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At least 271 records · Page 15Linked to original sources
PubMed1991

The influence of chromatin structure on the frequency of radiation-induced DNA strand breaks: a study using nuclear and nucleoid monolayers.

To assess the influence of chromatin structure on the frequency of radiation-induced DNA strand breaks, the alkaline unwinding technique was applied to nuclear and nucleoid monolayers. These chromatin substrates were prepared by treating human fibroblasts grown as monolayers with the nonionic detergent Triton X-100 and varying concentrations of cations. The chromatin structure was modified either by a stepwise removal of DNA-bound proteins by extraction in increasing concentrations of monovalent salt, or by the addition or deletion of mono- and divalent cations to condense or decondense the chromatin, respectively. It was found that the stepwise removal of DNA-bound proteins from the chromatin dramatically increased the frequency of radiation-induced DNA strand breaks. The DNA-bound proteins showed a qualitative difference in their ability to protect the DNA where proteins removed by salt concentrations above 1.0 M exerted the greatest protection. Furthermore, the frequency of radiation-induced DNA strand breaks was found to be 6 times lower in condensed chromatin than in decondensed chromatin and about 80 times lower than in protein-depleted chromatin. It is concluded that the presence of DNA-bound proteins and the folding of the chromatin into higher-order structures protect the DNA against radiation-induced strand breaks.

Cesium Radioisotopes↗
PubMed2001

Ssn6-Tup1 regulates RNR3 by positioning nucleosomes and affecting the chromatin structure at the upstream repression sequence.

The DNA damage inducible gene ribonucleotide reductase (RNR3) is regulated by a transcriptional repression mechanism by the recruitment of the Ssn6-Tup1 corepressor complex to its promoter by the sequence-specific DNA-binding protein Crt1. Ssn6-Tup1 is reported to represses transcription by interfering with transcription factors, recruiting histone deacetylases, and positioning nucleosomes at the promoter of its target genes. Two of the three mechanisms involve effects on chromatin structure, and therefore, we have delineated the nucleosomal structure of RNR3 in the repressed and derepressed state using multiple nuclease mapping strategies. A regular array of positioned nucleosomes is detected over the repressed RNR3 promoter that extends into the coding sequence. Treating cells with DNA damaging agents or deleting CRT1, SSN6, or TUP1 derepresses RNR3 transcription, and causes a dramatic disruption of nucleosome positioning over its promoter. Furthermore, derepression of RNR3 correlated with changes in nuclease sensitivity within the upstream repression sequence (URS) region. Specifically, the loss of a MNase-hypersensitive site, and the appearance of strong DNase I hypersensitivity, was observed over the URS. Interestingly, we find that the binding of Crt1 to the promoter in the absence of Ssn6 or Tup1 is insufficient for nucleosome positioning or regulating chromatin structure at the URS; thus, these two functions are strictly dependent upon Ssn6-Tup1. We propose that RNR3 is regulated by changes in nucleosome positioning and chromatin structure that are mediated by Ssn6, Tup1, and Crt1.

Blotting, Northern↗
PubMed2006

Myc influences global chromatin structure.

The family of myc proto-oncogenes encodes transcription factors (c-, N-, and L-Myc) that regulate cell growth and proliferation and are involved in the etiology of diverse cancers. Myc proteins are thought to function by binding and regulating specific target genes. Here we report that Myc proteins are required for the widespread maintenance of active chromatin. Disruption of N-myc in neuronal progenitors and other cell types leads to nuclear condensation accompanied by large-scale changes in histone modifications associated with chromatin inactivation, including hypoacetylation and altered methylation. These effects are largely reversed by exogenous Myc as well as by differentiation and are mimicked by the Myc antagonist Mad1. The first chromatin changes are evident within 6 h of Myc loss and lead to changes in chromatin structure. Myc widely influences chromatin in part through upregulation of the histone acetyltransferase GCN5. This study provides the first evidence for regulation of global chromatin structure by an oncoprotein and may explain the broad effects of Myc on cell behavior and tumorigenesis.

Acetylation↗
PubMed2006

Epigenetic regulation of chromatin structure and gene function by biotin.

Covalent modifications of histones are a crucial component of epigenetic events that regulate chromatin structures and gene function. Evidence exists that distinct lysine residues in histones are modified by covalent attachment of the vitamin biotin, catalyzed by biotinidase and holocarboxylase synthetase. Biotinylation of histones appears to be conserved across species. The following biotinylation sites were identified using both MS and enzymatic biotinylation of synthetic peptides: K9, K13, K125, K127, and K129 in histone H2A; K4, K9, and K18 in histone H3; and K8 and K12 in histone H4. Evidence was provided that biotinylated histone H4 is enriched in pericentromeric heterochromatin, and that biotinylation of histone H4 participates in gene silencing, mitotic condensation of chromatin, and the cellular response to DNA damage. Biotinylation of histones is a reversible process and depends on the exogenous biotin supply, but the identities of histone debiotinylases remain uncertain. We propose that some effects of biotin deficiency can be attributed to abnormal chromatin structures.

Biotin↗
PubMed2005

Redefining the relationship between sperm deoxyribonucleic acid fragmentation as measured by the sperm chromatin structure assay and outcomes of assisted reproductive techniques.

OBJECTIVE: To test the hypothesis that couples with sperm chromatin structure assay (SCSA) DNA fragmentation index (DFI) values >27% would not achieve pregnancy with assisted reproductive techniques (ART) and to investigate how DFI and high DNA stainability (HDS), as measured by the SCSA, affect fertilization, cleavage, implantation, and pregnancy rates in IVF cycles. DESIGN: Prospective clinical study. SETTING: Academic human reproduction laboratory. PATIENT(S): One hundred couples undergoing IVF with conventional insemination or intracytoplasmic sperm injection. INTERVENTION(S): Testing with SCSA was performed by SCSA Diagnostics (Brookings, South Dakota) on a semen aliquot taken from ejaculate used for ART. MAIN OUTCOME MEASURE(S): Relating total DFI and HDS to conventional semen parameters and cycle-specific outcomes after ART. RESULT(S): Nine of nineteen couples achieved clinical pregnancy when DFI was > or =27%, and 2 of 22 couples achieved clinical pregnancy when DFI was < or =9%. One of nine couples achieved clinical pregnancy with HDS >17%. The DFI was negatively correlated with sperm density (r = -0.23, P<.03) and motility (r = -0.55, P<.00), and HDS was negatively correlated with sperm density (r = -0.37, P<.00). CONCLUSION(S): Sperm chromatin structure assay failed to identify elevated DFI thresholds for negative pregnancy outcome after ART. Patients with low DFI (< or =9%) were least likely to become pregnant, which is also contradictory to SCSA marketing, which states that DFIs of < or =15% have excellent fertility potential. Patients with HDS > or =17% had low pregnancy rates, indicating decreased fertility potential, which deserves further investigation. Larger studies are necessary to confirm that low DFI is associated with decreased fertility and, if proved, might redefine the use of the SCSA in evaluating infertility.

Adult↗
PubMed2002

Differential chromatin structure within a tandem array 100 kb upstream of the maize b1 locus is associated with paramutation.

Recombination mapping defined a 6-kb region, 100 kb upstream of the transcription start site, that is required for B-I enhancer activity and paramutation-a stable, heritable change in transcription caused by allele interactions in maize (Zea mays). In this region, B-I and B' (the only b1 alleles that participate in paramutation) have seven tandem repeats of an 853-bp sequence otherwise unique in the genome; other alleles have one. Examination of recombinant alleles with different numbers of tandem repeats indicates that the repeats are required for both paramutation and enhancer function. The 6-kb region is identical in B-I and B', showing that epigenetic mechanisms mediate the stable silencing associated with paramutation. This is the first endogenous gene for which sequences required for paramutation have been defined and examined for methylation and chromatin structure. The tandem repeat sequences are more methylated in B-I (high expressing) relative to B' (low expressing), opposite of the typical correlation. Furthermore, the change in repeat methylation follows establishment of the B' epigenetic state. B-I has a more open chromatin structure in the repeats relative to B'. The nuclease hypersensitivity differences developmentally precede transcription, suggesting that the repeat chromatin structure could be the heritable imprint distinguishing the two transcription states.

Alleles↗
PubMed1986

An active chromatin structure acquired by translocated c-myc genes.

We used general sensitivity to DNase I digestion to analyze the chromatin structure of c-myc genes in seven murine plasmacytomas. In every case, the 3' portion of c-myc juxtaposed with C alpha displayed a much more DNase I-sensitive chromatin structure than untranslocated c-myc or, in one case analyzed, the reciprocally translocated 5' portion. Our data suggest the presence of regulatory sequences near the C alpha gene segment.

Animals↗
PubMed1996

Introduction of a DNA methyltransferase into Drosophila to probe chromatin structure in vivo.

The dam DNA methyltransferase gene from Escherichia coli was introduced into Drosophila in order to probe chromatin structure in vivo. Expression of the gene caused no visible defects or developmental delay even at high levels of active methylase. About half of each target site was found to be methylated in vivo, apparently reflecting a general property of chromatin packaged in nucleosomes. Although site-specific differences were detected, most euchromatic and heterochromatic sites showed comparable degrees of methylation, at least at high methylase levels. Methylase accessibility of a lacZ reporter gene subject to position-effect variegation throughout development was only slightly reduced, consistent with studies of chromatin accessibility in vitro. Silencing of lacZ during development differed from silencing of an adjacent white eye pigment reporter gene in the adult, as though chromatin structure can undergo dynamic alterations during development.

Animals↗
PubMed1985

Cell cycle variations in chromatin structure detected by DNase I.

We have recently developed a reproducible method for the use of DNase I as a sensitive probe of chromatin structure (Prentice, D A & Gurley, L R, Biochim biophys acta 740 (1983) 134) [12] and have used this probe to investigate chromatin structure during the interphase of the cell cycle. Chinese hamster cells (line CHO) were synchronized by: (1) mitotic detachment, to obtain M-phase cells; (2) isoleucine deprivation, to obtain G1-phase cells; and (3) sequential use of isoleucine deprivation followed by release into the presence of hydroxyurea, to obtain cells blocked at the start of S phase. The cells were released from the various blocking schemes and nuclei were isolated and digested with DNase I at various times. The digestion kinetics were monitored to detect possible changes in chromatin condensation through the cell cycle. The chromatin was much more accessible to DNase I in G1 phase than in S or G2 phase, with only small variations in structure detected in late G1 and very early S phase. From early S phase up to mitosis, the chromatin became increasingly condensed and inaccessible to DNase I action. These results support the concept of a chromatin condensation cycle during interphase as well as during mitosis.

Animals↗
PubMedDate not supplied

Effect of triplet repeat expansion on chromatin structure and expression of DMPK and neighboring genes, SIX5 and DMWD, in myotonic dystrophy.

Myotonic dystrophy (DM), an autosomal dominant neuromuscular disease, is associated with expansion of a polymorphic (CTG)n repeat in the 3'-untranslated region of the DM protein kinase (DMPK) gene. The repeat expansion results in decreased levels of DMPK mRNA and protein, but the mechanism for this decreased expression is unknown. Loss of a nuclease-hypersensitive site in the region of the repeat expansion has been observed in muscle and skin fibroblasts from DM patients, indicating a change in local chromatin structure. This change in chromatin structure has been proposed as a mechanism whereby the expression of DMPK and neighboring genes, sine oculis homeobox (Drosophila) homolog 5 (SIX5) and dystrophia myotonica-containing WD repeat motif (DMWD), might be affected. We have developed a polymerase chain reaction (PCR)-based method to assay the chromatin sensitivity of the region adjacent to the repeat expansion in somatic cell hybrids carrying either normal or affected DMPK alleles and show that hybrids carrying expanded alleles exhibit decreased sensitivity to PvuII digestion in this region. Semiquantitative multiplex reverse transcriptase PCR (RT/PCR) assays of gene expression from the chromosomes carrying the expanded alleles showed marked reduction of DMPK mRNA, partial inhibition of SIX5 expression from a congenital DM chromosome, and no reduction of DMWD mRNA. Nested RT/PCR analysis of DMPK mRNA from somatic cell hybrids carrying the repeat expansions revealed that most of the DMPK transcripts expressed from the expanded alleles lacked exons 13 and 14, whereas full-length transcripts were expressed predominantly from the normal alleles. These results suggest that the CTG repeat expansion leads to a decrease in DMPK mRNA levels by affecting splicing at the 3' end of the DMPK pre-mRNA transcript.

Cells, Cultured↗
PubMedDate not supplied

The sperm chromatin structure assay. Relationship with alternate tests of semen quality and heterospermic performance of bulls.

Data obtained by the sperm chromatin structure assay (SCSA) on spermatozoa from nine bulls were correlated with fertility, measured by heterospermic performance (-0.94, P less than 0.01) and by alternate tests of sperm quality, including motility, acrosome integrity, Sephadex filtration and morphology of spermatozoa (all significant at P less than 0.05 to P less than 0.01). The SCSA uses flow cytometry to determine the susceptibility of nuclear DNA to low pH-induced denaturation in situ as measured by the ratio of acridine orange binding to double- or single-stranded DNA. The error associated with multiple SCSA measurements was relatively low. The primary finding is that the assay of chromatin structure stability performed on killed spermatozoa was as highly correlated with the heterospermic performance of semen as the best of the classical tests for semen quality. The SCSA may therefore be a highly useful technique for evaluation of sperm quality.

Animals↗
PubMed1994

Formation and stability of higher order chromatin structures. Contributions of the histone octamer.

Unique roles have been identified for the histone octamer in the formation and stabilization of higher order chromatin structures. Histone octamers were assembled onto 12 tandem repeats of Lytechinus 5 S rDNA, at either saturating or subsaturating ratios. The extent of oligonucleosome folding and intermolecular association in divalent salts was monitored using analytical and differential sedimentation techniques. Saturated oligonucleosomes (12 nucleosomes/DNA) sedimented at 29 S in very low salt buffer. In 1.0-2.0 mM MgCl2, saturated oligonucleosomes formed a maximally folded 55 S structure whose extent of compaction was equivalent to that of classical higher order 30-nm diameter chromatin structures. These results are in marked contrast to those obtained previously in NaCl, where the maximally folded oligonucleosome species sedimented at only approximately 40 S (Hansen, J. C., Ausio, J., Stanik, V. H., and van Holde, K. E. (1989) Biochemistry 28, 9129-9136). Mg(2+)-dependent formation of the 55 S conformation was inhibited by histone octamer depletion; the maximum sedimentation coefficient observed for rDNA molecules containing 10-11 nucleosomes in 2.0 mM MgCl2 was only 40 S. Above 2.0 mM MgCl2, the equilibrium was progressively shifted toward formation of large associated oligonucleosome species. The implications of these results to the mechanism of chromatin folding and its relationship to the biological activity of the chromatin fiber are discussed.

Animals↗
PubMed1982

Alteration of chromatin structure induced by the binding of adriamycin, daunorubicin and ethidium bromide.

The results reported in this paper show the changes in chromatin structure caused by the binding of adriamycin (ADR), daunorubicin (DR) and ethidium bromide (EtdBr) to DNA in chromatin, either isolated or in nuclei or whole cells. Micrococcal nuclease was used as the structural probe of chromatin. The binding of the drugs to chromatin DNA induced two structural changes. First, it produced an unfolding of the overall chromatin structure as evidenced by the increased production of acid-soluble oligonucleotides for the drug-treated samples above the level of the control sample. Second, it caused a disruption of the core particle structure with increased production of DNA of subnucleosomal size and smearing of the nucleosome pattern. The effects were greatest for duanorubicin, followed by adriamycin and ethidium bromide.

Base Composition↗
PubMed2005

Analysis of chromatin structure in the control regions of the chlamydomonas HSP70A and RBCS2 genes.

We have used DNaseI and micrococcal nuclease sensitivity assays to determine the chromatin structures in the control regions of the Chlamydomonas reinhardtii HSP70A and RBCS2 genes. Both genes appear to be organized into nucleosome arrays, which exhibit shorter nucleosome repeat lengths than bulk chromatin. In HSP70A we have identified up to four confined DNaseI hypersensitive sites, three of them localize to the promoter region, a fourth one to the fourth intron. Three hypersensitive sites map close to putative heat shock elements, one close to a CCAAT-box. All hypersensitive sites are located to internucleosomal linkers. Alternative nucleosome positions at half-nucleosomal phasing were constitutively detected in the HSP70A promoter region, indicating local chromatin remodelling. Upon heat shock, dramatic changes in the nucleosome structure of HSP70A were detected that particularly affected the promoter, but also a region within the fourth intron. In contrast, light induction entailed no change in HSP70A chromatin. In the RBCS2 control region we identified a strong DNaseI hypersensitive site that maps close to a CCAAT-box. This site forms the boundary of a nucleosome array with a region of approximately 700 bp apparently devoid of nucleosomes. This study demonstrates that chromatin structure may be determined readily at fairly high resolution in Chlamydomonas, suggesting this organism as a well-suited model for studying the role of chromatin structure on gene expression in photosynthetic eukaryotes.

5' Flanking Region↗
PubMed1987

Human immunoglobulin kappa gene enhancer: chromatin structure analysis at high resolution.

The murine immunoglobulin kappa gene enhancer has previously been found to coincide with a region of altered chromatin structure reflected in a DNase I hypersensitivity site detectable on Southern blots of B-cell DNA. We examined the chromatin structure of the homologous region of human DNA using the high-resolution electroblotting method originally developed for genomic sequence analysis by G. Church and W. Gilbert (Proc. Natl. Acad. Sci. USA 81:1991-1995, 1984). Analysis of DNA isolated from cells treated in vivo with dimethyl sulfate revealed two B-cell-specific sites of enhanced guanine methylation. Both sites are located within perfect inverted repeats theoretically capable of forming cruciform structures; one of these repeats overlaps an enhancer core sequence. No enhancement or protection of guanine methylation was observed within sequences similar to sites of altered methylation previously described in the immunoglobulin heavy-chain enhancer. Treatment of isolated nuclei with DNase I or a variety of restriction endonucleases defined a B-cell-specific approximately 0.25-kilobase region of enhanced nuclease susceptibility similar to that observed in the murine kappa enhancer. The 130-base-pair DNA segment that shows high sequence conservation between human, mouse, and rabbit DNAs lies at the 5' end of the nuclease-susceptible region.

B-Lymphocytes↗
PubMed1991

Conserved chromatin structure in c-myc 5'flanking DNA after viral transduction.

The role of local sequence information in establishing the chromatin structure of the human c-myc upstream region (MUR) was investigated. Adeno-associated virus (AAV)-mediated gene transduction was used to introduce an additional unrearranged copy of the 2.4 kb HindIII-XhoI fragment of the MUR into a novel location in the genome in each of two cloned HeLa cell lines. The AAV-based rep- cap- viral vector SKMA used to transduce the MUR retained only 1.4 kb (24%) of the AAV genome and could accommodate inserts as large as 2.4 kb. SKMA was capable of infecting HeLa cells and integrating into the host genome at single copy number. Integration may have occurred at a preferred site in the HeLa genome, but this site was apparently distinct from the previously identified preferred AAV integration site on human chromosome 19. Indirect end-labelling was used to map DNase I and micrococcal nuclease (MNase) cleavage sites over the transduced c-myc sequences and the endogenous c-myc loci in infected HeLa cells. A similarly ordered chromatin domain, extending 5' from c-myc promoter P0, was found to exist at the transduced c-myc locus in each clone. The position and relative sensitivity of 13 MNase cleavage sites and five DNase I hypersensitive sites, originally identified at the endogenous MUR in non-transduced cells, were shown to be conserved when this DNA was moved to a new chromosome site. A conserved DNase I hypersensitive site also was mapped to the region between the left AAV terminal repeat and AAV promoter P5. These results suggest that the information required to establish the particular chromatin structure of the MUR resides within the local DNA sequence of that region.

Adenoviruses, Human↗
PubMed1993

Chromatin structure of the yeast FBP1 gene: transcription-dependent changes in the regulatory and coding regions.

We have studied the chromatin structure of the Saccharomyces cerevisiae FBP1 gene, which codes for fructose-1,6-bisphosphatase. A strong, constitutive, DNase I, micrococcal nuclease and S1 nuclease hypersensitive site is present close to the 3' end of the coding region. In the repressed state, positioned nucleosomes exist around this site, and subtle changes occur in this nucleosomal organization upon derepression. A DNase I hypersensitive region is located within the promoter between positions -540 and -400 and its extends towards the gene in the derepressed state, leading to an alteration of nucleosomal positioning. Psoralen crosslinking of chromatin, which is used for the first time to study the mobility of restriction fragments from an RNA polymerase II gene, revealed that part of the promoter is nucleosome-free, in accordance with the results of DNase I digestion. A model is presented that, based on the chromatin structure, puts forward the hypothesis that the promoter UAS is located between -540 and -340. Finally, psoralen crosslinking, as well as digestions with micrococcal nuclease or restriction endonucleases suggests that most if not all of the copies of the active FBP1 gene are covered by nucleosomes.

Chromatin↗
PubMed1996

Evidence that Spt6p controls chromatin structure by a direct interaction with histones.

Genetic analysis has implicated SPT6, an essential gene of Saccharomyces cerevisiae, in the control of chromatin structure. Mutations in SPT6 and particular mutations in histone genes are able to overcome transcriptional defects in strains lacking the Snf/Swi protein complex. Here it is shown that an spt6 mutation causes changes in chromatin structure in vivo. In addition, both in vivo and in vitro experiments provide evidence that Spt6p interacts directly with histones and primarily with histone H3. Consistent with these findings, Spt6p is capable of nucleosome assembly in vitro.

Chromatin↗