Analysis of expression profiles in stage II colon cancer according to the APC gene status
Expression Profiles in Stage II Colon Cancer According to APC Gene Status.
Sex, Age, Specimen part
View SamplesTristetraprolin is a vertebrate CCCH tandem zinc finger protein that can bind to and destabilize certain mRNAs containing AU-rich element binding sites. zfs1 is the single gene in the fission yeast, Schizosaccharomyces pombe, that encodes a protein containing the critical features of the tristetraprolin zinc finger domain. zfs1 has been linked to pheromone signal transduction control and to the coordination of mitosis, but no biological function has been ascribed to the zfs1 protein. Through a functional genomics approach we compared transcript levels in wild-type and zfs1-deficient S. pombe strains; those elevated in the zfs1-deficient strain were examined for the presence of potential tristetraprolin-like binding sites. One such potential target transcript was encoded by arz1, a gene encoding a protein of unknown function that contains armadillo repeats. arz1 mRNA decay was inhibited in the zfs1-deficient strain when it was expressed under the control of a thiamine-repressible promoter. Mutations within one AU-rich element present in the arz1 3-untranslated region protected this transcript from zfs1-promoted decay, whereas mutating another potential binding site had no effect. Binding assays confirmed a direct interaction between zfs1 and arz1 mRNA-based probes; this interaction was eliminated when key residues were mutated in either zfs1 zinc finger. zfs1 and its targets in S. pombe represent a useful model system for studies of zinc finger protein/AU-rich element interactions that result in mRNA decay.
Characterization of zfs1 as an mRNA-binding and -destabilizing protein in Schizosaccharomyces pombe.
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View SamplesTransient genetic modification of plant protoplasts is a straightforward and rapid technique for the analysis of numerous aspects of plant biology. One drawback in the analysis of transformed protoplast suspensions is that they are a heterogeneous mix of cells that have and have not been successfully transfected. To overcome this problem, we have developed a system that employs a fluorescent positive selection marker in combination with flow cytometric analysis as well as fluorescence activated cell sorting (FACS) to isolate responses in the transfected protoplasts exclusively. This recombinase-compatible system enables high-throughput screening of genetic circuitry. Moreover, the use of FACS allows in depth downstream analysis. Lastly, over-expression is an effective means to dissect regulatory networks, especially where redundancy exists. Here, this system has been applied to the study of auxin signaling in order to investigate reporter gene activation and genome-wide transcriptional changes in response to manipulation of the auxin-response network. We have transiently over-expressed dominant negative mutant isoforms of Aux/IAA transcription factors (IAA7mII and IAA19mII; Tiwari et al., 2001) in Arabidopsis Pwer::GFP root protoplasts, making use of a RFP fluorescent positive selection marker and FACS to isolate the dually labeled (IAAnmII expressing and Pwer::GFP-positive) cells. We have compared the transcriptional differences between an empty vector control, IAA7mII and IAA19mII protoplasts that had either been treated with 5microM IAA or mock-treated for 3 hours.
Positive fluorescent selection permits precise, rapid, and in-depth overexpression analysis in plant protoplasts.
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View SamplesHedgehog (Hh) signaling is critical for organogenesis, tissue homeostasis, and stem cell maintenance. Smoothened (SMO), the primary effector of Hh signaling, is expressed ectopically in human breast cancer, as well as in other cancers. Constitutive activation of SMO in mouse mammary glands leads to paracrine stimulation of proliferation, as well as hyperplasia. In canonical signaling, SMO functions via GLI transcription factor activation. However, recent data from Drosophila and mammalian cell lines indicate that SMO can function non-canonically as a G-protein coupled receptor (GPCR) by coupling to heterotrimeric G proteins, particularly those in the pertussis toxin (PTX)-sensitive G-alpha-i (Gai) class. Whether SMO functions as a GPCR in mammalian tissues in vivo is not known. Using genetically modified mouse models, we demonstrate here that SMO-induced stimulation of proliferation is PTX sensitive, and requires Gai2, but not Gai1 or Gai3. Our findings provide evidence for a non-canonical GPCR function of activated SMO in vivo, a finding that may have clinical significance given that most SMO-targeted agents were selected based largely on their ability to block canonical GLI-mediated transcription. Overall design: Primary mammary epithelial cell RNA was deep-sequenced from mT-mG/SmoM2;MMTV-Cre (EGFP), mT-mG/SmoM2;MMTV-Cre (tdTomato), and mT-mG/SmoM2;+ cells to examine the effects of SmoM2 overexpression in the mammary gland.
An essential role for Gα(i2) in Smoothened-stimulated epithelial cell proliferation in the mammary gland.
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View SamplesDisrupted skin barrier due to altered keratinocyte differentiation is common in pathologic conditions such as atopic dermatitis, ichthyosis and psoriasis. However, the molecular cascades governing keratinocyte terminal differentiation are still poorly understood. We have previously demonstrated that a dominant mutation in ZNF750 leads to a clinical phenotype that reminiscent of psoriasis and seborrheic dermatitis. We defined ZNF750 as a nuclear effector that is strongly activated in and essential for keratinocyte terminal differentiation. ZNF750 knockdown in HaCaT keratinocytes markedly reduced the expression of epidermal late differentiation markers, including gene subsets of epidermal differentiation complex and skin barrier formation such as FLG, LOR, SPINK5, ALOX12B and DSG1, known to be mutated in various human skin diseases. Furthermore, ZNF750 over-expression in undifferentiated cells induced terminal differentiation genes. Thus, ZNF750 is a regulator of keratinocyte terminal differentiation, and with its downstream targets can serve in future elucidation of therapeutics for common disease of skin barrier
ZNF750 is expressed in differentiated keratinocytes and regulates epidermal late differentiation genes.
Specimen part
View Samples40 QC single cells multiplexed using the CEL-Seq protocol Overall design: 40 cells from the QC
Quantification of cell identity from single-cell gene expression profiles.
Age, Subject
View SamplesThe Arabidopsis quiescent center (QC) is a small group of cells with low mitotic activity located at the center of the root stem cell niche. Its transcriptional profile was previously analyzed using two repeats of cells FACS isolated using the WOX5 marker.
Quantification of cell identity from single-cell gene expression profiles.
Specimen part
View SamplesThe organs of multicellular species are comprised of cell types that must function together to perform specific tasks. One critical organ function is responding to internal or external change but little is known about how responses are tailored to specific cell types or coordinated among them on a global level. Here we use cellular profiling of five Arabidopsis root cell types in response to a limiting resource, nitrogen, to uncover a vast and predominantly cell-specific response that was largely undetectable using traditional methods. These methods reveal a new class of cell-specific nitrogen responses. As a proof-of-principle, we dissected one cell-specific response circuit that mediates nitrogen-induced changes in root branching from pericycle cells. Thus, cellular response profiling links gene modules to discrete functions in specific cell types.
Cell-specific nitrogen responses mediate developmental plasticity.
Specimen part
View SamplesA critical step in regeneration is recreating the cellular identities and patterns of lost organs long after embryogenesis is complete. In plants, perpetual (indeterminate) organ growth occurs in apical stem cell niches, which have been shown to re-establish quickly when damaged or removed (1,2). Here we ask whether the machinery of perpetual organ growth, stem cell activity, is needed for the phase of regeneration that leads to replenishing lost cell identities and patterning, or, whether organ re-establishment enlists a wider group of pluripotent cells. We adapt a root tip regeneration system to Arabidopsis that permits us to assess the molecular and functional recovery of specific cell fates during organ regeneration. These results suggest a rapid restoration of missing cell fate and function in advance of the recovery of stem cell activity. Surprisingly, plants with mutations that fail to maintain stem cell activity were able to re-pattern their distal tip and re-specify lost cell fates. Thus, although stem cell activity is required to resume indeterminate growth (3), our results show it is not necessary for cell re-specification and patterning steps. This implies a regeneration mechanism that coordinates patterning of the whole organ, as in embryogenesis, but is initiated from different starting morphologies. 1. Feldman, L. J. Denovo Origin of Quiescent Center Regenerating Root Apices of Zea-Mays. Planta 128, 207-212 (1976). 2. Xu, J. et al. A molecular framework for plant regeneration. Science 311, 385-8 (2006). 3. Gordon, S. P. et al. Pattern formation during de novo assembly of the Arabidopsis shoot meristem. Development 134, 3539-48 (2007).
Organ regeneration does not require a functional stem cell niche in plants.
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View SamplesWe investigated the morphological roots decisions of Arabidopsis in a NO3- heterogeneous medium. To do so, we used the Split-Root System which is an experimental set up to assess root decisions in nutrient heterogeneous medium. Split-root plants have been subjected to three different treatments. Control KNO3 plants received KNO3 on both sides of the root system (C.NO3) and Control KCl plants received KCl on both sides (C.KCl) as a nitrogen deprivation treatment. 'Split' plants received KNO3 on one side (Sp.NO3) and KCl on the other side (Sp.KCl) of the root system to assess the root decision-making in a heterogeneous environment.
Nitrogen economics of root foraging: transitive closure of the nitrate-cytokinin relay and distinct systemic signaling for N supply vs. demand.
Specimen part, Treatment
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