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PubMed1989

Chromatin structure of transposon Tn903 cloned into a yeast plasmid.

Transposon Tn903 contains the APH gene for kanamycin resistance, which is active in yeast [A. Jiménez and J. Davies (1980) Nature (London) 287, 869-871] and is flanked by two inverted repeats (IR) 1057 bp long. When plasmid pAJ50, carrying Tn903 and the 2-microns circle origin of replication, is cloned into Saccharomyces cerevisiae, nucleosomes are assembled in vivo on the prokaryotic DNA of the transposon. Indirect end labeling revealed that three nucleosomes are preferentially positioned on symmetrical sequences from both IRs. DNase I digestion also confirmed that the chromatin structure is symmetrical in both IRs. This suggests that sequence determinants are decisive for chromatin structure in these regions. We have calculated the rotational and translational fits [H. R. Drew and C. R. Calladine (1987) J. Mol. Biol. 195, 143-173] for the Tn903 sequence and the results indicate that the nucleosome positioning on the IRs is sequence-directed. Nucleosome deposition on the APH gene also occurs, but no clear positioning exists. Some sequence preference for positioning nucleosomes on the promoter can be predicted, especially from the translational fit. Experimental data indicate, however, that nucleosomes are absent from the promoter. Therefore, chromatin can be organized on prokaryotic DNA in a manner that resembles the typical eukaryotic chromatin structure.

Chromatin↗
PubMed1984

Changes in histone H1 content and chromatin structure of cells blocked in early S phase by 5-fluorodeoxyuridine and aphidicolin.

We have measured changes in histone H1 content and changes in chromatin structure of Chinese hamster (line CHO) cells blocked in early S phase by sequential use of isoleucine deprivation and blockade with 5-fluorodeoxyuridine or aphidicolin. Both the H1:core histone ratio in isolated nuclei and the H1 content of the cell are reduced 20-60%, depending on the duration of the block. The new deoxyribonucleic acid (DNA) synthesized during S-phase block has a shorter nucleosome repeat length than that of bulk chromatin, but it is nearly equally resistant as bulk DNA to attack by micrococcal nuclease. During the time that H1 content is decreasing, bulk chromatin also undergoes structural changes so that its nucleosome cores appear to be more closely packed along the DNA chain. The losses in H1 content and changes in chromatin structure are similar to those reported for cells blocked in early S phase by hydroxyurea [D'Anna, J. A., & Prentice, D. A. (1983) Biochemistry 22, 5631-5640]. The results suggest that losses of H1 and changes in chromatin structure are general events which occur when the elongation of initiated replicons or the joining of intermediate-sized DNA fragments is retarded during replication. They are consistent with the notions that H1 is lost from initiated replicons and/or the loss of H1 is part of an alarm response in the cell which might facilitate events leading to gene amplification.

Animals↗
PubMed1998

Permeabilized Arabidopsis protoplasts provide new insight into the chromatin structure of plant alcohol dehydrogenase genes.

New data from permeabilized protoplasts have expanded our view of the 5'DNase I hypersensitive area of the Arabidopsis Adh gene derived from nuclei. DNase I hypersensitivity analyses conducted with permeabilized protoplasts from Arabidopsis cell cultures indicates that there are four distinct sites of hypersensitivity centered around positions -425, -325, -200, and -60. The hypersensitive site at -200 coincides with an in vitro hypersensitive site created by purified transcription factors bound to a G-box element. The G-box is a functional cis element that plays a role in the signal transduction of hypoxia and other stresses in Adh. The data presented in this paper support the notion that G-box-related elements may also play a role in defining chromatin structure. The new Arabidopsis data are discussed within the context of what is known about the chromatin structures and regulation of two other plant Adh genes; maize Adh1 and Adh2. The chromatin of the maize Adh1 promoter is divided into a region that is constitutively hypersensitive to DNase I (-700 to -160) and an inducibly hypersensitive region (-140 and -40). There are several sequence elements within the hypersensitive regions bound by proteins in vivo. The anaerobic response element is the most well characterized and functions in the detection of hypoxia. The maize Adh2 gene promoter is constitutively hypersensitive to DNase I, with the exception of a small region that extends to include the TATA box as the gene becomes active. Several cis elements in the Adh2 promoter are bound by factors in vivo. One, at -160, is a functional element that acts as an activator in vascular tissue. The overall goal of our research with the Adh genes from maize and Arabidopsis is to gain further insight into the relationships between the regulation of gene transcription and chromatin structure in plants as it is clear that all the necessary components that characterize regulated gene activity may not be found simply by elucidating the linear sequence of nucleotides that lie 5' to the protein coding regions and finding proteins capable of binding the promoter in vitro.

Alcohol Dehydrogenase↗
PubMed1988

Studies of acetylcholine receptor subunit gene expression: chromatin structural changes during myogenesis.

Myogenesis proceeds stepwise from pluripotential stem cell to differentiated myotube. The precise number of transitions that occur along the developmental pathway remains to be determined. We examined the myogenic pathway as modelled by mouse mesodermal stem cell and muscle cell lines for stage-specific alterations in the chromatin structure of the acetylcholine receptor delta and gamma subunit genes. We reasoned that such an analysis would allow us to observe either the primary events in the activation of these muscle-specific genes or processes secondary to the binding of muscle-specific regulatory proteins. We probed chromatin structure with DNase I (deoxyribonuclease I) and precisely mapped to the 5' ends of the delta and gamma genes DNase I hypersensitive (DH) sites whose induction is unique to each myogenic stage. Putative mesodermal stem cells have the simplest pattern of DH sites with no sites near the 5' ends of the delta and gamma genes, whereas differentiated myotubes express the most complex pattern; the myoblast pattern is intermediate and of two types. In muscle cell lines where differentiation must be induced the myoblasts have a simple pattern (one more site than stem cells); in muscle lines where differentiation is spontaneous the myoblasts express a complex pattern of DH sites (one less site than myotubes). Inducible myoblasts seem to be arrested in an earlier step in the myogenic pathway than spontaneously differentiating myoblasts. Thus, myogenic activation of acetylcholine receptor subunit genes appears to be a stepwise process that can be detected by chromatin structural changes specific to four distinct stages of muscle development: stem cell, early myoblast, late myoblast, and differentiated myotube.

Animals↗
PubMed2000

Structure and functional analysis of the human STAT3 gene promoter: alteration of chromatin structure as a possible mechanism for the upregulation in cisplatin-resistant cells.

STAT3 is involved in the signal transduction activated by various cytokines and growth factors. We found that the STAT3 gene is overexpressed in cisplatin-resistant cells. We isolated a genomic fragment containing the 5'-portion of the human STAT3 gene using a bubble PCR method. Using the bubble PCR product as a probe, one genomic clone was isolated. The nucleotide sequence of the first exon and the 1800 base pairs (bps) preceding it was determined. The promoter region of the human STAT3 gene is highly homologous to the corresponding region of the mouse STAT3 gene; several potential factor binding sites such as CRE/ATF, SBE, and GC boxes are also well conserved between human and mouse. A transient expression assay using the luciferase reporter gene showed that the sequence from -403 to +102 possesses maximal promoter activity, and transcription of the STAT3 gene was significantly higher in cisplatin-resistant cells than in parental cisplatin-sensitive cells. Deletion of the region between -261 and -167 resulted in significant loss of promoter activity in both parental and cisplatin-resistant cells. In vivo footprint analysis revealed several protein bindings; however, no significant differences were observed between drug-sensitive and drug-resistant cells. MNase digestion revealed that several open or active nucleosomes were only detected in cisplatin-resistant cells. These results suggest that STAT3 promoter function in a highly structured chromatin environment requires a complex interaction of several transcriptional factors.

Base Sequence↗
PubMed1990

Chromatin structure of Drosophila melanogaster ribosomal genes.

The chromatin structure of ribosomal genes of D. melanogaster has been studied by crosslinking proteins to DNA. We found that a number of histone contacts with DNA through histidine in the approximately 1 kb-long region surrounding the transcription initiation site, coding regions and the region of 240 bp-long repeats from the intergenic spacers (Alu-repeats) were weakened as compared to the inactive chromatin of the type II insertion. A protein with the molecular mass of 50 kDa (p50), associated with all DNA sequences analysed, has been discovered. Another protein with molecular mass of about 70 kDa (p70) has been found to be specific only for the Alu-repeats.

Animals↗
PubMed1983

Chromatin structure of simian virus 40-pBR322 recombinant plasmids in COS-1 cells.

To study the nucleoprotein structure formed by recombinant plasmid DNA in mammalian cells, nuclei were isolated from COS-1 cells after transfection with a recombinant (pJI1) containing pBR322 sequences and a segment of simian virus 40 containing information for a nuclease-sensitive chromatin structure. The nuclei were incubated with DNase I. DNA fragments which were the size of linear pJI1 DNA were isolated, redigested with restriction enzymes, fractionated by electrophoresis, and detected by hybridization with nick-translated segments prepared from the plasmid DNA. Two DNase I-sensitive sites were detected in the simian virus 40 portion of the plasmid at the same sites that were DNase I sensitive in simian virus 40 chromatin prepared late after infection of African green monkey kidney (BSC-1) cells. One site extended from the viral origin of replication to approximately nucleotide 40. The 21-base pair repeated sequences were relatively DNase I resistant. A second site occurred over the single copy of the 72-base pair segment present in this plasmid. These results indicate that the nuclease-sensitive chromatin structure does not depend on the presence of viral structural proteins. In addition, late viral proteins added to pJI1-transfected COS-1 cells by superinfection with simian virus 40 caused no change in the distribution of DNase I-sensitive sites in plasmid chromatin. Analysis of transfected plasmid DNA may provide a general method applicable to the study of the chromatin structure of cloned segments of DNA.

Animals↗
PubMed1996

Three different states of the chromatin structure of the mouse peripherin gene.

The chromatin structure of the mouse peripherin gene domain was analyzed in peripherin-positive and -negative cell lines. At least nine DNase I hypersensitive sites (HSS) are present within the 20-kb peripherin domain in the mouse neuroblastoma cell lines which express peripherin. Three of them are situated in intron I and intron III, the others being distributed within the 5' flanking region up to -5.5 kb. The presence of these sites was also investigated in the peripherin chromatin domain of peripherin-negative cell lines. Two other types of HSS distribution were observed along the peripherin gene according to the category of cell considered: constantly peripherin-negative cells, or negative cells arising from transiently peripherin-expressing precursors. From comparison of HSS patterns in these cell lines with those of neuroblastoma cells, it can be predicted that HSS located in the region -1500/+800 bp participate in cell-specific expression of the mouse peripherin gene.

3T3 Cells↗
PubMed1998

Role of histone H1 as an architectural determinant of chromatin structure and as a specific repressor of transcription on Xenopus oocyte 5S rRNA genes.

We explore the role of histone H1 as a DNA sequence-dependent architectural determinant of chromatin structure and of transcriptional activity in chromatin. The Xenopus laevis oocyte- and somatic-type 5S rRNA genes are differentially transcribed in embryonic chromosomes in vivo depending on the incorporation of somatic histone H1 into chromatin. We establish that this effect can be reconstructed at the level of a single nucleosome. H1 selectively represses oocyte-type 5S rRNA genes by directing the stable positioning of a nucleosome such that transcription factors cannot bind to the gene. This effect does not occur on the somatic-type genes. Histone H1 binds to the 5' end of the nucleosome core on the somatic 5S rRNA gene, leaving key regulatory elements in the promoter accessible, while histone H1 binds to the 3' end of the nucleosome core on the oocyte 5S rRNA genes, specifically blocking access to a key promoter element (the C box). TFIIIA can bind to the somatic 5S rRNA gene assembled into a nucleosome in the presence of H1. Because H1 binds with equivalent affinities to nucleosomes containing either gene, we establish that it is the sequence-selective assembly of a specific repressive chromatin structure on the oocyte 5S rRNA genes that accounts for differential transcriptional repression. Thus, general components of chromatin can determine the assembly of specific regulatory nucleoprotein complexes.

Animals↗
PubMed1996

In vivo chromatin structure of the murine interleukin-5 gene region: a new intact cell system.

The study of chromatin involvement in the regulation of gene expression has traditionally required the isolation of nuclei. However, cell fractionation techniques are subject to redistribution of proteins during the isolation procedure, which prevents rigorous physiologically relevant analysis. To eliminate the need to isolate nuclei and to analyze chromatin structures in vivo in response to agents regulating murine interleukin-5 (IL-5) gene activation, we have established a novel lysolecithin permeabilized intact cell system for suspension cell types, in this case T cells. Nuclear integrity of permeabilized cells is demonstrated by nuclear transport assays using confocal laser scanning microscopy. Results are identical in unstimulated and stimulated T cells, indicating that the chromatin structure after activation is not the result of gross alterations in nuclear protein transport properties. Potential new IL-5 gene regulatory regions are identified by DNase I hypersensitivity mapping. Our lysolecithin permeabilized intact cell system is amenable to physiologically relevant analysis of responses to signaling pathways at the level of chromatin, nuclear protein translocation and possibly other cellular functions in a variety of suspension and adherent cell types.

Animals↗
PubMed1995

Stimulation of transcription accompanying relaxation of chromatin structure in cells overexpressing high mobility group 1 protein.

We developed murine C-127 cell lines that stationarily overexpress high mobility group (HMG) proteins 1 and 2 by transfecting them with the bovine papilloma virus plasmid carrying their respective cDNA sequences. Using these cell lines, we examined the effects of these HMG proteins on the modulation of chromatin structure that accompanied transcription. The levels of HMG1 mRNA and protein in cells overexpressing HMG1 protein were enhanced about 7- and 3-fold, respectively, in comparison with control cells, whereas those in cells overexpressing HMG2 protein were enhanced about 17- and 9-fold. The expression of reporter genes transfected into the cells was enhanced approximately 2-fold in cells overexpressing HMG1, but not HMG2, in comparison with those in control cells, irrespective of the sources of the genes and promoters. The minichromosome derived from the reporter plasmid in cells overexpressing HMG1 protein was more susceptible to micrococcal nuclease digestion than those in cells overexpressing HMG2 protein and control cells. The enhanced accessibility to micrococcal nuclease was not restricted to the expressing gene and promoter but involved the entire minichromosome, suggesting that the enhancement of gene expression resulted from changes in the condensation of the entire minichromosomal region by HMG1 protein. Minichromosomes in cells overexpressing HMG contained enhanced amounts of the respective HMG proteins and simultaneously reduced amounts of histone H1s. These results suggest that HMG1 and -2 proteins have different functions in the modulation of chromatin structure, and that HMG1 protein may sustain the structure of the respective gene to ensure that its activity as a template is expressed fully. These observations on the modulation of chromatin structure accompanying gene transcription in cells overexpressing HMG protein may provide important information on the function of these proteins.

Animals↗
PubMed1995

Programming of a repressed but committed chromatin structure during early development.

The determination of chromatin for transcription during early development as well as the requirement for trans-acting factors during this period has been analysed in Xenopus. Basal transcription is repressed both during oogenesis and after the mid-blastula transition (MBT), and transactivators are required to relieve this repression. In contrast, transactivators cannot overcome the generalized transcriptional repression which occurs in embryos before MBT. However, they do bind to promoters leading to a repressed but preset chromatin structure. Experiments involving the pre-binding of TATA binding protein (TBP) or of the strong transactivator GAL4-VP16 further show that there is no limiting factor before the MBT, and that it is the recruitment and stabilization of the basal transcription machinery and not of transactivators which is repressed during early development. This multi-step process in gene activation, with activation of promoters temporally uncoupled from their commitment, may be of importance in the regulation of early embryonic events by providing molecular signposts for future determinations.

Animals↗
PubMed1983

Modulation of chromatin structure associated with derepression of the acid phosphatase gene of Saccharomyces cerevisiae.

We have analyzed the chromatin structure of a phosphate-repressible acid phosphatase gene (PHO5) within yeast nuclei. Under derepressed conditions (low Pi media), the gene is much more sensitive to either DNAse I or micrococcal nuclease digestion than is the repressed gene. We have mapped DNase I hypersensitive sites unique to the active gene near the 5'-end of the acid phosphatase mRNA and within a region presumed to function in the regulation of the gene by Pi. Although the gene is packaged into regularly spaced nucleosomes, no detectable phase relationship exists between nucleosomes and DNA sequence under derepressed conditions, whereas in the repressed state the nucleosomes occur in one predominant phase. These results demonstrate reversible changes in the chromatin structure of a eukaryotic gene system that directly correlate with the functional state of the gene.

Acid Phosphatase↗
PubMed1992

Different chromatin structures along the spacers flanking active and inactive Xenopus rRNA genes.

The accessibility of DNA in chromatin to psoralen was assayed to compare the chromatin structure of the rRNA coding and spacer regions of the two related frog species Xenopus laevis and Xenopus borealis. Isolated nuclei from tissue culture cells were photoreacted with psoralen, and the extent of cross-linking in the different rDNA regions was analyzed by using a gel retardation assay. In both species, restriction fragments from the coding regions showed two distinct extents of cross-linking, indicating the presence of two types of chromatin, one that contains nucleosomes and represents the inactive gene copies, and the other one which is more cross-linked and corresponds to the transcribed genes. A similar cross-linking pattern was obtained with restriction fragments from the enhancer region. Analysis of fragments including these sequences and the upstream portions of the genes suggests that active genes are preceded by nonnucleosomal enhancer regions. The spacer regions flanking the 3' end of the genes gave different results in the two frog species. In X. borealis, all these sequences are packaged in nucleosomes, whereas in X. laevis a distinct fraction, presumably those flanking the active genes, show a heterogeneous chromatin structure. This disturbed nucleosomal organization correlates with the presence of a weaker terminator at the 3' end of the X. laevis genes compared with those of X. borealis, which allows polymerases to transcribe into the downstream spacer.

Animals↗
PubMed2002

Effects of fast neutrons on chromatin: dependence on chromatin structure.

The effects of fast neutrons (10-100 Gy) on chromatin extracted from normal (liver of Wistar rats) and tumor (Walker carcinosarcoma maintained on Wistar rats) tissues were compared. The spectroscopic assays used were (i) chromatin intrinsic fluorescence, (ii) time-resolved fluorescence of chromatin - proflavine complexes, and (iii) fluorescence resonance energy transfer (FRET) between dansyl chloride and acridine orange coupled to chromatin. For both normal and tumor chromatin, the intensity of intrinsic fluorescence specific for acidic and basic proteins decreased with increasing dose. The relative contributions of the excited-state lifetime of proflavine bound to chromatin were reduced upon fast-neutron irradiation, indicating a decrease in the proportion of chromatin DNA available for ligand binding. The Forster energy transfer efficiencies were also modified by irradiation. These effects were larger for chromatin from tumor tissue. In the range 0-100 Gy, fast neutrons induced alterations in DNA and acidic and basic proteins, as well as in global chromatin structure. The radiosensitivity of chromatin extracted from tumor tissue seems to be higher than that of chromatin extracted from normal tissue, probably because of its higher euchromatin (loose)--heterochromatin (compact) ratio.

Algorithms↗
PubMed1996

A developmentally modulated chromatin structure at the mouse immunoglobulin kappa 3' enhancer.

Transcription of the mouse immunoglobulin kappa gene is controlled by two enhancers: the intronic enhancer (Ei) that occurs between the joining (J kappa) and constant (C kappa) exons and the 3' enhancer (E3') located 8.5 kb downstream of the gene. To understand the role of E3' in the activation of the mouse immunoglobulin kappa gene, we studied its chromatin structure in cultured B-cell lines arrested at various stages of differentiation. We found that 120 bp of the enhancer's transcriptional core becomes DNase I hypersensitive early in B-cell development. Genomic footprinting of pro-B and pre-B cells localized this chromatin alteration to B-cell-specific protections at the region including the direct repeat (DR) and the sequence downstream of the DR (DS), the PU.1-NFEM-5 site, and the core's E-box motif, identifying bound transcription factors prior to kappa gene rearrangement. Early footprints were, however, not detected at downstream sites proposed to play a negative role in transcription. The early chromatin structure persisted through the mature B-cell stage but underwent a dramatic shift in plasma cells, correlating with the loss of guanosine protection within the DR-DS junction and the appearance of novel footprints at a GC-rich motif upstream and the NF-E1 (YY1/delta)-binding site downstream. Gel shift analysis demonstrated that the DR-DS junction is bound by a factor with properties similar to those of BSAP (B-cell-specific activator protein). These results reveal developmental-stage-specific changes in the composition of nuclear factors bound to E3', clarify the role of factors that bind constitutively in vitro, and point to the differentiation of mature B cells to plasma cells as an important transitional point in the function of this enhancer. The observed changes in nuclear factor composition were accompanied by the rearrangement of positioned nucleosomes that flank the core region, suggesting a role for both nuclear factors and chromatin structure in modulating kappa E3' function during B-cell development. The functional implications of the observed chromatin alterations are discussed in the context of recent studies on kappa E3' and the factors that bind to it.

Animals↗
PubMed2006

Common chromatin structures at breakpoint cluster regions may lead to chromosomal translocations found in chronic and acute leukemias.

The t(9;22) BCR/ABL fusion is associated with over 90% of chronic myelogenous and 25% of acute lymphocytic leukemia. Chromosome 11q23 translocations in acute myeloid and lymphoid leukemia cells demonstrate myeloid lymphoid leukemia (MLL) fusions with over 40 gene partners, like AF9 and AF4 on chromosomes 9 and 4, respectively. Therapy-related leukemia is associated with the above gene rearrangements following the treatment with topoisomerase II (topo II) inhibitors. BCR, ABL, MLL, AF9 and AF4 have defined patient breakpoint cluster regions. Chromatin structural elements including topo II and DNase I cleavage sites and scaffold attachment sites have previously been shown to closely associate with the MLL and AF9 breakpoint cluster regions, implicating these elements in non-homologous recombination (NHR). In this report, using cell lines and primary cells, chromatin structural elements were analyzed in BCR, ABL and AF4 and, for comparison, in MLL2, which is a homolog to MLL, but not associated with chromosome translocations. Topo II and DNase I cleavage sites associated with all breakpoint cluster regions, whereas SARs associated with ABL and AF4, but not with BCR. No close breakpoint clustering with the topo II/DNase I sites were observed; however, a statistically significant 5' or 3' distribution of patient breakpoints to the topo II DNase I sites was found, implicating DNA repair and exonucleases. Although MLL2 was expressed in all cell lines tested, except for the presence of one DNAse I site in the promoter, no other structural elements were found in MLL2. A NHR model presented demonstrates the importance of chromatin structure in chromosome translocations involved with leukemia.

Acute Disease↗
PubMed1976

Relaxation of chromatin structure by ethidium bromide binding: determined by viscometry and histone dissociation studies.

The effects of ethidium bromide intercalation on chromatin structure were monitored by viscometry and analysis of histone dissociation. Investigation of the NaCl concentration dependence of chromatin viscosity showed that the reduced viscosity (etared) was very low up to 0.4 M NaCl and increased gradually when the salt concentration was raised further. In chromatin intercalated by ethidium bromide, etared was not significantly different at low salt concentrations (up to 0.2 M NaCl). However, when the salt concentration was raised further, the viscosity response curve increased sharply to reach viscosities about 4-5 times higher than those for nonintercalated chromatin. The increase in viscosity was proportional to the increase in fluorescence intensity, when the ratio of ethidium bromide to DNA mucleotide was raised. The transition of intercalated chromatin into the relaxed form was reversible, dependent on the nature of the electrolyte and cooperative, as indicated by the small increase in salt concentration required to obtain chromatin relaxation. Investigation of the NaCl concentration dependence of histone dissociation revealed that total histones and each individual histone fraction were released from intercalated chromatin at much reduced NaCl concentrations. The midpoints of the dissociation curves of the individual histones ranged from 0.30 to 0.45 M NaCl and fell within the same range where the drastic viscosity change occurred. These results indicate that intercalation of ethidium bromide labilizes chromatin structure to relaxation by moderately elevated salt concentrations. It is suggested that the labilization is caused by changes in the DNA helix conformation due to dye intercalation decreasing the stability of histone-DNA interactions.

Animals↗