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PLoS Genetics Jan 2012Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a...
Chromatin undergoes major remodeling around DNA double-strand breaks (DSB) to promote repair and DNA damage response (DDR) activation. We recently reported a high-resolution map of γH2AX around multiple breaks on the human genome, using a new cell-based DSB inducible system. In an attempt to further characterize the chromatin landscape induced around DSBs, we now report the profile of SMC3, a subunit of the cohesin complex, previously characterized as required for repair by homologous recombination. We found that recruitment of cohesin is moderate and restricted to the immediate vicinity of DSBs in human cells. In addition, we show that cohesin controls γH2AX distribution within domains. Indeed, as we reported previously for transcription, cohesin binding antagonizes γH2AX spreading. Remarkably, depletion of cohesin leads to an increase of γH2AX at cohesin-bound genes, associated with a decrease in their expression level after DSB induction. We propose that, in agreement with their function in chromosome architecture, cohesin could also help to isolate active genes from some chromatin remodelling and modifications such as the ones that occur when a DSB is detected on the genome.
Topics: Cell Cycle Proteins; Cell Line; Chondroitin Sulfate Proteoglycans; Chromatin Assembly and Disassembly; Chromosomal Proteins, Non-Histone; DNA Breaks, Double-Stranded; DNA Damage; DNA Repair; DNA-Binding Proteins; Gene Expression Regulation; Histones; Homologous Recombination; Humans; Nuclear Proteins; Phosphoproteins; Tamoxifen; Transcription Initiation Site; Cohesins
PubMed: 22275873
DOI: 10.1371/journal.pgen.1002460 -
Neuroscience Letters Feb 2016Recent evidence reported that activation of the mechanistic target of rapamycin complex 1 (mTORC1) induces terminal differentiation of neural stem cells (NSCs) in the...
Recent evidence reported that activation of the mechanistic target of rapamycin complex 1 (mTORC1) induces terminal differentiation of neural stem cells (NSCs) in the neonatal subventricular zone (SVZ), but did not affect their proliferation. Here, we investigated whether such an effect of hyperactive mTORC1 would be recapitulated in young adults following removal of the negative mTORC1 regulator TSC1as seen in the neurological disorder tuberous sclerosis complex, TSC. Conditional mTORC1 activation in NSCs of 3-4 weeks old mice resulted in the generation of proliferative (Ki67+) cells and newborn neuroblasts. However, hyperactive mTORC1 did not induce NSCs to proliferate, consistent with the findings that mTORC1 induces symmetric division and differentiation of slow-cycling NSCs into proliferative daughter cells. Taken together these data suggest that hyperactivity of mTORC1 could lead to the progressive loss of NSCs over time.
Topics: Animals; Cell Differentiation; Cell Proliferation; Mechanistic Target of Rapamycin Complex 1; Mice, Transgenic; Multiprotein Complexes; Neural Stem Cells; Neurogenesis; TOR Serine-Threonine Kinases; Tamoxifen
PubMed: 26812181
DOI: 10.1016/j.neulet.2015.12.042 -
European Journal of Immunology Dec 2000T lymphocyte stimulation leading to interleukin-2 (IL-2) expression requires activation of protein kinase C (PKC); however, the relevant PKC isoform(s) have not yet been...
T lymphocyte stimulation leading to interleukin-2 (IL-2) expression requires activation of protein kinase C (PKC); however, the relevant PKC isoform(s) have not yet been systematically defined. Here we examine seven major T cell expressed PKC isoforms (PKCalpha, delta, epsilon, zeta, nu, theta and iota) and identify PKCtheta to be essential for IL-2 expression (via the critical NF-AT and NF-kappaB enhancer) in Jurkat T cells. Employing a conditionally activated PKCtheta estrogen-receptor fusion mutant, a de novo synthesis-independent transactivation of JNK2 was established. Based on mRNA in situ hybridization to mouse whole body sections, PKCtheta was found to be highly expressed in lymphoid organs but also skeletal muscle and the nervous system. PKCtheta function appears to be cell-type specific, since its isoenzyme-selective function was not observed in ectopic expression studies, employing COS-1 or NIH3T3 cells. These results confirm PKCtheta to be the prime target for the activating effect of phorbol ester in T cell signaling and suggest that gene expression as well as gene function of PKCtheta is strictly controlled by the cell type.
Topics: 3T3 Cells; Animals; COS Cells; Humans; Interleukin-2; Isoenzymes; Jurkat Cells; Mice; Mitogen-Activated Protein Kinase 9; Mitogen-Activated Protein Kinases; Promoter Regions, Genetic; Protein Biosynthesis; Protein Kinase C; RNA, Messenger; T-Lymphocytes; Tamoxifen
PubMed: 11169407
DOI: 10.1002/1521-4141(200012)30:12<3645::AID-IMMU3645>3.0.CO;2-# -
Genesis (New York, N.Y. : 2000) May 2006Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we...
Conditional Cre-mediated recombination has emerged as a robust method of introducing somatic genetic alterations in an organ-specific manner in the mouse. Here, we generated and characterized mice harboring a 4-hydroxytamoxifen (OHT)-inducible Cre recombinase-estrogen receptor fusion transgene under the control of the melanocyte-specific tyrosinase promoter, designated Tyr::CreER(T2). Cre-mediated recombination was induced in melanocytes in a spatially and temporally controlled manner upon administration of OHT and was documented in embryonic melanoblasts, follicular bulb melanocytes, dermal dendritic melanocytes, epidermal melanocytes of tail skin, and in putative melanocyte stem cells located within the follicular bulge. Functional evidence suggestive of recombination in follicular melanocyte stem cells included the presence of Cre-mediated recombination in follicular bulb melanocytes 1 year after topical OHT administration, by which time several hair cycles have elapsed and the melanocytes residing in this location have undergone multiple rounds of apoptosis and replenishment. These Tyr:: CreER(T2) transgenic mice represent a useful resource for the evaluation of melanocyte developmental genetics, the characterization of melanocyte stem cell function and dynamics, and the construction of refined mouse models of malignant melanoma.
Topics: Animals; Central Nervous System; Embryo, Mammalian; Enhancer Elements, Genetic; Female; Gene Expression Regulation; Integrases; Melanocytes; Mice; Mice, Transgenic; Monophenol Monooxygenase; Pregnancy; Promoter Regions, Genetic; Receptors, Estrogen; Recombination, Genetic; Stem Cells; Tamoxifen
PubMed: 16676322
DOI: 10.1002/dvg.20205 -
Proceedings of the National Academy of... Jul 2004Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent...
Artificial molecular switches that modulate protein activities in response to synthetic small molecules would serve as tools for exerting temporal and dose-dependent control over protein function. Self-splicing protein elements (inteins) are attractive starting points for the creation of such switches, because their insertion into a protein blocks the target protein's function until splicing occurs. Natural inteins, however, are not known to be regulated by small molecules. We evolved an intein-based molecular switch that transduces binding of a small molecule into the activation of an arbitrary protein of interest. Simple insertion of a natural ligand-binding domain into a minimal intein destroys splicing activity. To restore activity in a ligand-dependent manner, we linked protein splicing to cell survival or fluorescence in Saccharomyces cerevisiae. Iterated cycles of mutagenesis and selection yielded inteins with strong splicing activities that highly depend on 4-hydroxytamoxifen. Insertion of an evolved intein into four unrelated proteins in living cells revealed that ligand-dependent activation of protein function is general, fairly rapid, dose-dependent, and posttranslational. Our directed-evolution approach therefore evolved small-molecule dependence in a protein and also created a general tool for modulating the function of arbitrary proteins in living cells with a single cell-permeable, synthetic small molecule.
Topics: Amebicides; Directed Molecular Evolution; Estrogen Antagonists; Gentamicins; Ligands; Models, Molecular; Protein Splicing; Recombinant Fusion Proteins; Saccharomyces cerevisiae; Saccharomyces cerevisiae Proteins; Tamoxifen
PubMed: 15247421
DOI: 10.1073/pnas.0402762101 -
Nucleic Acids Research Mar 1998In mice transgenesis through oocyte injection or DNA recombination in embryonal stem (ES) cells allows mutations to be introduced into the germline. However, the...
In mice transgenesis through oocyte injection or DNA recombination in embryonal stem (ES) cells allows mutations to be introduced into the germline. However, the earliest phenotype of the introduced mutation can eclipse later effects. We show in mice that site-specific genomic recombination can be induced in a selected cell type, B lymphocytes, at a chosen time. This precision of somatic mutagenesis was accomplished by limiting expression of a Cre recombinase-estrogen receptor fusion protein to B lymphocytes by use of tissue-specific elements in the promoter of the transgene employed. The expressed fusion protein remained inactive until derepressed by systemic administration of an exogenous ligand for the estrogen receptor, 4-OH-tamoxifen. Upon derepression the Cre recombinase enzyme deleted specific DNA segments, flanked by loxP sites, in B lymphocytes only. The efficiency of recombination in cells expressing the fusion protein could be varied from low levels to >80%, depending on the dose of ligand administered. Our work presents a paradigm applicable to other uses of site-specific recombination in somatic mutagenesis where both temporal and spatial regulation are desired.
Topics: Animals; B-Lymphocytes; Cell Line; Female; Gene Deletion; Gene Expression; Integrases; Ligands; Mice; Mice, Inbred C57BL; Mice, Inbred CBA; Mice, Transgenic; Mutagenesis; Receptors, Estrogen; Recombinant Fusion Proteins; Recombination, Genetic; Tamoxifen; Viral Proteins
PubMed: 9490788
DOI: 10.1093/nar/26.6.1427 -
Nucleic Acids Research Jun 2001The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases...
The use of the site-specific DNA recombinases FLP and Cre is well-established in a broad range of organisms. Here we investigate the applicability of both recombinases to the Xenopus system where they have not been analyzed yet. We show that injection of FLP mRNA triggers the excision of an FLP recombination target (FRT)-flanked green fluorescent protein (GFP) sequence in a coinjected reporter construct inducing the expression of a downstream beta-galactosidase gene (lacZ). The FLP-mediated gene activation can be controlled in Xenopus embryos by injecting a mRNA encoding a fusion of FLP to the mutant ligand binding domain of the human estrogen receptor whose activity is dependent on 4-hydroxytamoxifen. We also demonstrate that a Cre reporter injected into fertilized eggs is fully recombined by Cre recombinase before zygotic gene transcription initiates. Our results indicate that in Xenopus embryos Cre is more effective than FLP in recombining a given quantity of reporter molecules. Finally, we present FLP-inducible double reporter systems encoding two fluorescence proteins (EYFP, ECFP, DsRed or GFP). These novel gene expression systems enable the continuous analysis of two reporter activities within living embryos and are expected to allow cell-lineage studies based on recombinase-mediated DNA rearrangement in transgenic Xenopus lines.
Topics: Animals; Cytomegalovirus; DNA Nucleotidyltransferases; Embryo, Nonmammalian; Gene Expression Regulation; Green Fluorescent Proteins; Humans; Integrases; Lac Operon; Luminescent Proteins; Plasmids; Promoter Regions, Genetic; Recombination, Genetic; Tamoxifen; Transcriptional Activation; Viral Proteins; Xenopus laevis
PubMed: 11376165
DOI: 10.1093/nar/29.11.e53 -
Development (Cambridge, England) Jan 2011Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here,...
Molecular genetics approaches in zebrafish research are hampered by the lack of a ubiquitous transgene driver element that is active at all developmental stages. Here, we report the isolation and characterization of the zebrafish ubiquitin (ubi) promoter, which drives constitutive transgene expression during all developmental stages and analyzed adult organs. Notably, ubi expresses in all blood cell lineages, and we demonstrate the application of ubi-driven fluorophore transgenics in hematopoietic transplantation experiments to assess true multilineage potential of engrafted cells. We further generated transgenic zebrafish that express ubiquitous 4-hydroxytamoxifen-controlled Cre recombinase activity from a ubi:cre(ERt2) transgene, as well as ubi:loxP-EGFP-loxP-mCherry (ubi:Switch) transgenics and show their use as a constitutive fluorescent lineage tracing reagent. The ubi promoter and the transgenic lines presented here thus provide a broad resource and important advancement for transgenic applications in zebrafish.
Topics: Animals; Animals, Genetically Modified; Enzyme Activation; Integrases; Promoter Regions, Genetic; Tamoxifen; Transgenes; Ubiquitin; Zebrafish
PubMed: 21138979
DOI: 10.1242/dev.059345 -
ACS Chemical Biology Apr 2017The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor...
The use of coumarin caged molecules has been well documented in numerous photocaging applications including for the spatiotemporal control of Cre-estrogen receptor (Cre-ERT2) recombinase activity. In this article, we report that 4-hydroxytamoxifen (4OHT) caged with coumarin via a conventional ether linkage led to an unexpected photo-Claisen rearrangement which significantly competed with the release of free 4OHT. The basis for this unwanted reaction appears to be related to the coumarin structure and its radical-based mechanism of uncaging, as it did not occur in ortho-nitrobenzyl (ONB) caged 4OHT that was otherwise linked in the same manner. In an effort to perform design optimization, we introduced a self-immolative linker longer than the ether linkage and identified an optimal linker which allowed rapid 4OHT release by both single-photon and two-photon absorption mechanisms. The ability of this construct to actively control Cre-ERT2 mediated gene modifications was investigated in mouse embryonic fibroblasts (MEFs) in which the expression of a green fluorescent protein (GFP) reporter dependent gene recombination was controlled by 4OHT release and measured by confocal fluorescence microscopy and flow cytometry. In summary, we report the implications of this photo-Claisen rearrangement in coumarin caged compounds and demonstrate a rational linker strategy for addressing this unwanted side reaction.
Topics: Animals; Cells, Cultured; Chromatography, Liquid; Coumarins; Kinetics; Mice; Photochemistry; Selective Estrogen Receptor Modulators; Spectrum Analysis; Tamoxifen
PubMed: 28191924
DOI: 10.1021/acschembio.6b00999 -
ELife Feb 2014In mouse hairy skin, lanceolate complexes associated with three types of hair follicles, guard, awl/auchene and zigzag, serve as mechanosensory end organs. These...
In mouse hairy skin, lanceolate complexes associated with three types of hair follicles, guard, awl/auchene and zigzag, serve as mechanosensory end organs. These structures are formed by unique combinations of low-threshold mechanoreceptors (LTMRs), Aβ RA-LTMRs, Aδ-LTMRs, and C-LTMRs, and their associated terminal Schwann cells (TSCs). In this study, we investigated the organization, ultrastructure, and maintenance of longitudinal lanceolate complexes at each hair follicle subtype. We found that TSC processes at hair follicles are tiled and that individual TSCs host axonal endings of more than one LTMR subtype. Electron microscopic analyses revealed unique ultrastructural features of lanceolate complexes that are proposed to underlie mechanotransduction. Moreover, Schwann cell ablation leads to loss of LTMR terminals at hair follicles while, in contrast, TSCs remain associated with hair follicles following skin denervation in adult mice and, remarkably, become re-associated with newly formed axons, indicating a TSC-dependence of lanceolate complex maintenance and regeneration in adults. DOI: http://dx.doi.org/10.7554/eLife.01901.001.
Topics: Animals; Cell Death; Denervation; Diphtheria Toxin; Genotype; Hair Follicle; Mechanoreceptors; Mechanotransduction, Cellular; Mice; Mice, Transgenic; Microscopy, Electron, Transmission; Nerve Regeneration; Phenotype; Schwann Cells; Tamoxifen
PubMed: 24569481
DOI: 10.7554/eLife.01901