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Results for “Chromatin structure”

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At least 55 records · Page 3Linked to original sources
PubMed1999

Targeted remodeling of human beta-globin promoter chromatin structure produces increased expression and decreased silencing.

The chromatin structure of the human beta-globin gene locus assumes a transcriptionally-active conformation in erythroid cells. One feature of this chromatin reorganization is the formation of DNase 1 hypersensitive sites in the regions of active globin gene promoters. This reorganization requires the globin locus control region and is associated with normal expression of the beta-like globin genes. To determine whether it is possible to artificially enhance the opening of the chromatin structure of a minimal beta-globin promoter, we placed a 101bp, erythroid-specific DNase 1 hypersensitive site-forming element (HSFE) immediately upstream of the beta-globin promoter and gene. This element includes binding sites for NF-E2, AP-1, GATA-1 and Sp-1. Constructs were stably transfected into murine erythroleukemia cells and promoter chromatin structure and gene expression were analyzed. The HSFE induced an area of enhanced DNase 1 hypersensitivity extending from the transcriptional start site to -300bp of the artificial promoter and significantly increased the proportion of beta-globin promoters in an open chromatin configuration. This remodeling of promoter chromatin structure resulted in 3-fold increases in beta-globin gene transcription and induction, and inhibited long-term beta-globin gene silencing. These results indicate that a relatively small cis-acting element is able to enhance remodeling of promoter chromatin structure resulting in increased beta-globin gene expression.

Chromatin↗
PubMed2004

The pituitary-specific transcription factor, Pit-1, can direct changes in the chromatin structure of the prolactin promoter.

The chromatin structure of a promoter is an important determinant of its transcriptional activity. Many promoters are assembled into repressive polynucleosomal arrays that are subsequently remodeled to allow for the activation of gene expression. This study addresses the contribution of a single transcription factor, Pit-1, in orchestrating the chromatin structure of the prolactin gene. Utilizing an in vivo reconstitution system, we found that Pit-1 can bind to multiple sites in the chromatin-assembled 5'-flanking region of the prolactin gene and activate transcription from the chromatin-assembled template. Interestingly, Pit-1 was able to substantially alter micrococcal nuclease digestion of the prolactin 5'-flanking region, and the results are consistent with presence of a translationally positioned nucleosome on the prolactin promoter. Changes in micrococcal nuclease digestion were also observed with a truncated Pit-1 mutant containing only the DNA-binding domain. As the truncation mutant was unable to activate transcription from the chromatin-assembled template, the ability of Pit-1 to alter chromatin structure of the prolactin gene is not dependent on transcriptional activation. We propose that Pit-1 likely plays a role in altering chromatin to facilitate recruitment and subsequent transcriptional activation by additional factors.

5' Flanking Region↗
PubMed1986

The salt dependence of chicken and yeast chromatin structure. Effects on internucleosomal organization and relation to active chromatin.

The ionic strength dependences of yeast and chicken erythrocyte chromatin structure have been examined by analysis of nuclear DNase I and Staphylococcal nuclease digestions done under various salt and divalent cation concentrations. The basic features of yeast DNase I profiles (intracore/intercore patterns and their 5-base pair offset) remain present under all conditions tested. However, there are changes in specific parts of the patterns. In very low salt, the intercore DNase I pattern is enhanced; even very small intercore bands can be detected. Staphylococcal nuclease intracore cleavage becomes prominent. Increasing salt enhances the large DNase I intracore bands and the frequency of spacer cleavage for both nucleases. Thus, yeast has a salt-dependent higher order structure: a chromatin fiber with a prominent spacer/core distinction in (physiological) salt; a fiber with a decreased distinction between spacer and core, i.e. a more uniform fiber, in very low salt. The salt-dependent bulk changes resemble single gene chromatin changes during gene expression and may provide a model for that process. Above bands 16.5-17.5, chicken and yeast intercore patterns are coincident. Thus, at least a fraction of chicken chromatin has discrete length spacers like yeast does. This fraction may be significant, for the prominence of the intercore pattern, and hence the apparent abundance of discrete spacers, can be significantly enhanced by digestion in very low salt. The major differences between the two chromatins are in the intracore/intercore transition region: the region is larger and more complex in chicken; ionic strength changes affect the chicken transition region more strongly. Since this region of the profile corresponds to digestion near the ends of the core, that part of the nucleosome must differ in structure and in conformational flexibility in the two chromatins.

Animals↗
PubMed2001

The gypsy insulator of Drosophila affects chromatin structure in a directional manner.

Chromatin insulators are thought to regulate gene expression by establishing higher-order domains of chromatin organization, although the specific mechanisms by which these sequences affect enhancer-promoter interactions are not well understood. Here we show that the gypsy insulator of Drosophila can affect chromatin structure. The insulator itself contains several DNase I hypersensitive sites whose occurrence is dependent on the binding of the Suppressor of Hairy-wing [Su(Hw)] protein. The presence of the insulator in the 5' region of the yellow gene increases the accessibility of the DNA to nucleases in the promoter-proximal, but not the promoter-distal, region. This increase in accessibility is not due to alterations in the primary chromatin fiber, because the number and position of the nucleosomes appears to be the same in the presence or absence of the insulator. Binding of the Su(Hw) protein to insulator DNA is not sufficient to induce changes in chromatin accessibility, and two domains of this protein, presumed to be involved in interactions with other insulator components, are essential for this effect. The presence of Modifier of mdg4 [Mod(mdg4)] protein, a second component of the gypsy insulator, is required to induce these alterations in chromatin accessibility. The results suggest that the gypsy insulator affects chromatin structure and offer insights into the mechanisms by which insulators affect enhancer-promoter interactions.

Animals↗
PubMed2001

Chromatin structure and phaseolin gene regulation.

Chromatin structure, the organized packaging of DNA with histones in the nucleus, is now seen as a dynamic fabric that changes with development. Here, we use studies on the phaseolin (phas) gene that encodes a seed protein to show how chromatin structure interacts with the transcription machinery to accomplish rigorous spatial regulation of expression. In leaf and other vegetative tissues, a nucleosome is rotationally and translationally positioned over an ensemble of three phased TATA boxes, denying access to TBP. Current interest focuses on the mechanisms by which this architecture is remodeled during embryogenesis. The transcription factor PvALF is intrinsically involved, as are other non-histone proteins and abscisic acid. These concepts, and the possible modular nature of phas expression, are summarized together with speculations concerning the re-establishment of the nucleosome over the phas promoter during terminal stages of embryogenesis.

Chromatin↗
PubMed2025

Chromatin structures from integrated AI and polymer physics model.

The physical organization of the genome in three-dimensional space regulates many biological processes, including gene expression and cell differentiation. Three-dimensional characterization of genome structure is critical to understanding these biological processes. Direct experimental measurements of genome structure are challenging; computational models of chromatin structure are therefore necessary. We develop an approach that combines a particle-based chromatin polymer model, molecular simulation, and machine learning to efficiently and accurately estimate chromatin structure from indirect measures of genome structure. More specifically, we introduce a new approach where the interaction parameters of the polymer model are extracted from experimental Hi-C data using a graph neural network (GNN). We train the GNN on simulated data from the underlying polymer model, avoiding the need for large quantities of experimental data. The resulting approach accurately estimates chromatin structures across all chromosomes and across several experimental cell lines despite being trained almost exclusively on simulated data. The proposed approach can be viewed as a general framework for combining physical modeling with machine learning, and it could be extended to integrate additional biological data modalities. Ultimately, we achieve accurate and high-throughput estimations of chromatin structure from Hi-C data, which will be necessary as experimental methodologies, such as single-cell Hi-C, improve.

Chromatin↗
PubMed1997

Histone gene expression and chromatin structure during spermatogenesis.

The chromatin of male germ cells is restructured throughout spermatogenesis. Analysis of differential histone protein patterns at specific stages of spermatogenesis may contribute towards an understanding of the changes in chromatin structure and function during this differentiation process. The most striking changes in histone patterns occur at the stage of pachytene spermatocytes when most of the linker H1 histones are replaced by the testis specific subtype H1t. In addition, replacement of core histone subtypes is observed at this stage. These structural changes precede the reorganization of chromatin at haploid stages when histones are replaced first by transition proteins and then by protamines.

Animals↗
PubMed1983

DNAase I and cellular factors that affect chromatin structure.

The use of DNAase I as a probe of chromatin structure is frequently fraught with problems of irreproducibility. We have recently evaluated this procedure, documented the sources of the problems, and standardized the method for reproducible results (Prentice and Gurley (1983) Biochim. Biophys. Acta 740, 134-144). We have now used this probe to detect differences in chromatin structure between cells blocked (1) in G1 phase by isoleucine deprivation, or (2) in early S phase by sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea. The cells blocked in G1 phase have easily-digestible chromatin, while cells blocked in early S phase have chromatin which is much more resistant to DNAase I. These differences were found to be the result of diffusible factors found in the cytoplasm and nuclei of G1- and S-phase cells, respectively. The G1 cells contained a cytoplasmic factor which modulates the chromatin structure of S-phase nuclei to a more easily digestible state, while cells blocked in S phase contain a nuclear factor which modulates the chromatin structure of G1 nuclei to a state more resistant to digestion. DNAase I is much more sensitive to these cell cycle-specific chromatin changes than is micrococcal nuclease. The results indicate that, under controlled conditions, DNAase I should be a valuable probe for detecting chromatin structural changes associated with cell cycle traverse, differentiation, development, hormone action and chemical toxicity.

Animals↗
PubMed1998

Coculture of human sperm with bovine oviduct epithelial cells decreases sperm chromatin structural changes seen during culture in media alone.

OBJECTIVE: To compare sperm chromatin structural changes seen in media only culture or in coculture with bovine oviduct epithelial cells. DESIGN: Three freshly ejaculated and three cryopreserved sperm samples in media culture or in oviduct epithelial cell coculture. Sperm in each treatment were evaluated by the sperm chromatin structure assay during a 72-hour time course. SETTING: An academic research laboratory. PATIENT(S): Normospermic donors with children. INTERVENTION(S): Semen collection through masturbation after 48 hours of abstinence. MAIN OUTCOME MEASURE(S): The sperm chromatin structure assay using flow cytometry to detect the susceptibility of sperm in either treatment to denaturation of DNA in situ. RESULT(S): The sperm chromatin structure assay data differed for sperm type (fresh or cryopreserved), over time, and between treatments within 6 hours of culture. In oviduct epithelial cell coculture, fresh sperm chromatin structure assay values for fresh sperm were stable, whereas in control medium higher chromatin degeneration levels were seen by 10 hours. For cryopreserved sperm, chromatin degeneration had increased by 1 hour postthaw in both treatments, although levels were higher in the control treatment thereafter. CONCLUSION(S): Sperm chromatin structural changes occur over time in culture. Such changes were observed within 2 hours for cryopreserved sperm. Coculture of sperm with oviduct epithelial cells results in a stabilizing effect for sperm against chromatin changes.

Animals↗
PubMed2004

Methods for Analyzing the Role of DNA Methylation and Chromatin Structure in Regulating T Lymphocyte Gene Expression.

Chromatin structure, determined in part by DNA methylation, is established during differentiation and prevents expression of genes unnecessary for the function of a given cell type. We reported that DNA methylation and chromatin structure contributes to lymphoid-specific ITGAL (CD11a) and PRF1 (perforin) expression. We used bisulfite sequencing to compare methylation patterns in the ITGAL promoter and 5' flanking region of T cells and fibroblasts, and in the PRF1 promoter and upstream enhancer of CD4+ and CD8+ T cells with fibroblasts. The effects of methylation on promoter function were tested using regional methylation of reporter constructs, and confirmed by DNA methyltransferase inhibition. The relationship between DNA methylation and chromatin structure was analyzed by DNaseI hypersensitivity. Herein we described the methods and results in greater detail.

Journal Article↗
PubMed1995

[The application of halo assay in analysis of cell chromatin structure].

Halo assay, a method for quantitative analysis of chromatin structure in cell, was developed recently. Due to visualization, feasibility, rapidity, and sensitivity of the method, it has been applied in radiobiology and cancer research. According to previous reports, with some improvement, this method has been established in our laboratory. Employing halo assay, the chromatin structure of two kinds of cell strain with similar genetic background and different radiosensitivity was analyzed and the results show that radiosensitive strain has looser chromatin structure than radioresistant one. It is possible that this difference is related to their different radiosensitivity, thus it's a proof for the relation of chromatin structure and radiosensitivity.

Animals↗
PubMed1979

Relaxation of chromatin structure induced by ethidium binding. Involvement of the intercalation process.

In this paper we study the effects of the binding of ethidium on the structure of chromatin, using micrococcal nuclease as a structural probe. This binding induces two structural changes of chromatin either isolated or in the nuclei. (a) An unfolding of the overall structure which results in an activation of the rate of degradation by the nuclease. (b) A disorganisation of the core particle structure which has the effect of unwrapping the DNA from the histone core, this disruption can go on so far as to leave only 90 base pairs. By comparing the bindings of ethidium and tetramethylethidium, we conclude that the first type of structural change is due to an electrostatic effect and does not depend upon intercalation. On the other hand, the second one is due to the intercalation process and to the change of topological constraints on the DNA that such a process involves.

Animals↗
PubMedDate not supplied

An erythroid-specific chromatin opening element reorganizes beta-globin promoter chromatin structure and augments gene expression.

In erythroid tissues the chromatin structure of the beta-globin gene locus is extensively remodeled. Changes include the formation of DNase I hypersensitive sites (HSs) over the promoters of actively expressed genes. To test the hypothesis that such "opening" of promoter chromatin structure is important for beta-globin gene expression, we placed a 101-bp erythroid-specific hypersensitive-site forming element (HSFE) from the core of LCR HS4 immediately upstream of a minimal beta-globin gene promoter. We then studied the effects of this element alone and in combination with other cis-acting elements on globin gene chromatin structure and gene expression in MEL cells and transgenic mice. Single or tandem HSFEs increased the size of the portion of the promoter accessible to DNase digestion, increased the proportion of promoters in an accessible conformation, and increased gene expression approximately 5-fold. These were equivalent to expression levels attained using a 2.8-kb microLCR construct. Inclusion of the LCR HS2 enhancer did not increase expression further. In transgenic mouse fetal liver cells the HSFE increased average expression 2.5-fold compared to the minimal promoter alone. These results indicate that a small cis-acting element is capable of remodeling local beta-globin promoter chromatin structure and producing expression similar to that seen with a microLCR construct.

Animals↗
PubMed1992

Ribosomal gene clusters are uniquely proportioned between open and closed chromatin structures in both tomato leaf cells and exponentially growing suspension cultures.

The accessibility of regulatory molecules to specific DNA sequences and chromatin regions in the nucleus is crucial to gene expression. In this study, we examined the chromatin structure in tomato leaf cells and in exponentially growing tomato cell suspension cultures. The structure of ribosomal chromatin was investigated by micrococcal nuclease and psoralen photocrosslinking. We showed that ribosomal genes in tomato are folded into two distinct types of chromatin: an open chromatin conformation and a closed nucleosomecontaining chromatin. In contrast to previous findings in Friend cells, where half of the ribosomal genes were found to be complexed within an inactive chromatin structure, we demonstrated that the canonical nucleosome-containing chromatin is present in the majority (approximately 80%) of the tomato rRNA-encoding DNA clusters. The minor open chromatin population (approximately 20% of the ribosomal genes) could be detected only after analysis following psoralen crosslinking. The relative amounts of the two ribosomal chromatin structures are similar in stationary and exponentially growing cells. This suggests that the proportions of open and closed chromatin structures present in either stationary or exponentially growing tomato cells are not dependent on the transcriptional process.

Journal Article↗
PubMed2002

Effects of estrogen receptor and estrogen on the chromatin structure in estrogen receptor stable transfectants.

The estrogen receptor (ER), a member of the nuclear hormone receptor superfamily recruits coactivators that modify local chromatin structure. Here we investigated the effect of the estrogen receptor and estrogen on the global chromatin structure and the local chromatin structure of the progesterone receptor gene during the process of transcriptional activation using Rat1+ER cells stably expressing the estrogen receptor. The total chromatin was more accessible to DNasel in Rat1+ER cells than in the parental estrogen receptor-negative Rat1 cells. After 18 h of estrogen treatment, total chromatin was more dispersed in Rat1+ER cells than in Rat1 cells. The chromatin structure of the progesterone receptor gene was more sensitive to DNasel in Rat1+ER cells than in Rat1 cells. However, the chromatin structure of the progesterone receptor gene did not change further on estrogen treatment. Our results suggest that under certain circumstances unoccupied estrogen receptors may play some role in reorganizing the repressive chromatin structure to induce gene activation.

Animals↗
PubMed1984

Gamma radiation as a probe of chromatin structure: damage to and repair of active chromatin in the metaphase chromosome.

Cobalt-60 gamma radiation has been employed as a means of preferentially damaging actively transcribing chromatin within interphase and metaphase Chinese hamster V79-379 lung fibroblasts. The single-strand size distribution and break frequency of bulk 3H-labeled DNA have been compared to those same parameters for active sequences, i.e., sequences complementary to 125I-labeled poly(A+)RNA. The results show that (a) sequences active during interphase are more sensitive than inactive sequences to single-strand break formation by gamma radiation even when the chromatin is condensed in metaphase, (b) repair of strand breaks in the bulk DNA is slower in metaphase than in interphase cells, but (c) during metaphase, repair is faster in active sequences than in the bulk DNA. Furthermore, this study demonstrates that chromatin structure can be probed within intact cells by a method which circumvents isolation of nuclei or chromatin and the use of exogenous nucleases.

Animals↗
PubMed1975

A model for chromatin structure.

A model for chromatin structure is presented. (a) Each of four histone species, H2A (IIbl or f2a2), H2B (IIb2 or f2b), H3 (III or f3) and H4 (IV or f2al) can form a parallel dimer. (b) These dimers can form two tetramers, (H2A)2(H2b)2 and (H3)2(H4)2. (C) These two tetramers bind a segment of DNA and condense it into a "C" segments. (d) The adjacent segments, termed extended or "E" segments, are bound by histone H1 (I or fl) for the major fraction of chromatin; the other "E" regions can be either bound by non-histone proteins or free of protein binding. (e) The binding of histones causes a structural distortion of the DNA which, depending upon the external conditions, may generate the formation of either an open structure with a heterogeneous and non-uniform supercoil or a compact structure with a string of beads. The model is supported by experimental data on histone-histone interaction, histone-DNA interaction and histone subunit-DNA interaction.

Binding Sites↗
PubMed1991

Transactivation functions facilitate the disruption of chromatin structure by estrogen receptor derivatives in vivo.

The activation of gene transcription by nuclear receptors is invariably associated with alterations in chromatin structure at hormone-responsive elements of target genes. To identify the molecular functions underlying receptor-mediated chromatin structure alterations we have evaluated the effects of DNA binding and transactivation of estrogen receptor derivatives on the promoter chromatin structure of estrogen-responsive reporter minichromosomes in Saccharomyces cerevisiae. We report here that the DNase I-hypersensitive chromatin structure at the promoter region is not simply a consequence of estrogen receptor binding to estrogen-responsive elements but is greatly enhanced by transactivation functions. These chromatin structure alterations are dependent on the presence of more than one estrogen-responsive element as well as downstream promoter sequences and appear to be correlated with transcriptional competence of the promoter. Our results imply that a disruption of chromatin structure at promoters is associated with the establishment of active transcription complexes. Since RNA polymerase cannot initiate transcription on nucleosomal DNA in vitro (Lorch, Y., Lapointe, J.W., and Kornberg, R.D. (1987) Cell 49, 203-210) this local disruption of chromatin structure may represent a nucleosome-free window, allowing initiation to occur in vivo.

Base Sequence↗