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Results for “Promoter dynamics”

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PubMedDate not supplied

Molecular dynamics and nuclear receptor function.

The development of live cell and biochemical analysis methods has led to an increase in our understanding of the dynamic regulation of transcription. Live single cell studies using photobleaching techniques indicate that many proteins have a high nuclear mobility. Pioneering work using promoter array systems based on the lac operon or the mouse mammary tumor virus promoter enabled the study of chromatin structure, promoter occupancy and protein-chromatin interaction dynamics in relation to transcription. Chromatin immunoprecipitation (ChIP)-based assays allow an exhaustive analysis of the temporal recruitment of proteins to an endogenous promoter and provide evidence of cyclic protein-protein and protein-promoter interactions. Although reflecting different timescales, both ChIP and live cell studies indicate a highly dynamic control of transcription that until now has gone undetected and unappreciated.

Animals↗

PubMed1991

Analysis of hydride transfer and cofactor fluorescence decay in mutants of dihydrofolate reductase: possible evidence for participation of enzyme molecular motions in catalysis.

A remarkable correlation has been discovered between fluorescence lifetimes of bound NADPH and rates of hydride transfer among mutants of dihydrofolate reductase (DHFR) from Escherichia coli. Rates of hydride transfer from NADPH to dihydrofolate change by a factor of 1,000 for the series of mutant enzymes. Since binding constants for the initial complex between coenzyme and DHFR change by only a factor of 10, the major portion of the change in hydride transfer must be attributed to losses in transition-state stabilization. The time course of fluorescence decay for NADPH bound to DHFR is biphasic. Lifetimes ranging from 0.3 to 0.5 ns are attributed to a solvent-exposed dihydronicotinamide conformation of bound coenzyme which is presumably not active in catalysis, while decay times (tau 2) in the range of 1.3 to 2.3 ns are assigned to a more tightly bound species of NADPH in which dihydronicotinamide is sequestered from solvent. It is this slower component that is of interest. Ternary complexes with three different inhibitors, methotrexate, 5-deazafolate, and trimethoprim, were investigated, along with the holoenzyme complex; 3-acetylNADPH was also investigated. Fluorescence polarization decay, excitation polarization spectra, the temperature variation of fluorescence lifetimes, fluorescence amplitudes, and wavelength of absorbance maxima were measured. We suggest that dynamic quenching or internal conversion promotes decay of the excited state in NADPH-DHFR. When rates of hydride transfer are plotted against the fluorescence lifetime (tau 2) of tightly bound NADPH, an unusual correlation is observed. The fluorescence lifetime becomes longer as the rate of catalysis decreases for most mutants studied. However, the fluorescence lifetime is unchanged for those mutations that principally alter the binding of dihydrofolate while leaving most dihydronicotinamide interactions relatively undisturbed. The data are interpreted in terms of possible dynamic motions of a flexible loop region in DHFR which closes over both substrate and coenzyme binding sites. These motions could lead to faster rates of fluorescence decay in holoenzyme complexes and, when correlated over time, may be involved in other motions which give rise to enhanced rates of catalysis in DHFR.

Catalysis↗

PubMed2003

Mpi recombinase globally modulates the surface architecture of a human commensal bacterium.

The mammalian gut represents a complex and diverse ecosystem, consisting of unique interactions between the host and microbial residents. Bacterial surfaces serve as an interface that promotes and responds to this dynamic exchange, a process essential to the biology of both symbionts. The human intestinal microorganism, Bacteroides fragilis, is able to extensively modulate its surface. Analysis of the B. fragilis genomic sequence, together with genetic conservation analyses, cross-species cloning experiments, and mutational studies, revealed that this organism utilizes an endogenous DNA inversion factor to globally modulate the expression of its surface structures. This DNA invertase is necessary for the inversion of at least 13 regions located throughout the genome, including the promoter regions for seven of the capsular polysaccharide biosynthesis loci, an accessory polysaccharide biosynthesis locus, and five other regions containing consensus promoter sequences. Bacterial DNA invertases of the serine site-specific recombinase family are typically encoded by imported elements such as phage and plasmids, and act locally on a single region of the imported element. In contrast, the conservation and unique global regulatory nature of the process in B. fragilis suggest an evolutionarily ancient mechanism for surface adaptation to the changing intestinal milieu during commensalism.

Bacterial Capsules↗

PubMed2000

Genotypic differences in the hepatitis B virus core promoter and precore sequences during seroconversion from HBeAg to anti-HBe.

Hepatitis B virus (HBV) strains from anti-HBe positive patients often show specific mutations in the precore gene, the core promoter region, or both. The dynamics of seroconversion in relation to the appearance of these mutations has not been studied and compared between defined HBV genotypes. Samples from patients followed during seroconversion from HBeAg to anti-HBe were amplified by polymerase chain reaction (PCR), sequenced and genotyped. Among 16 sets of samples, 6 belonged to genotype A, 6 to genotype D, 2 to genotype B, 1 to genotype C, and 1 to genotype E. Whereas strains from genotypes B, C and E showed changes in the core promoter, precore codon 28 or both, genotype A and D strains displayed a different pattern. In 4 of 6 anti-HBe positive samples from genotype A, the precore had a wild-type sequence while the core promoter sequence showed a specific TGA mutation. In another genotype A strain a precore stop mutation was preceded by a mutation in codon 15, thus conserving base-pairing at the pregenomic RNA level in this region. In contrast, all genotype D strains showed wild-type sequences in both the core promoter and precore codon 28 in pre- and post-seroconversion samples. Thus, in 8 patients with a mean follow-up time of 17 months, wild-type sequences in both the core promoter and precore codon 28 were found after seroconversion to anti-HBe. This study also confirmed, for genotype D, that HBeAg seroconversion often occurs earlier than genomic conversion.

Adolescent↗

PubMed2006

Dynamic interactions of chromatin-related mesenchymal modulator, a chromodomain helicase-DNA-binding protein, with promoters in osteoprogenitors.

The newly identified protein chromatin-related mesenchymal modulator (CReMM) is expressed by marrow stromal progenitors in vivo and ex vivo. CReMM belongs to a recently identified subgroup of chromodomain helicase-DNA-binding proteins composed of multiple domains including chromodomains, SNF2/ATPase, helicase-C domain, SANT, and A/T-hook-DNA binding domain. Chromatin immunoprecipitation assay was applied to follow the dynamics of CReMM binding to A/T-rich regions on promoters of genes that play a role in osteoblast maturation. CReMM interaction with BMP4 and biglycan promoters in the marrow stromal cells was challenged with transforming growth factor-beta. Treatment with 17beta-estradiol enhanced the binding to estrogen receptor and abolished binding to the prolactin receptor promoters; CReMM interaction with osteocalcin promoter was identified constantly. CReMM binding to the analyzed endogenous promoters suggests its direct role in the transcriptional program activated during osteogenic cell differentiation, which may be a useful tool for following the molecular mechanism of the "stemness" of mesenchymal cells.

Base Sequence↗

PubMed1996

Agonist stimulation increases the turnover rate of beta 2AR-bound palmitate and promotes receptor depalmitoylation.

We have characterized the dynamic nature of beta 2-adrenergic receptor palmitoylation in Sf9 cells. Under basal conditions, the turnover of receptor-bound palmitate is rapid (half-life = 9.8 +/- 1.8 min) compared to the turnover rate of the receptor protein itself (half-life = 109 +/- 10 min). This suggests that an equilibrium between the palmitoylated and nonpalmitoylated forms of the receptor exists at resting state. Stimulation of the receptor by the agonist isoproterenol reduces the half-life of the beta 2-adrenergic receptor-bound palmitate by 1.8 fold without affecting the turnover rate of the receptor itself. Upon sustained stimulation, this increased palmitate turnover rate shifted the equilibrium toward the nonpalmitoylated form of the receptor, suggesting that prolonged activation either increases the rate of depalmitoylation or prevents receptor palmitoylation. Consistent with the latter possibility, pretreatment of cells with agonist, prior to metabolic labeling, reduced the incorporation of [3H]palmitate into the beta 2-adrenergic receptor by more than 80%. This suggests a link between receptor desensitization occurring upon sustained agonist stimulation and the decrease in receptor palmitoylation. Supporting this hypothesis, mutation of PKA phosphorylation sites known to be involved in receptor desensitization abolished the agonist-promoted reduction in palmitate incorporation. We have previously reported that palmitoylation of the beta 2-adrenergic receptor is important in controlling receptor phosphorylation by PKA [Moffett, S., et al. (1993) EMBO J. 12, 349-356; Moffett, S., et al. (1996) J. Biol. Chem. 271, 21490-21497]. The present study now demonstrates that the receptor palmitoylation state is regulated by agonist stimulation and suggests the existence of concerted reciprocal regulatory interactions between palmitoylation and phosphorylation upon sustained receptor stimulation.

Adrenergic beta-2 Receptor Agonists↗

PubMed1997

Stathmin: a tubulin-sequestering protein which forms a ternary T2S complex with two tubulin molecules.

Stathmin is an important regulatory protein thought to control the dynamics of microtubules through the cell cycle in a phosphorylation-dependent manner. Here we show that stathmin interacts with two molecules of dimeric alphabeta-tubulin to form a tight ternary T2S complex, sedimenting at 7.7 S. This complex appears in slow association-dissociation equilibrium in the analytical ultracentrifuge. The T2S complex is formed under a variety of ionic conditions, either from GTP- or GDP-tubulin or from the tubulin-colchicine complex. The S16/25/38/63E mutated stathmin in contrast is in rapid equilibrium with tubulin in the T2S complex. The T2S complex cannot polymerize in microtubules nor in ring oligomers. Stathmin acts as a pure tubulin-sequestering protein via formation of the T2S complex. It does not act directly on microtubule ends to promote catastrophe nor enhance microtubule dynamics.

Animals↗

PubMed2025

Extrachromosomal DNA-Driven Oncogene Dosage Heterogeneity Promotes Rapid Adaptation to Therapy in MYCN-Amplified Cancers.

UNLABELLED: Extrachromosomal DNA (ecDNA) amplification enhances intercellular oncogene dosage variability and accelerates tumor evolution by violating foundational principles of genetic inheritance through its asymmetric mitotic segregation. Spotlighting high-risk neuroblastoma, we demonstrate how ecDNA amplification undermines the clinical efficacy of current therapies in cancers with extrachromosomal MYCN amplification. Integrating theoretical models of oncogene copy number-dependent fitness with single-cell ecDNA quantification and phenotype analyses, we reveal that ecDNA copy-number heterogeneity drives phenotypic diversity and determines treatment sensitivity through mechanisms unattainable by chromosomal oncogene amplification. We demonstrate that ecDNA copy number directly influences cell fate decisions in cancer cell lines, patient-derived xenografts, and primary neuroblastomas, illustrating how extrachromosomal oncogene dosage-driven phenotypic diversity offers a strong evolutionary advantage under therapeutic pressure. Furthermore, we identify senescent cells with reduced ecDNA copy numbers as a source of treatment resistance in neuroblastomas and outline a strategy for their targeted elimination to improve the treatment of MYCN-amplified cancers. SIGNIFICANCE: ecDNA-driven tumor genome evolution provides a major challenge to curative cancer therapies. We demonstrate that ecDNA copy-number dynamics drives treatment resistance by promoting oncogene dosage-dependent phenotypic heterogeneity in MYCN-amplified cancers. Exploiting phenotype-specific vulnerabilities of ecDNA cells, therefore, presents a powerful strategy to overcome treatment resistance. See related commentary by Korsah, p. 1979.

Humans↗

PubMed2005

Ligand-specific dynamics of the progesterone receptor in living cells and during chromatin remodeling in vitro.

Progesterone receptor (PR), a member of the nuclear receptor superfamily, is a key regulator of several processes in reproductive function. We have studied the dynamics of the interaction of PR with a natural target promoter in living cells through the use of fluorescence recovery after photobleaching (FRAP) analysis and also have characterized the dynamics of the interaction of PR with the mouse mammary tumor virus (MMTV) promoter reconstituted into chromatin in vitro. In photobleaching experiments, PR in the presence of the agonist R5020 exhibits rapid exchange with the MMTV promoter in living cells. Two PR antagonists, RU486 and ZK98299, have opposite effects on receptor dynamics in vivo. In the presence of RU486, PR binds to the promoter and is exchanged more slowly than the agonist-activated receptor. In contrast, PR bound to ZK98299 is not localized to the promoter and exhibits higher mobility in the nucleoplasm than the agonist-bound receptor. Significantly, PR bound to R5020 or RU486 can recruit the SWI/SNF chromatin remodeling complex to the promoter, but PR activated with ZK98299 cannot. Furthermore, we found ligand-specific active displacement of PR from the MMTV promoter during chromatin remodeling in vitro and conclude that the interaction of PR with chromatin is highly dynamic both in vivo and in vitro. We propose that factor displacement during chromatin remodeling is an important component of receptor mobility and that ligand-specific interactions with remodeling complexes can strongly influence receptor nuclear dynamics and rates of exchange with chromatin in living cells.

Cell Nucleus↗

PubMed2007

Prolyl-hydroxylase inhibition and HIF activation in osteoblasts promotes an adipocytic phenotype.

Bone is a dynamic environment where cells sense and adapt to changes in nutrient and oxygen availability. Conditions associated with hypoxia in bone are also associated with bone loss. In vitro hypoxia (2% oxygen) alters gene expression in osteoblasts and osteocytes and induces cellular changes including the upregulation of hypoxia inducible factor (HIF) levels. Our studies show that osteoblasts respond to hypoxia (2% oxygen) by enhancing expression of genes associated with adipocyte/lipogenesis phenotype (C/EBPbeta, PPARgamma2, and aP2) and by suppressing expression of genes associated with osteoblast differentiation (alkaline phosphatase, AP). Hypoxia increased HIF protein levels, hypoxic response element (HRE) binding, and HRE-reporter activity. We also demonstrate that prolyl-hydroxylases 2 and 3 (PHD2, PHD3), one of the major factors coordinating HIF degradation under normoxic but not hypoxic conditions, are induced in osteoblasts under hypoxic conditions. To further determine the contribution of PHDs and upregulated HIF activity in modulating osteoblast phenotype, we treated osteoblasts with a PHD inhibitor, dimethyloxaloylglycine (DMOG), and maintained cells under normoxic conditions. Similar to hypoxic conditions, HRE reporter activity was increased and adipogenic gene expression was increased while osteoblastic genes were suppressed. Taken together, our findings indicate a role for PHDs and HIFs in the regulation of osteoblast phenotype.

3T3 Cells↗

PubMed1997

The role of self-organization in the promotion of resilience in maltreated children.

The dynamic nature of resilience necessitates that children from high-risk backgrounds who are functioning adaptively despite experiences of adversity must be examined over time. In the current investigation, the adaptation of school-age maltreated and nonmaltreated socioeconomically disadvantaged children was examined over 3 consecutive years. In accord with predictions, a higher percentage of nonmaltreated children than of maltreated children were found to be resilient. Moreover, a higher percentage of maltreated than of nonmaltreated children were shown to exhibit functioning consistently in the low adaptive range. Differential predictors of resilience were found in maltreated and nonmaltreated children. Specifically, for maltreated children, positive self-esteem, ego resilience, and ego overcontrol predicted resilient functioning, whereas relationship features were more influential for nonmaltreated children. These findings are discussed in relation to the unfolding of resilient self-organizational strivings in maltreated and nonmaltreated children.

Adaptation, Psychological↗

PubMed2005

Mucin dynamics and microbial populations in chicken small intestine are changed by dietary probiotic and antibiotic growth promoter supplementation.

The mucous layer that covers the intestinal absorptive surface acts as a barrier against bacterial translocation. The chicken gut contains a diverse bacterial population which interacts with the mucous layer. In this report, we studied the effect of changing the intestinal microbial populations on mucin dynamics by feeding 1-d-old chicks a control diet or that diet containing either antibiotic growth promoter (AGP) or a probiotic product for 14 d. Dietary AGP increased the proportions of Bifidobacterium species in the duodenum compared with the other groups. In AGP-fed chicks, the villous surface area was increased in the jejunum, goblet cell density was greater in the jejunum and ileum, and mucin glycoprotein levels in the duodenum were lower than in the other groups (P < 0.05). Feeding AGP increased the expression of mucin mRNA in the jejunum and ileum compared with controls. The dietary probiotic increased the proportion of Lactobacillus species in the ileum compared with the controls (P < 0.05) and significantly enlarged the goblet cell "cup" area throughout the small intestine compared with the other groups. Expression of mucin mRNA and the levels of mucin glycoprotein were greater in the jejunum of the probiotic-fed chicks compared with controls (P < 0.05). Neither the probiotic nor AGP treatments affected the thickness of the mucous adherent layer. These results indicate that both probiotic and AGP altered processes of mucin biosynthesis and/or degradation mediated via changes in the intestinal bacterial populations. These modifications in mucin dynamics influence gut function and health and may change nutrient uptake.

Animal Feed↗

PubMed1993

Strategies for maintenance of health-promoting behaviors.

With our understanding of health expanding to include a dynamic of well-being, definitions of health vary, and activities associated with health promotion and disease prevention often overlap. Consequently, studies of health-promotion research include risk reduction. Most nursing studies are descriptive and cross-sectional, and although other disciplines report more intervention efforts, the same theoretic, definitional, and measurement issues exist in all studies. Women, older adults, and those better educated report a greater number of health-promoting behaviors and engage in a healthier lifestyle. An individual's view of health may affect his or her motivation to perform health-promoting behaviors. Removal of barriers, creation of supportive environments, and a strong sense of self-efficacy are important aspects of adoption and maintenance of health-promoting behaviors. Development of self-efficacy should be an integral part of health-promotion programs as increases in self-efficacy have been shown to precede the adoption and maintenance of health-promoting behaviors. Goal setting, contracting, and other behavioral techniques can help an individual develop competence in self-regulation of behavior. Outcomes of research must be congruent with the long-range view that promotion of health implies. Short-term outcomes may continue to be specific, but longer-term indicators will be more comprehensive. Measurement difficulties include evaluation of large-scale programs over extended periods, accounting for positive change in samples in which some persons already maintain health-promoting behaviors and proxy measures for future health. Future attention must be directed toward promoting healthy lifestyles and development of "wellness" thinking.

Attitude to Health↗

PubMed2000

Herpes simplex virus-mediated expression of the axonal protein tau in human model neurons (NT2-N cells).

The establishment of axonal-somatodendritic polarity is an important event during neuronal development. The analysis of the underlying molecular events requires experimental models that display characteristic steps in the development of polarity and that are accessible for experimental manipulations. Here we show that human model neurons (NT2-N cells) can be efficiently infected with an amplicon-based herpes simplex virus (HSV) system that expresses the axonal microtubule-associated protein tau. We demonstrate that the neurons express a high level of exogenous tau, which persists for several days, thus allowing us to analyze the morphological effects of the expressed protein. The intracellular interactions of tau and the effects on the microtubule structure of infected neurons, which were processed for immunocytochemistry, were determined using laser scanning microscopy (LSM). Exogenous tau expression does not result in an increased axon growth of the neurons but promotes neuronal microtubule assembly as indicated by an increased amount of total microtubule polymer as well as a labile, detyrosinated microtubule subpopulation. In contrast, tau expression does not induce a significant microtubule stabilization as judged from the quantitation of acetylated microtubule staining 24 hours after infection. The data demonstrate that HSV-mediated expression of proteins in human model neurons provides a useful system for analysis of the effect of neuronal proteins on the morphology and cytoskeletal organization of terminally differentiated polar neurons. In addition, it suggests a role for tau as a factor which locally promotes tubulin polymerization while the dynamics of axonal microtubules are preserved.

Axons↗

PubMed1999

The establishment and maintenance of DNA methylation patterns in mouse somatic cells.

Somatic cell DNA methylation patterns in mammals are established during embryonic development and are then maintained somewhat faithfully for the remainder of the individual's lifetime. Pattern formation can be divided into a series of linked steps that include demethylation, de novo methylation, methylation spreading, methylation blocking, and maintenance methylation. In this review, these steps will be combined to present a model for the formation and maintenance of a methylation pattern in the 5' region of the mouse Aprt gene. This model suggests that an apparently 'stable' methylation pattern results from a dynamic equilibrium between forces that promote and inhibit methylation spreading.

Adenine Phosphoribosyltransferase↗

PubMed2006

Behavioural considerations for hand hygiene practices: the basic building blocks.

Hand hygiene is considered to be the most effective measure to prevent microbial pathogen cross-transmission and healthcare-associated infections. In October 2005, the World Health Organization (WHO) World Alliance for Patient Safety launched the first Global Patient Safety Challenge 2005-2006, 'Clean Care is Safer Care', to tackle healthcare-associated infection on a large scale. Within the Challenge framework, international infection control experts and consultative taskforces met to develop new WHO Guidelines on Hand Hygiene in Healthcare. The taskforce was asked to explore aspects underlying hand hygiene behaviour that may influence its promotion among healthcare workers. The dynamics of behavioural change are complex and multi-faceted, but are of vital importance when designing a strategy to improve hand hygiene compliance. A reflection on challenges to be met and areas for future research are also proposed.

Attitude of Health Personnel↗

PubMed2004

Integrative radiation carcinogenesis: interactions between cell and tissue responses to DNA damage.

Tissue function requires coordinated multicellular behavior as a consequence of diverse signals integrated through the tissue microenvironment; importantly, these cell-cell and cell-microenvironment interactions also actively suppress cancer. Ionizing radiation (IR) elicits a well-defined cellular response to DNA damage that mediates the fate of the individual cell, concomitantly with a less well-characterized overarching tissue stress response that coordinates the response of multiple cell types via microenvironment signaling. We have now shown that these programs to reestablish homeostasis intersect via mutual regulation by transforming growth factor beta1 (TGF beta 1), which acts as an extracellular sensor and signal of stress. In this review, the concept that this type of functional integration of cell and tissue stress response programs is essential to cancer suppression will be discussed. Our experiments using IR, and several recent studies that experimentally manipulate stromal TGF beta, show that disruption of microenvironment signaling actively promotes malignant progression. Understanding the dynamic interactions between tissue and cell stress responses will be necessary for an accurate assessment of cancer risk and may also provide targets for prevention.

Animals↗

PubMed1996

Role of Tim23 as voltage sensor and presequence receptor in protein import into mitochondria.

Tim23, an essential component of the protein import machinery of the inner membrane of mitochondria (TIM complex), forms dimers that display a dynamic behavior. Dimer formation is promoted by the membrane potential delta psi. Binding of a matrix targeting sequence to Tim23 triggers dimer dissociation. Monomeric Tim23 is present when a preprotein chain is in transit across the TIM complex. Dimerization of Tim23 is dependent on the second half of its N-terminal hydrophilic domain, which is exposed to the intermembrane space. This segment contains a heptad leucine repeat motif with a predicted capacity for dimer formation. We propose that Tim23 exerts a key function in protein import: Tim23 dimers formed in response to delta psi act as receptors for matrix targeting sequences on the surface of the inner membrane. The ensuring dissociation of Tim23 dimer triggers opening of the TIM channel and insertion of the preprotein.

Amino Acid Sequence↗