Knockdown and Overexpression of Cbx4 wild type, Cbx4 chromodomain mutant, and Cbx4 sumoylation mutant in human epidermal stem cells
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ABSTRACT:
ORGANISM(S): Homo sapiens
SUBMITTER: Salvador Benitah
PROVIDER: E-GEOD-31147 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex-1 (PRC1)-associated protein, maintains human epidermal stem cells slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its anti-senescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors, such as Ezh2, Dnmt1, and Bmi1, to prevent the activate stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. Primary human keratinocytes were infected with control vector pBABE, Cbx4 wildtype and a chromodomain mutant version of Cbx4 (F11A and W35L), both fused to a Estrogen Receptor to render their activaty inducible by addition of 4OHT to the media. Cells were cultured for 5 days (after infection and selection) under 4OHT treatment, after which total RNA was collected. For the knockdown, the pRetroSuperPuro was used as a control vector, in which a shRNA sequence for Cbx4 was introduced to interfere with Cbx4 expression. Cell were cultured in the same way as for the overexpression experiments.
2011-08-01 | E-GEOD-31093 | biostudies-arrayexpress
Project description:Human epidermal stem cells transit from a slow cycling to an actively proliferating state to contribute to homeostasis. Both stem cell states differ in their cell cycle profiles but must remain guarded from differentiation and senescence. Here we show that Cbx4, a Polycomb Repressive Complex-1 (PRC1)-associated protein, maintains human epidermal stem cells slow-cycling and undifferentiated, while protecting them from senescence. Interestingly, abrogating the polycomb activity of Cbx4 impairs its anti-senescent function without affecting stem cell differentiation, indicating that differentiation and senescence are independent processes in human epidermis. Conversely, Cbx4 inhibits stem cell activation and differentiation through its SUMO ligase activity. Global transcriptome and chromatin occupancy analyses indicate that Cbx4 regulates modulators of epidermal homeostasis and represses factors, such as Ezh2, Dnmt1, and Bmi1, to prevent the activate stem cell state. Our results suggest that distinct Polycomb complexes balance epidermal stem cell dormancy and activation, while continually preventing senescence and differentiation. Primary human keratinocytes were infected with control vector pBABE, Cbx4 wildtype and a Sumoylation mutant version of Cbx4 (the two Sumo Interaction Motifs were deleted), both fused to a Estrogen Receptor to render their activaty inducible by addition of 4OHT to the media. Cells were cultured for 5 days (after infection and selection) under 4OHT treatment, after which total RNA was collected.
2011-08-01 | E-GEOD-31094 | biostudies-arrayexpress
Project description:We designed a custom microarray to profile the expression and used it to measure the expression of 9929 human lncRNAs manually-annotated by the GENCODE group as part of the ENCODE consortium. One microarray hybridisation per biological sample.
2012-02-01 | E-GEOD-34894 | biostudies-arrayexpress
Project description:Surveying epidermal stem cell homeostais over a 45-hour time course 45h time course with 10 samples over triplicates (x2 Agilent arrays)
2013-09-06 | E-GEOD-50631 | biostudies-arrayexpress
Project description:Activation-induced cytidine deaminase (AID) is essential for class switch recombination (CSR) and somatic hypermutation (SHM). Its deregulated expression acts as a genomic mutator that can contribute to the development of various malignancies. During treatment with imatinib mesylate (IM), patients with chronic myeloid leukemia (CML) often develop hypogammaglobulinemia, the mechanism of which has not yet been clarified. Here, we provide evidence that CSR upon B cell activation is apparently inhibited by IM through downregulation of AID. Furthermore, expression of E2A, a key transcription factor for AID induction, was markedly suppressed by IM. These results elucidate not only the underlying mechanism of IM-induced hypogammaglobulinemia but also its potential efficacy as an AID suppressor. We investigated that class switch recombination(CSR) upon B cell activation is apparently inhibited by imatinib mesylate(IM) through downregulation of activation-induced cytidine deaminase(AID). Furthermore, expression of E2A was markedly suppressed by IM. To elucidate the more detailed pathway, we performed the microarray analysis. Microarray analysis was performed on splenocytes cultured for 72 h in the presence or absence of 10 µM Imatinib. The mouse splenocytes were cultured for 72 h with or without 10 µM Imatinib (IM) in conditioning medium containing IL-4 and LPS. RNA from 1X10^6 splenocytes used for microarray analysis was isolated using the RNeasy Mini Kit (50) (Qiagen, Hilden, Germany). Gene expression microarray analysis was performed using one-color microarray-based gene-expression analysis (Agilent Technologies, Santa Clara, CA, USA) according to the manufacturer’s instructions. After scanning, expression values for the genes were determined using GeneSpringGX software.
2012-02-08 | E-GEOD-35559 | biostudies-arrayexpress
Project description:A major challenge in Down syndrome (DS) is to understand how the extra-dose of functional chromosome 21 (HSA21) genetic elements can impact on the tissue-specific transcriptome to contribute to phenotypic alterations. MiRNAs are post-transcriptional modulators with genome-wide regulatory effects. Five microRNAs have been identified in HSA21 that are present in triple copy in DS individuals. Interestingly, in the Ts65Dn mouse model of DS two of these miRNAs, miR-155 and miR-802, are also triplicated resulting in its overexpression. In the current work, we have developed a lentiviral miRNA-sponge genetic strategy for miR-155 and miR-802 (Lv-miR155-802T) to identify novel mRNA targets involved in hippocampal function. Hippocampal injection of the lentiviral sponge in Ts65Dn mice reduced miR-155 and miR-802 overexpression. Noticeable lentiviral sponge rescued the expression of the miRNA predicted targets showing the potential of the strategy to identify miRNA dosage-sensitive genes with potential involvement in DS-hippocampal phenotypes. Euploid and trisomic adult mice were bilaterally injected at the level of the ventral hippocampus at selected coordinates (AP=-3.3mm, L=+/- 3mm, DV=-3.3mm and -2.3mm relative to bregma). Up to 108 transducing units (3µl of viral suspensions of Lv-Contol or Lv-miR155-802T) were injected into each hemisphere at a rate of 0.2 µl/min, under the precise control of an infusion pump (Ultramicropump, World Precision Instruments). Mice were euthanized for hippocampus collection at day 23 after administration. Transcriptome of hippocampus of euploid and trisomic mice treated with Lv-Control or Lv-miR155-802T was analysed using an Agilent SurePrint G3 Mouse gene expression 8x60K Microarray (ID 028005). A total RNA 100 ng, obtained using miRNeasy Mini Ki (QIAGEN), were labeled using LowInputQuick Amp Labeling kit (Agilent 5190-2305) following manufacturer instructions. Briefly, mRNA was reverse transcribed in the presence of T7-oligo-dT primer to produce cDNA. cDNA was then in vitro transcribed with T7 RNA polymerase in the presence of Cy3-CTP to produce labeled cRNA. The labeled cRNA was hybridized to the Agilent SurePrint G3 Mouse gene expression 8x60K Microarray (ID 028005) according to the manufacturer's protocol. The arrays were washed, and scanned on an Agilent G2565CA microarray scanner at 100% PMT and 3µm resolution. Intensity data was extracted using the Feature Extraction software (Agilent). Replicates from each genotypes and treatment group were distributed as follows: EU+Lv-Contol n=4, EU+Lv-miR155-802T n=4, TS+Lv-Contol n=5, TS+Lv-miR155-802T n=3.
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