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PubMed2004

DNaseI hypersensitivity analysis of chromatin structure.

Transcriptionally inactive DNA is packaged into condensed chromatin such that it is unavailable to the transcription initiation complex. Activation of the silenced genes during processes such as differentiation first requires that the chromatin structure be remodeled into a transcriptionally permissive configuration, with the DNA "exposed" and accessible to transcription factors. The change in chromatin structure associated with transcriptional competence can be detected as increased sensitivity of the exposed DNA to digestion with DNaseI. This increased susceptibility is referred to as DnaseI-hypersensitivity. DNaseI hypersensitive sites are often located in the recognition sites for transcription factors, including promoters and enhancers. This chapter describes the protocols necessary to perform and analyze DNaseI hypersensitivity assays, a technique becoming increasingly important given the rapid advances in our understanding of the chromatin remodeling processes.

Blotting, Southern↗
PubMed1986

Chromatin structure of the cytochrome P-450c gene changes following induction.

The chromatin structure of cytochrome P-450c and P-450d genes, which in the liver are highly inducible by 3-methylcholanthrene, was studied in normal and carcinogen-treated rats by using a cDNA probe specific for P-450c and a genomic probe that recognizes both genes. Digestion with micrococcal nuclease revealed that the active genes are not present in the typical 200 base pair nucleosomal structure. Gene induction is associated with a rearrangement of the nuclear organization of the genes. By use of indirect end-label hybridization, three DNase I hypersensitive sites were mapped, one in the 5'-terminal region and two in the 3' region of the P-450c gene. Gene induction, by treatment with 3-methylcholanthrene, changes the location of the DNase I site present in the 5' region without affecting the sites present in the 3' region. Rat thymus chromatin does not contain these DNase I hypersensitive sites, suggesting that, in the liver, the chromatin structure is altered so as to allow tissue-specific expression of the P-450c gene. The chromatin structure of the highly inducible P-450c gene is compared to that of the P-450m gene, which is induced to a significantly smaller extent and is constitutively expressed.

Animals↗
PubMed1980

On the chromatin structure of Trypanosoma cruzi.

The chromatin structure of Trypanosoma cruzi was investigated. It was found that, as in other eukaryotes, the chromatin is organized in repeating units, the nucleosomes containing about 200 base pairs of DNA associated with histones. While there is no difference in the DNA size in nucleosomes from T. cruzi and from rat liver nuclei, the histone population of T. cruzi differs in various aspects. Of particular interest is the presence of two basic proteins, possibly histone H1 subcomponents, with very high electrophoretic mobilities.

Animals↗
PubMed2004

Generic features of tertiary chromatin structure as detected in natural chromosomes.

Knowledge of tertiary chromatin structure in mammalian interphase chromosomes is largely derived from artificial tandem arrays. In these model systems, light microscope images reveal fibers or beaded fibers after high-density targeting of transactivators to insertional domains spanning several megabases. These images of fibers have lent support to chromonema fiber models of tertiary structure. To assess the relevance of these studies to natural mammalian chromatin, we identified two different approximately 400-kb regions on human chromosomes 6 and 22 and then examined light microscope images of interphase tertiary chromatin structure when the regions were transcriptionally active and inactive. When transcriptionally active, these natural chromosomal regions elongated, yielding images characterized by a series of adjacent puncta or "beads", referred to hereafter as beaded images. These elongated structures required transcription for their maintenance. Thus, despite marked differences in the density and the mode of transactivation, the natural and artificial systems showed similarities, suggesting that beaded images are generic features of transcriptionally active tertiary chromatin. We show here, however, that these images do not necessarily favor chromonema fiber models but can also be explained by a radial-loop model or even a simple nucleosome affinity, random-chain model. Thus, light microscope images of tertiary structure cannot distinguish among competing models, although they do impose key constraints: chromatin must be clustered to yield beaded images and then packaged within each cluster to enable decondensation into adjacent clusters.

Base Sequence↗
PubMed2004

Global chromatin structure of 45,000 base pairs of chromosome III in a- and alpha-cell yeast and during mating-type switching.

Directionality of yeast mating-type switching has been attributed to differences in chromatin structure for the left arm of chromosome III. We have mapped the structure of approximately 45 kbp of the left arm of chromosome III in a and alpha cells in logarithmically growing cultures and in a cells during switching. Distinctive features of chromatin structure were the occurrence of DNase I-hypersensitive sites in the promoter region of nearly every gene and some replication origins and the presence of extended regions of positioned nucleosomes in approximately 25% of the open reading frames. Other than the recombination enhancer, chromatin structures were identical in the two cell types. Changes in chromatin structure during switching were confined to the recombination enhancer. This unbiased analysis of an extended region of chromatin reveals that significant features of organized chromatin exist for the entire region, and these features are largely static with respect to mating type and mating-type switching. Our analysis also shows that primary chromatin structure does not cause the documented differences in recombinational frequency of the left arm of chromosome III in yeast a and alpha cells.

Chromatin↗
PubMed1984

Chromatin structure and nucleic acid synthesis in human lymphocyte activation by phytohemagglutinin.

Alterations in chromatin structure were explored as a function of time following phytohemagglutinin (PHA) activation of human lymphocytes. Nuclear refringency (related to chromatin status), ultrastructural changes, and modifications of nucleic acid synthesis were compared. Chromatin dispersion was observed within 20 min of stimulation, with a concomitant increase in [3H]uridine incorporation; both were inhibited by actinomycin D. Recondensation of chromatin had occurred at 60 min when RNA synthesis was still increasing. The early chromatin dispersion thus was a reversible step and one of the most striking cellular events during the first phase of PHA activation. Late chromatin dispersion of blastic cells during the second phase of PHA response (after 24 h) was related to DNA synthesis in mitotic cells. This step was also inhibited by actinomycin D. These results suggest a relationship between the morphological changes in chromatin structure and the induction of RNA and DNA syntheses. The regulation of transcriptional and replicational states of the nucleus is discussed in terms of modifications of chromatin structure, considering PHA-induced calcium and cyclic nucleotide metabolism and interleukin II receptor expression.

Adult↗
PubMed1993

Hydroxyurea exposure alters mouse testicular kinetics and sperm chromatin structure.

The effects of hydroxyurea (HU) on testicular cell kinetics and sperm chromatin differentiation were investigated in mice. Whole testis, minced testicular cell suspensions and caudal epididymal sperm cells were obtained at 8 and 29 days after i.p. injections containing 0, 25, 50, 100, 200, 400 and 500 mg/kg HU x 5 days. Testis weights were unaffected by 25 mg/kg HU while 500 mg/kg caused up to a 50% loss of testicular weight by 29 days. Flow cytometrically measured acridine-orange (AO) stained testicular cells revealed altered population ratios at the highest dosages at 8 days and for all dosages except 25 mg/kg HU at 29 days. At 8 days, 400-500 mg/kg HU caused a near depletion of tetraploid cells. Flow cytometry of AO stained sperm, previously treated with acid to potentially induce DNA denaturation, was used to follow the shift from normal chromatin structure to an abnormal form with increased sensitivity to DNA denaturation in situ. The extent of DNA denaturation was quantitated for each cell by the computer-derived value alpha t, alpha t = [red/(red+green) fluorescence]. The flow cytometry measures, standard deviation of alpha t (SD alpha t), mean of alpha t (X alpha t) and cells outside the main peak of alpha t (COMP alpha t), gave similar dose response curves to the sperm head morphology assay. SD alpha t was more sensitive than the X alpha t as a measure of HU-induced alteration of chromatin structure. The major conclusions reached are that HU inhibits DNA synthesis, probably by inhibiting ribonucleotide reductase, causing maturation depletion of pachytene spermatocytes and, subsequently, depletion of meiotic daughter cells and differentiated cell types leading to mature sperm. This inhibition of DNA synthesis is related to an alteration of sperm chromatin structure and abnormal sperm head morphology.

Animals↗
PubMed1997

CpG methylation remodels chromatin structure in vitro.

One of the mechanisms proposed to explain how CpG methylation effects gene repression invokes a DNA methylation-determined chromatin structure. Previous work implied that this DNA modification does not influence nucleosome formation in vitro, thus current models propose that certain non-histone proteins or a preferential affinity by linker histones for methylated DNA may mediate changes in chromatin structure. We have reinvestigated whether CpG methylation alters the chromatin structure of reconstitutes comprising only core histones and DNA. We find that DNA methylation prevents the histone octamer from interacting with an otherwise high affinity positioning sequence in the promoter region of the chicken adult beta-globin gene. This exclusion is attributed to methylation-determined changes in DNA structure within a triplet of CpG dinucleotides. In the affected nucleosome, this sequence motif is located 1.5 helical turns from the dyad axis and is oriented towards the histone core. These findings establish that DNA methylation does have the capacity to modulate chromatin structure directly, at its most fundamental level. Furthermore, our observations strongly suggest that a very limited number of nucleotides can make a decisive contribution to the translational positioning of nucleosomes.

Animals↗
PubMed1985

Electron probe x-ray microanalysis studies on the ionic environment of nuclei and the maintenance of chromatin structure.

We have demonstrated the application of electron probe X-ray microanalysis to studies aimed at determining the role of the ionic environment in the maintenance of a stable chromatin structure during interphase. Our results indicate that X-ray microanalysis can be used to accurately measure the concentration of ions, such as Na+ and K+ within subcellular compartments. We also present evidence from our X-ray microanalysis studies to show that only a portion of the K+ in the interphase nucleus is in a free ionic state and that the rest of the intranuclear K+ is adsorbed to intranuclear macromolecules. The stable structural state of interphase chromatin was correlated with the maintenance of a normal range in the chemical activities of monovalent ions in the oocyte nucleus. Finally, we demonstrated that the chemical activity of the monovalent ions probably increases beyond this normal interphase range as the chromatin in the nucleus condenses into discrete chromosomes during the first meiotic division in the amphibian oocyte. The above observations support the hypothesis of Kellermayer et al. (1984) that the maintenance of chromatin structure in the nucleus of living cells requires a balance between the presence or absence of mobile nuclear proteins and the chemical activity of monovalent cations. It also suggests that an elevation of the chemical activity of the monovalent cations above a normal range will result in the destabilization of interphase chromatin structure. It is hypothesized that an elevation of monovalent cations may be directly involved in the structural and functional changes in chromatin which are known to take place at the time of meiosis and mitosis.

Animals↗
PubMed1997

Analysis of chromatin structure in vivo.

A number of important nuclear processes including replication, recombination, repair, and transcription involve the interaction of soluble nuclear proteins with DNA assembled as chromatin. Recent progress in a number of experimental systems has focused attention on the influence chromatin structure may exert on gene regulation in eukaryotes. With the advent of new technologies for the analysis of chromatin structure in vivo, studies evaluating the influence of chromatin structure on gene transcription have become feasible for a number of systems. This article serves as an introduction to the use of restriction endonucleases to define nucleosomal organization and characterize changes in this organization that accompany transcriptional activation in vivo. The procedure includes the isolation of intact transcriptionally competent nuclei, limited digestion with specific restriction endonucleases, and purification of the DNA. This DNA serves as the substrate for a linear amplification using single primers that generate enzyme-specific DNA fragments, which are then resolved by electrophoresis. Specific examples related to our studies of the influence of chromatin structure on steroid hormone regulation of transcription from the mouse mammary tumor virus promoter are provided to illustrate this technique and several novel variations. Alternative methods for analysis of chromatin architecture using DNase I, micrococcal nuclease, permanganate, and methidiumpropyl-EDTA-iron(II) are also described. Through the use of these methodologies one is able to determine both the translational and the rotational positions for a given nucleosome as well as quantify changes at a specific nucleosome in response to regulatory and developmental signals.

Animals↗
PubMed2006

Osmotic stress-dependent repression is mediated by histone H3 phosphorylation and chromatin structure.

Histone H3 phosphorylation has been linked to various environmental stress responses and specific chromatin structure. The role of H3 phosphorylation in the osmotic stress response was investigated on the mouse mammary tumor virus (MMTV) promoter in different chromatin configurations. Hormone-dependent transcription from the MMTV promoter is repressed by osmotic stress when the promoter is integrated and has a normal chromatin structure. However, when the MMTV promoter is transiently transfected, the chromatin structure is less organized, and hormone induction is not affected by osmotic stress. On the integrated MMTV promoter, phosphorylation of histone H3 serine 10 and 28 increases in response to osmotic stress, but the transient promoter shows no change. Hormone-dependent glucocorticoid receptor binding is reduced on the repressed promoter, and elevated H3 phosphorylation is temporally correlated with maximal MMTV repression Additionally, the protein kinase C inhibitor rottlerin, but not other kinase inhibitors, blocks both histone H3 phosphorylation and osmotic repression of MMTV transcription. Glucocorticoid receptor binding is inversely correlated with H3 phosphorylation, suggesting that displacement of the glucocorticoid receptor from the promoter is due to H3 phosphorylation and is the mechanism for the osmotic repression of hormone-dependent transcription.

Acetophenones↗
PubMed2000

A statistical-mechanical model for regulation of long-range chromatin structure and gene expression.

In eukaryotic organisms, organization of chromatin is considered to play a role in transcriptional regulation by limiting the accessibility of a gene to the transcription machinery. It is not fully understood, however, how chromatin around a particular locus can be specifically altered to allow transcription. This paper introduces a statistical-mechanical model of chromatin to illustrate a potential mechanism. The model, which is mathematically equivalent to the one-dimensional Ising model of magnetic systems, explains how gene regulatory DNA sequences can affect the chromatin structure over a long distance in cis. The main assumption of the model is cooperativity of histone H1 in binding to the nucleosome array. This cooperativity results in a long-range correlation of histone H1 distribution along the chromatin. Due to this long-range correlation, a gene regulatory element, such as a transcriptional enhancer, may lead to depletion of histone H1 over a large region of chromatin thereby increasing the accessibility of the gene. The model provides a mechanism for a sensitive genetic switch and explains several aspects of gene regulation and chromatin structure.

Animals↗
PubMedDate not supplied

[DNA degradation in vivo in dying thymocytes, model for the study of chromatin structure].

Properties of chromatin fragments which are formed as a result of in vivo DNA degradation in thymocytes of gamma-irradiated rats were studied. It is shown that under these conditions chromatin breakdown is accounted for by internucleosomal DNA scission without proteolysis and disturbance of nucleosome structure as judged from how they are split by DNAase I. Amount of mononucleosomes and their dimers, trimers and tetramers is in average about 10% of the fragments formed, and that of oligomers composed of about 8--10 nucleosomes equals about 80%. Core-particles and subnucleosomal fragments are never observed. Chromatin fragments of different size have been purified and their composition was studied. Electrophoresis in 3% PAAG--0.5% agarose divides mononucleosomes into two fractions. The less mobile fraction contains all histones, non-histone proteins and DNA fragments of 178 base pairs long. More mobile mononucleosomes do not contain at least fragments of 178 base pairs long. More mobile mononucleosomes do not contain at least one of the histone H1 subfraction and the size of their DNA is 168 base pairs. Chromatin fragments of different size contain both similar and different non-histone proteins. In mononucleosomes and their dimers, trimers and tetramers proteins are found which are absent in chromatin fragments of high molecular weight. It is assumed that mononucleosomes and their dimers, trimers and tetramers are products of active chromatin degradation and that high molecular weight oligomers are supernucleosomal structures of inactive chromatin.

Animals↗
PubMed1994

Ethidium bromide intercalation and chromatin structure: a spectropolarimetric analysis.

Native chromatin has been characterized at different ionic strengths, according to different methods of preparation, by means of circular dichroism and ethidium bromide intercalation. For a more precise interpretation of these results, a series of analogous experiments on phagic DNA, as a function of its circularization, and on mononucleosomes have been performed. Results are discussed here in terms of high order chromatin structure, DNA supercoiling, and topological constraints.

Animals↗
PubMed1990

Thyroid hormone and dietary carbohydrate interact to regulate rat liver S14 gene transcription and chromatin structure.

Dietary carbohydrate and thyroid hormone (T3) interact to regulate rat liver S14 gene expression. The molecular basis for this interaction was examined by analysis of hepatic mRNAS14 levels, S14 gene transcription, and chromatin structure. While starvation of euthyroid rats inhibited hepatic S14 gene transcription greater than or equal to 90%, sucrose administration induced mRNAS14 and S14 transcription to 82% of euthyroid-fed levels within 4 h. In contrast, administration of sucrose or T3 to starved hypothyroid animals restored S14 gene transcription to only 30% of euthyroid-fed values. Both T3 and sucrose were required to restore S14 run-on activity and mRNAS14 to euthyroid-fed levels within 4 h. Thus, T3 and sucrose interact synergistically and rapidly to induce S14 gene transcription. Analysis of S14 chromatin structure showed that starvation of hypothyroid rats inhibited the formation of three DNase I-hypersensitive sites flanking the 5'-end of the S14 gene (Hss-1 at -65 to -265 base pairs; Hss-2 at -1.2 kilobases and Hss-3 at -2.67 kilobases). The loss of these sites correlated with the repression of S14 gene transcription in starved hypothyroid rats. Whereas administration of sucrose to starved hypothyroid rats consistently induced the Hss-1 and Hss-2 sites, T3 consistently induced all three DNase I-hypersensitive sites. Yet, neither treatment alone induced S14 gene transcription to euthyroid levels. The combination of T3 and sucrose induced no additional change in S14 chromatin structure over that induced by T3 alone. Thus, regulation of S14 chromatin structure alone is not the sole mechanism by which these stimuli regulate S14 gene transcription. We speculate that the synergistic regulation of S14 gene transcription by T3 and dietary carbohydrate involves a complex interaction between factors which regulate the accessibility of putative cis-regulatory elements through changes in chromatin structure and the regulation of "transcription factors" which interact with these elements.

Animals↗
PubMedDate not supplied

[Supranucleosomal levels of chromatin structure: a nucleodisome].

Dinucleosome periodicity of DNA fragmentation produced by DNAase I in nuclei of erythroid cells replacing its lysine-rich histone H1 by erythrocyte-specific fraction H5 has been electrophoretically investigated. It was found that the double-nucleosome repeat becomes more definite as the content of H5 increases. Electrophoretic analysis of DNA fragments generated by DNAse I reveals a "nucleodisome" structure of chromatin not only is condensed inactive nuclei of mature erythrocytes but also in chromatin of transcriptionally-active cells possessing a usual lysine-rich histone H1 such as rat thymus cells or CHO cell line. It seems that the nucleodisome structure is the specific feature of any chromatin. The nuclease of another specificity (DNAse II) is also able to reveal dinucleosome periodicity in chromatin of both erythrocytes and sea urchin sperm. Comparison of the results of DNA cutting in nuclei of these cells allows us to establish that the pattern of chromatin fragmentation by DNAses I and II is very similar, if not identical. On the basis of data obtained a mechanism of nucleodisome splitting off is suggested.

Animals↗
PubMed2000

The mouse mammary tumor virus promoter adopts distinct chromatin structures in human breast cancer cells with and without glucocorticoid receptor.

Steroid receptors represent a class of transcription regulators that act in part by overcoming the often repressive nature of chromatin to modulate gene activity. The mouse mammary tumor virus (MMTV) promoter is a useful model for studying transcriptional regulation by steroid hormone receptors in the context of chromatin. The chromatin architecture of the promoter prevents the assembly of basal transcription machinery and binding of ubiquitous transcription factors. However, in human breast carcinoma T47D cells lacking the glucocorticoid receptor (GR), but expressing the progesterone receptor (PR), nucleosome B (nuc B) assumes a constitutively hypersensitive chromatin structure. This correlation led us to test the hypothesis that the chromatin structure of nuc B was dependent on GR expression in T47D cells. To examine this possibility, we stably co-transfected the MMTV promoter and the GR into T47D cells that lacked both the GR and the PR. We found that in T47D cells that lack both the GR and the PR or express only the GR, nuc B assumes a constitutively "open" chromatin structure, which allows hormone independent access by restriction endonucleases and transcription factors. These results suggest that in GR(+)/pr(-) T47D cells, the MMTV chromatin structure permits GR transcriptional activation, independent of chromatin remodeling.

Blotting, Western↗
PubMed1986

Chromatin structure in scrapie and Alzheimer's disease.

Scrapie affected brains exhibit a number of pathological features in common with the human neurodegenerative condition, Alzheimer's disease. The present report describes studies on chromatin structure seen in these two disease processes. Chromatin associated proteins influence transcriptional activity of DNA through an effect upon chromatin structure. We examined chromatin structure by: measuring the capacity of the enzyme micrococcal nuclease to release mono- and dinucleosomes from isolated nuclei and measuring DNA-histone interactions by examining the effect of ambient tonicity upon the release of chromatin proteins. In two strains of mice infected with two strains of scrapie agent there was reduced accessibility to micrococcal nuclease and an increased content on dinucleosomes of the histone H1 and H1(0) types. These changes precede clinical signs of scrapie and resemble those found in the human conditions of Alzheimer's and Pick's disease. Scrapie mouse brain differs from Alzheimer brain in that scrapie does not alter histone-DNA interactions as monitored by ionically induced histone release from chromatin. Despite similarities, the scrapie agent appears to operate upon different molecular mechanisms than those found in Alzheimer's disease.

Alzheimer Disease↗