DNA-histone Complexes Research Articles

Boswellic acids trigger apoptosis via a pathway dependent on caspase-8 activation but independent on Fas/Fas ligand interaction in colon cancer HT-29 cells.

Boswellic acids are the effective components of gum resin of Boswellia serrata, which has anti-inflammatory properties. Recent studies on brain tumors and leukemic cells indicate that boswellic acids may have antiproliferative and apoptotic effects with the mechanisms being not studied in detail. We studied their antiproliferative and apoptotic effects on colon cancer cells and the pathway leading to apoptosis. HT-29 cells were treated with beta-boswellic acid (BA), keto-beta-boswellic acid (K-BA) and acetyl-keto-beta-boswellic acid (AK-BA), respectively. Apoptosis was determined by flow cytometry, by cytoplasmic DNA-histone complex and the activity of caspase-3. The cleavage of poly-(ADP-ribose)-polymerase (PARP) and expression of Fas were examined by western blot. Specific caspase inhibitors, polyclonal Fas antibody, and antagonistic Fas antibody ZB4 were employed to elucidate apoptotic pathways. DNA synthesis and cell viability were examined. Both K-BA and AK-BA increased cytoplasmic DNA-histone complex dose-dependently and increased pre-G(1) peak in flow cytometer analysis, with the effects of AK-BA being stronger than K-BA. BA only increased the formation of DNA-histone complex at a high concentration. K-BA and AK-BA increased caspase-8, caspase-9 and caspase-3 activities accompanied by cleavage of PARP. The effects of AK-BA on formation of cytoplasmic DNA histone and on caspase-3 activation were 3.7- and 3.4-fold, respectively, more effective than those induced by camptothecin. The apoptosis induced by AK-BA was inhibited completely by caspase-3 or caspase-8 inhibitor and partially by caspase-9 inhibitor. ZB4 blocked exogenous Fas ligand-induced apoptosis, but had no effect on AK-BA-induced apoptosis. AK-BA had no significant effect on expression of Fas. Apart from apoptotic effect, these acids also inhibited [(3)H]thymidine incorporation and cell viability to different extent. In conclusion, boswellic acids, particularly AK-BA and K-BA have antiproliferative and apoptotic effects in human HT-29 cells. The apoptotic effect is mediated via a pathway dependent on caspase-8 activation but independent of Fas/FasL interaction.

Differential effect of neonatal thymectomy on systemic and organ-specific autoimmune disease.

Thymectomy on day 3 after birth (d3tx) depletes CD4+CD25+ regulatory T cells leading to multiple independent organ-specific autoimmune diseases. However, systemic autoimmune disease such as systemic lupus erythematosus has not been reported in d3tx mice. Herein, we investigate the effect of d3tx on spontaneous autoantibody response and immune complex glomerulonephritis (GN) in the lupus-prone (SWR x NZB)F1 (SNF1) mice. The d3tx SNF1 mice developed accelerated antibody responses to double-stranded DNA and DNA-histone complexes, and an increased frequency of activated CD4+ T cells. Unexpectedly, the renal histopathology and mortality from GN were significantly ameliorated in d3tx SNF1 mice, which concomitantly exhibited a Th2-biased antibody response. By 16 weeks, the d3tx mice had higher levels of total and autoantigen-specific IgG1, and at 12 months, the autoantigen-specific IgG2a was significantly below that of the sham thymectomized mice. These differences corresponded with reduction in total and autoantigen-specific IgG2a in the renal eluates of the d3tx mice. In addition, while all the mice had immune complex deposition in the mesangium and peripheral capillary loops of the glomeruli, IgG2a and C3 deposits restricted to the mesangial regions alone were more frequent in d3tx mice. D3tx SNF1 mice, protected from lupus-like GN, developed organ-specific autoimmune responses and diseases including prostatitis, orchitis and oophoritis, and antibodies to prostate, cardiac and skeletal muscle antigens. Therefore, d3tx paradoxically protects SNF1 mice from genetically prone lupus-like GN, yet promotes de novo organ-specific autoimmunity.

Archaeal Histone Tetramerization Determines DNA Affinity and the Direction of DNA Supercoiling

DNA binding and the topology of DNA have been determined in complexes formed by >20 archaeal histone variants and archaeal histone dimer fusions with residue replacements at sites responsible for histone fold dimer:dimer interactions. Almost all of these variants have decreased affinity for DNA. They have also lost the flexibility of the wild type archaeal histones to wrap DNA into a negative or positive supercoil depending on the salt environment; they wrap DNA into positive supercoils under all salt conditions. The histone folds of the archaeal histones, HMfA and HMfB, from Methanothermus fervidus are almost identical, but (HMfA)(2) and (HMfB)(2) homodimers assemble into tetramers with sequence-dependent differences in DNA affinity. By construction and mutagenesis of HMfA+HMfB and HMfB+HMfA histone dimer fusions, the structure formed at the histone dimer:dimer interface within an archaeal histone tetramer has been shown to determine this difference in DNA affinity. Therefore, by regulating the assembly of different archaeal histone dimers into tetramers that have different sequence affinities, the assembly of archaeal histone-DNA complexes could be localized and used to regulate gene expression.

Tapping and contact mode imaging of native chromosomes and extraction of genomic DNA using AFM tips

It is very important both in medicine and biology to clarify the chromosomal structure to understand its functions. In a standard cytogenetic procedure, chromosomes are often fixed in a mixture of acetic acid and methanol. This process most likely changes the mechanical property of chromosomes. We adopted a method to prepare native and unfixed chromosomes from mouse 3T3 cells and used tapping and contact mode atomic force microscopy (AFM) to image and manipulate them. Modified AFM tips were used to image chromosomes in contact mode in air, and then the chromosome samples were immobilized on a substrate and placed in a buffer solution to pull out DNA–histone complexes from them after they were optimally treated with trypsin. From the AFM images, we could see several bands and granular structures on chromosomes. We obtained force curves indicating long fiber extensions from native chromosomes both with low (in high concentration of NaCl) and high forces (physiological conditions). The result suggested that the degree of chromosome condensation decreased in high concentration of salt. It agrees with the known fact of histone H1 dissociation in a high concentration of salt. We intend to pull out DNA–histone complexes from chromosomes for later molecular operations on them using an AFM.

Bcl-xL expression after contusion to the rat spinal cord.

After contusion-derived spinal cord injury, (SCI) there is localized tissue disruption and energy failure that results in early necrosis and delayed apoptosis, events that contribute to chronic central pain in a majority of patients. We assessed the extent of contusion-induced apoptosis of neurons in a known central pain-signaling pathway, the spinothalamic tract (STT), which may be a contributor to SCI-induced pain. We observed the loss of STT cells and localized increase of DNA fragmentation and cytoplasmic histone-DNA complexes, which suggested potential apoptotic changes among STT neurons after SCI. We also showed SCI-associated changes in the expression of the antiapoptotic protein Bcl-xL, especially among STT cells, consistent with the hypothesis that Bcl-xL regulates the extent of apoptosis after SCI. Apoptosis in the injured spinal cord correlated well with prompt decreases in Bcl-xL protein levels and Bcl-xL/Bax protein ratios at the contusion site. We interpret these results as evidence that regulation of Bcl-xL may play a role in neural sparing after spinal injury and pain-signaling function.

Visualization of reconstituted solenoid chromatin structure by tapping mode atomic force microscopy

AbstractIn this paper, tapping mode atomic force microscopy was applied to visualize the higher order chromatin structure reconstituted from calf thymus DNA and calf thymus whole histone in vitro. All the revealed reconstituted chromatin structures consisted of similar well‐defined subunits. In some of the chromatin structures, four to six subunits per turn were wrapped right‐handed to form a flattened solenoid with a height distribution of 12 ± 2 nm; and the other Y‐ and V‐shaped chromatin structures were formed by aggregation of these solenoids, which presented similar heights as the solenoids. Typical atomic force microscopy (AFM) images revealed relatively regular solenoid structure (containing six nucleosomes per turn) with a ratio of diameter to pitch of 3 : 1, which is in good agreement with that of 30 : 11 in the chromatin solenoid model proposed by Finch and Klug. Bio‐gel elution was used to separate reconstituted chromatin from uncombined histone particles, which proved to be helpful for the AFM imaging of the higher order chromatin structure with nearly no disturbance of histone particles and salt in the background. The AFM observations showed that the higher order chromatin structure was preserved well enough during the bio‐gel elution process. The cause for the stability of higher order chromatin structure during the bio‐gel elution was analysed. Electron microscopy analysis and circular dichroism spectroscopy investigation of the DNA–histone complexes indicated the formation of ordered aggregates. Copyright © 2001 John Wiley & Sons, Ltd.

Comparative analysis of anti-histone and anti-chromatin antibody specificity in lupus erythematosus cell-positive and -negative sera and their relation to disease activity

Anti-histone and anti-chromatin antibody responses play a central role in the autoimmune response of systemic lupus erythematosus (SLE). Furthermore, anti-histone H1 antibodies are essential for the formation of the lupus erythematosus cell (LEC) phenomenon. In this study, the binding properties of LEC+ and LEC- SLE sera to chromatin-associated nuclear antigens (histones H1, H2A, H2B, H3, H4; complexes of H2A-H2B, [H2A-H2B]-DNA, H1-DNA; total and H1-stripped chromatin; native and denatured DNA) were investigated. In addition, sera from patients with drug-induced lupus (by procainamide, hydralazine, or quinidine), as well as from patients with rheumatoid arthritis and osteoarthritis, were assessed. Enzyme-linked immunosorbent assay was used to detect specific antibody binding. Mirroring the important role of histone H1 in the formation of LE cells, anti-histone H1 reactivity was 8-fold higher in LEC+ sera than in LEC- sera. In addition, reactivities to most of the other antigens tested, i.e., other histones and histone-DNA complexes as well as chromatin and DNA, were significantly higher in LEC+ sera than in LEC- sera. All but 1 serum sample from the patients with drug-induced lupus were negative for LE cell formation as well as for anti-histone H1 reactivity, but displayed high antibody reactivities to histone-DNA complexes, including chromatin. Sera from patients with rheumatoid arthritis and osteoarthritis did not show significant binding to these antigens. When comparing the clinical features of LEC+ and LEC-SLE patients, severe organ involvement, including nephritis and central nervous system involvement, was common in the LEC+ group, but rare in the LEC- group. A positive LE cell phenomenon not only correlated with the presence of high anti-histone H1 antibody levels in SLE, but also indicated serologically and clinically active disease with major organ involvement.

Transcription Through Chromatin

The eukaryotic genome is organized in DNA–histone complexes called chromatin. The nucleosome core (∼150 bp DNA tightly wrapped around the histone octamer in two superhelical coils) is a minimal unit of chromatin organization. Chromatin is designed to allow both tight DNA packaging and proper functioning and regulation of enzymes such as DNA and RNA polymerases. The mechanisms making DNA accessible for enzymes of DNA metabolism in a regulated manner are beginning to emerge. Thus the histone octamer has a remarkable ability to survive transcription by stepping around the transcribing polymerase without ever going free in solution. Moreover, nucleosomes can present a serious barrier for transcribing polymerases that dramatically decreases the rate of transcription and is probably regulated in vivo. The mechanism of transcription through the nucleosomal barrier is discussed in detail.

Salt-Induced DNA-Histone Complexation

We study numerically the binding of one semiflexible charged polymer onto an oppositely charged sphere. Using parameters appropriate for DNA-histone complexes, we find complete wrapping for intermediate salt concentrations only, in agreement with experiments. For high salt concentrations, a strongly discontinuous dewrapping occurs. For low salt concentrations, we find multiple conformational transitions, leading to an extended DNA configuration. The wrapped states are characterized by spontaneously broken rotational and mirror symmetries, giving rise to four distinct structures.

PARTICIPATION OF DNA STRUCTURE ON SPERM CHROMATIN ORGANIZATION

The in vitro interaction between purified bovine liver and sperm DNA with somatic histones, to form nucleosomes, and with bovine and salmon protamines were studied. DNAse or microccocal nuclease digestion of liver DNA-histone reassociated chromatin produced the expected polynucleosome type of fragments. Electrophoretic patterns of digested sperm-DNA nucleosomes were different. Micrococcal nuclease digestion produced mainly fragments smaller than 100 bp and some nucleosome-type particles. Under DNAse activity most of the products were smaller than 100 bp, indicating an increased susceptibility of the sperm DNA-histone complexes to the hydrolytic activity of both nucleases, particularly toward DNAse I. This differential susceptibility was confirmed by sucrose gradient spectrophotometric analysis. Acridine orange (AO) staining of histone-DNA reassociated nucleosomes showed significant differences in fluorescence intensity, sperm DNA-histone complexes being almost twice as fluorescent as liver DNA-histone complexes. On the contrary, liver DNA/protamine complexes stained with AO were consistently more fluorescent than sperm DNA-protamine complexes. Finally, no differences in either fluorescence intensity or spectra were observed when liver and sperm DNA were stained with AO after interaction with salmon protamines. The data suggest that sperm DNA has important structural characteristics that differentiates it from somatic DNA. These differences seem to be species specific and must surely play an important role on the determination of the dramatic sequence of that participates sperm chromatin organization.

Kinetics of hallmark biochemical changes in paclitaxel-induced apoptosis.

Apoptosis is associated with cascades of biochemical changes, including caspase activation, cleavage of poly-ADP-ribose polymerase (PARP), and fragmentation of genomic DNA. Knowledge of the kinetics of these changes in drug-induced apoptosis is important for designing pharmacodynamic studies. We have shown that the slow manifestation of apoptosis contributes to the delayed pharmacological effects of paclitaxel (Cancer Res. 58:2141-2148, 1998). The present study examined the timing of the biochemical changes in paclitaxel-induced apoptosis in human prostate PC3 cancer cells. After treatment with 20 nM paclitaxel, the fraction of cells that detached from the culture flask increased with time to reach 68% at the end of the 96-hour experiment. In contrast, the control samples showed <1% detachment. The attached and detached paclitaxel-treated cells showed different biochemical properties. The detached cells exhibited the full spectrum of apoptotic changes, whereas the attached cells only showed activation of caspase-3-like proteases but not PARP cleavage, DNA fragmentation, nor release of DNA fragments to the cytoplasm. Activation of caspases in the attached cells was several-fold lower and occurred at a later time (ie, 24 vs 12 hours) compared to the detached cells. In the detached cells, caspase activation was first detected at 12 hours and peaked at 36 hours, whereas PARP cleavage was first detected at 24 hours and was completed prior to 72 hours. In contrast, the extent of internucleosomal DNA fragmentation and the release of DNA-histone complex to the cytoplasm (both were first detected at 24 hours) were cumulative over time up to the last time point of 96 hours. In summary, in paclitaxel-induced apoptosis, caspase activation was followed with a 12-hour lag time by PARP cleavage, internucleosomal DNA fragmentation, and release of DNA-histone complex to the cytoplasm. There was no detectable lag time between PARP cleavage and DNA fragmentation. The observation that only the detached cells but not the attached cells showed the full spectrum of apoptotic changes suggests that detachment is either a part of the initiation/execution phases of apoptosis and/or is required for their completion.

Formaldehyde Cross-Linking for Studying Nucleosomal Dynamics

Methods are described for the utilization of formaldehyde as a reversible cross-linking agent for the characterization of protein–protein and protein–DNA interactions. The methods include a description of procedures to: (1) isolate and characterize transcriptionally active chromatin from cells cross-linked with formaldehyde; (2) study histone mobility during replication and transcription by the characterization of the formaldehyde-cross-linked histone octamer that is isolated from cells labeled with density-labeled amino acids; and (3) cross-link thein vitroreconstituted histone–DNA complex in order to maintain its structural state during subsequent characterization. Included in these methods are procedures for a second dimensional analysis of protein–protein cross-links in which the monomer components are electrophoretically resolved in the second dimension. The methods also include procedures to selectively reverse protein–DNA cross-links while maintaining the protein–protein cross-links. Potential artifacts are also discussed; i.e., data are presented which indicate that the helical pitch of DNA can be altered if the ionic strength is not properly controlled. The stability of the cross-linked nucleosome in the presence of altered pH or salt/urea concentrations is described in order to indicate that there are limitations to procedures that can be used for the subsequent characterization of the cross-linked complex.

ANTHERIDIAL CHROMATIN CONDENSATION FACTOR FROM MALE SEX ORGANS OFCHARA TOMENTOSA

Incubation of roots (Melandrium noctiflorum) in crude extracts obtained from maturing male sex organs ofChara tomentosaresults in supercondensation of mitotic chromosomes. The observed changes, including a number of ‘secondary effects’, confirm precisely with all those kinds of alterations which are typical of M-phase cells in antheridial filaments during their progression from the proliferative period of development towards terminal differentiation into sperm cells. Biological assays performed with proteinaceous extracts of maturing antheridia indicate that the activity is confined merely to a fraction containing low molecular (4.5kDa) peptides, termed as ACCF (antheridial chromatin condensation factor). Microchemical tests showed the acidic character of ACCF and its apparent ability to form cross-links with histones and DNA-histone complexes, mediated presumably by disulfide bridges. The characteristic punctate distribution of vesicles within the cytoplasm suggests the internalization of rhodamine-isothiocyanate labelled ACCF (ACCF-TRITC) by endocytosis, implying possible sequestering and nuclear translocation upon release from the endosomal/lysosomal compartment of the cell.

Sieving study of chromatin and histone-DNA complex by porous hollow fiber membranes

Permeation of chromatin isolated from human HeLa S3 cells and histone-DNA complex through regenerated cellulose hollow fibers was investigated as a model study of DNA removal for drug manufacturing using membrane technology. It was found that the permeation of histone-DNA and chromatin through the membranes having a mean pore diameter of 15 nm was substantially lower than the permeation of free DNA which did not complex with proteins based on the concentration determination of the feed and permeate solutions using UV spectroscopy. Direct observation to determine the existence of chromatin and histone-DNA complex in the permeate solution was also performed using atomic force microscopic imaging. No chromatin and chromatin-like structures were found in the examination of 10 areas of 1.0×1.0 μm on the mica which had been adsorbed from the solution permeated through the membranes. Thus, the membranes having a mean pore diameter of 15 nm most likely reject the chromatin and histone-DNA complex that are observed to have an apparent width of 25–30 nm based on atomic force microscopic imaging.

Autoantibodies to Nucleosomes and Histone–DNA Complexes

Individuals with systemic autoimmune diseases develop autoantibodies to nucleosome antigens. For many years, investigators have devoted much effort to precisely mapping epitopes on individual chromatin components. This approach, however, overlooks the existence of determinants that result from multimolecular interactions among nucleosome elements, such as DNA and histones. Anti-nucleosome antibodies can recognize a variety of complex epitopes and are especially prevalent in spontaneous and drug-induced lupus. Using numerous monoclonal anti-nucleosome antibodies obtained from autoimmune mice, we have further characterized these determinants and sequenced the variable region genes of these autoantibodies. I herein review these studies and their implications for the origin of antinuclear autoantibodies.

Detection of polyanion-restricted anti-histone antibodies in patients with systemic lupus erythematosus.

The nature of the antibodies responsible for lupus erythematosus (LE) cells in systemic lupus erythematosus (SLE) remains obscure. We examined whether polyanion-restricted anti-histone antibodies were present in serum of patients with SLE using Western blotting analysis. Dextran sulfate or alginate was used as a polyanion compound in place of DNA. Antibodies which recognized dextran sulfate-histone complexes were present in serum of patients with SLE (17/34, 50%). These antibodies were detected in most SLE patients positive for LE cells (17/18, 94%) but not in those negative for LE cells or in patients with other collagen diseases. Similar results were obtained using alginate-histone complexes as antigens for Western blotting analysis. The antibodies to dextran sulfate-histone or alginate-histone complexes in serum of SLE patients were completely absorbed by treatment of serum with DNA-histone complexes, while they were unaffected by treatment with DNA only. The presence of antibodies to free histones and dextran sulfate-histone complexes did not seem to be related to the titer of anti-single stranded DNA antibody and anti-double stranded DNA antibody. We demonstrated the presence of polyanion-restricted antibodies in SLE, which may be responsible for the LE factor.

Down-regulation of apoptosis-related bcl-2 but not bcl-xL or bax proteins in multidrug-resistant MCF-7/Adr human breast cancer cells.

Recent studies have shown that high levels of the apoptosis-related proteins bcl-2 and bcl-xL increase, while over-expression of bcl-xs or bax decreases, resistance to drugs that induce apoptosis in some human cancer cells. In the present report, we investigated whether expression of these apoptosis-related proteins correlates with changes in the degree of resistance to apoptosis induced by doxorubucin, taxol, vincristine and VP-16 and contributes to the development of acquired resistance in multidrug-resistant MCF-7/Adr breast cancer cells. In this study, high levels of bcl-xL and bax proteins are detected in both MCF-7 and MCF-7/Adr cells. In contrast, bcl-2 protein is down-regulated about 10-fold in MCF-7/Adr cells compared with MCF-7 cells. RT-PCR analysis showed that MCF-7/Adr cells express approximately 2-fold less bcl-2 mRNA than MCF-7 cells. Moreover, 4-24 hr cycloheximide treatment of MCF-7 and MCF-7/Adr cells did not affect the expression of bcl-2 protein, indicating that this protein is very stable in both cell lines. Our results suggest that bcl-2 expression is modulated partly by transcriptional, but mainly by post-transcriptional, mechanisms. Despite the down-regulation of bcl-2 in MCF-7/Adr cells and equal levels of bcl-x, and bax proteins in both cell lines, cytoplasmic DNA-histone complexes induced by doxorubucin, taxol, vincristine and VP-16 indicate that MCF-7/Adr cells are highly resistant to apoptosis. Moreover, treatments of MCF-7/Adr cells with P-glycoprotein (P-gp) modulators, cyclosporin A and verapamil increased doxorubicin and vincristine-induced DNA fragmentation about 1.4- and 2.5-fold, indicating that P-gp is involved in the development of resistance to chemotherapy-induced apoptosis in this cell line.

1,25-Dihydroxyvitamin D3 induces morphological and biochemical markers of apoptosis in MCF-7 breast cancer cells.

1,25-Dihydroxyvitamin D3 [1,25(OH)2(D)3], the active metabolite of vitamin D, is a potent inhibitor of breast cancer cell growth both in vivo and in vitro. To complement data which documents the anti-proliferative effects of 1,25(OH)2(D)3, we assessed the role of apoptosis in vitamin D-mediated growth arrest of MCF-7 cells. Time course studies indicated that 100 nM 1,25(OH)2(D)3 significantly reduces MCF-7 cell numbers within 48 h of treatment. Morphological assessment demonstrated that MCF-7 cells treated with 1,25(OH)2(D)3 for 48 h exhibit characteristic apoptotic features, including cytoplasmic condensation, pyknotic nuclei, condensed chromatin and nuclear matrix re-organization. In situ end labelling with terminal transferase indicated that cells exhibiting apoptotic morphology in 1,25(OH)2(D)3-treated cultures were positive for DNA strand breaks. These morphological features of apoptosis were accompanied by an increase in the cell death rate assessed as soluble DNA-histone complexes indicative of DNA fragmentation. To complement the morphological data, we assessed the temporal expression of two proteins which have been associated with apoptosis in mammary cells and tumors. The steady state mRNA levels for TRPM-2/clusterin and cathepsin B mRNA were significantly up-regulated in MCF-7 cells treated with 1,25(OH)2(D)3 compared to control cells. Time-dependent increases in the expression of TRPM-2/clusterin and cathepsin B proteins were detected by Western blotting in 1,25(OH)2(D)3-treated cells. These findings indicate that, in addition to its anti-proliferative effects, 1,25(OH)2(D)3 activates the apoptotic cell death pathway in MCF-7 breast cancer cells.

The central role of chromatin in autoimmune responses to histones and DNA in systemic lupus erythematosus.

To gain insight into the mechanisms of autoantibody induction, sera from 40 patients with systemic lupus erythematosus (SLE) were tested by ELISAs for antibody binding to denatured individual histones, native histone-histone complexes, histone-DNA subnucleosome complexes, three forms of chromatin, and DNA. Whole chromatin was the most reactive substrate, with 88% of the patients positive. By chi-square analysis, only the presence of anti-(H2A-H2B), anti-[(H2A-H2B)-DNA], and antichromatin were correlated with kidney disease measured by proteinuria > 0.5 g/d. SLE patients could be divided into two groups based on their antibody-binding pattern to the above substrates. Antibodies from about half of the patients reacted with chromatin and the (H2A-H2B)-DNA subnucleosome complex but displayed very low or no reactivity with native DNA or the (H3-H4)2-DNA subnucleosome complex. An additional third of the patients had antibody reactivity to chromatin, as well as to both subnucleosome structures and DNA. Strikingly, all sera that bound to any of the components of chromatin also bound to whole chromatin, and adsorption with chromatin removed 85-100% of reactivity to (H2A-H2B)-DNA, (H3-H4)2-DNA, and native DNA. Individual sera often bound to several different epitopes on chromatin, with some epitopes requiring quaternary protein-DNA interactions. These results are consistent with chromatin being a potent immunogenic stimulus in SLE. Taken together with previous studies, we suggest that antibody activity to the (H2A-H2B)-DNA component signals the initial breakdown of immune tolerance whereas responses to (H3-H4)2-DNA and native DNA reflect subsequent global loss of tolerance to chromatin.

Transcription: In tune with the histones

The regulation of transcription in eukaryotes relies upon the histone proteins in several essential ways. The incorporation of the binding sites for the basal transcriptional machinery into nucleosomes serves to repress transcription. Specific regulatory molecules other than the basal transcriptional machinery exist that can associate with nucleosomal DNA and initiate a chain of events that disrupt repressive histone-DNA complexes. The main players in this story have been defined physically and genetically and include positioned nucleosomes, interactions of the histone tetramer (H3-H4)2 with DNA, the N-terminal tails of histones H3 and H4, and a large general activator complex. How they fit together biochemically is yet to be defined. The genetic data demonstrate that the disruption of histone-DNA complexes plays a major role in the induction of transcription from many genes. However, not all genes are repressed by nucleosome assembly: certain promoters make use of the staged assembly of chromatin in vivo and a rapid and tight association of transacting factors with promoter elements to remain constitutively active. Moreover, nucleosome assembly is not necessarily repressive, since the folding of DNA by the histones can facilitate the activation of genes by bringing widely separated regulatory elements into juxtaposition. Thus, histones provide the necessary infrastructure for the correct and efficient operation of the transcriptional machinery; however, their exact contributions to the transcriptional regulation of an individual gene may depend on the spatial distribution of regulatory elements, the transcription factors involved, and the three-dimensional folding of DNA that they direct.