Nuclease hypersensitive regions with adjacent positioned nucleosomes mark the gene boundaries of the PHO5/PHO3 locus in yeast.

The chromatin structure of two tandemly linked acid phosphatase genes (PHO5 and PHO3) from Saccharomyces cerevisiae was analyzed under conditions at which the strongly regulated PHO5 gene is repressed. Digestion experiments with DNase I, DNase II, micrococcal nuclease and restriction nucleases reveal the presence of five hypersensitive sites at the PHO5/PHO3 locus, two of them upstream of PHO5 at distances of 920 and 370 bp, one in between the two genes and two downstream of PHO3. Specifically positioned nucleosomes are located next to these hypersensitive sites as shown by indirect end-labeling experiments. The positions deduced from these experiments could be verified by monitoring the accessibility of various restriction sites to the respective nucleases. Sites within putative linker regions were about 50-60% susceptible, whereas sites located within nucleosome cores were resistant. Hybridizing micrococcal nuclease digests to a probe from in between the two upstream hypersensitive sites leads to an interruption of an otherwise regular nucleosomal DNA pattern. This shows directly that these hypersensitive sites represent gaps within ordered nucleosomal arrays.     

Chromatin structure at the replication origins and transcription-initiation regions of the ribosomal RNA genes of Tetrahymena

The chromatin structure of regulatory regions of the extrachromosomal rRNA genes of Tetrahymena thermophila was probed by nuclease treatment of isolated nuclei. The chromatin near the origins of replication contains hypersensitive sites for micrococcal nuclease, DNAase I, and DNAase II. These sites persist in starved cells, consistent with the origins' being maintained in an altered chromatin structure independent of DNA replication. The region between the two origins of replication is organized into a phased array of seven nucleosomes, the fourth of which is centered at the axis of symmetry of the palindromic rDNA. The entire transcribed region and 150 bp upstream from the initiation site are generally accessible to nucleases; any histone proteins associated with these regions are clearly not in a highly organized nucleosomal array as seen in the central region. Comparison of the chromatin structures of the central spacer of T. thermophila and T. pyriformis rDNA reveals that deletion or insertion of DNA has occurred in increments of 200 bp. This is taken to imply that there are constraints on the evolution of spacer DNA sequences at the level of the nucleosome.     

Unusual chromatin in human telomeres.

We report that human telomeres have an unusual chromatin structure characterized by diffuse micrococcal nuclease patterns. The altered chromatin manifested itself only in human telomeres that are relatively short (2 to 7 kb). In contrast, human and mouse telomeres with telomeric repeat arrays of 14 to 150 kb displayed a more canonical chromatin structure with extensive arrays of tightly packed nucleosomes. All telomeric nucleosomes showed a shorter repeat size than bulk nucleosomes, and telomeric mononucleosomal particles were found to be hypersensitive to micrococcal nuclease. However, telomeric nucleosomes were similar to bulk nucleosomes in the rate at which they sedimented through sucrose gradients. We speculate that mammalian telomeres have a bipartite structure with unusual chromatin near the telomere terminus and a more canonical nucleosomal organization in the proximal part of the telomere.     

Chromatin structure in a region of a yeast transposable element regulating adjacent gene expression.

The chromatin structure of a portion of yeast transposable elements known to be responsible for regulation of the expression of the adjacent HIS4 gene has been investigated, using the nuclease probe micrococcal nuclease. Yeast strains containing Ty917 or derivatives of this element that possess either a His-, weak His+, or strong His+ phenotype were examined. The chromatin at the Ty/HIS4 junction region was accessible to micrococcal nuclease. A partial nucleosome ladder was observed upon digestion with micrococcal nuclease indicating the presence of three phased nucleosomes located in Ty sequences upstream of the HIS4 gene. Phased nucleosomes could not be detected upstream of the HIS4 gene in wild-type cells. These data suggest that nucleosomal structure is not a major contributor to Ty917-regulated adjacent gene expression at HIS4.     

Non-random arrangement of nucleosomes in satellite I containing chromatin of rat liver.

The location of nucleosomes on the nucleotide sequence of rat satellite I DNA was investigated using micrococcal nuclease, exonuclease III, and restriction nucleases as tools. Hae III cleaved the satellite DNA containing chromatin very preferentially in the linker region. Nucleosomes were found predominantly in three defined positions on the 370 bp satellite I monomer unit. This type of arrangement occurs on not more than half of the satellite DNA containing chromatin while the rest of this chromatin is arranged differently. The arrangement of nucleosomes with high probability in preferred frames and with low probability in less preferred frames may be a general phenomenon which can be discussed as a possible mechanism to modulate sequence recognition.     

DNase I hypersensitive regions correlate with a site-specific endogenous nuclease activity on the r-chromatin of Tetrahymena

A novel nuclease activity have been detected at three specific sites in the chromatin of the spacer region flanking the 5'-end of the ribosomal RNA gene from Tetrahymena. The endogenous nuclease does not function catalytically in vitro, but is in analogy with the DNA topoisomerases activated by strong denaturants to cleave DNA at specific sites. The endogenous cleavages have been mapped at positions +50, -650 and -1100 relative to the 5'-end of the pre-35S rRNA. The endogenous cleavage sites are associated with micrococcal nuclease hypersensitive sites and DNase I hypersensitive regions. Thus, a single well-defined micrococcal nuclease hypersensitive site is found approximately 130 bp upstream from each of the endogenous cleavages. Clusters of defined sites, the majority of which fall within the 130 bp regions defined by vicinal micrococcal nuclease and endogenous cleavages, constitute the DNase I hypersensitive regions.     

In vivo topography of Rap1p-DNA complex at Saccharomyces cerevisiae TEF2 UAS(RPG) during transcriptional regulation

We have analyzed in detail the structure of RAP1-UAS(RPG) complexes in Saccharomyces cerevisiae cells using multi-hit KMnO(4), UV and micrococcal nuclease high-resolution footprinting. Three copies of the Rap1 protein are bound to the promoter simultaneously in exponentially growing cells, as shown by KMnO(4) multi-hit footprinting analysis, causing extended and diagnostic changes in the DNA structure of the region containing the UAS(RPG). Amino acid starvation does not cause loss of Rap1p from the complex; however, in vivo UV-footprinting reveals the occurrence of structural modifications of the complex. Moreover, low-resolution micrococcal nuclease digestion shows that the chromatin of the entire region is devoid of positioned nucleosomes but is susceptible to changes in accessibility to the nuclease upon amino acid starvation. The implications of these results for the mechanism of Rap1p action are discussed.     

Positioning of nucleosomes in satellite I-containing chromatin of rat liver

The location of nucleosomes on rat satellite I DNA has been investigated using a new approach. Nucleosome cores were prepared from rat liver nuclei with micrococcal nuclease, exonuclease III and nucleases S1. From the total population of core DNA fragments the satellite-containing fragments were isolated by molecular cloning and the complete sequence of 50 clones was determined. The location of nucleosomes along the satellite sequence was found to be non-random. Our results show that nucleosomes occupy a number of positions on satellite I DNA. About 35 to 50% of all nucleosomes are positioned in two corresponding major sites, the remainder in about 16 less preferred sites. The major nucleosome positions are apparently strictly defined with the precision of a single base-pair. These results were confirmed by other approaches, including restriction nuclease digestion experiments. There are good indications of a defined long-range organization of the satellite chromatin fiber in two or more oligonucleosomal arrays with distinct nucleosome configurations.     

Site-specific phasing in the chromatin of the rDNA in Dictyostelium discoideum

The rDNA in Dictyostelium discoideum is organized in linear, extrachromosomal, palindromic dimers of approximately 88 X 10(3) bases in length. The dimers are repeated about 90 times per haploid genome. Using indirect end-labeling, we have mapped micrococcal nuclease and DNAase I-sensitive sites in the chromatin near the rDNA telomeres. This region is 3' to the 36 S rRNA coding region and contains a single 5 S rRNA cistron but is primarily non-coding. We have observed somewhat irregularly spaced but specific phasing of nuclease-sensitive sites relative to the underlying DNA sequence. Comparison of the sites in chromatin with those in naked DNA reveals an unusual and striking pattern: the sites in naked DNA that are attacked most readily by both nucleases, presumably because of the specificity of the nucleases for certain sequences or physical characteristics of the DNA, appear to be the same sites that are most protected in chromatin. This pattern extends over most of a 10(4) base region, from the sequence immediately distal to the 36 S rRNA coding region and extending to the terminus. Although much of the sequence-specific phasing is irregularly spaced, salt extraction data are consistent with the presence of nucleosomes. In addition, phasing in the terminal region may be directed partially by proteins that do not bind DNA as tightly as do core histones. We present a model for phasing in spacer regions in which the sequence preferences of nucleases such as micrococcal nuclease and DNAase I may be useful tools in predicting nucleosome placement.     

Accessibility of some regions of DNA in chromatin (chicken erythrocytes) to single strand-specific nucleases.

The susceptibility of the DNA in chromatin to single strand-specific nucleases was examined using nuclease P1, mung bean nuclease, and venom phosphodiesterase. A stage in the reaction exists where the size range of the solubilized products is similar for each of the three nucleases and is nearly independent of incubation time. During this stage, the chromatin fragments sediment in the range of 30 to 100 S and contain duplex DNA ranging from 1 to 10 million daltons. Starting with chromatin depleted of histones H1 and H5 similar fragments are generated. In both cases these nucleoprotein fragments are reduced to nucleosomes and their multimers by micrococcal nuclease. Thus, chromatin contains a limited number of DNA sites which are susceptible to single strand-specific nucleases. These sites occur at intervals of 8 to 80 nucleosomes and are distributed throughout the chromatin. Nucleosome monomers, dimers, or trimers were not observed at any stage of single strand-specific nuclease digestion of nuclei, H1- and H5-depleted chromatin, or micrococcal nuclease-generated oligonucleosomes. Each of the three nucleases converted mononucleosomes (approximately 160 base pairs) to nucleosome cores (approximately 140 base pairs) probably by exonucleolytic action that was facilitated by the prior removal of H1 and H5. The minichromosome of SV40 is highly resistant to digestion by nuclease P1.     

DNA-histone interactions are sufficient to position a single nucleosome juxtaposing Drosophila Adh adult enhancer and distal promoter.

The alcohol dehydrogenase gene (Adh) of Drosophila melanogaster is transcribed from two tandem promoters in distinct developmental and tissue-specific patterns. Both promoters are regulated by separate upstream enhancer regions. In its wild-type context the adult enhancer specifically stimulates only the distal promoter, approximately 400 bp downstream, and not the proximal promoter, which is approximately 700 bp further downstream. Genomic footprinting and micrococcal nuclease analyses have revealed a specifically positioned nucleosome between the distal promoter and adult enhancer. In vitro reconstitution of this nucleosome demonstrated that DNA-core histone interactions alone are sufficient to position the nucleosome. Based on this observation and sequence periodicities in the underlying DNA, the mechanism of positioning appears to involve specific DNA structural features (ie flexibility or curvature). We have observed this nucleosome positioned early during development, before tissue differentiation, and before non-histone protein-DNA interactions are established at the distal promoter or adult enhancer. This nucleosome positioning element in the Adh regulatory region could be involved in establishing a specific tertiary nucleoprotein structure that facilitates specific cis-element accessibility and/or distal promoter-adult enhancer interactions.