scholarly journals E-ChRPs: Engineered Chromatin Remodeling Proteins for Precise Nucleosome Positioning

2018 ◽  
Author(s):  
DA Donovan ◽  
JG Crandall ◽  
OGB Banks ◽  
ZD Jensvold ◽  
LE McKnight ◽  
...  
Keyword(s):  
Chromatin Remodeling ◽  
Dna Sequences ◽  
Structural Changes ◽  
Nucleosome Positioning ◽  
Dna Binding Proteins ◽  
Chromatin Structural ◽  
Development And Validation ◽  
Dependent Processes

SummaryRegulation of chromatin structure is essential for controlling the access of DNA to factors that require association with specific DNA sequences. The ability to alter chromatin organization in a targeted manner would provide a mechanism for directly manipulating DNA-dependent processes and should provide a means to study direct consequences of chromatin structural changes. Here we describe the development and validation of engineered chromatin remodeling proteins (E-ChRPs) for inducing programmable changes in nucleosome positioning by design. We demonstrate that E-ChRPs function both in vivo and in vitro to specifically reposition target nucleosomes and entire nucleosomal arrays, and possess the ability to evict native DNA-binding proteins through their action. E-ChRPs can be designed with a range of targeting modalities, including the SpyCatcher and dCas9 moieties, resulting in high versatility and enabling diverse future applications. Thus, engineered chromatin remodeling proteins represent a simple and robust means to probe regulation of DNA-dependent processes in different chromatin contexts.

scholarly journals Specialization of the chromatin remodeler RSC to mobilize partially-unwrapped nucleosomes

eLife ◽  
2020 ◽  
Vol 9 ◽  
Author(s):  
Alisha Schlichter ◽  
Margaret M Kasten ◽  
Timothy J Parnell ◽  
Bradley R Cairns
Keyword(s):  
Ribosomal Protein ◽  
Chromatin Remodeling ◽  
Dna Sequences ◽  
Dna Binding Proteins ◽  
Ribosomal Protein Genes ◽  
Expression Of Genes ◽  
Chromatin Remodeling Complexes ◽  
Hmg Protein ◽  
Better Than

SWI/SNF-family chromatin remodeling complexes, such as S. cerevisiae RSC, slide and eject nucleosomes to regulate transcription. Within nucleosomes, stiff DNA sequences confer spontaneous partial unwrapping, prompting whether and how SWI/SNF-family remodelers are specialized to remodel partially-unwrapped nucleosomes. RSC1 and RSC2 are orthologs of mammalian PBRM1 (polybromo) which define two separate RSC sub-complexes. Remarkably, in vitro the Rsc1-containing complex remodels partially-unwrapped nucleosomes much better than does the Rsc2-containing complex. Moreover, a rsc1Δ mutation, but not rsc2Δ, is lethal with histone mutations that confer partial unwrapping. Rsc1/2 isoforms both cooperate with the DNA-binding proteins Rsc3/30 and the HMG protein, Hmo1, to remodel partially-unwrapped nucleosomes, but show differential reliance on these factors. Notably, genetic impairment of these factors strongly reduces the expression of genes with wide nucleosome-deficient regions (e.g., ribosomal protein genes), known to harbor partially-unwrapped nucleosomes. Taken together, Rsc1/2 isoforms are specialized through composition and interactions to manage and remodel partially-unwrapped nucleosomes.

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 Archaeal Nucleosome Positioning by CTG Repeats

1999 ◽  
Vol 181 (3) ◽  
pp. 1035-1038 ◽  
Author(s):  
Kathleen Sandman ◽  
John N. Reeve
Keyword(s):  
Dna Binding ◽  
Dna Sequences ◽  
Binding Sites ◽  
Shape Recognition ◽  
Nucleosome Positioning ◽  
Dna Binding Proteins ◽  
Ctg Repeats ◽  
Dna Shape ◽  
Ctg Repeat

ABSTRACT DNA shape recognition determines the preferred binding sites for sequence-independent DNA binding proteins, and here we document that archaeal histones assemble archaeal nucleosomes in vitro centered preferentially within (CTG)6 and (CTG)8repeats, close to junctions with flanking mixed-sequence DNA. Archaeal nucleosomes were not positioned by (CTG)4-, (CTG)5-, or (CTG)3AA(CTG)3-containing DNA sequences. The features of CTG repeat-containing sequences that direct eucaryal nucleosome positioning may also be similarly recognized by archaeal histones.

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.

scholarly journals Specialization of the Chromatin Remodeler RSC to Mobilize Partially-Unwrapped Nucleosomes

2019 ◽  
Author(s):  
Alisha Schlichter ◽  
Margaret M. Kasten ◽  
Timothy J. Parnell ◽  
Bradley R. Cairns
Keyword(s):  
Ribosomal Protein ◽  
Chromatin Remodeling ◽  
Dna Sequences ◽  
Dna Binding Proteins ◽  
Ribosomal Protein Genes ◽  
Expression Of Genes ◽  
Chromatin Remodeling Complexes ◽  
Hmg Protein ◽  
Better Than

AbstractSWI/SNF-family chromatin remodeling complexes, such as S. cerevisiae RSC, slide and eject nucleosomes to regulate transcription. Within nucleosomes, stiff DNA sequences confer spontaneous partial unwrapping, prompting whether and how SWI/SNF-family remodelers are specialized to remodel partially-unwrapped nucleosomes. RSC1 and RSC2 are orthologs of mammalian PBRM1 (polybromo) which define two separate RSC sub-complexes. Remarkably, in vitro the Rsc1-containing complex remodels partially-unwrapped nucleosomes much better than does the Rsc2-containing complex. Moreover, a rsc1Δ mutation, but not rsc2Δ, is lethal with histone mutations that confer partial unwrapping. Rsc1/2 isoforms both cooperate with the DNA-binding proteins Rsc3/30 and the HMG protein, Hmo1, to remodel partially-unwrapped nucleosomes, but show differential reliance on these factors. Notably, genetic impairment of these factors strongly reduces the expression of genes with wide nucleosome-deficient regions (e.g. ribosomal protein genes), known to harbor partially-unwrapped nucleosomes. Taken together, Rsc1/2 isoforms are specialized through composition and interactions to manage and remodel partially-unwrapped nucleosomes.

scholarly journals Homotypic cooperativity and collective binding are determinants of bHLH specificity and function

2019 ◽  
Vol 116 (32) ◽  
pp. 16143-16152 ◽  
Author(s):  
Christian A. Shively ◽  
Jiayue Liu ◽  
Xuhua Chen ◽  
Kaiser Loell ◽  
Robi D. Mitra
Keyword(s):  
Transcription Factor ◽  
Dna Sequences ◽  
Dna Binding Proteins ◽  
Eukaryotic Cells ◽  
Sequence Information ◽  
Genome Wide ◽  
Important Open Problem ◽  
And Function

Eukaryotic cells express transcription factor (TF) paralogues that bind to nearly identical DNA sequences in vitro but bind at different genomic loci and perform different functions in vivo. Predicting how 2 paralogous TFs bind in vivo using DNA sequence alone is an important open problem. Here, we analyzed 2 yeast bHLH TFs, Cbf1p and Tye7p, which have highly similar binding preferences in vitro, yet bind at almost completely nonoverlapping target loci in vivo. We dissected the determinants of specificity for these 2 proteins by making a number of chimeric TFs in which we swapped different domains of Cbf1p and Tye7p and determined the effects on in vivo binding and cellular function. From these experiments, we learned that the Cbf1p dimer achieves its specificity by binding cooperatively with other Cbf1p dimers bound nearby. In contrast, we found that Tye7p achieves its specificity by binding cooperatively with 3 other DNA-binding proteins, Gcr1p, Gcr2p, and Rap1p. Remarkably, most promoters (63%) that are bound by Tye7p do not contain a consensus Tye7p binding site. Using this information, we were able to build simple models to accurately discriminate bound and unbound genomic loci for both Cbf1p and Tye7p. We then successfully reprogrammed the human bHLH NPAS2 to bind Cbf1p in vivo targets and a Tye7p target intergenic region to be bound by Cbf1p. These results demonstrate that the genome-wide binding targets of paralogous TFs can be discriminated using sequence information, and provide lessons about TF specificity that can be applied across the phylogenetic tree.

scholarly journals Phosphorylation of multiple CD3 zeta tyrosine residues leads to formation of pp21 in vitro and in vivo. Structural changes upon T cell receptor stimulation.

1992 ◽  
Vol 267 (5) ◽  
pp. 3375-3381 ◽  
Author(s):  
S Koyasu ◽  
D.J. McConkey ◽  
L.K. Clayton ◽  
S Abraham ◽  
B Yandava ◽  
...  
Keyword(s):  
T Cell ◽  
T Cell Receptor ◽  
Structural Changes ◽  
Cell Receptor ◽  
Receptor Stimulation ◽  
Tyrosine Residues

scholarly journals Engineering nucleosomes for generating diverse chromatin assemblies

2021 ◽  
Author(s):  
Zenita Adhireksan ◽  
Deepti Sharma ◽  
Phoi Leng Lee ◽  
Qiuye Bao ◽  
Sivaraman Padavattan ◽  
...  
Keyword(s):  
Dynamic Properties ◽  
Dna Nanostructures ◽  
Macromolecular Assemblies ◽  
Maximum Resolution ◽  
Different Types ◽  
Chromatin Structural ◽  
Dna Fragment

Abstract Structural characterization of chromatin is challenging due to conformational and compositional heterogeneity in vivo and dynamic properties that limit achievable resolution in vitro. Although the maximum resolution for solving structures of large macromolecular assemblies by electron microscopy has recently undergone profound increases, X-ray crystallographic approaches may still offer advantages for certain systems. One such system is compact chromatin, wherein the crystalline state recapitulates the crowded molecular environment within the nucleus. Here we show that nucleosomal constructs with cohesive-ended DNA can be designed that assemble into different types of circular configurations or continuous fibers extending throughout crystals. We demonstrate the utility of the method for characterizing nucleosome compaction and linker histone binding at near-atomic resolution but also advance its application for tackling further problems in chromatin structural biology and for generating novel types of DNA nanostructures. We provide a library of cohesive-ended DNA fragment expression constructs and a strategy for engineering DNA-based nanomaterials with a seemingly vast potential variety of architectures and histone chemistries.

scholarly journals Interaction of Lactoferrin with Unsaturated Fatty Acids: In Vitro and In Vivo Study of Human Lactoferrin/Oleic Acid Complex Cytotoxicity

Materials ◽  
2021 ◽  
Vol 14 (7) ◽  
pp. 1602
Author(s):  
Anna Elizarova ◽  
Alexey Sokolov ◽  
Valeria Kostevich ◽  
Ekaterina Kisseleva ◽  
Evgeny Zelenskiy ◽  
...  
Keyword(s):  
Oleic Acid ◽  
Structural Changes ◽  
Unsaturated Fatty Acids ◽  
Structural Features ◽  
Control Group ◽  
Acid Complex ◽  
Hepatoma 22A ◽  
Constitutive Parameters

As shown recently, oleic acid (OA) in complex with lactoferrin (LF) causes the death of cancer cells, but no mechanism(s) of that toxicity have been disclosed. In this study, constitutive parameters of the antitumor effect of LF/OA complex were explored. Complex LF/OA was prepared by titrating recombinant human LF with OA. Spectral analysis was used to assess possible structural changes of LF within its complex with OA. Structural features of apo-LF did not change within the complex LF:OA = 1:8, which was toxic for hepatoma 22a cells. Cytotoxicity of the complex LF:OA = 1:8 was tested in cultured hepatoma 22a cells and in fresh erythrocytes. Its anticancer activity was tested in mice carrying hepatoma 22a. In mice injected daily with LF-8OA, the same tumor grew significantly slower. In 20% of animals, the tumors completely resolved. LF alone was less efficient, i.e., the tumor growth index was 0.14 for LF-8OA and 0.63 for LF as compared with 1.0 in the control animals. The results of testing from 48 days after the tumor inoculation showed that the survival rate among LF-8OA-treated animals was 70%, contrary to 0% rate in the control group and among the LF-treated mice. Our data allow us to regard the complex of LF and OA as a promising tool for cancer treatment.

scholarly journals Brown Spiders’ Phospholipases-D with Potential Therapeutic Applications: Functional Assessment of Mutant Isoforms

Biomedicines ◽  
2021 ◽  
Vol 9 (3) ◽  
pp. 320
Author(s):  
Thaís Pereira da Silva ◽  
Fernando Jacomini de Castro ◽  
Larissa Vuitika ◽  
Nayanne Louise Costacurta Polli ◽  
Bruno César Antunes ◽  
...  
Keyword(s):  
Structural Changes ◽  
Capillary Permeability ◽  
Structural Data ◽  
Histopathological Analysis ◽  
Amino Acid Residues ◽  
Wild Type ◽  
Antigenic Properties ◽  
Harmful Effects

Phospholipases-D (PLDs) found in Loxosceles spiders’ venoms are responsible for the dermonecrosis triggered by envenomation. PLDs can also induce other local and systemic effects, such as massive inflammatory response, edema, and hemolysis. Recombinant PLDs reproduce all of the deleterious effects induced by Loxosceles whole venoms. Herein, wild type and mutant PLDs of two species involved in accidents—L. gaucho and L. laeta—were recombinantly expressed and characterized. The mutations are related to amino acid residues relevant for catalysis (H12-H47), magnesium ion coordination (E32-D34) and binding to phospholipid substrates (Y228 and Y228-Y229-W230). Circular dichroism and structural data demonstrated that the mutant isoforms did not undergo significant structural changes. Immunoassays showed that mutant PLDs exhibit conserved epitopes and kept their antigenic properties despite the mutations. Both in vitro (sphingomyelinase activity and hemolysis) and in vivo (capillary permeability, dermonecrotic activity, and histopathological analysis) assays showed that the PLDs with mutations H12-H47, E32-D34, and Y228-Y229-W230 displayed only residual activities. Results indicate that these mutant toxins are suitable for use as antigens to obtain neutralizing antisera with enhanced properties since they will be based on the most deleterious toxins in the venom and without causing severe harmful effects to the animals in which these sera are produced.

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