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 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 Long-Range Transcriptional Control of theIl2Gene by an Intergenic Enhancer

2015 ◽  
Vol 35 (22) ◽  
pp. 3880-3891 ◽  
Author(s):  
Parul Mehra ◽  
Andrew D. Wells
Keyword(s):  
Autoimmune Disease ◽  
Long Range ◽  
Transcriptional Control ◽  
Regulatory Element ◽  
Interleukin 2 ◽  
Regulatory Region ◽  
Dependent Manner ◽  
Regulatory Architecture

Interleukin-2 (IL-2) is a potent cytokine with roles in both immunity and tolerance. Genetic studies in humans and mice demonstrate a role forIl2in autoimmune disease susceptibility, and for decades the proximalIl2upstream regulatory region has served as a paradigm of tissue-specific, inducible gene regulation. In this study, we have identified a novel long-range enhancer of theIl2gene located 83 kb upstream of the transcription start site. This element can potently enhanceIl2transcription in recombinant reporter assaysin vitro, and the native region undergoes chromatin remodeling, transcribes a bidirectional enhancer RNA, and loops to physically interact with theIl2genein vivoin a CD28-dependent manner in CD4+T cells. Thiscisregulatory element is evolutionarily conserved and is situated near a human single-nucleotide polymorphism (SNP) associated with multiple autoimmune disorders. These results indicate that the regulatory architecture of theIl2locus is more complex than previously appreciated and suggest a novel molecular basis for the genetic association ofIl2polymorphism with autoimmune disease.

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 Topoisomerases I and II facilitate condensin DC translocation to organize and repress X chromosomes in C. elegans

2021 ◽  
Author(s):  
Ana Karina Morao ◽  
Jun Kim ◽  
Daniel Obaji ◽  
Siyu Sun ◽  
Sevinc Ercan
Keyword(s):  
Long Range ◽  
X Chromosome ◽  
Molecular Motors ◽  
Topoisomerase Ii ◽  
Dna Looping ◽  
Loop Formation ◽  
X Chromosomes ◽  
C Elegans ◽  
And Control

Condensin complexes are evolutionarily conserved molecular motors that translocate along DNA and form loops. While condensin-mediated DNA looping is thought to direct the chain-passing activity of topoisomerase II to separate sister chromatids, it is not known if topological constraints in turn regulate loop formation in vivo. Here we applied auxin inducible degradation of topoisomerases I and II to determine how DNA topology affects the translocation of an X chromosome specific condensin that represses transcription for dosage compensation in C. elegans (condensin DC). We found that both topoisomerases colocalize with condensin DC and control its movement at different genomic scales. TOP-2 depletion hindered condensin DC translocation over long distances, resulting in accumulation around its X-specific recruitment sites and shorter Hi-C interactions. In contrast, TOP-1 depletion did not affect long-range spreading but resulted in accumulation of condensin DC within expressed gene bodies. Both TOP-1 and TOP-2 depletions resulted in X chromosome transcriptional upregulation indicating that condensin DC translocation at both scales is required for its function in gene repression. Together the distinct effects of TOP-1 and TOP-2 on condensin DC distribution revealed two distinct modes of condensin DC association with chromatin: long-range translocation that requires decatenation/unknotting of DNA and short-range translocation across genes that requires resolution of transcription-induced supercoiling.

scholarly journals SBE6: a novel long-range enhancer involved in driving sonic hedgehog expression in neural progenitor cells

Open Biology ◽  
2016 ◽  
Vol 6 (11) ◽  
pp. 160197 ◽  
Author(s):  
Nezha S. Benabdallah ◽  
Philippe Gautier ◽  
Betul Hekimoglu-Balkan ◽  
Laura A. Lettice ◽  
Shipra Bhatia ◽  
...  
Keyword(s):  
Progenitor Cells ◽  
Long Range ◽  
Sonic Hedgehog ◽  
Neural Progenitor Cells ◽  
Embryonic Stem ◽  
Regulatory Elements ◽  
Neural Progenitor ◽  
Chromatin Profiling

The expression of genes with key roles in development is under very tight spatial and temporal control, mediated by enhancers. A classic example of this is the sonic hedgehog gene ( Shh ), which plays a pivotal role in the proliferation, differentiation and survival of neural progenitor cells both in vivo and in vitro. Shh expression in the brain is tightly controlled by several known enhancers that have been identified through genetic, genomic and functional assays. Using chromatin profiling during the differentiation of embryonic stem cells to neural progenitor cells, here we report the identification of a novel long-range enhancer for Shh—Shh-brain-enhancer-6 (SBE6)—that is located 100 kb upstream of Shh and that is required for the proper induction of Shh expression during this differentiation programme. This element is capable of driving expression in the vertebrate brain. Our study illustrates how a chromatin-focused approach, coupled to in vivo testing, can be used to identify new cell-type specific cis -regulatory elements, and points to yet further complexity in the control of Shh expression during embryonic brain development.

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 Possible Existence of Optical Communication Channels in the Brain

2016 ◽  
Author(s):  
Sourabh Kumar ◽  
Kristine Boone ◽  
Jack Tuszyński ◽  
Paul Barclay ◽  
Christoph Simon
Keyword(s):  
Quantum Entanglement ◽  
Long Range ◽  
Optical Communication ◽  
Communication Channels ◽  
Optical Communication Channels ◽  
Photonic Waveguides ◽  
The Brain ◽  
Physical Modalities

Given that many fundamental questions in neuroscience are still open, it seems pertinent to explore whether the brain might use other physical modalities than the ones that have been discovered so far. In particular it is well established that neurons can emit photons, which prompts the question whether these biophotons could serve as signals between neurons, in addition to the well-known electro-chemical signals. For such communication to be targeted, the photons would need to travel in waveguides. Here we show, based on detailed theoretical modeling, that myelinated axons could serve as photonic waveguides, taking into account realistic optical imperfections. We propose experiments, both in vivo and in vitro, to test our hypothesis. We discuss the implications of our results, including the question whether photons could mediate long-range quantum entanglement in the brain.

scholarly journals Ca2+ and Membrane Potential Transitions During Action Potentials Are Self-Similar to Each Other and to Variability of AP Firing Intervals Across the Broad Physiologic Range of AP Intervals During Autonomic Receptor Stimulation

2021 ◽  
Vol 12 ◽  
Author(s):  
Dongmei Yang ◽  
Christopher H. Morrell ◽  
Alexey E. Lyashkov ◽  
Syevda Tagirova Sirenko ◽  
Ihor Zahanich ◽  
...  
Keyword(s):  
Long Range ◽  
Power Laws ◽  
Cholinergic Receptor ◽  
Receptor Stimulation ◽  
Long Range Correlations ◽  
Clock System ◽  
Self Similar ◽  
Kinetic Transitions

Ca2+ and Vm transitions occurring throughout action potential (AP) cycles in sinoatrial nodal (SAN) cells are cues that (1) not only regulate activation states of molecules operating within criticality (Ca2+ domain) and limit-cycle (Vm domain) mechanisms of a coupled-clock system that underlies SAN cell automaticity, (2) but are also regulated by the activation states of the clock molecules they regulate. In other terms, these cues are both causes and effects of clock molecular activation (recursion). Recently, we demonstrated that Ca2+ and Vm transitions during AP cycles in single SAN cells isolated from mice, guinea pigs, rabbits, and humans are self-similar (obey a power law) and are also self-similar to trans-species AP firing intervals (APFIs) of these cells in vitro, to heart rate in vivo, and to body mass. Neurotransmitter stimulation of β-adrenergic receptor or cholinergic receptor–initiated signaling in SAN cells modulates their AP firing rate and rhythm by impacting on the degree to which SAN clocks couple to each other, creating the broad physiologic range of SAN cell mean APFIs and firing interval variabilities. Here we show that Ca2+ and Vm domain kinetic transitions (time to AP ignition in diastole and 90% AP recovery) occurring within given AP, the mean APFIs, and APFI variabilities within the time series of APs in 230 individual SAN cells are self-similar (obey power laws). In other terms, these long-range correlations inform on self-similar distributions of order among SAN cells across the entire broad physiologic range of SAN APFIs, regardless of whether autonomic receptors of these cells are stimulated or not and regardless of the type (adrenergic or cholinergic) of autonomic receptor stimulation. These long-range correlations among distributions of Ca2+ and Vm kinetic functions that regulate SAN cell clock coupling during each AP cycle in different individual, isolated SAN cells not in contact with each other. Our numerical model simulations further extended our perspectives to the molecular scale and demonstrated that many ion currents also behave self-similar across autonomic states. Thus, to ensure rapid flexibility of AP firing rates in response to different types and degrees of autonomic input, nature “did not reinvent molecular wheels within the coupled-clock system of pacemaker cells,” but differentially engaged or scaled the kinetics of gears that regulate the rate and rhythm at which the “wheels spin” in a given autonomic input context.

scholarly journals Functional analyses of a low-penetrance risk variant rs6702619/1p21.2 associating with colorectal cancer in Polish population

2019 ◽  
Author(s):  
Malgorzata Statkiewicz ◽  
Natalia Maryan ◽  
Maria Kulecka ◽  
Urszula Kuklinska ◽  
Jerzy Ostrowski ◽  
...  
Keyword(s):  
Colorectal Cancer ◽  
Long Range ◽  
Cancer Biology ◽  
Polish Population ◽  
Nucleotide Polymorphisms ◽  
Chromosome Conformation ◽  
Functional Studies ◽  
Functional Analyses

Several studies employed the genome-wide association (GWA) analysis of single-nucleotide polymorphisms (SNPs) to identify susceptibility regions in colorectal cancer (CRC). However, the functional studies exploring the role of associating SNPs with cancer biology are limited. Herein, using chromatin immunoprecipitation assay (ChIP), reporter assay and chromosome conformation capture sequencing (3C-Seq) augmented with publically available genomic and epigenomic databases we aimed to define the function of rs6702619/1p21.2 region associated with CRC in the Polish population. Using ChIP we confirmed that rs6702619 region is occupied by a CTCF, a master regulator of long-range genomic interactions, and is decorated with enhancer-like histone modifications. The enhancer blocking assay revealed that rs6702619 region acts as an insulator with activity dependent on the SNP genotype. Finally, a 3C-Seq survey indicated more than a hundred loci in the rs6702619 locus interactome, including GNAS gene that is frequently amplified in CRC. Taken together, we showed that the CRC-associated rs6702619 region has in vitro and in vivo properties of an insulator that demonstrates long-range physical interactions with CRC-relevant loci.

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