The organization of oligonucleosomes in yeast

Szent-Györgyi, Christopher; Isenberg, Irvin

doi:10.1093/nar/11.11.3717

Cited by 30 publications

(11 citation statements)

References 54 publications

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“…For example, in contrast to mammalian chromatin, yeast chromatin exhibits no significant difference in the DNase I sensitivity of transcriptionally active and inactive regions (28). Furthermore, isolated yeast mononucleosomes tend not to be stable (29,30), while those of higher eurkaryotes are sufficiently stable to allow them to be crystallized (26). One reported biochemical difference between the yeast and mammalian chromatins that could conceivably explain the difference in structural flexibility is the fact that histone H1 may be absent in yeast (31).…”

Section: Discussionmentioning

confidence: 99%

Thermal unwinding of simian virus 40 transcription complex DNA.

Lutter

1989

Proc. Natl. Acad. Sci. U.S.A.

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Two long-standing questions in the control of eukaryotic gene expression have been how the structure of transcribing chromatin compares with that of nontranscribing chromatin and how chromatin structure differs among various eukaryotic organisms. We have addressed aspects of these two questions by characterizing the rotational flexibility of the DNA of the simian virus 40 (SV40) transcription complex. When transcription complex samples are incubated with topoisomerase at 0C or 37C, the DNA of the 37C sample is unwound by 1.8 turns relative to that of the 0C sample. This amount of unwinding is similar to that observed for bulk, untranscribed SV40 minichromosome DNA, indicating that the chromatin structure of a transcribed gene resembles that of a nontranscribed gene in the degree of constraint that it imposes on its DNA. However, this amount of unwinding differs substantially from the value observed for yeast plasmid chromatin DNA, suggesting that yeast chromatin differs significantly from mammalian chromatin in this fundamental property.Substantial progress has been made in describing structural details of bulk eukaryotic chromatin, but as yet little is known about the structural details of that minor fraction of chromatin that is transcribed (1-3). The work of Weintraub and Groudine (4) demonstrates that some structural difference exists because the enzyme DNase I digests the DNA of transcriptionally active chromatin more rapidly than that of transcriptionally inactive chromatin. This indicates that active chromatin has a more "open" or accessible structure, suggesting that the DNA-histone interactions may perhaps be less stable than those of inactive chromatin.Recent results from studies of yeast plasmid chromatin (5, 6) would seen to support this view. The topology of the DNA of yeast plasmid chromatin was found to change with temperature in an amount that is --70% the value exhibited by bare DNA. This substantial rotational flexibility of the yeast chromatin DNA differs markedly from that of the DNA of the simian virus 40 (SV40) minichromosome (7,8), which has been shown to unwind by only 25% [or =--1.5 turns per 5243 base pairs (bp)] of the value for bare DNA. However, the bulk chromatin of SV40 is predominantly inactive transcriptionally: only ==1% of the SV40 minichromosomes in an infected cell appear to be transcriptionally active (9). Yeast 2-,um plasmid chromatin, on the other hand, is thought for the most part to be transcriptionally active. Noting these functional differences, Saavedra and Huberman (5) have suggested that the relatively flexible structure that they observe in the yeast 2-,um minichromosome may be a fundamental feature of the transcriptionally active subclass of eukaryotic chromatin. By this proposal, the transcribed fraction of mammalian chromatin would be expected to exhibit the enhanced DNA flexibility characteristic of the yeast chromatin.We have carried out experiments here that directly test this proposal by analyzing the topological properties of that fraction of SV40 m...

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Section: Discussionmentioning

confidence: 99%

Thermal unwinding of simian virus 40 transcription complex DNA.

Lutter

1989

Proc. Natl. Acad. Sci. U.S.A.

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“…Nuclei were isolated and then digested with micrococcal nuclease or DNase I (Worthington); DNA was purified as described previously (67,85) with modifications as detailed by Weiss and Simpson (93). Protein-free DNA controls were obtained by either digesting purified, previously undigested DNA with a 50-fold-lower concentration of enzyme or by digesting a PCR product.…”

Section: Methodsmentioning

confidence: 99%

High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating Type Locus HMLα

Weiß

Simpson

1998

Molecular and Cellular Biology

120

125

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Genetic studies have suggested that chromatin structure is involved in repression of the silent mating type loci in Saccharomyces cerevisiae. Chromatin mapping at nucleotide resolution of the transcriptionally silent HML␣ and the active MAT␣ shows that unique organized chromatin structure characterizes the silent state of HML␣. Precisely positioned nucleosomes abutting the silencers extend over the ␣1 and ␣2 coding regions. The HO endonuclease recognition site, nuclease hypersensitive at MAT␣, is protected at HML␣. Although two precisely positioned nucleosomes incorporate transcription start sites at HML␣, the promoter region of the ␣1 and ␣2 genes is nucleosome free and more nuclease sensitive in the repressed than in the transcribed locus. Mutations in genes essential for HML silencing disrupt the nucleosome array near HML-I but not in the vicinity of HML-E, which is closer to the telomere of chromosome III. At the promoter and the HO site, the structure of HML␣ in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MAT␣. The discontinuous chromatin structure of HML␣ contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer. Punctuation at HML␣ may be necessary for higher-order structure or karyoskeleton interactions. The unique chromatin architecture of HML␣ may relate to the combined requirements of transcriptional repression and recombinational competence.Transcriptional repression of the silent-mating-type loci is fundamental for the haploid yeast life cycle. The a or ␣ mating type is determined by expression of master regulatory genes of the active MAT locus near the centromere of chromosome III. Identical genes present at the HM (haploid mating) loci near the telomeres of the same chromosome, HML carrying ␣ information and HMR bearing a information, are not transcribed, thus preserving the unique mating type. The HM loci serve as donors during the gene interconversion event that allows a homothallic haploid cell to switch mating type, ensuring a diploid population in the wild. In addition to transcriptional repression, the DNA of the silenced HM loci is protected from HO endonuclease, which makes a double-strand break at the MAT HO site to initiate mating-type switching (40,46,55,75).Silencing of the HM-mating-type loci in Saccharomyces cerevisiae is remarkably similar to long-term, epigenetic inactivation of specific genomic domains in complex eukaryotes. Xchromosome inactivation (33) and gene imprinting (82) in mammalian cells, telomeric silencing (22) in yeast, and position effect variegation (reviewed by Henikoff [28]) in Drosophila melanogaster are examples of such parallel situations. Epigenetic states are thought to be achieved by chromosomal condensation into heterochromatin. The molecular events leading to such position-dependent, gene-independent transcriptional repression of a chromosomal region are not well understood. Silencing mechanisms in yeast are likely to involve a r...

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“…Yeasts were grown in yeast extract-peptone-dextrose at 30°C to mid-log phase (optical density at 600 nm with 1-cm light path, ϳ1). Nuclei were isolated and digested with micrococcal nuclease or DNase I (Worthington), and DNA was purified as described previously (37,44,51). Protein-free DNA control samples were obtained by digesting purified, previously undigested DNA with a 50-foldlower concentration of enzyme or by digesting a DNA sample obtained by PCR amplification of yeast genomic DNA.…”

Section: ϫ4mentioning

confidence: 99%

High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating-Type Locus HMRa

Ravindra

Weiß

Simpson

1999

Molecular and Cellular Biology

114

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Genetic and biochemical evidence implicates chromatin structure in the silencing of the two quiescent mating-type loci near the telomeres of chromosome III in yeast. With high-resolution micrococcal nuclease mapping, we show that the HMRa locus has 12 precisely positioned nucleosomes spanning the distance between the E and I silencer elements. The nucleosomes are arranged in pairs with very short linkers; the pairs are separated from one another by longer linkers of ϳ20 bp. Both the basic amino-terminal region of histone H4 and the silent information regulator protein Sir3p are necessary for the organized repressive chromatin structure of the silent locus. Compared to HMRa, only small differences in the availability of the TATA box are present for the promoter in the cassette at the active MATa locus. Features of the chromatin structure of this silent locus compared to the previously studied HML␣ locus suggest differences in the mechanisms of silencing and may relate to donor selection during mating-type interconversion.The silencing of the haploid mating-type loci is a critical requirement for the yeast life cycle (10). Mating types in the yeast Saccharomyces cerevisiae are defined by a set of genes expressed at the active MAT locus near the center of chromosome III. MATa and MAT␣ differ by approximately 750-bp regions, designated Ya and Y␣, respectively, which contain the promoters for genes encoding the master regulatory proteins that define the unique mating type of the cell. Strains with the MATa allele express the a1 and a2 genes, while strains with the MAT␣ allele express the ␣1 and ␣2 genes. In addition to the active MAT locus, two almost identical HM loci are located near the telomeres of chromosome III. HML␣ is near the left telomere, while HMRa resides near the right telomere. These loci are transcriptionally silent and make no direct contribution to mating type. Rather, they serve as donors during yeast mating-type interconversion, or switching (7).The switching event is initiated by expression of the HO endonuclease. This enzyme recognizes and cleaves doublestranded DNA at a site at MAT. The break is repaired by replacing it with the Y region of one of the HM loci, usually that with information of the opposite mating type (18,22,27,41). Interestingly, identical HO sites present at the silent loci are not recognized by the endonuclease. Thus, DNA at the silent mating-type loci is present in a unique state, in which it is invisible to the endonuclease and transcription machinery but completely competent to participate in a recombinational event.Extensive genetic studies (20) led to a model in which proteins binding to cis-acting DNA elements flanking the loci, a number of interacting, non-DNA binding proteins, and histones cooperate to form a repressive, heterochromatin-like structure that packages DNA in a presumably inaccessible format. Heterochromatic condensation has been implicated in position effect variegation in Drosophila melanogaster (9) and X-chromosome inactivation (13) and gene imprinting...

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The organization of oligonucleosomes in yeast

Cited by 30 publications

References 54 publications

Thermal unwinding of simian virus 40 transcription complex DNA.

Thermal unwinding of simian virus 40 transcription complex DNA.

High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating Type Locus HMLα

High-Resolution Structural Analysis of Chromatin at Specific Loci: Saccharomyces cerevisiae Silent Mating-Type Locus HMRa

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