scholarly journals DNA looping and the helical repeat in vitro and in vivo: effect of HU protein and enhancer location on Hin invertasome assembly.

1993 ◽  
Vol 12 (6) ◽  
pp. 2503-2512 ◽  
Author(s):  
M.J. Haykinson ◽  
R.C. Johnson
Keyword(s):  
Dna Looping ◽  
Hu Protein

scholarly journals TAF7L modulates brown adipose tissue formation

eLife ◽  
2014 ◽  
Vol 3 ◽  
Author(s):  
Haiying Zhou ◽  
Bo Wan ◽  
Ivan Grubisic ◽  
Tommy Kaplan ◽  
Robert Tjian
Keyword(s):  
Adipose Tissue ◽  
Brown Adipose Tissue ◽  
Core Promoter ◽  
Uncoupling Protein ◽  
Dna Looping ◽  
Chromatin Interactions ◽  
Brown Adipose ◽  
Important Regulator

Brown adipose tissue (BAT) plays an essential role in metabolic homeostasis by dissipating energy via thermogenesis through uncoupling protein 1 (UCP1). Previously, we reported that the TATA-binding protein associated factor 7L (TAF7L) is an important regulator of white adipose tissue (WAT) differentiation. In this study, we show that TAF7L also serves as a molecular switch between brown fat and muscle lineages in vivo and in vitro. In adipose tissue, TAF7L-containing TFIID complexes associate with PPARγ to mediate DNA looping between distal enhancers and core promoter elements. Our findings suggest that the presence of the tissue-specific TAF7L subunit in TFIID functions to promote long-range chromatin interactions during BAT lineage specification.

scholarly journals Quantitation of the DNA tethering effect in long-range DNA looping in vivo and in vitro using the Lac and   repressors

2013 ◽  
Vol 111 (1) ◽  
pp. 349-354 ◽  
Author(s):  
D. G. Priest ◽  
L. Cui ◽  
S. Kumar ◽  
D. D. Dunlap ◽  
I. B. Dodd ◽  
...  
Keyword(s):  
Long Range ◽  
Dna Looping

scholarly journals Reconcilingin vitroandin vivoactivities of engineered, LacI-based repressor proteins: Contributions of DNA looping and operator sequence variation

2018 ◽  
Author(s):  
Sudheer Tungtur ◽  
Kristen M. Schwingen ◽  
Joshua J. Riepe ◽  
Chamitha J. Weeramange ◽  
Liskin Swint-Kruse
Keyword(s):  
Amino Acid ◽  
Dna Looping ◽  
Published Data ◽  
Binding Affinities ◽  
Ligand Specificity ◽  
Primary Operator ◽  
Repressor Proteins ◽  
Operator Sequence

AbstractOne way to create new components for synthetic transcription circuits is to re-purpose naturally occurring transcription factor proteins and their cognate DNA operators. For the proteins, re-engineering can be accomplished via domain recombination (to create chimeric regulators) and/or amino acid substitutions. The resulting activities of new protein regulators are often assessedin vitrousing a representative operator. However, when functioningin vivo, transcription factors can interact with multiple operators. We comparedin vivoandin vitroresults for two LacI-based transcription repressor proteins, their mutational variants, and four operator sequences. The two sets of repressor variants differed in their overallin vivorepression, even though theirin vitrobinding affinities for the primary operator spanned the same range. Here, we show that the offset can be explained by different abilities to simultaneously bind and “loop” two DNA operators. Furtherin vitrostudies of the looping-competent repressors were carried out to measure binding to a secondary operator sequence. Surprisingly, binding to this operator was largely insensitive to amino acid changes in the repressor protein.In vitroexperiments with additional operators and analyses of published data indicates that amino acid changes in these repressor proteins leads to complicated changes in ligand specificity. These results raise new considerations for engineering components of synthetic transcription circuits and – more broadly – illustrate difficulties encountered when trying to extrapolate information about specificity determinant positions among protein homologs.

scholarly journals Effect of Different Crowding Agents on the Architectural Properties of the Bacterial Nucleoid-Associated Protein HU

2020 ◽  
Vol 21 (24) ◽  
pp. 9553
Author(s):  
Szu-Ning Lin ◽  
Gijs J.L. Wuite ◽  
Remus T. Dame
Keyword(s):  
Local Concentration ◽  
Complex Interplay ◽  
Binding Properties ◽  
In Vitro System ◽  
Hu Protein ◽  
Tethered Particle Motion ◽  
Bacterial Nucleoid ◽  
Dna Binding Properties

HU is a nucleoid-associated protein expressed in most eubacteria at a high amount of copies (tens of thousands). The protein is believed to bind across the genome to organize and compact the DNA. Most of the studies on HU have been carried out in a simple in vitro system, and to what extent these observations can be extrapolated to a living cell is unclear. In this study, we investigate the DNA binding properties of HU under conditions approximating physiological ones. We report that these properties are influenced by both macromolecular crowding and salt conditions. We use three different crowding agents (blotting grade blocker (BGB), bovine serum albumin (BSA), and polyethylene glycol 8000 (PEG8000)) as well as two different MgCl2 conditions to mimic the intracellular environment. Using tethered particle motion (TPM), we show that the transition between two binding regimes, compaction and extension of the HU protein, is strongly affected by crowding agents. Our observations suggest that magnesium ions enhance the compaction of HU–DNA and suppress filamentation, while BGB and BSA increase the local concentration of the HU protein by more than 4-fold. Moreover, BGB and BSA seem to suppress filament formation. On the other hand, PEG8000 is not a good crowding agent for concentrations above 9% (w/v), because it might interact with DNA, the protein, and/or surfaces. Together, these results reveal a complex interplay between the HU protein and the various crowding agents that should be taken into consideration when using crowding agents to mimic an in vivo system.

scholarly journals Control of the Arabinose Regulon in Bacillus subtilisby AraR In Vivo: Crucial Roles of Operators, Cooperativity, and DNA Looping

2001 ◽  
Vol 183 (14) ◽  
pp. 4190-4201 ◽  
Author(s):  
Luı́s Jaime Mota ◽  
Leonor Morais Sarmento ◽  
Isabel de Sá-Nogueira
Keyword(s):  
Regulatory Gene ◽  
Regulatory Elements ◽  
In Vitro Transcription ◽  
Dna Looping ◽  
Major Protein ◽  
Flexible Control ◽  
Transcription System ◽  
Single Operator

ABSTRACT The proteins involved in the utilization of l-arabinose by Bacillus subtilis are encoded by thearaABDLMNPQ-abfA metabolic operon and by thearaE/araR divergent unit. Transcription from the ara operon, araE transport gene, andaraR regulatory gene is induced by l-arabinose and negatively controlled by AraR. The purified AraR protein binds cooperatively to two in-phase operators within thearaABDLMNPQ-abfA (ORA1 and ORA2) and araE (ORE1 and ORE2) promoters and noncooperatively to a single operator in the araR (ORR3) promoter region. Here, we have investigated how AraR controls transcription from theara regulon in vivo. A deletion analysis of theara promoters region showed that the five AraR binding sites are the key cis-acting regulatory elements of their corresponding genes. Furthermore, ORE1-ORE2 and ORR3 are auxiliary operators for the autoregulation ofaraR and the repression of araE, respectively. Analysis of mutations designed to prevent cooperative binding of AraR showed that in vivo repression of the ara operon requires communication between repressor molecules bound to two properly spaced operators. This communication implicates the formation of a small loop by the intervening DNA. In an in vitro transcription system, AraR alone sufficed to abolish transcription from thearaABDLMNPQ-abfA operon and araEpromoters, strongly suggesting that it is the major protein involved in the repression mechanism of l-arabinose-inducible expression in vivo. The ara regulon is an example of how the architecture of the promoters is adapted to respond to the particular characteristics of the system, resulting in a tight and flexible control.

scholarly journals DNA looping by protamine follows a nonuniform spatial distribution

2021 ◽  
Author(s):  
Ryan B. McMillan ◽  
Victoria D. Kuntz ◽  
Luka M. Devenica ◽  
Hilary Bediako ◽  
Ashley R. Carter
Keyword(s):  
Spatial Distribution ◽  
Dna Origami ◽  
Dna Looping ◽  
Loop Formation ◽  
Force Microscopy ◽  
Atomic Force ◽  
Multivalent Cation ◽  
Structural Maintenance Of Chromosomes

ABSTRACTDNA looping plays an important role in cells in both regulating and protecting the genome. Often, studies of looping focus on looping by prokaryotic transcription factors like lac repressor or by structural maintenance of chromosomes (SMC) proteins such as condensin. Here, however, we are interested in a different looping method whereby multivalent cations (charge≥+3), such as protamine proteins, neutralize the DNA, causing it to form loops and toroids. We considered two previously proposed mechanisms for DNA looping by protamine. In the first mechanism, protamine stabilizes spontaneous DNA fluctuations, forming randomly distributed loops along the DNA. In the second mechanism, protamine binds and bends the DNA to form a loop, creating a distribution of loops that is biased by protamine binding. To differentiate between these mechanisms, we imaged both spontaneous and protamine-induced loops on short-length (≤ 1 μm) DNA fragments using atomic force microscopy (AFM). We then compared the spatial distribution of the loops to several model distributions. A random looping model, which describes the mechanism of spontaneous DNA folding, fit the distribution of spontaneous loops, but it did not fit the distribution of protamine-induced loops. Specifically, it overestimated the number of loops that form at the ends of the molecule and failed to predict a peak in the spatial distribution of loops at an intermediate location along the DNA. An electrostatic multibinding model, which was created to mimic the bind-and-bend mechanism of protamine, was a better fit of the distribution of protamine-induced loops. In this model, multiple protamines bind to the DNA electrostatically within a particular region along the DNA to coordinate the formation of a loop. We speculate that these findings will impact our understanding of protamine’s in vivo role for looping DNA into toroids and the mechanism of DNA condensation by multivalent cations more broadly.SIGNIFICANCEDNA looping is important in a variety of both in vivo functions (e.g. gene regulation) and in vitro applications (e.g. DNA origami). Here, we sought a mechanistic understanding of DNA looping by multivalent cations (≥+3), which condense DNA into loops and toroids. One such multivalent cation is the protein protamine, which condenses DNA in sperm. We investigated the mechanism for loop formation by protamine and found that the experimental data was consistent with an electrostatic multibinding model in which two protamines bind electrostatically to the DNA within a 50-nm region to form a loop. This model is likely general to all multivalent cations and may be helpful in applications involving toroid formation or DNA nanoengineering.

scholarly journals Transcriptional regulation of FoxO3 gene by glucocorticoids in murine myotubes

2016 ◽  
Vol 310 (7) ◽  
pp. E572-E585 ◽  
Author(s):  
Taiyi Kuo ◽  
Patty H. Liu ◽  
Tzu-Chieh Chen ◽  
Rebecca A. Lee ◽  
Jenny New ◽  
...  
Keyword(s):  
Gene Expression ◽  
Gene Transcription ◽  
Dna Looping ◽  
Micrococcal Nuclease ◽  
Metabolic Effects ◽  
C2c12 Myotubes ◽  
Chromatin Structural ◽  
Genomic Regions

Glucocorticoids and FoxO3 exert similar metabolic effects in skeletal muscle. FoxO3 gene expression was increased by dexamethasone (Dex), a synthetic glucocorticoid, both in vitro and in vivo. In C2C12 myotubes the increased expression is due to, at least in part, the elevated rate of FoxO3 gene transcription. In the mouse FoxO3 gene, we identified three glucocorticoid receptor (GR) binding regions (GBRs): one being upstream of the transcription start site, −17kbGBR; and two in introns, +45kbGBR and +71kbGBR. Together, these three GBRs contain four 15-bp glucocorticoid response elements (GREs). Micrococcal nuclease (MNase) assay revealed that Dex treatment increased the sensitivity to MNase in the GRE of +45kbGBR and +71kbGBR upon 30- and 60-min Dex treatment, respectively. Conversely, Dex treatment did not affect the chromatin structure near the −17kbGBR, in which the GRE is located in the linker region. Dex treatment also increased histone H3 and/or H4 acetylation in genomic regions near all three GBRs. Moreover, using chromatin conformation capture (3C) assay, we showed that Dex treatment increased the interaction between the −17kbGBR and two genomic regions: one located around +500 bp and the other around +73 kb. Finally, the transcriptional coregulator p300 was recruited to all three GBRs upon Dex treatment. The reduction of p300 expression decreased FoxO3 gene expression and Dex-stimulated interaction between distinct genomic regions of FoxO3 gene identified by 3C. Overall, our results demonstrate that glucocorticoids activated FoxO3 gene transcription through multiple GREs by chromatin structural change and DNA looping.

scholarly journals Transcriptional Organization and Regulatory Elements of a Pseudomonas sp. Strain ADP Operon Encoding a LysR-Type Regulator and a Putative Solute Transport System

2012 ◽  
Vol 194 (23) ◽  
pp. 6560-6573 ◽  
Author(s):  
Ana Isabel Platero ◽  
Manuel García-Jaramillo ◽  
Eduardo Santero ◽  
Fernando Govantes
Keyword(s):  
Mutational Analysis ◽  
Cyanuric Acid ◽  
Transcriptional Start Site ◽  
Regulatory Elements ◽  
Dna Looping ◽  
Activation Process ◽  
Binding Assays ◽  
Content Type

ABSTRACTTheatzS-atzT-atzU-atzV-atzWgene cluster of thePseudomonassp. strain ADP atrazine-degradative plasmid pADP-1, which carries genes for an outer membrane protein and the components of a putative ABC-type solute transporter, is located downstream fromatzR, which encodes the LysR-type transcriptional regulator of the cyanuric acid-degradative operonatzDEF. Here we describe the transcriptional organization of these genes. Our results show that all six genes are cotranscribed from the PatzRpromoter to form theatzRSTUVWoperon. A second, stronger promoter, PatzT, is found withinatzSand directs transcription of the four distal genes. PatzTis σNdependent, activated by NtrC in response to nitrogen limitation with the aid of IHF, and repressed by AtzR. A combination ofin vivomutational analysis and primer extension allowed us to locate the PatzTpromoter and map the transcriptional start site. Similarly, we used deletion and point mutation analyses, along within vivoexpression studies andin vitrobinding assays, to locate the NtrC, IHF, and AtzR binding sites and address their functionality. Our results suggest a regulatory model in which NtrC activates PatzTtranscription via DNA looping, while AtzR acts as an antiactivator that diminishes expression by interfering with the activation process.

Induction and Differentiation of Dental Epithelium in Vivo and in Vitro

1982 ◽  
Vol 40 ◽  
pp. 142-145
Author(s):  
E. J. Kollar
Keyword(s):  
Connective Tissue ◽  
Oral Mucosa ◽  
Basal Lamina ◽  
Functional Derivatives ◽  
Specific Source ◽  
Dental Epithelium ◽  
Connective Tissue Stroma

The differentiation and maintenance of many specialized epithelial structures are dependent on the underlying connective tissue stroma and on an intact basal lamina. These requirements are especially stringent in the development and maintenance of the skin and oral mucosa. The keratinization patterns of thin or thick cornified layers as well as the appearance of specialized functional derivatives such as hair and teeth can be correlated with the specific source of stroma which supports these differentiated expressions.

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