Poly(dA-dT) Promoter Elements Increase the Equilibrium Accessibility of Nucleosomal DNA Target Sites

ABSTRACT Polypurine tracts are important elements of eukaryotic promoters. They are believed to somehow destabilize chromatin, but the mechanism of their action is not known. We show that incorporating an A16 element at an end of the nucleosomal DNA and further inward destabilizes histone-DNA interactions by 0.1 ± 0.03 and 0.35 ± 0.04 kcal mol−1, respectively, and is accompanied by 1.5- ± 0.1-fold and 1.7- ± 0.1-fold increases in position-averaged equilibrium accessibility of nucleosomal DNA target sites. These effects are comparable in magnitude to effects of A16 elements that correlate with transcription in vivo, suggesting that our system may capture most of their physiological role. These results point to two distinct but interrelated models for the mechanism of action of polypurine tract promoter elements in vivo. Given a nucleosome positioned over a promoter region, the presence of a polypurine tract in that nucleosome's DNA decreases the stability of the DNA wrapping, increasing the equilibrium accessibility of other DNA target sites buried inside that nucleosome. Alternatively (if nucleosomes are freely mobile), the presence of a polypurine tract provides a free energy bias for the nucleosome to move to alternative locations, thereby changing the equilibrium accessibilities of other nearby DNA target sites.

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Tissue specific expression of avian vitellogenin gene is correlated with DNA hypomethylation and in vivo specific protein-DNA interactions

Philosophical Transactions of the Royal Society of London Series B Biological Sciences ◽

10.1098/rstb.1990.0007 ◽

1990 ◽

Vol 326 (1235) ◽

pp. 231-240 ◽

Cited By ~ 13

Keyword(s):

Promoter Region ◽

Gene Activation ◽

Specific Protein ◽

Egg Laying ◽

Dna Hypomethylation ◽

Dna Interactions ◽

Specific Expression ◽

Protein Dna Interactions ◽

Vitellogenin Gene

The avian vitellogenin gene is expressed only in the liver of egg-laying hens. It can, however, be activated in immature chicks or roosters by oestradiol. Parallel to the onset of transcription, there is a demethylation of specific mCpGs in the promoter region and in the oestrogen response element (ERE). The methylation pattern in the promoter region is hormone and expression specific, whereas in the ERE it is only hormone and not organ specific. The demethylation occurring in the promoter region is correlated with the appearance of DNase I hypersensitivity sites and changes in the specific protein-DNA interactions. In vivo genomic footprinting of the ERE with varying concentrations of dimethylsulphate revealed, upon gene activation, only minor changes in the protein-DNA interaction. We present evidence that there is another protein that binds with high affinity to the ERE, besides the oestrogen receptor.

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Spontaneous Access of Proteins to Buried Nucleosomal DNA Target Sites Occurs via a Mechanism That Is Distinct from Nucleosome Translocation

Molecular and Cellular Biology ◽

10.1128/mcb.22.20.7147-7157.2002 ◽

2002 ◽

Vol 22 (20) ◽

pp. 7147-7157 ◽

Cited By ~ 90

Author(s):

J. D. Anderson ◽

A. Thåström ◽

J. Widom

Keyword(s):

Conformational Changes ◽

Restriction Enzymes ◽

Nucleosome Positioning ◽

The Other ◽

Energetic Cost ◽

Rrna Gene ◽

Nucleosomal Dna ◽

Target Sites

ABSTRACT Intrinsic nucleosome dynamics termed “site exposure” provides spontaneous and cooperative access to buried regions of nucleosomal DNA in vitro. Two different mechanisms for site exposure have been proposed, one based on nucleosome translocation, the other on dynamic nucleosome conformational changes in which a stretch of the nucleosomal DNA is transiently released off the histone surface. Here we report on three experiments that distinguish between these mechanisms. One experiment investigates the effects on the accessibilities of restriction enzyme target sites inside nucleosomes when extra DNA (onto which the nucleosome may move at low energetic cost) is appended onto one end. The other two experiments test directly for nucleosome mobility under the conditions used to probe accessibility to restriction enzymes: one on a selected nonnatural nucleosome positioning sequence, the other on the well-studied 5S rRNA gene nucleosome positioning sequence. We find from all three assays that restriction enzymes gain access to sites throughout the entire length of the nucleosomal DNA without contribution from nucleosome translocation. We conclude that site exposure in nucleosomes in vitro occurs via a nucleosome conformational change that leads to transient release of a stretch of DNA from the histone surface, most likely involving progressive uncoiling from an end. Recapture at a distal site along DNA that has partially uncoiled would result in looped structures which are believed to contribute to RNA polymerase elongation and may contribute to spontaneous or ATP-driven nucleosome mobility. Transient open states may facilitate the initial entry of transcription factors and enzymes in vivo.

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Characterisation of the Interaction among Oil-In-Water Nanocapsules and Mucin

Biomimetics ◽

10.3390/biomimetics5030036 ◽

2020 ◽

Vol 5 (3) ◽

pp. 36

Author(s):

Mar Collado-González ◽

Gurmeet Kaur ◽

Yadira González-Espinosa ◽

Rebecca Brooks ◽

Francisco M. Goycoolea

Keyword(s):

Light Scattering ◽

Bacterial Biofilm ◽

Bioactive Molecules ◽

Field Flow Fractionation ◽

Mucosal Surfaces ◽

Oil In Water ◽

Target Sites ◽

The Stability

Mucins are glycoproteins present in all mucosal surfaces and in secretions such as saliva. Mucins are involved in the mucoadhesion of nanodevices carrying bioactive molecules to their target sites in vivo. Oil-in-water nanocapsules (NCs) have been synthesised for carrying N,N′-(di-m-methylphenyl)urea (DMTU), a quorum-sensing inhibitor, to the oral cavity. DMTU-loaded NCs constitute an alternative for the treatment of plaque (bacterial biofilm). In this work, the stability of the NCs after their interaction with mucin is analysed. Mucin type III from Sigma-Aldrich has been used as the mucin model. Mucin and NCs were characterised by the multi-detection asymmetrical flow field-flow fractionation technique (AF4). Dynamic light scattering (DLS) and ζ-potential analyses were carried out to characterise the interaction between mucin and NCs. According to the results, loading DMTU changes the conformation of the NC. It was also found that the synergistic interaction between mucin and NCs was favoured within a specific range of the mucin:NC ratio within the first 24 h. Studies on the release of DMTU in vitro and the microbial activity of such NCs are ongoing in our lab.

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Formation and Structure of Casein Micelles in Lactating Mammary Tissue

Proceedings, annual meeting, Electron Microscopy Society of America ◽

10.1017/s0424820100067741 ◽

1970 ◽

Vol 28 ◽

pp. 150-151

Author(s):

Robert J. Carroll ◽

Marvin P. Thompson ◽

Harold M. Farrell

Keyword(s):

Size Distribution ◽

G Protein ◽

Milk Proteins ◽

Mammary Tissue ◽

Colloidal System ◽

Casein Micelles ◽

The Stability

Milk is an unusually stable colloidal system; the stability of this system is due primarily to the formation of micelles by the major milk proteins, the caseins. Numerous models for the structure of casein micelles have been proposed; these models have been formulated on the basis of in vitro studies. Synthetic casein micelles (i.e., those formed by mixing the purified αsl- and k-caseins with Ca2+ in appropriate ratios) are dissimilar to those from freshly-drawn milks in (i) size distribution, (ii) ratio of Ca/P, and (iii) solvation (g. water/g. protein). Evidently, in vivo organization of the caseins into the micellar form occurs in-a manner which is not identical to the in vitro mode of formation.

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Kinetics of Various 99mTc-Sn-Pyrophosphate Compounds in the Rat

Nuklearmedizin ◽

10.1055/s-0037-1620623 ◽

1977 ◽

Vol 16 (04) ◽

pp. 157-162 ◽

Cited By ~ 1

Author(s):

C. Schümichen ◽

B. Mackenbrock ◽

G. Hoffmann

Keyword(s):

Normal Saline ◽

Half Life ◽

Compound A ◽

Short Half Life ◽

Low Concentrations ◽

The Stability ◽

Kinetics Of ◽

In Vitro Stability

SummaryThe bone-seeking 99mTc-Sn-pyrophosphate compound (compound A) was diluted both in vitro and in vivo and proved to be unstable both in vitro and in vivo. However, stability was much better in vivo than in vitro and thus the in vitro stability of compound A after dilution in various mediums could be followed up by a consecutive evaluation of the in vivo distribution in the rat. After dilution in neutral normal saline compound A is metastable and after a short half-life it is transformed into the other 99mTc-Sn-pyrophosphate compound A is metastable and after a short half-life in bone but in the kidneys. After dilution in normal saline of low pH and in buffering solutions the stability of compound A is increased. In human plasma compound A is relatively stable but not in plasma water. When compound B is formed in a buffering solution, uptake in the kidneys and excretion in urine is lowered and blood concentration increased.It is assumed that the association of protons to compound A will increase its stability at low concentrations while that to compound B will lead to a strong protein bond in plasma. It is concluded that compound A will not be stable in vivo because of a lack of stability in the extravascular space, and that the protein bond in plasma will be a measure of its in vivo stability.

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