Robert T. Simpson

Chromatin reconstituted from tandemly repeated cloned DNA fragments and core histones: a model system for study of higher order structure.

We describe a model system for study of chromatin structure at levels above that of the nucleosome. A series of fragments with lengths ranging from 172 to 207 bp tandemly repeated three to greater than 50 times was prepared; each repeat contains the region important in forming a positioned core particle on a sea urchin 5S rRNA gene upon in vitro association with histones. The tandemly repeated sequences can be studied as linear DNA fragments or as relaxed or supercoiled circular molecules. A number of criteria indicate that nucleosomes position correctly on all the tandemly repeated elements. Measurement of the change in linking number per core particle led to a value of -1.0. Both length and repeat number dependent changes in conformation of the nucleoproteins are observed. We discuss the possibility that some ordered higher level chromatin structure can form with DNA and core histones alone.

Nucleosome Positioning: Occurrence, Mechanisms, and Functional Consequences

Publisher Summary This chapter describes the salient features of chromatin structure involved in positioning, reviews examples of non-random location of nucleosomes, examines mechanisms that have been proposed for positioning and experimental tests of several of these, and discusses some recent efforts to ascertain whether positioning could and/or does have any effect on the function of DNA in chromatin. There is no experimental evidence for, and sound theoretical arguments against, positioning of nucleosomes for most of any eukaryotic genome. However, strong evidence is accumulating for positioning of some nucleosomes. Almost any mechanism that has been postulated for the positioning has found experimental support in some system. It seems clear that there will be hierarchies in the strength of positioning signals and mechanisms that may lead to variability in the location of nucleosomes in different cell types. However, a positioned nucleosome is located in a precise site relative to DNA sequence in all cells of a given population. In this situation, any particular DNA sequence in the region of the positioned nucleosome would always lie in the same relationship to histones-in the linker or in the core particle, in the central or the peripheral region of the core particle, etc.

Nucleosomes are positioned with base pair precision adjacent to the alpha 2 operator in Saccharomyces cerevisiae.

Analysis of the chromatin structure of minichromosomes containing the binding site for the yeast alpha 2 repressor protein by indirect end‐labeling has previously indicated that nucleosomes are stably positioned over sequences adjacent to the alpha 2 operator in the presence of the repressor. Development of a primer extension assay for nucleosome position now allows a more detailed examination of the location of these nucleosomes relative to the operator sequence, and indicates that nucleosomes are precisely and stably positioned both translationally and rotationally over sequences adjoining the operator. In addition, this assay enables analysis of the chromatin structure of single copy, genomic sequences. Chromatin structures determined for two genes regulated by alpha 2, STE6 and BAR1, are consistent with nucleosomes precisely positioned downstream of the operator sequence, incorporating promoter elements, in alpha cells but not in a‐cells. The location of these nucleosomes relative to the operator sequence is highly analogous to that observed in the minichromosome. The stability of the nucleosomes adjacent to the operator together with the precision of their location suggests that they may play a role in repression of a specific gene expression by alpha 2. Further, the primer extension assay allows a comparison of the structure of these positioned nucleosomes formed in vivo to that previously described for core particles reconstituted in vitro.

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

ABSTRACT 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 HMLα and the activeMATα shows that unique organized chromatin structure characterizes the silent state of HMLα. Precisely positioned nucleosomes abutting the silencers extend over the α1 and α2 coding regions. The HO endonuclease recognition site, nuclease hypersensitive at MATα, is protected atHMLα. Although two precisely positioned nucleosomes incorporate transcription start sites at HMLα, the promoter region of the α1 and α2 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 HMLα in Sir protein and histone H4 N-terminal deletion mutants is identical to that of the transcriptionally active MATα. The discontinuous chromatin structure of HMLα contrasts with the continuous array of nucleosomes found at repressed a-cell-specific genes and the recombination enhancer. Punctuation at HMLα may be necessary for higher-order structure or karyoskeleton interactions. The unique chromatin architecture of HMLα may relate to the combined requirements of transcriptional repression and recombinational competence.

Yeast alpha 2 repressor positions nucleosomes in TRP1/ARS1 chromatin.

The yeast alpha 2 repressor suppresses expression of a-mating-type-specific genes in haploid alpha and diploid a/alpha cell types. We inserted the alpha 2-binding site into the multicopy TRP1/ARS1 yeast plasmid and examined the effects of alpha 2 on the chromatin structure of the derivative plasmids in alpha cells, and a/alpha cells. Whereas no effect on nucleosome position was observed in a cells, nucleosomes were precisely and stably positioned over sequences flanking the alpha 2 operator in alpha and a/alpha cells. In addition, when the alpha 2 operator was located upstream of the TRP1 gene, an extended array of positioned nucleosomes was formed in alpha cells and a/alpha cells, with formation of a nucleosome not present in a cells, and TRP1 mRNA production was substantially reduced. These data indicate that alpha 2 causes a positioning of nucleosomes over sequences proximal to its operator in TRP1/ARS1 chromatin and suggest that changes in chromatin structure may be related to alpha 2 repression of cell-type-specific genes.

Effects of cycloheximide on chromatin biosynthesis

In the presence of sufficient cycloheximide, puromycin or NaCl to quantitatively inhibit protein synthesis in HeLa cells, thymidine incorporation continues at 20% of control rates for 60 to 90 minutes, after which incorporation gradually ceases. Both DNA and protein synthesis revert to control rates in about five minutes after removal of cycloheximide. DNA synthesis in the presence of cycloheximide appears to be a continuation of the replicative process by several criteria. The persistent DNA synthesis in the presence of cycloheximide is abolished by hydroxyurea, which does not inhibit repair synthesis, while ethidium bromide, an inhibitor of mitochondrial DNA synthesis, is without effect. Nuclear DNA is not nicked during incubation in cycloheximide. Low molecular weight Okazaki fragments (4 to 5 S) are both synthesized and processed to high molecular weight DNA in cells treated with cycloheximide. Replication forks, identified in alkaline CsCl gradients by incorporation of bromodeoxyuridine as a density marker just before the addition of cycloheximide, are selectively labeled with radioactive thymidine during DNA synthesis. In the presence of cycloheximide the maturation of DNA intermediates into high molecular weight DNA is defective. All size classes of DNA fragments, normally present during progression of low to high molecular weight DNA, are demonstrable in cells preincubated in cycloheximide for prolonged periods. However, 21 S fragments, intermediate in size between Okazaki pieces and mature, high molecular weight DNA, accumulate in cells treated with cycloheximide, demonstrating a defect in maturation of the 21 S intermediates into high molecular weight DNA. After removal of the cycloheximide, the 21 S DNA fragments are processed to high molecular weight DNA at a significantly impaired rate, requiring about three hours for completion of chain growth as compared to 40 to 60 minutes in controls. The slowed growth of DNA fragments synthesized in the presence of cycloheximide following drug removal is not due to persisting effects of cyeloheximide since DNA synthesis immediately following removal of the drug has chain growth rates similar to that of controls. Pools of chromatin proteins exist in HeLa cells, as demonstrated by a brief, labeled amino acid pulse followed by a chase with cycloheximide. The specific activity of chromatin proteins increases significantly during 60 minutes of cycloheximide inhibition. Histone f2a1 accumulates preferentially during this chase period, suggesting that a supply of this highly conserved histone might be requisite to continued replication. Comparison of chromatin synthesized during cycloheximide treatment with pulse-labeled control chromatin has provided insight into the mechanism of assembly of proteins and DNA into the nucleoprotein complex. The DNA of ch-chromatin† is more susceptible to nuclease digestion than control chromatin, suggesting that it is deficient in protein content. Upon reversal of cycloheximide inhibition, the recovery of nuclease digestibility of ch-chromatin to control values takes two to three hours, a time similar to that required for conversion of the corresponding 21 S chDNA fragments to high molecular weight DNA. Briefly pulse-labeled (30 to 60 s) DNA in control chromatin also has an enhanced susceptibility to nuclease digestion of the same degree as found in ch-ehromatin. The time of recovery of increased nuclease susceptibility of newly made chromatin DNA (via protein addition) to control levels is about 10 to 15 minutes and corresponds to the time required for synthesis of replicon-sized units of DNA. In addition to being nuclease-sensitive, both cycloheximide and newly synthesized (30 to 60 s) chromatin have lighter buoyant densities in CsCl gradients than bulk chromatin. This property exists for only one to two minutes in controls and is probably due to structural properties distinct from those rendering nuclease sensitivity. Limit digests of chromatin by micrococcal nuclease yield a characteristic pattern of polynucleotides when resolved in polyacrylamide gels. The radioactivity profiles of limit digest polynucleotides from control and ch-chromatin are identical, indicating that pre-existing chromatin proteins remain in place on newly replicated DNA in the same fashion as in mature chromatin.

Direct study of DNA-protein interactions in repressed and active chromatin in living cells.

Current methods for analysis of chromatin architecture are invasive, utilizing chemicals or nucleases that damage DNA, making detection of labile constituents and conclusions about true in vivo structure problematic. We describe a sensitive assay of chromatin structure which is performed in intact, living yeast. The approach utilizes expression of SssI DNA methyltransferase (MTase) in Saccharomyces cerevisiae to provide an order‐of‐magnitude increase in resolution over previously introduced MTases. Combining this resolution increase with the novel application of a PCR‐based, positive chemical display of modified cytosines provides a significant advance in the direct study of DNA‐protein interactions in growing cells that enables quantitative footprinting. The validity and efficacy of the strategy are demonstrated in mini‐chromosomes, where positioned nucleosomes and a labile, operator‐bound repressor are detected. Also, using a heterologous system to study gene activation, we show that in vivo hormone occupancy of the estrogen receptor is required for maximal site‐specific DNA binding, whereas, at very high receptor‐expression levels, hormone‐independent partial occupancy of an estrogen‐responsive element was observed. Receptor binding to a palindromic estrogen‐responsive element leads to a footprint with strand‐specific asymmetry, which is explicable by known structural information.

The organized chromatin domain of the repressed yeast a cell‐specific gene STE6 contains two molecules of the corepressor Tup1p per nucleosome

In yeast α cells the a cell‐specific genes STE6 and BAR1 are packaged as gene‐sized chromatin domains of positioned nucleosomes. Organized chromatin depends on Tup1p, a corepressor that interacts with the N‐terminal regions of H3 and H4. If Tup1p functions to organize or stabilize a chromatin domain, the protein might be expected to be present at a level stoichiometric with nucleosomes. Chromatin immunoprecipitation assays using Tup1p antibodies showed Tup1p to be associated with the entire genomic STE6 coding region. To determine stoichiometry of Tup1p associated with the gene, a yeast plasmid containing varying lengths of the STE6 gene including flanking control regions and an Escherichia coli lac operator sequence was constructed. After assembly into chromatin in vivo in Saccharomyces cerevisiae, minichromosomes were isolated using an immobilized lac repressor. In these experiments, Tup1p was found to be specifically associated with repressed STE6 chromatin in vivo at a ratio of about two molecules of the corepressor per nucleosome. These observations strongly suggest a structural role for Tup1p in repression and constrain models for organized chromatin in repressive domains.

Interplay of yeast global transcriptional regulators Ssn6p-Tup1p and Swi-Snf and their effect on chromatin structure.

Transcriptional regulation in yeast involves a number of general trans‐acting factors affecting chromatin structure. The Swi–Snf complex is required for expression of a large number of genes and has the ability to remodel chromatin in vitro. The Ssn6p–Tup1p repressor complex may be involved in chromatin organization through the interaction with pathway‐specific DNA‐binding proteins. To study the interplay of these factors and their effect on chromatin we have analyzed SUC2 chromatin structure in wild‐type cells and in strains bearing combinations of ssn6/tup1 and swi1 mutations. We have mapped nucleosome positioning of the repressed gene in wild‐type cells using primer extension methodology, allowing base pair resolution, and have analyzed details of chromatin remodeling in the derepressed state. In ssn6 or tup1 mutants under repressing conditions the observed changes in SUC2 chromatin structure may be suppressed by the swi1 mutation, suggesting that Ssn6p–Tup1p is not required for the establishment of nucleosome positioning at the SUC2 promoter. Our data indicate the involvement of chromatin remodeling factors distinct from the Swi–Snf complex in SUC2 transcriptional regulation and suggest that Swi–Snf may antagonize Ssn6p–Tup1p by controlling remodeling activity. We also show that a relatively high level of SUC2 transcription can coexist with positioned nucleosomes.

A Transcriptionally Active tRNA Gene Interferes with Nucleosome Positioning in Vivo

Incorporation into a positioned nucleosome of a cis-acting element essential for replication in Saccharomyces cerevisiae disrupts the function of the element in vivo [R. T. Simpson, Nature (London) 343:387-389, 1990]. Furthermore, nucleosome positioning has been implicated in repression of transcription by RNA polymerase II in yeast cells. We have now asked whether the function of cis-acting elements essential for transcription of a gene transcribed by RNA polymerase III can be similarly affected. A tRNA gene was fused to either of two nucleosome positioning signals such that the predicted nucleosome would incorporate near its center the tRNA start site and essential A-box element. These constructs were then introduced into yeast cells on stably maintained, multicopy plasmids. Competent tRNA genes were transcribed in vivo and were not incorporated into positioned nucleosomes. Mutated, inactive tRNA genes were incorporated into nucleosomes whose positions were as predicted. This finding demonstrates that the transcriptional competence of the tRNA gene determined its ability to override a nucleosome positioning signal in vivo and establishes that a hierarchy exists between cis-acting elements and nucleosome positioning signals.