Positioning of a Nucleosome on Mouse Satellite DNA Inserted into a Yeast Plasmid Is Determined by Its DNA Sequence and an Adjacent Nucleosome

It has earlier been shown that multiple positioning of nucleosomes on mouse satellite DNA is determined by its nucleotide sequence. To clarify whether other factors, such as boundary ones, can affect the positionings, we modified the environment of satellite DNA monomer by inserting it into a yeast plasmid between inducible GalCyc promoter and a structural region of the yeast FLP gene. We have revealed that the positions of nucleosomes on satellite DNA are identical to those detected upon reconstruction in vitro. The positioning signal (GAAAAA sequence) of satellite DNA governs nucleosome location at the adjacent nucleotide sequence as well. Upon promoter induction the nucleosome, translationally positioned on the GalCyc promoter, transfers to the satellite DNA and its location follows the positioning signal of the latter. Thus, the alternatives of positioning of a nucleosome on satellite DNA are controlled by its nucleotide sequence, though the choice of one of them is determined by the adjacent nucleosome.     

Nucleosome positioning in the <Emphasis Type="Italic">NPTII</Emphasis> neomycin phosphotransferase gene on a yeast plasmid in the repressed and active states

A yeast plasmid was constructed to contain a hybrid GAL-CYC promoter, the NPTII neomycin phosphotransferase gene, and the FRT sequence between them. The CYC part of the GAL-CYC promoter harbored four upstream activating sequences (UASs) and two close TATA boxes. NPTII was efficiently expressed upon induction with galactose, conferring G418 resistance on yeast cells. Nucleosome positioning was studied in repressed and induced NPTII in transformed cells. A stable positioning of three nucleosomes was detected under repressive conditions (growth on glucose). Two nucleosomes were on the CYC part of the promoter, one including both of the TATA boxes. The third nucleosome overlapped the FRT sequence and the start of the NPTII coding region. Each of the three nucleosomes displayed multiple positions, suggesting their sliding along DNA. After induction of NPTII expression with galactose, a sliding of two nucleosomes was detected, exposing the TATA box and a long promoter segment. The 5′-distal nucleosome moved closer to the UASs, bringing them closer to the TATA box, which was assumed to facilitate the assembly of the preinitiation complex. The two nucleosomes slid independently of each other. The second nucleosome moved towards the FRT sequence and repositioned at its nucleosome positioning signal. Galactose-induced expression did not affect the nucleosome positioning in the coding region of NPTII. Unidirectional sliding and repositioning were detected without induction after deacetylase inhibition with trichostatin A. Basal NPTII expression was observed without activation of the GAL-CYC promoter and after a spatial uncoupling of the coding sequence and promoter via gene inversion and was probably driven by the FRT TATA-like element, which is in the region permanently exposed in vivo.     

High-Resolution Mapping of Sequence-Directed Nucleosome Positioning on Genomic DNA

We have mapped in vitro nucleosome positioning on the sheep β-lactoglobulin gene using high-throughput sequencing to characterise the DNA sequences recovered from reconstituted nucleosomes. This methodology surpasses previous approaches for coverage, accuracy and resolution and, most importantly, offers a simple yet rapid and relatively inexpensive method to characterise genomic DNA sequences in terms of nucleosome positioning capacity. We demonstrate an unambiguous correspondence between in vitro and in vivo nucleosome positioning around the promoter of the gene; identify discrete, sequence-specific nucleosomal structures above the level of the canonical core particle—a feature that has implications for regulatory protein access and higher-order chromatin packing; and reveal new insights into the involvement of periodically organised dinucleotide sequence motifs of the type GG and CC and not AA and TT, as determinants of nucleosome positioning—an observation that supports the idea that the core histone octamer can exploit different patterns of sequence organisation, or structural potential, in the DNA to bring about nucleosome positioning.     

FLP-mediated recombination in the vector mosquito, Aedes aegypti.

The activity of a yeast recombinase, FLP, on specific target DNA sequences, FRT, has been demonstrated in embryos of the vector mosquito, Aedes aegypti. In a series of experiments, plasmids containing the FLP recombinase under control of a heterologous heat-shock gene promoter were co-injected with target plasmids containing FRT sites into preblastoderm stage mosquito embryos. FLP-mediated recombination was detected between (i) tandem repeats of FRT sites leading to the excision of specific DNA sequences and (ii) FRT sites located on separate plasmids resulting in the formation of heterodimeric or higher order multimeric plasmids. In addition to FRT sites originally isolated from the yeast 2 microns plasmid, a number of synthetic FRT sites were also used. The synthetic sites were fully functional as target sites for recombination and gave results similar to those derived from the yeast 2 microns plasmid. This successful demonstration of yeast FLP recombinase activity in the mosquito embryo suggests a possible future application of this system in establishing transformed lines of mosquitoes for use in vector control strategies and basic studies.     

FLP-mediated recombination of FRT sites in the maize genome.

Molecular evidence is provided for genomic recombinations in maize cells induced by the yeast FLP/FRT site-specific recombination system. The FLP protein recombined FRT sites previously integrated into the maize genome leading to excision of a selectable marker, the neo gene. NPTII activity was not observed after the successful recombination process; instead, the gusA gene was activated by the removal of the blocking DNA fragment. Genomic sequencing in the region of the FRT site (following the recombination reaction) indicated that a precise rearrangement of genomic DNA sequences had taken place. The functional FLP gene could be either expressed transiently or after stable integration into the maize genome. The efficiency of genomic recombinations was high enough that a selection for recombination products, or for FLP expression, was not required. The results presented here establish the FLP/FRT site-specific recombination system as an important tool for controlled modifications of maize genomic DNA.     

Nucleosome positioning based on the sequence word composition

The DNA of all eukaryotic organisms is packaged into nucleosomes (a basic repeating unit of chromatin). A nucleosome consists of histone octamer wrapped by core DNA and linker histone H1 associated with linker DNA. It has profound effects on all DNA-dependent processes by affecting sequence accessibility. Understanding the factors that influence nucleosome positioning has great help to the study of genomic control mechanism. Among many determinants, the inherent DNA sequence has been suggested to have a dominant role in nucleosome positioning in vivo. Here, we used the method of minimum redundancy maximum relevance (mRMR) feature selection and the nearest neighbor algorithm (NNA) combined with the incremental feature selection (IFS) method to identify the most important sequence features that either favor or inhibit nucleosome positioning. We analyzed the words of 53,021 nucleosome DNA sequences and 50,299 linker DNA sequences of Saccharomyces cerevisiae. 32 important features were abstracted from 5,460 features, and the overall prediction accuracy through jackknife cross-validation test was 76.5%. Our results support that sequence-dependent DNA flexibility plays an important role in positioning nucleosome core particles and that genome sequence facilitates the rapid nucleosome reassembly instead of nucleosome depletion. Besides, our results suggest that there exist some additional features playing a considerable role in discriminating nucleosome forming and inhibiting sequences. These results confirmed that the underlying DNA sequence plays a major role in nucleosome positioning.     

Differential core histone binding behavior: RNA polymerase I promoter region vs 5S rDNA positioning DNA sequences

In order to further characterize the previously observed disruptive effect of the RNA polymerase I promoter sequence (Pol I) from Acanthamoeba castellanii on tandemly repeated 5S rDNA positioning sequences from sea urchin (Lytechinus variegatus), we compared the histone-binding ability of the isolated 199-bp Pol I promoter region to that of the 208-bp 5S rDNA and that of nucleosome core particle sequences isolated from chicken erythocytes. We found the 5S rDNA positioning sequence to be more efficient at forming nucleosomes than the RNA polymerase I promoter sequence. Nevertheless, examination of the free-DNA half-depletion points during the titrations suggested that twice as much histone had bound to RNA polymerase I promoter sequence as to the 5S nucleosome-positioning or core particle sequences. DNA bending analysis suggested two potential DNA bending loci in the RNA polymerase I promoter, whereas only one such locus was predicted for the 5S positioning sequence. Such mixed bending signals on the RNA polymerase I promoter could favor non-nucleosomal deposition of histones on these sequences.     

Nucleosome formation potential of eukaryotic DNA: calculation and promoters analysis.

MOTIVATION: A rapid growth in the number of genes with known sequences calls for developing automated tools for their classification and analysis. It became clear that nucleosome packaging of eukaryotic DNA is very important for gene functioning. Automated computer tools for characterization of nucleosome packaging density could be useful for studying of gene regulation and genome annotation. RESULTS: A program for constructing nucleosome formation potential profiles of eukaryotic DNA sequences was developed. Nucleosome packaging density was analyzed for different functional types of human promoters. It was found that in promoters of tissue-specific genes, the nucleosome formation potential was essentially higher than in genes expressed in many tissues, or housekeeping genes. Hence, capability of nucleosome positioning in the promoter region may serve as a factor regulating gene expression. AVAILABILITY: The program for nucleosome sites recognition is included into the GeneExpress system; section 'DNA Nucleosomal Organization', http://wwwmgs.bionet.nsc.ru/mgs/programs/recon/.     

The core histone tail domains contribute to sequence-dependent nucleosome positioning

The precise positioning of nucleosomes plays a critical role in the regulation of gene expression by modulating the DNA binding activity of trans-acting factors. However, molecular determinants responsible for positioning are not well understood. We examined whether the removal of the core histone tail domains from nucleosomes reconstituted with specific DNA fragments led to alteration of translational positions. Remarkably, we find that removal of tail domains from a nucleosome assembled on a DNA fragment containing a Xenopus borealis somatic-type 5S RNA gene results in repositioning of nucleosomes along the DNA, including two related major translational positions that move about 20 bp further upstream with respect to the 5S gene. In a nucleosome reconstituted with a DNA fragment containing the promoter of a Drosophila alcohol dehydrogenase gene, several translational positions shifted by about 10 bp along the DNA upon tail removal. However, the positions of nucleosomes assembled with a DNA fragment known to have one of the highest binding affinities for core histone proteins in the mouse genome were not altered by removal of core histone tail domains. Our data support the notion that the basic tail domains bind to nucleosomal DNA and influence the selection of the translational position of nucleosomes and that once tails are removed movement between translational positions occurs in a facile manner on some sequences. However, the effect of the N-terminal tails on the positioning and movement of a nucleosome appears to be dependent on the DNA sequence such that the contribution of the tails can be masked by very high affinity DNA sequences. Our results suggest a mechanism whereby sequence-dependent nucleosome positioning can be specifically altered by regulated changes in histone tail-DNA interactions in chromatin.     

Nucleosomal location of the STE6 TATA box and Mat alpha 2p-mediated repression.

It has been proposed that yeast MATa cell-specific genes are repressed in MAT alpha cells by the Mat alpha 2p repressor-directed placement of a nucleosome in a position that incorporates the TATA box of the MATa-specific gene close to the nucleosomal pseudodyad. In this study, we address this proposal directly with a series of plasmids designed to place the MATa-specific STE6 TATA box at different locations in a nucleosome and in the internucleosomal linker. These plasmids contain different lengths of synthetic random DNA between the Mat alpha 2p operator and the TATA box of the STE6 promoter, which is located upstream of a lacZ reporter gene in a multicopy plasmid. We show that in MAT alpha cells, a nucleosome is retained in an identical translational frame relative to the Mat alpha 2p operator in all the constructs investigated, irrespective of the sequence of the DNA wrapped onto the histone octamer. This result shows that the nucleosomal organization of the STE6 promoter in MAT alpha cells is not conferred by the sequence of the promoter itself. No expression of the lacZ reporter gene was detectable in MAT alpha cells in any of the constructs, even with the TATA box located in a short internucleosomal linker. These data indicate that repression of MATa-specific genes in MAT alpha cells does not require the precise translational placement of the TATA box close to the nucleosomal pseudodyad; the gene remains repressed when the TATA box is located within the investigated 250-bp region in the organized chromatin domain abutting the Mat alpha 2p operator in MAT alpha cells and may remain repressed with the TATA box located anywhere within this organized repression domain.     

Nucleosome Positioning by Sequence,State of the Art and Apparent Finale

Abstract

All major suggestions about the nucleosome positioning sequence pattern(s) are overviewed. Two basic binary periodical patterns are well established: in purine/pyrimidine alphabet—YRRRRRYYYYYR and in strong/weak alphabet-SWWWWWSSSSSW. Their merger in four-letter alphabet sequence coincides with first ever complete matrix of nucleosome DNA bendability derived from very large database of nucleosome DNA sequences. Its simplified linear form is CGGAAATTTCCG. Several independent ways of derivation of the same pattern are described. It appears that the pattern represents an ultimate solution of long-standing problem of nucleosome positioning, and provides simple means for nucleosome mapping on sequences with single-base resolution.     

Cloning and structure of the human immune interferon-gamma chromosomal gene.

Two clones containing the human immune interferon-gamma (IFN-gamma) chromosomal gene were isolated from a human DNA library present in lambda Charon4A phage. DNA from these clones specified biologically active interferon upon injection into the nuclei of Xenopus laevis oocytes. Analysis of the clones revealed that they were derived from the same chromosomal segment. Restriction fragments that hybridized with 32P-labeled cDNA probes were subcloned into plasmids and the complete sequence of the IFN-gamma gene was determined. Unlike IFNs-alpha and -beta, IFN-gamma does contain introns. Their presence was also revealed by electron microscopy. It is intriguing that the smallest of the three introns is located just in the middle of the Glu-Glu sequence which is conserved among all three forms of interferon at approximately the same position. The promoter region was found to contain a prototype TATA box, many palindromic structures and several repeating sequences and two symmetrical structures. Particularly interesting was the existence of two sequences homologous to those present in the chicken albumin and the human IFN-beta gene promoter region. A sequence GTGTTG common to several other genes was found in the region approximately 10 nucleotides downstream from the polyadenylation site.