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PubMed2003

Effects of genomic context and chromatin structure on transcription-coupled and global genomic repair in mammalian cells.

It has been long recognized that in mammalian cells, DNA damage is preferentially repaired in the transcribed strand of transcriptionally active genes. However, recently, we found that in Chinese hamster ovary (CHO) cells, UV-induced cyclobutane pyrimidine dimers (CPDs) are preferentially repaired in both the transcribed and the non-transcribed strand of exon 1 of the dihydrofolate reductase (DHFR) gene. We mapped CPD repair at the nucleotide level in the transcriptionally active DHFR gene and the adjacent upstream OST gene, both of which have been translocated to two chromosomal positions that differ from their normal endogeneous positions. This allowed us to study the role of transcription, genomic context and chromatin structure on repair. We found that CPD repair in the transcribed strand is the same for endogenous and translocated DHFR genes, and the order of repair efficiency is exon 1 > exon 2 > exon 5. However, unlike the endogenous DHFR gene, efficient repair of CPDs in the non-transcribed strand of exon 1 is not observed in the translocated DHFR gene. CPDs are efficiently repaired in the transcribed strand in endogenous and translocated OST genes, which indicates that efficient repair in exon 1 of the non-transcribed strand of the endogenous DHFR gene is not due to the extension of transcription-coupled repair of the OST gene. Using micrococcal nuclease digestion, we probed the chromatin structure in the DHFR gene and found that chromatin structure in the exon 1 region of endogenous DHFR is much more open than at translocated loci. These results suggest that while transcription-coupled repair is transcription dependent, global genomic repair is greatly affected by chromatin structure.

Animals↗
PubMed1994

Chromatin structure modulation in Saccharomyces cerevisiae by centromere and promoter factor 1.

CPF1 is an abundant basic-helix-loop-helix-ZIP protein that binds to the CDEI motif in Saccharomyces cerevisiae centromeres and in the promoters of numerous genes, including those encoding enzymes of the methionine biosynthetic pathway. Strains lacking CPF1 are methionine auxotrophs, and it has been proposed that CPF1 might positively influence transcription at the MET25 and MET16 genes by modulating promoter chromatin structure. We test this hypothesis and show that the regions surrounding the CDEI motifs in the MET25 and MET16 promoters are maintained in a nucleosome-free state and that this requires the entire CPF1 protein. However, the chromatin structure around the CDEI motifs does not change on derepression of transcription and does not correlate with the methionine phenotype of the cell. An intact CDEI motif but not CPF1 is required for transcriptional activation from a region of the MET25 upstream activation sequence. Our results suggest that CPF1 functions to modulate chromatin structure around the CDEI motif but that these changes at the MET25 and MET16 promoters do not explain how CPF1 functions to maintain methionine-independent growth. The presence of CPF1-dependent chromatin structures at these promoters leads to a weak repression of transcription.

Amino Acid Sequence↗
PubMed1987

Site-specific demethylation and normal chromatin structure of the human dihydrofolate reductase gene promoter after transfection into CHO cells.

The effect of in vitro methylation on the function and chromatin structure of the human dihydrofolate reductase (DHFR) promoter linked to the DHFR coding sequences (minigene) was studied after DNA-mediated gene transfer into DHFR- CHO cells. Methylation of HhaI sites reduced the transforming frequency to about 10% of control, whereas methylation of HpaII sites had a less significant effect. The integrated genes were demethylated at specific sites in the promoter sequence, namely, HpaII sites around -57 base pairs from the major start site for transcription and HhaI sites around +9, +24, or both. All other HpaII or HhaI sites in the DHFR coding region or in the plasmid sequences remained consistently methylated. The DHFR minigene, after methylation with HhaI methylase, was also introduced without selection by cotransfection with pSV2neo and selection for neor clones in G418. Preferential demethylation of the same sites was observed even without selection for the DHFR+ phenotype. Analysis of the chromatin structure of the integrated minigene revealed characteristic proximal and distal hypersensitive regions of the promoter, as previously observed in human cells. Correctly initiated DHFR mRNA was detected in all of the transformants studied. These results suggest that formation of the characteristic chromatin structure is an intrinsic property of the DHFR promoter sequence and that demethylation of specific sites accompanies gene expression.

Animals↗
PubMed2003

Role of the promoter in maintaining transcriptionally active chromatin structure and DNA methylation patterns in vivo.

Establishment and maintenance of differential chromatin structure between transcriptionally competent and repressed genes are critical aspects of transcriptional regulation. The elements and mechanisms that mediate formation and maintenance of these chromatin states in vivo are not well understood. To examine the role of the promoter in maintaining chromatin structure and DNA methylation patterns of the transcriptionally active X-linked HPRT locus, 323 bp of the endogenous human HPRT promoter (from position -222 to +102 relative to the translation start site) was replaced by plasmid sequences by homologous recombination in cultured HT-1080 male fibrosarcoma cells. The targeted cells, which showed no detectable HPRT transcription, were then assayed for effects on DNase I hypersensitivity, general DNase I sensitivity, and DNA methylation patterns across the HPRT locus. In cells carrying the deletion, significantly diminished DNase I hypersensitivity in the 5' flanking region was observed compared to that in parental HT-1080 cells. However, general DNase I sensitivity and DNA methylation patterns were found to be very similar in the mutated cells and in the parental cells. These findings suggest that the promoter and active transcription play a relatively limited role in maintaining transcriptionally potentiated epigenetic states.

Base Sequence↗
PubMed1991

An ectopic copy of the Drosophila ftz associated SAR neither reorganizes local chromatin structure nor hinders elution of a chromatin fragment from isolated nuclei.

In vitro assays using detergent extracted nuclei allow the operational definition of SARs--specific sequences in the chromosome which are thought to interact with a structural matrix or scaffold. This interaction results in the formation of large stable protein-DNA complexes. We have used P-element transformation to introduce a characterized SAR into the Drosophila genome. The standard assay, which uses detergent extracted nuclei, shows that the ectopic SAR is indeed bound to the scaffold. However, in unextracted nuclei, a chromatin fragment containing the SAR sequence is eluted from the nucleus as readily as a fragment which lacks an SAR. Furthermore, an analysis of the accessibility of the neighbouring chromosomal restriction sites in unextracted nuclei indicates that the introduction of this ectopic SAR does not influence the local structure of chromatin. We conclude that the ectopic SAR site is not attached to a nuclear scaffold or matrix in vivo.

Animals↗
PubMed2005

Psoralen photocrosslinking, a tool to study the chromatin structure of RNA polymerase I--transcribed ribosomal genes.

The chromatin structure of RNA polymerase I--transcribed ribosomal DNA (rDNA) is well characterized. In most organisms, i.e., lower eukaryotes, plants, and animals, only a fraction of ribosomal genes are transcriptionally active. At the chromatin level inactive rDNA is assembled into arrays of nucleosomes, whereas transcriptionally active rDNA does not contain canonical nucleosomes. To separate inactive (nucleosomal) and active (non-nucleosomal) rDNA, the technique of psoralen photocrosslinking has been used successfully both in vitro and in vivo. In Saccharomyces cerevisiae, the structure of rDNA chromatin has been particularly well studied during transcription and during DNA replication. Thus, the yeast rDNA locus has become a good model system to study the interplay of all nuclear DNA processes and chromatin. In this review we focused on the studies of chromatin in ribosomal genes and how these results have helped to address the fundamental question: What is the structure of chromatin in the coding regions of genes?

Chromatin↗
PubMed1999

Effects of H1 histone variant overexpression on chromatin structure.

The importance of histone H1 heterogeneity and total H1 stoichiometry in chromatin has been enigmatic. Here we report a detailed characterization of the chromatin structure of cells overexpressing either H1(0) or H1c. Nucleosome spacing was found to change during cell cycle progression, and overexpression of either variant in exponentially growing cells results in a 15-base pair increase in nucleosome repeat length. H1 histones can also assemble on chromatin and influence nucleosome spacing in the absence of DNA replication. Overexpression of H1(0) and, to a lesser extent, H1c results in a decreased rate of digestion of chromatin by micrococcal nuclease. Using green fluorescent protein-tagged H1 variants, we show that micrococcal nuclease-resistant chromatin is specifically enriched in the H1(0) variant. Overexpression of H1(0) results in the appearance of a unique mononucleosome species of higher mobility on nucleoprotein gels. Domain switch mutagenesis revealed that either the N-terminal tail or the central globular domain of the H1(0) protein could independently give rise to this unique mononucleosome species. These results in part explain the differential effects of H1(0) and H1c in regulating chromatin structure and function.

3T3 Cells↗
PubMed1988

In vitro and in vivo activation of B-lymphocytes: a flow cytometric study of chromatin structure employing 7-aminoactinomycin D.

The chromatin structure of a diploid precursor B-cell line (REH), in vitro-stimulated normal B-lymphocytes, and reactive and malignant lymph node B-lymphocytes was studied by staining formaldehyde-fixed, permeabilized cells with the DNA-specific fluorophore 7-aminoactinomycin D (7-AMD) and measuring single-cell fluorescence by flow cytometry. Resting peripheral blood B- and T-lymphocytes (G0 cells) bound low amounts of 7-AMD (7-AMD- phenotype), while G1 REH cells and purified B-cells stimulated with anti-mu + B-cell growth factor bound nearly twice as much 7-AMD (7-AMD+ phenotype). 7-AMD binding increased up to threefold and the differences in binding between G0 and G1 cells were nearly abolished when nuclei were isolated prior to fixation or when fixed whole cells were treated with DNase 1. 7-AMD binding increased in parallel with autofluorescence and approximately linearly with time during the G0-G1 transition of in vitro stimulated B-cells, as was determined by simultaneous measurements of 7-AMD fluorescence and autofluorescence or fluorescence of fluorescein isothiocyanate-labeled antibodies to the early activation antigen 4F2 and to the transferrin receptor. In cell suspensions from lymph node biopsies, the 7-AMD+ phenotype was a property of tumor cells in patients with high grade non-Hodgkin's lymphoma (H-NHL, Kiel classification, 5/5); cells with this phenotype were only found in one of nine low grade non-Hodgkin's lymphoma samples (L-NHL, 1/9). The other (8/9) L-NHL samples and the reactive lymph node contained only 7-AMD- cells. All tumors were diploid. The correlation observed between 7-AMD binding and DNase 1 susceptibility of DNA in chromatin (P less than 0.001) suggests that 7-AMD binding is a marker of general transcriptional activity. Surprisingly, the percentage of tumor cells in S phase did not correlate significantly with 7-AMD stainability (P = 0.07), while the light scattering (cell size) of G0/G1 cells was highly correlated to 7-AMD binding (P less than 0.001).

B-Lymphocytes↗
PubMed2003

A human globin enhancer causes both discrete and widespread alterations in chromatin structure.

Gene activation requires alteration of chromatin structure to facilitate active transcription complex formation at a gene promoter. Nucleosome remodeling complexes and histone modifying complexes each play unique and interdependent roles in bringing about these changes. The role of distant enhancers in these structural alterations is not well understood. We studied nucleosome remodeling and covalent histone modification mediated by the beta-globin locus control region HS2 enhancer at nucleosome-level resolution throughout a 5.5-kb globin gene model locus in vivo in K562 cells. We compared the transcriptionally active locus to one in which HS2 was inactivated by mutations in the core NF-E2 sites. In contrast to inactive templates, nucleosomes were mobilized in discrete areas of the active locus, including the HS2 core and the proximal promoter. Large differences in restriction enzyme accessibility between the active and inactive templates were limited to the regions of nucleosome mobilization, which subsumed the DNase I hypersensitive sites. In contrast to this discrete pattern, histone H3 and H4 acetylation and H3 K4 methylation were elevated across the entire active locus, accompanied by depletion of linker histone H1. The coding region of the gene differed from the regulatory regions, demonstrating both nucleosome mobilization and histone hyperacetylation, but lacked differences in restriction enzyme accessibility between transcriptionally active and inactive genes. Thus, although the histone modification pattern we observe is consistent with the spreading of histone modifying activity from the distant enhancer, the pattern of nucleosome mobilization is more compatible with direct contact between an enhancer and promoter.

Acetylation↗
PubMed1983

Chromatin structure and transcriptional activity of an X-linked heat shock gene in drosophila pseudoobscura.

Nuclease digestion of isolated nuclei was used to test whether differential chromatin structure exists for a dosage-compensated heat shock gene in Drosophila pseudoobscura. No differences were observed in nuclease sensitivity at this locus in males and females, either under heat shock or non-heat shock conditions, using micrococcal nuclease or DNase I. Although the higher level of nuclease sensitivity characterized by the induced state was removed when nuclei were prepared in high salt (0.45 M sodium chloride), this procedure did not reveal covert differences in X-linked chromatin structure between males and females. However, a clear difference was observed in the nuclease sensitivity at low level (uninduced) and high level (heat-induced) expression of the X-linked heat shock gene, suggesting that the same gene transcribed at two steady state rates can have different chromatin structures.

Animals↗
PubMed1990

Diabetes-related changes in chromatin structure of brain, liver, and intestinal epithelium.

To determine whether diabetes alters chromatin structure in vivo, fluorometric analysis of alkali-induced DNA unwinding was carried out in various tissues of streptozocin-induced diabetic rats and genetically obese diabetic (db/db) mice. When zero-order kinetics were used to analyze the data, the percentage of double-stranded DNA (%dsDNA) unwinding in brain, liver, and intestinal epithelium of diabetic rats maintained for 4 wk was significantly reduced compared with vehicle-injected control rats (%dsDNA 0.37 +/- 0.05 vs. 0.73 +/- 0.02 for brain, 0.59 +/- 0.1 vs. 0.84 +/- 0.02 for liver, and 0.58 +/- 0.07 vs. 0.90 +/- 0.13 for intestinal epithelium). Insulin treatment of diabetic rats normalized the rate of DNA unwinding in liver (0.82 +/- 0.09 %dsDNA/min) and intestinal epithelium (1.05 +/- 0.09 %dsDNA/min), but the increase in the unwinding rate of brain DNA (0.51 +/- 0.06 %dsDNA/min) did not achieve control values. Similarly, alkali-induced DNA unwinding was significantly slower in brain and liver of db/db mice compared with homozygote controls. When first-order kinetics were used to analyze the data, fractional rate constants of DNA unwinding in brain and liver of diabetic rats or mice were significantly smaller than observed in nondiabetic control animals. The fractional rate constant of DNA unwinding in intestinal epithelium was not altered with diabetes. We conclude that chronic uncontrolled hyperglycemia can alter chromatin structure in vivo.

Animals↗
PubMed1987

Heterogeneity of sperm nuclear chromatin structure and its relationship to bull fertility.

The relationship between sperm nuclear chromatin structure and fertility was evaluated in two groups of Holstein bulls: Group 1, 49 mature bulls, and Group 2, 18 young bulls. Fertility ratings had been estimated for Group 1 and nonreturn rates were known for Group 2. Semen samples were measured by the sperm chromatin structure assay (SCSA): sperm were treated to induce partial in situ DNA denaturation, stained with acridine orange, and evaluated by flow cytometry. Acridine orange intercalated into double-stranded DNA emits green fluorescence upon excitation with 488 nm light, and red fluorescence when associated with single-stranded DNA. An index of DNA denaturation per cell is provided by alpha-t [alpha t = red/(red + green) fluorescence]. The standard deviation (SD alpha t), coefficient of variation (CV alpha t) and proportion of cells outside the main population (COMP alpha t) of the alpha t distribution quantify the extent of denaturation for a sample. Intraclass correlations of the alpha t values were high (greater than or equal to 0.70), based on four collections obtained over several years from Group 1 bulls. Negative correlations were obtained between fertility ratings and both SD alpha t (-0.58, p less than 0.01) and COMP alpha t (-0.40, p less than 0.01) in Group 1, and between nonreturn rates and both SD alpha t (-0.65, p less than 0.01) and COMP alpha t (-0.53, p less than 0.05) in Group 2. These data suggest that the SCSA will be of value for identification of low fertility sires and poor quality semen samples.

Aging↗
PubMedDate not supplied

Chromatin structure and endonuclease sensitivity in human leukemic cell lines.

The relationship between chromatin structure and endonuclease sensitivity was investigated. The cells used in this study were a) human myelogenous leukemic cell lines (HL-60, ML-I, U-937, THP-I) (Group I), which produced internucleosomal DNA cleavage, and b) human T-cell leukemia (MOLT-4), erythroleukemia (K562), glioblastoma (T98G, U87MG) and glioma (KG-1-C) cell lines (Group II), which produced no internucleosomal DNA cleavage, upon treatment with various apoptosis-inducing agents. When the nuclei, isolated from these cells were digested with micrococcal nuclease, chromatin DNA was cleaved into oligonucleosomal units. Although sensitivity to micrococcal nuclease considerably differed from cell to cell, Group I cells were generally more sensitive to micrococcal nuclease digestion than Group II cells. Similar sensitivity to DNase I was observed in both groups of cells. Acid-urea polyacrylamide gel electrophoresis of histone fractions from control and apoptosing HL-60 cells (induced either by hydrogen peroxide or UV irradiation) revealed no significant change in the relative composition of five major histones, indicating the absence of selective degradation of histone HI, but rather the nonspecific degradation of many nuclear proteins. These data suggest a difference in a chromatin structure between Group I and II cells, which might result in the selective production of internucleosomal DNA cleavage only in Group I cells.

Apoptosis↗
PubMed1983

Human fetal to adult hemoglobin switching: changes in chromatin structure of the beta-globin gene locus.

We have investigated the chromatin structure of the chromosomal DNA regions containing the human G gamma-, A gamma-, delta-, and beta-globin structural genes in both fetal and adult erythropoietic tissues and in two human erythroleukemia cells lines before and after induction. Our results indicate that DNase I introduces specific cuts into the beta-globin gene cluster in erythroid cells but not in leukocytes. The predominant sites are located at the 5' sides of the G gamma-, A gamma-, delta-, and beta-globin genes, within 200 base pairs of the respective cap sites. Examination of fetal liver cells has revealed the presence of hypersensitive sites at the 5' side of all four genes, whereas analysis of adult bone marrow has revealed the characteristic sites near the delta- and beta-globin genes but no hypersensitive sites at the 5' termini of the G gamma- or A gamma-globin genes. The presence of delta and beta hypersensitive sites in fetal cells suggests that the increment in expression of the delta and beta genes during development most likely involves the modulation of another pathway to gene expression. Using isolated nuclei from HEL and K562 cells, we have found that the G gamma, A gamma, delta, and beta genes are preferentially sensitive [relative to the pro-alpha2(I) collagen gene] to mild digestion with DNase I, whereas these genes are as resistant as collagen genes in cells that do not express globin. These findings are discussed within the context of chromatin structural correlates of hemoglobin switching.

Adult↗
PubMed1989

[Automated morphometric evaluation of the chromatin structure of liver cell nuclei after vagotomy].

The morphometric analysis of the interphase chromatine structure of the hepatic cells nuclei was carried out on the automated TV installation for the quantitative analysis of images "IBAS-2" (by the OPTON firm, the FRG) according to 50 optical and geometric parameters during various periods (1.2 and 4 weeks) after the vagotomy operation. It is determined that upper-molecular organisation of chromatine undergoes the biggest changes one week after operation, and changes of granular component are more informative than changes of the nongranular component (with the difference 15-20%). It was also revealed that chromatine components differ in tinctorial properties, which are evidently dependent on physicochemical characteristics of the chromatine under various functional conditions of the cell. As a result of the correlation analysis the group of morphometric indices of chromatine structure was revealed, which are highly correlated with level of transcription activity of chromatine during various terms after denervation. The correlation quotient of these parameters is 0.85-0.97. The summing up: vagus denervation of the liver causes changes in the morphofunctional organisation of the chromatine.

Animals↗
PubMed2002

Increased DNA damage in sperm from leukocytospermic semen samples as determined by the sperm chromatin structure assay.

OBJECTIVE: To determine DNA damage as measured by the sperm chromatin structure assay (SCSA) in subsets of human spermatozoa at different stages of maturation in patients who are undergoing infertility evaluation. DESIGN: Prospective study. SETTING: Andrology laboratory at a tertiary care hospital. PATIENT(S): Fifty-six patients undergoing infertility evaluation. Patients with normal semen parameters (n = 17), abnormal semen parameters (n = 29), leukocytospermia (n = 10), and a group of healthy fertile men (n = 18) were included in the study. INTERVENTION(S): None. MAIN OUTCOME MEASURE(S): The shift of green (native DNA) to red (denatured, single-stranded DNA) fluorescence was measured and quantified using the expression alpha(t) (red fluorescence/[red + green fluorescence] per cell). Sperm DNA damage was examined in subsets of spermatozoa isolated by a three-step density gradient. The DNA damage was correlated with classic semen characteristics. RESULT(S): Leukocyte concentration in semen was directly correlated with chromatin alterations in immature and mature sperm. Leukocyte concentration in semen was also directly correlated with immature germ cell concentration and the percentage of abnormal forms in semen. CONCLUSION(S): The increase in chromatin alterations and DNA damage in sperm, as defined by the sperm chromatin structure assay from leukocytospermic samples may be related to alterations in the regulation of spermatogenesis.

Centrifugation, Density Gradient↗
PubMed1985

Chromatin structure of a 3-methylcholanthrene-induced cytochrome P-450 gene.

Plasmids carrying fragments of a cytochrome P-450 gene, inducible by 3-methylcholanthrene, were used to study the chromatin structure of this gene in the liver of normal and carcinogen-treated rats. Digestion with micrococcal nuclease revealed that the gene is not present in the typical 200 base pair nucleosomal structure. By use of indirect end-label hybridization, four DNase I hypersensitive sites were mapped in the 5'-terminal region of the gene. An S1 nuclease sensitive site is located close to a DNase I site. Gene induction by treatment with 3-methylcholanthrene does not result in detectable changes in the DNase I hypersensitive sites. Rat thymus chromatin does not contain DNase I hypersensitive sites in the P-450 gene, suggesting that in the liver the chromatin structure is altered so as to allow tissue-specific expression of the gene. This paper is the first study on the chromatin structure of a gene coding for a member of the cytochrome P-450 family of enzymes. The implications of our results to the understanding of gene regulation of the P-450 genes are discussed.

Animals↗
PubMed2006

Cooperativeness of the higher chromatin structure of the beta-globin locus revealed by the deletion mutations of DNase I hypersensitive site 3 of the LCR.

High-level transcription of the globin genes requires the enhancement by a distant element, the locus control region (LCR). Such long-range regulation in vivo involves spatial interaction between transcriptional elements, with intervening chromatin looping out. It has been proposed that the clustering of the HS sites of the LCR, the active globin genes, as well as the remote 5' hypersensitive sites (HSs) (HS-60/-62 in mouse, HS-110 in human) and 3'HS1 forms a specific spatial chromatin structure, termed active chromatin hub (ACH). Here we report the effects of the HS3 deletions of the LCR on the spatial chromatin structure of the beta-globin locus as revealed by the chromatin conformation capture (3C) technology. The small HS3 core deletion (0.23 kb), but not the large HS3 deletion (2.3 kb), disrupted the spatial interactions among all the HS sites of the LCR, the beta-globin gene and 3'HS1. We have previously demonstrated that the large HS3 deletion barely impairs the structure of the LCR holocomplex, while the structure is significantly disrupted by the HS3 core deletion. Taken together, these results suggest that the formation of the ACH is dependent on a largely intact LCR structure. We propose that the ACH indeed is an extension of the LCR holocomplex.

Animals↗