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PubMed1986

Diet-mediated alteration of chromatin structure.

Higher-order chromatin structure and the process of transcription are related. The significance of a nutritional state's altering chromatin structure lies in the potential role of that nutritional state in the regulation of gene expression. In rats short-term feeding of semisynthetic diets varying in the proportion of carbohydrate, protein, or fat alters the configuration of liver chromatin as measured by sensitivity to micrococcal nuclease (EC 3.1.31.1). A carbohydrate-rich, fat-free diet increases the sensitivity of rat liver chromatin to micrococcal nuclease and decreases the nucleosome repeat length. In contrast, a protein-free diet or a diet deficient in magnesium or zinc decreases the sensitivity of liver nuclear chromatin to micrococcal nuclease. Diet-mediated mechanisms that alter chromatin structure are now unknown, but the continued study of nutritional interaction with the genome should identify the responsible features as well as their significance to gene function.

Animals↗

PubMed1999

Higher-order chromatin structure: looping long molecules.

Chromatin structure plays a variety of roles in eukaryotes, ranging from the structural organization of the genome to the facilitation of transcription factors and remodeling of individual gene promoters. Higher-order chromatin structure typically refers to those structural features of the genome that serve to facilitate large-scale condensation and packaging. It is becoming increasingly clear, however, that large-scale features that create loop domains play an important role in the management and functional organization of the genome as well. Recently, plant models have made significant contributions to our understanding of higher-order chromatin structures in eukaryotes.

Animals↗

PubMed1999

Nuclear chromatin variations in human spermatozoa undergoing swim-up and cryopreservation evaluated by the flow cytometric sperm chromatin structure assay.

The sperm chromatin structure assay (SCSA) is a flow cytometric (FCM) technique which exploits the metachromatic properties of Acridine Orange to monitor the susceptibility of sperm chromatin DNA to in-situ acid denaturation. SCSA was used to study the chromatin structure variations of human spermatozoa in semen, both before and after swim-up and after cryopreservation. Semen samples were provided by 19 healthy normozoospermic subjects attending pre-marriage checks. Each sample was divided into three aliquots: the first aliquot was evaluated without further treatment, the second underwent swim-up, and the third was stored according to standard cryopreservation techniques in liquid nitrogen at -196 degrees C. Samples were also analysed by light and fluorescence microscopy (after Acridine Orange staining to evaluate the number of green fluorescent sperm heads), and by computer-assisted semen analysis. The results showed that post-rise spermatozoa represent a subpopulation characterized by a general improvement of the morphological (reduction of the percentage of abnormal forms and heads, increase of the green head sperm percentage) and kinetic parameters. This subpopulation also exhibited improved chromatin structure properties, confirming that these cells have the best structural and functional characteristics, indicative of optimal fertilizing ability. On the other hand, overall sperm quality deteriorates after cryopreservation. When thawed spermatozoa underwent an additional swim-up round, a general improvement of nuclear maturity was seen in the post-rise spermatozoa.

Adult↗

PubMed1998

Template activating factor-I remodels the chromatin structure and stimulates transcription from the chromatin template.

To study the mechanisms of replication and transcription on chromatin, we have been using the adenovirus DNA complexed with viral basic core proteins, called Ad core. We have identified template activating factor (TAF)-I from uninfected HeLa cells as the factor that stimulates replication and transcription from the Ad core. The nuclease sensitivity assays have revealed that TAF-I remodels the Ad core, thereby making transcription and replication apparatus accessible to the template DNA. To examine whether TAF-I remodels the chromatin consisting of histones, the chromatin structure was reconstituted on the DNA fragment with core histones by the salt dialysis method. The transcription from the reconstituted chromatin was completely repressed, while TAF-I remodeled the chromatin and stimulated the transcription. TAF-I was found to interact with histones. Furthermore, it was shown that TAF-I is capable not only of disrupting the chromatin structure but also of preventing the formation of DNA-histone aggregation and transferring histones to naked DNA. The possible function of TAF-I in conjunction with a histone chaperone activity is discussed.

Adenoviruses, Human↗

PubMed2004

Thymocyte apoptosis induced by various compounds including YO-2 is accompanied by a change in chromatin structure.

To elucidate the role of chromatin structure in DNA fragmentation during apoptosis, we have examined whether chromatin structural change is observed after treatment with proapoptotic compounds. Analysis of the circular dichroism (CD) spectrum of the soluble chromatin from dexamethasone-treated thymocytes revealed a decrease in alpha-helical content. Mifepristone, an antagonist of glucocorticoid receptor, prevented both the change in chromatin structure and DNA fragmentation induced by dexamethasone. The effect of YO-2 [trans-4-aminomethylcyclohexanecarbonyl-l-(O-picolyl)tyrosine-n-octylamide], which possesses proapoptotic action, on chromatin structure was also examined. Judging from the CD spectrum of the soluble chromatin from YO-2-treated thymocytes, the structure was changed by this compound as well as by dexamethasone. A decrease in alpha-helical content was also observed in cells treated with etoposide, which is used clinically as an anticancer agent. These results suggest that the change in chromatin structure is likely to be an important process in DNA fragmentation of cells undergoing apoptosis.

Animals↗

PubMed1992

Involvement of the SIN4 global transcriptional regulator in the chromatin structure of Saccharomyces cerevisiae.

We have cloned and sequenced the SIN4 gene and determined that SIN4 is identical to TSF3, identified as a negative regulator of GAL1 gene transcription (S. Chen, R.W. West, Jr., S.L. Johnson, H. Gans, and J. Ma, submitted for publication). Yeast strains bearing a sin4 delta null mutation have been constructed and are temperature sensitive for growth and display defects in both negative and positive regulation of transcription. Transcription of the CTS1 gene is reduced in sin4 delta mutants, suggesting that Sin4 functions as a positive transcriptional regulator. Additionally, a Sin4-LexA fusion protein activates transcription from test promoters containing LexA binding sites. The sin4 delta mutant also shows phenotypes common to histone and spt mutants, including suppression of delta insertion mutations in the HIS4 and LYS2 promoters, expression of promoters lacking upstream activation sequence elements, and decreased superhelical density of circular DNA molecules. These results suggest that the sin4 delta mutation may alter the structure of chromatin, and these changes in chromatin structure may affect transcriptional regulation.

Amino Acid Sequence↗

PubMed1998

Chromatin structures of goat and sheep beta-lactoglobulin gene differ.

Different levels of the major milk protein beta-lactoglobulin are found in evolutionarily related ruminant species: with sheep milk containing as much as three times the concentration in goat milk. In an attempt to understand why these differences exist, we have characterised, using DNaseI as a probe of structure, the chromatin surrounding the goat beta-lactoglobulin promoter and compared it to that of the sheep homologue. The goat gene displays a mammary-specific chromatin pattern, which is reformed on expressing goat beta-lactoglobulin transgenes. This implies that this chromatin structure is sequence dependent and suggests that it plays a role in regulating beta-lactoglobulin gene expression. This pattern differs from that seen on the ovine beta-lactoglobulin gene in lactating sheep mammary chromatin. Thus, even between highly related species, the transcriptional mechanisms regulating activity of a gene can differ.

Animals↗

PubMed1993

[Changes in the chromatin structure under the influence of intranuclear bacteria with antihistone activity].

Computed TV morphodensitometry was used to examine the integration of bacteria into the human nuclear genome. The bacterial strains with antihistone activity were shown to be incorporated into the structure of chromatin structure of an epithelial cell with its network organization impaired. The microscopic analysis of cultured Hep 2 cells by manual scanning indicated that genetically active bacteria were incorporated into the nucleus and nucleolus.

Cell Nucleus↗

PubMed1999

In vivo methods to analyze chromatin structure.

A groundswell of interest in chromatin structure and its role in regulating the function of DNA in transcription, replication, recombination and repair has developed in the past decade. Fueled by genetic observations of effects of histone mutations on transcription and identification of genes whose products must alter chromatin structure as they affect gene activity, this subject leapt to the forefront in the past two years with the correlation of certain transcription factors with enzymes that post-translationally modify histones and are presumed to alter chromatin structure thereby. Surprisingly few experimental reports have actually addressed chromatin structure. In part, this may be related to the technical difficulties of traditional approaches to structure inference. Methods have become available recently for assessment of various aspects of chromatin structure in vivo. Study in intact cells may limit potential problems resulting from loss of components or rearrangement of structures and simplify analysis by eliminating the need for isolation of organelles.

Animals↗

PubMed1977

Salt and divalent cations affect the flexible nature of the natural beaded chromatin structure.

A natural chromatin containing simian virus 40 (SV40) DNA and histone has been used to examine changes in chromatin structure caused by various physical and chemical treatments. We find that histone H1 depleted chromatin is more compact in solutions of 0.15M NaCl or 2 mM MgCl2 than in 0.01 M NaCl or 0.6M NaCL, and is compact in 0.01 M NaCl solutions if histone H 1 is present. Even high concentrations of urea did not alter the fundamental beaded structure, consisting of 110A beads of 200 base pair content, each joined by thin DNA bridges of 50 base pairs. The physical bead observed by EM therefore contains more DNA than the 140 base pair "core particle". The natural variation in the bridge length is consistent with the broad bands observed after nuclease digestion of chromatin. Chromatin prepared for EM without fixation containing long 20A to 30A fibers possibly complexed with protein.

Cations, Divalent↗

PubMed1992

Ligand dependence of estrogen receptor induced changes in chromatin structure.

To determine whether the human estrogen receptor requires ligand to bind to its cognate estrogen receptor element (ERE) in vivo, we have examined the structure of chromatin at a chromosomally integrated ERE-URA3 reporter gene in yeast, and the influence of ligand bound and ligand free estrogen receptors on that structure. Using indirect end-labelling to map DNaseI and micrococcal nuclease sensitive sites, we found that receptor induced alterations in chromatin structure were completely dependent upon the presence of estradiol. These same alterations in chromatin structure were induced by a truncated estrogen receptor with both TAF-1 and TAF-2 transactivation functions deleted, suggesting that DNA binding per se disrupts chromatin structure. These results support models in which the estrogen receptor requires ligand to bind to the ERE in vivo.

Chromatin↗

PubMed2002

Cooperation between complexes that regulate chromatin structure and transcription.

Chromatin structure creates barriers for each step in eukaryotic transcription. Here we discuss how the activities of two major classes of chromatin-modifying complexes, ATP-dependent remodeling complexes and HAT or HDAC complexes, might be coordinated to create a DNA template that is accessible to the general transcription apparatus.

Acetyltransferases↗

PubMed2001

Participation of histones and histone-modifying enzymes in cell functions through alterations in chromatin structure.

Alterations in the chromatin structure are preferentially involved in the regulation of cell functions, including gene expression, in eukaryotes. Three types of mechanisms, by which the alterations are caused have been reported: (i) variants of histone subtypes, (ii) chromatin remodeling, and (iii) post-translational modification. This review focuses mainly on the first and third mechanisms, especially on the acetylation of core histones, one of the third mechanisms. Using the gene targeting technique for the DT40 chicken B cell line, we systematically generated a number of mutants, respectively, devoid of particular genes encoding histones and histone deacetylase(s) (HDACs). Most of the H1 and core histone variants should be involved positively or negatively in the transcription regulation of particular genes. Of the chicken HDACs (chHDACs), chHDAC-2 controls the amount of the IgM H-chain at the steps of both transcription and alternative pre-mRNA processing, and chHDAC-3 is essential for cell viability, whereas chHDAC-1 merely affects gene expression in DT40 cells. These results indicate that HDAC family members should participate, in combination with one another, and/or histone acetyltransferase(s) (HATs), in the acetylation of core histones that regulates gene expression through alterations in the chromatin structure.

Acetyltransferases↗

PubMed1992

Variation in radiation-induced formation of DNA double-strand breaks as a function of chromatin structure.

The influence of chromatin structure on induction of DNA double-strand breaks (DSBs) by X radiation was studied in DNA from CHO cells. Whole cells, nuclei with condensed or relaxed chromatin, and deproteinized DNA in agarose plugs were irradiated and DSB formation was measured as a decrease in the length of DNA by nondenaturing, pulsed-field, agarose gel electrophoresis. The yield of DSBs in deproteinized DNA (2.3 x 10(-10) DSBs Da-1 Gy-1) was observed to be 70 times greater than the yield of DSBs (3.1 x 10(-12) DSBs Da-1 Gy-1) observed in DNA in the intact cell nucleus. Organization of DNA into the basic nucleosome repeat structure and condensation of the chromatin fiber into higher-order structure protected DNA from DSB induction by factors of 8.3 and 4.5, respectively. An additional twofold protection of DNA in fully condensed chromatin occurred in the intact cell nucleus. Since this protection did not appear to involve chromatin structure, we speculate that this additional protection may result from the association of soluble protein and nonprotein sulfhydryls with DNA in the intact cell nucleus. The results are consistent with the organization of nuclear DNA into both basic nucleosome repeat structure and higher-order chromatin structure providing significant protection against DSB induction.

Animals↗

PubMed1995

A novel cis-acting centromeric DNA element affects S. pombe centromeric chromatin structure at a distance.

The chromatin structure of the central core region of Schizosaccharomyces pombe centromeric DNA is unusual. This distinctive chromatin structure is associated only with central core sequences in a functional context and is modulated by a novel cis-acting DNA element (centromere enhancer) within the functionally critical K centromeric repeat, which is found in multiple copies in all three S. pombe centromeres. The centromere enhancer alters central core chromatin structure from a distance and in an orientation-independent manner without altering the nucleosomal packaging of sequences between the enhancer and the central core. These findings suggest a functionally relevant structural interaction between the enhancer and the centromeric central core brought about by DNA looping.

Blotting, Southern↗

PubMed1997

Ligation-mediated PCR for chromatin-structure analysis of interphase and metaphase chromatin.

Chromatin structure is becoming increasingly recognized as important for a full understanding of gene function and cell memory. With regard to cell memory, which involves the transfer of chromatin-encoded epigenetic information from one cell generation to another, the detailed structure of metaphase chromatin is of crucial importance. In this paper we describe methods for the use of dimethyl sulfate, DNase I, and potassium permanganate for in vivo footprinting and chromatin analysis, with special emphasis on studies of metaphase cells. We review the use of ligation-mediated PCR for the analysis of chromatin, including the human phosphoglycerate kinase promoter, and also report initial studies of a matrix attachment region near the human beta-interferon gene.

Cell Line↗

PubMed1985

Evidence for chromatin structure as a regulatory determinant in HLA-DR alpha gene expression.

We examined the possibility that one mechanism for controlling HLA-DR alpha gene expression involves the alteration of chromatin structure. Chromatin structure was analyzed by measuring the susceptibility of DR alpha genes in intact nuclei to nuclease treatment. We first examined a somatic cell hybrid of a T-lymphoblastoid cell line (LCL) and a B-LCL, since the DR alpha gene, which is inactive in the T-LCL parent, is expressed in the hybrid, thus providing a system to study DR alpha gene induction. The hybrid line 174 X CEM.T1 contains and expresses solely the DR alpha gene from the T-LCL parent, since the DR alpha gene from the B-LCL parent, 174, is deleted. Using cytoplasmic dot blot analysis and RNA-DNA Northern hybridization, we detected DR alpha-specific transcripts in the hybrid, but not in the parental lines, indicating activation of the DR alpha gene in the hybrid. The transcribed DR alpha gene from the hybrid was compared with the untranscribed gene from the T-LCL parental line, and an association between DR alpha gene expression and increased sensitivity to DNase I was observed. A switch in the chromatin structure of the DR alpha gene from a closed to an open configuration apparently occurred in this hybrid. Such a change is associated with DR alpha gene expression. Comparison of a DR-positive B-LCL and an isogenic DR-negative T-LCL also showed that the chromatin of the former is more sensitive to DNase I digestion. There were no restriction enzyme fragment length differences between the DR alpha genes from 174 X CEM.T1 and CEMR, indicating that the process of somatic cell hybridization did not result in DNA rearrangement or translocation.

Cell Line↗

PubMed1985

The effect of salt extraction on the structure of transcriptionally active genes; evidence for a DNAseI-sensitive structure which could be dependent on chromatin structure at levels higher than the 30 nm fibre.

The procedure developed by Lawson and Cole (Biochemistry, 1979, 18 2161-2166) for removing lysine-rich histones from nuclei at low pH also quantitatively extracts proteins HMG14 and 17. The effect of this low pH extraction on the DNAseI-sensitive structures of active genes in avian red blood cells has been investigated. No major perturbation of a developmentally regulated DNAseI hypersensitive site in the beta-globin domain and at the 5' end of the alpha D gene was seen. The overall DNAseI-sensitive conformation of the beta A-globin gene (relative to the ovalbumin gene) is minimally affected by pH3 salt extraction, but there is some loss of sensitivity of the alpha D gene. Removal of HMG proteins at neutral pH had no effect on the sensitivity of active genes in erythroid or fibroblast nuclei. These results, together with those carried out on DNAseI sensitivity and HMG binding to monomer nucleosomes, indicate that there is a major structural feature of active genes responsible for DNAseI-sensitivity which is independent of HMG proteins or nucleosome core particle structure but may be dependent on higher order chromatin structures.

Animals↗