohistones were obtained by mixing in given conditions acid extracted histones and eukaryotic DNA. The histone/DNA ratio (w/w) was in the range 0.35 - 0.95. With the four histones (H2A2B) we have been able to obtain subunits (nucleosomes or upsilon-bodies). The variation of cirsular dichroism signal with temperature at 280 nm was measured to follow structural changes of the DNA inside the complex. The true change of ellipticity (see article) of histone-bound DNA regions, is similar for reconstituted nucleohistone and H1-depleted chromatin, and is therefore a physical probe of the presence of nucleosomes.     

Nucleosome assembly on CTG triplet repeats

Expansion of CTG repeat sequences is associated with several human genetic diseases. We have examined the consequences of CTG repeat expansion for nucleosome assembly and positioning. Short CTG repeats are found within the most favored DNA sequences yet defined for nucleosome assembly. We find that as few as six CTG repeats will facilitate nucleosome assembly to a similar extent as the 50 or more repeats found in disease genes. Thus an increase in nucleosome stability on expansion of existing triplet repeats is unlikely to explain the acquisition of the disease phenotype. However, the CTG repeat sequence is efficiently wrapped around the histone octamer, preferring to associate with histones at the nucleosomal dyad. Thus short segments CTG repeat sequence will facilitate the assembly of a stable positioned nucleosome which might contribute to the expansion phenomenon and the functional organization of chromatin.     

Value of laparoscopy in ascites of undetermined origin

It is generally assumed that anti-dsDNA antibodies play an important role in the pathogenesis of lupus nephritis. This is mainly based on the facts that an increase in anti-dsDNA titer often precedes onset of renal disease, that immune deposits are present in glomeruli and that eluates of glomeruli are enriched for anti-dsDNA. This led to the classical concept that deposition of DNA-anti-DNA complexes incites the glomerular inflammation. However, important pieces of evidence are lacking to support this hypothesis. Free, naked, DNA is not present in the circulation. The existence of DNA/anti-DNA complexes is highly questionable and injection of these complexes hardly leads to glomerular localization. As an alternative concept cross-reactivity of anti-dsDNA with glomerular constituents like heparan sulfate (HS) and laminin has been proposed. However, subsequent research has indicated that this cross-reactivity is due to nucleosomal antigens (histones and DNA) complexed to the auto-antibodies. The cationic histone part of the complex is responsible for the binding to the anionic HS. This binding also occurs in vivo since renal perfusion of nucleosome complexed antibodies leads to abundant binding of auto-antibodies to the GBM, while enzymatic removal of HS from the GBM, decreases this binding considerably. Non-complexed antibodies did not bind at all. This mechanism of binding is also consistent with the decrease of HS staining in the GBM in human and murine lupus due to masking of HS with nucleosome-complexed auto-antibodies. Furthermore the presence of histones and nucleosomes in glomerular deposits in lupus nephritis was recently shown. Elution of auto-antibodies from glomeruli not only showed anti-dsDNA but also anti-nucleosome specificities. Nucleosomes are not only important for the induction of glomerular lesions, but there is now also increasing evidence that the nucleosome is the auto-antigen that drives the auto-immune response in SLE. There is ample evidence that this response is antigen-driven and T cell dependent. However immunization with DNA in general fails to induce pathogenic anti-dsDNA antibodies. Recently, in SLE T helper cells were identified specific for nucleosomes. These nucleosome specific T helper cells were not only able to induce anti-nucleosome antibodies but also anti-dsDNA and anti-histone antibodies. This is in line with the finding that in SLE nucleosome specific antibodies are formed. These antibodies react exclusively with nucleosomes and not with its constituents DNA or histones. The formation of these nucleosome specific antibodies precedes the development of anti-dsDNA or anti-histone suggesting that the loss of tolerance for nucleosomes is a primary event. The systemic release of nucleosomes is due to an aberrant apoptosis. There is now growing evidence that apoptosis is disturbed both in certain murine lupus models as well as in human lupus. In conclusion, nucleosomes seem to play a central role in the induction and the effector phase of SLE.     

In vitro reconstitution of Artemia satellite chromatin

We report the characterization of an in vitro chromatin assembly system derived from Artemia embryos and its application to the study of AluI-113 satellite DNA organization in nucleosomes. The system efficiently reconstitutes chromatin templates by associating DNA, core histones, and H1. The polynucleosomal complexes show physiological spacing of repeat length 190 +/- 5 base pairs, and the internucleosomal distances are modulated by energy-using activities that contribute to the dynamics of chromatin conformation. The assembly extract was used to reconstitute tandemly repeated AluI-113 sequences. The establishment of preferred histone octamer/satellite DNA interactions was observed. In vitro, AluI-113 elements dictated the same nucleosome translational localizations as found in vivo. Specific rotational constraints seem to be the central structural requirement for nucleosome association. Satellite dinucleosomes showed decreased translational mobility compared with mononucleosomes. This could be the consequence of interactions between rotationally positioned nucleosomes separated by linker DNA of uniform length. AluI-113 DNA led to weak cooperativity of nucleosome association in the proximal flanking regions, which decreased with distance. Moreover, the structural properties of satellite chromatin can spread, thus leading to a specific organization of adjacent nucleosomes.     

In vitro binding of H1 histone subtypes to nucleosomal organized mouse mammary tumor virus long terminal repeat promotor

The binding of all known linker histones, named H1a through H1e, including H1(0) and H1t, to a model chromatin complex based on a DNA fragment containing the mouse mammary tumor virus long terminal repeat promotor was systematically studied. As for the histone subtype H1b, we found a dissociation constant of 8-16 nM to a single mononucleosome (210 base pairs), whereas the binding constant of all other subtypes varied between 2 and 4 nM. Most of the H1 histones, namely H1a, H1c, H1d/e, and H1(0), completely aggregate polynucleosomes (1.3 kilobase pairs, 6 nucleosomes) at 270-360 nM, corresponding to a molar ratio of six to eight H1 molecules per reconstituted nucleosome. To form aggregates with the histones H1t and H1b, however, greater amounts of protein were required. Furthermore, our results show that specific types of in vivo phosphorylation of the linker histone tails influence both the binding to mononucleosomes and the aggregation of polynucleosomes. S phase-specific phosphorylation with one to three phosphate groups at specific sites in the C terminus influences neither the binding to a mononucleosome nor the aggregation of polynucleosomes. In contrast, highly phosphorylated H1 histones with four to five phosphate groups in the C and N termini reveal a very high binding affinity to a mononucleosome but a low chromatin aggregation capability. These findings suggest that specific S phase or mitotic phosphorylation sites act independently and have distinct functional roles.     

Small slipped register genetic instabilities in Escherichia coli in triplet repeat sequences associated with hereditary neurological diseases

Genetic instability investigations on three triplet repeat sequences (TRS) involved in human hereditary neurological diseases (CTG.CAG, CGG.CCG, and GAA.TTC) revealed a high frequency of small expansions or deletions in 3-base pair registers in Escherichia coli. The presence of G to A polymorphisms in the CTG.CAG sequences served as reporters for the size and location of these instabilities. For the other two repeat sequences, length determinations confirmed the conclusions found for CTG.CAG. These studies were conducted in strains deficient in methyl-directed mismatch repair or nucleotide excision repair in order to investigate the involvement of these postreplicative processes in the genetic instabilities of these TRS. The observation that small and large instabilities for (CTG.CAG)175 fall into distinct size classes (1-8 repeats and approximate multiples of 41 repeats, respectively) leads to the conclusion that more than one DNA instability process is involved. The slippage of the complementary strands of the TRS is probably responsible for the small deletions and expansions in methyl-directed mismatch repair-deficient and nucleotide excision repair-deficient cells. A model is proposed to explain the observed instabilities via strand misalignment, incision, or excision, followed by DNA synthesis and ligation. This slippage-repair mechanism may be responsible for the small expansions in type 1 hereditary neurological diseases involving polyglutamine expansions. Furthermore, these observations may relate to the high frequency of small deletions versus a lower frequency of large instabilities observed in lymphoblastoid cells from myotonic dystrophy patients.     

Chromatin assembly in a yeast whole-cell extract

A simple in vitro system that supports chromatin assembly was developed for Saccharomyces cerevisiae. The assembly reaction is ATP-dependent, uses soluble histones and assembly factors, and generates physiologically spaced nucleosomes. We analyze the pathway of histone recruitment into nucleosomes, using this system in combination with genetic methods for the manipulation of yeast. This analysis supports the model of sequential recruitment of H3/H4 tetramers and H2A/H2B dimers into nucleosomes. Using a similar approach, we show that DNA ligase I can play an important role in template repair during assembly. These studies demonstrate the utility of this system for the combined biochemical and genetic analysis of chromatin assembly in yeast.     

Binding of actinomycin D to DNA oligomers of CXG trinucleotide repeats

Actinomycin D (ACTD) binding propensities of DNA with CXG trinucleotide repeats were investigated using oligomers of the form d[AT(CXG)n = 2-4AT] and their corresponding heteroduplexes, where X = A, C, G, or T. These oligonucleotides contain -CXGCXG-, -CXGCXGCXG-, and -CXGCXGCXGCXG- units that can form homoduplexes containing one, two, and three GpC binding sites, respectively, with flanking X/X mismatches. The corresponding heteroduplexes contain these same sites with flanking Watson-Crick base pairs. It was found that oligomers with X = G exhibit weak ACTD affinities whereas those with X not equal to G and n = 3 exhibit unusually strong ACTD binding affinities with binding constants ranging from 2.3 x 10(7) to 3.3 x 10(7) M-1 and binding densities of approximately 1 drug molecule/strand (or 2/duplex). These binding affinities are considerably higher than those of their shorter and longer counterparts and are about 2- and 10-fold stronger than the corresponding CAG.CTG and CGG.CCG heteroduplexes, respectively. The CTG-containing oligomer d[AT(CTG)3AT] stands out as unique in having its ACTD dissociation kinetics being dominated by a strikingly slow process with a characteristic time of 205 min at 20 degrees C, which is 100-fold slower than d[AT(CAG)3AT], nearly 10-fold slower than the corresponding heteroduplex, and considerably slower than d[AT(CTG)2AT] (63 min) and d[AT(CTG)4AT] (16 min). The faster dissociation rate of the n = 4 oligomer compared to its n = 2 counterpart is in apparent contrast with the observed 10-fold stronger ACTD binding affinity of the former. It was also found that d[AT(CCG)3AT] exhibits the slowest dissociation rate of the CGG/CCG series, being more than an order of magnitude slower than that of its heteroduplex (tau slow of 43 vs 2 min). The finding that a homoduplex d[AT-CXG-CXG-CXG-AT]2 can bind two ACTD molecules tightly is significant since it was thought unlikely for two consecutive GpC sites separated by a single T/T mismatch to do so.