scholarly journals Nucleosome positioning on large tandem DNA repeats of the '601' sequence engineered in Saccharomyces cerevisiae

2021 ◽  
Author(s):  
Astrid Lancrey ◽  
Alexandra Joubert ◽  
Evelyne Duvernois-Berthet ◽  
Etienne Routhier ◽  
Saurabh Raj ◽  
...  
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequence ◽  
Dna Sequences ◽  
Nucleosome Occupancy ◽  
Nucleosome Positioning ◽  
Dna Repeats ◽  
Repeat Array ◽  
Synthetic Dna

The so-called 601 DNA sequence is often used to constrain the position of nucleosomes on a DNA molecule in vitro. Although the ability of the 147 base pair sequence to precisely position a nucleosome in vitro is well documented, in vivo application of this property has been explored only in a few studies and yielded contradictory conclusions. Our goal in the present study was to test the ability of the 601 sequence to dictate nucleosome positioning in Saccharomyces cerevisiae in the context of a long tandem repeat array inserted in a yeast chromosome. We engineered such arrays with three different repeat size, namely 167, 197 and 237 base pairs. Although our arrays are able to position nucleosomes in vitro as expected, analysis of nucleosome occupancy on these arrays in vivo revealed that nucleosomes are not preferentially positioned as expected on the 601-core sequence along the repeats and that the measured nucleosome repeat length does not correspond to the one expected by design. Altogether our results demonstrate that the rules defining nucleosome positions on this DNA sequence in vitro are not valid in vivo, at least in this chromosomal context, questioning the relevance of using the 601 sequence in vivo to achieve precise nucleosome positioning on designer synthetic DNA sequences.

scholarly journals Sequence-based prediction of single nucleosome positioning and genome-wide nucleosome occupancy

2012 ◽  
Vol 109 (38) ◽  
pp. E2514-E2522 ◽  
Author(s):  
Thijn van der Heijden ◽  
Joke J.F.A. van Vugt ◽  
Colin Logie ◽  
John van Noort
Keyword(s):  
Dna Sequences ◽  
Nucleosome Occupancy ◽  
Correlation Coefficients ◽  
Simple Algorithm ◽  
Nucleosome Positioning ◽  
Mechanics Model ◽  
Genome Wide ◽  
Sequence Effects

Nucleosome positioning dictates eukaryotic DNA compaction and access. To predict nucleosome positions in a statistical mechanics model, we exploited the knowledge that nucleosomes favor DNA sequences with specific periodically occurring dinucleotides. Our model is the first to capture both dyad position within a few base pairs, and free binding energy within 2 kBT, for all the known nucleosome positioning sequences. By applying Percus’s equation to the derived energy landscape, we isolate sequence effects on genome-wide nucleosome occupancy from other factors that may influence nucleosome positioning. For both in vitro and in vivo systems, three parameters suffice to predict nucleosome occupancy with correlation coefficients of respectively 0.74 and 0.66. As predicted, we find the largest deviations in vivo around transcription start sites. This relatively simple algorithm can be used to guide future studies on the influence of DNA sequence on chromatin organization.

Active nucleosome positioning beyond intrinsic biophysics is revealed by in vitro reconstitution

2012 ◽  
Vol 40 (2) ◽  
pp. 377-382 ◽  
Author(s):  
Philipp Korber
Keyword(s):  
Dna Sequence ◽  
Nucleosome Occupancy ◽  
Nucleosome Positioning ◽  
Major Theme ◽  
Promoter Regions ◽  
Genome Wide ◽  
Nucleosome Formation ◽  
Dna Binders

Genome-wide nucleosome maps revealed well-positioned nucleosomes as a major theme in eukaryotic genome organization. Promoter regions often show a conserved pattern with an NDR (nucleosome-depleted region) from which regular nucleosomal arrays emanate. Three mechanistic contributions to such NDR-array-organization and nucleosome positioning in general are discussed: DNA sequence, DNA binders and DNA-templated processes. Especially, intrinsic biophysics of DNA sequence preferences for nucleosome formation was prominently suggested to explain the majority of nucleosome positions (‘genomic code for nucleosome positioning’). Nonetheless, non-histone factors that bind DNA with high or low specificity, such as transcription factors or remodelling enzymes respectively and processes such as replication, transcription and the so-called ‘statistical positioning’ may be involved too. Recently, these models were tested for yeast by genome-wide reconstitution. DNA sequence preferences as probed by SGD (salt gradient dialysis) reconstitution generated many NDRs, but only few individual nucleosomes, at their proper positions, and no arrays. Addition of a yeast extract and ATP led to dramatically more in vivo-like nucleosome positioning, including regular arrays for the first time. This improvement depended essentially on the extract and ATP but not on transcription or replication. Nucleosome occupancy and close spacing were maintained around promoters, even at lower histone density, arguing for active packing of nucleosomes against the 5′ ends of genes rather than statistical positioning. A first extract fractionation identified a direct, specific, necessary, but not sufficient role for the RSC (remodels the structure of chromatin) remodelling enzyme. Collectively, nucleosome positioning in yeast is actively determined by factors beyond intrinsic biophysics, and in steady-state rather than at equilibrium.

scholarly journals Transcription of alpha-specific genes in Saccharomyces cerevisiae: DNA sequence requirements for activity of the coregulator alpha 1.

1993 ◽  
Vol 13 (11) ◽  
pp. 6866-6875 ◽  
Author(s):  
D C Hagen ◽  
L Bruhn ◽  
C A Westby ◽  
G F Sprague
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Binding Site ◽  
Dna Sequences ◽  
Point Mutations ◽  
Transcription Activation ◽  
Specific Gene ◽  
Binding Assays ◽  
Weak Binding

Transcription activation of alpha-specific genes in Saccharomyces cerevisiae is regulated by two proteins, MCM1 and alpha 1, which bind to DNA sequences, called P'Q elements, found upstream of alpha-specific genes. Neither MCM1 nor alpha 1 alone binds efficiently to P'Q elements. Together, however, they bind cooperatively in a manner that requires both the P' sequence, which is a weak binding site for MCM1, and the Q sequence, which has been postulated to be the binding site for alpha 1. We analyzed a collection of point mutations in the P'Q element of the STE3 gene to determine the importance of individual base pairs for alpha-specific gene transcription. Within the 10-bp conserved Q sequence, mutations at only three positions strongly affected transcription activation in vivo. These same mutations did not affect the weak binding to P'Q displayed by MCM1 alone. In vitro DNA binding assays showed a direct correlation between the ability of the mutant sequences to form ternary P'Q-MCM1-alpha 1 complexes and the degree to which transcription was activated in vivo. Thus, the ability of alpha 1 and MCM1 to bind cooperatively to P'Q elements is critical for activation of alpha-specific genes. In all natural alpha-specific genes the Q sequence is adjacent to the degenerate side of P'. To test the significance of this geometry, we created several novel juxtapositions of P, P', and Q sequences. When the Q sequence was opposite the degenerate side, the composite QP' element was inactive as a promoter element in vivo and unable to form stable ternary QP'-MCM1-alpha 1 complexes in vitro. We also found that addition of a Q sequence to a strong MCM1 binding site allows the addition of alpha 1 to the complex. This finding, together with the observation that Q-element point mutations affected ternary complex formation but not the weak binding of MCM1 alone, supports the idea that the Q sequence serves as a binding site for alpha 1.

Simian virus 40 major late promoter: an upstream DNA sequence required for efficient in vitro transcription

1984 ◽  
Vol 4 (1) ◽  
pp. 133-141
Author(s):  
J Brady ◽  
M Radonovich ◽  
M Thoren ◽  
G Das ◽  
N P Salzman
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Simian Virus 40 ◽  
Promoter Sequence ◽  
In Vitro Transcription ◽  
Simian Virus ◽  
Upstream Promoter ◽  
Upstream Promoter Sequence

We have previously identified an 11-base DNA sequence, 5'-G-G-T-A-C-C-T-A-A-C-C-3' (simian virus 40 [SV40] map position 294 to 304), which is important in the control of SV40 late RNA expression in vitro and in vivo (Brady et al., Cell 31:625-633, 1982). We report here the identification of another domain of the SV40 late promoter. A series of mutants with deletions extending from SV40 map position 0 to 300 was prepared by nuclease BAL 31 treatment. The cloned templates were then analyzed for efficiency and accuracy of late SV40 RNA expression in the Manley in vitro transcription system. Our studies showed that, in addition to the promoter domain near map position 300, there are essential DNA sequences between nucleotide positions 74 and 95 that are required for efficient expression of late SV40 RNA. Included in this SV40 DNA sequence were two of the six GGGCGG SV40 repeat sequences and an 11-nucleotide segment which showed strong homology with the upstream sequences required for the efficient in vitro and in vivo expression of the histone H2A gene. This upstream promoter sequence supported transcription with the same efficiency even when it was moved 72 nucleotides closer to the major late cap site. In vitro promoter competition analysis demonstrated that the upstream promoter sequence, independent of the 294 to 304 promoter element, is capable of binding polymerase-transcription factors required for SV40 late gene transcription. Finally, we show that DNA sequences which control the specificity of RNA initiation at nucleotide 325 lie downstream of map position 294.

scholarly journals The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage.

1985 ◽  
Vol 5 (11) ◽  
pp. 3274-3279 ◽  
Author(s):  
R M Gronostajski ◽  
P D Sadowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Flp Recombinase ◽  
Specific Target ◽  
Target Sites ◽  
Dna Strands ◽  
2 Micron Plasmid ◽  
Dna Strand

The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

scholarly journals Spt4 Facilitates the Movement of RNA Polymerase II through the +2 Nucleosomal Barrier

2021 ◽  
Author(s):  
Ülkü Uzun ◽  
Thomas Brown ◽  
Harry Fischl ◽  
Andrew Angel ◽  
Jane Mellor
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Rna Polymerase ◽  
Rna Polymerase Ii ◽  
Elongation Factor ◽  
Nucleosome Positioning ◽  
Gene Body ◽  
Precise Position ◽  
Transcription Elongation Factor

AbstractSpt4 is a transcription elongation factor, with homologues in organisms with nucleosomes. Structural and in vitro studies implicate Spt4 in transcription through nucleosomes, yet the in vivo function of Spt4 is unclear. Here we assessed the precise position of Spt4 during transcription and the consequences of loss of Spt4 on RNA polymerase II (RNAPII) dynamics and nucleosome positioning in Saccharomyces cerevisiae. In the absence of Spt4, the spacing between gene-body nucleosomes increases and RNAPII accumulates upstream of the nucleosomal dyad, most dramatically at nucleosome +2. Spt4 associates with elongating RNAPII early in transcription and its association dynamically changes depending on nucleosome positions. Together, our data show that Spt4 regulates early elongation dynamics, participates in co-transcriptional nucleosome positioning, and promotes RNAPII movement through the gene-body nucleosomes, especially the +2 nucleosome.

scholarly journals Modulating the properties of DNA-SWCNT sensors using chemically modified DNA

2021 ◽  
Author(s):  
Alice J. Gillen ◽  
Benjamin P. Lambert ◽  
Alessandra Antonucci ◽  
Daniel Molina-Romero ◽  
Ardemis A. Boghossian
Keyword(s):  
Dna Sequence ◽  
Fluorescence Intensity ◽  
Dna Sequences ◽  
Attractive Alternative ◽  
Laser Exposure ◽  
Chemically Modified ◽  
Modified Dna ◽  
Penetration Depths

AbstractProperties of SWCNT-based sensors such as brightness and detection capabilities strongly depend on the characteristics of the wrapping used to suspend the nanotubes. In this study, we explore ways to modify the properties of DNA-SWCNT sensors by using chemically modified DNA sequences, with the aim of creating sensors more suitable for use in in vivo and in vitro applications. We show that both the fluorescence intensity and sensor reactivity are strongly impacted not only by the chemical modification of the DNA but also by the method of preparation. In the absence of modifications, the sensors prepared using MeOH-assisted surfactant exchange exhibited higher overall fluorescence compared to those prepared by direct sonication. However, we demonstrate that the incorporation of chemical modifications in the DNA sequence could be used to enhance the fluorescence intensity of sonicated samples. We attribute these improvements to both a change in dispersion efficiency as well as to a change in SWCNT chirality distribution.Furthermore, despite their higher intensities, the response capabilities of sensors prepared by MeOH-assisted surfactant exchange were shown to be significantly reduced compared to their sonicated counterparts. Sonicated sensors exhibited a globally higher turn-on response towards dopamine compared to the exchanged samples, with modified samples retaining their relative intensity enhancement. As the increases in fluorescence intensity were achieved without needing to alter the base sequence of the DNA wrapping or to add any exogenous compounds, these modifications can - in theory - be applied to nearly any DNA sequence to increase the brightness and penetration depths of a variety of DNA-SWCNT sensors without affecting biocompatibility or reducing the near-limitless sequence space available. This makes these sensors an attractive alternative for dopamine sensing in vitro and in vivo by enabling significantly higher penetration depths and shorter laser exposure times.

The FLP recombinase of the Saccharomyces cerevisiae 2 microns plasmid attaches covalently to DNA via a phosphotyrosyl linkage

1985 ◽  
Vol 5 (11) ◽  
pp. 3274-3279
Author(s):  
R M Gronostajski ◽  
P D Sadowski
Keyword(s):  
Saccharomyces Cerevisiae ◽  
Dna Sequences ◽  
Flp Recombinase ◽  
Specific Target ◽  
Target Sites ◽  
Dna Strands ◽  
2 Micron Plasmid ◽  
Dna Strand

The FLP recombinase, encoded by the 2 micron plasmid of Saccharomyces cerevisiae, promotes efficient recombination in vivo and in vitro between its specific target sites (FLP sites). It was previously determined that FLP interacts with DNA sequences within its target site (B. J. Andrews, G. A. Proteau, L. G. Beatty, and P. D. Sadowski. Cell 40:795-803, 1985), generates a single-stranded break on both DNA strands within the FLP site, and remains covalently attached to the 3' end of each break. We now show that the FLP protein is bound to the 3' side of each break by an O-phosphotyrosyl residue and that it appears that the same tyrosyl residue(s) is used to attach to either DNA strand within the FLP site.

scholarly journals Absence of MeCP2 binding to non-methylated GT-rich sequences in vivo

2020 ◽  
Vol 48 (7) ◽  
pp. 3542-3552 ◽  
Author(s):  
John C Connelly ◽  
Justyna Cholewa-Waclaw ◽  
Shaun Webb ◽  
Verdiana Steccanella ◽  
Bartlomiej Waclaw ◽  
...  
Keyword(s):  
Dna Sequence ◽  
Dna Sequences ◽  
Cytosine Methylation ◽  
Epigenetic Mark ◽  
Primary Role ◽  
Flanking Sequence ◽  
Primary Determinant ◽  
Human Neuronal Cells

Abstract MeCP2 is a nuclear protein that binds to sites of cytosine methylation in the genome. While most evidence confirms this epigenetic mark as the primary determinant of DNA binding, MeCP2 is also reported to have an affinity for non-methylated DNA sequences. Here we investigated the molecular basis and in vivo significance of its reported affinity for non-methylated GT-rich sequences. We confirmed this interaction with isolated domains of MeCP2 in vitro and defined a minimal target DNA sequence. Binding depends on pyrimidine 5′ methyl groups provided by thymine and requires adjacent guanines and a correctly orientated A/T-rich flanking sequence. Unexpectedly, full-length MeCP2 protein failed to bind GT-rich sequences in vitro. To test for MeCP2 binding to these motifs in vivo, we analysed human neuronal cells using ChIP-seq and ATAC-seq technologies. While both methods robustly detected DNA methylation-dependent binding of MeCP2 to mCG and mCAC, neither showed evidence of MeCP2 binding to GT-rich motifs. The data suggest that GT binding is an in vitro phenomenon without in vivo relevance. Our findings argue that MeCP2 does not read unadorned DNA sequence and therefore support the notion that its primary role is to interpret epigenetic modifications of DNA.

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