Lysine methylation of histones is associated with both transcriptionally active chromatin and with silent chromatin, depending on what residue is modified. Histone methyltransferases and demethylases ensure that histone methylations are dynamic and can vary depending on cell cycle- or developmental stage. KDM4A demethylates H3K36me3, a modification enriched in the 3end of active genes. The genomic targets and the role of KDM4 proteins in development remain largely unknown.
Gene regulation by the lysine demethylase KDM4A in Drosophila.
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View SamplesThis SuperSeries is composed of the SubSeries listed below.
Activated stress response pathways within multicellular aggregates utilize an autocrine component.
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View SamplesMammalian cells were grown as multicellular aggregates (spheroids) in an effort to determine the signaling events required for two cellular transformations states; primary foreskin fibroblasts (HFF-2) and glioblastoma cancer (T98G) cells, to survive at room temperature under oxygen and nutrient-deprived conditions for extended periods of time (2 weeks) and subsequently grown out from the arrested state as adherent monolayers. HFF-2 cells were cultured in DMEM supplemented with 15% fetal bovine serum and 5% carbon dioxide humidified air at 37 degrees C. T98G cells were cultured in EMEM with 10% FBS, 5% non-essential amino acids and 5% carbon dioxide humidified air at 37 degreesC. Monolayers were grown in T-185 flasks to 60% confluency then split into T-185 flasks coated with a 1% agarose mix in a 2:1 media/water ratio. Cells were suspended in 30 ml of supplemented media and grown for 4 days in order to form multicellular spheroids as described previously by our group (J. Cell. Physiol., 206 [2006] 526-536; see GSE1364 and GSE1455 for similar experiments with HEK293 cells). The suspension was removed from the flasks and centrifuged (1500 x g, 2 min) and the media removed. The pellet was returned to the flasks and then placed in vacuum bags (Dri-shield 2000 moisture barrier bag from Surmount Inc., USA; Cat. number 70068), which were sealed immediately under vacuum (Deni Magic Vac, Champion model; Keystone Manufacturing, USA). Vacuum-sealed flasks were stored for 2 weeks (in the dark) at room temperature. Recovery was initiated by removing the flask from the bag and resuspending the spheroids in supplemented media and placing the flasks in a 5% CO2/humidified air incubator maintained at 37 degreesC. Timepoints for transcriptional analysis were monolayer (control), 4 day growth spheroids, 2 week stored spheroids and 7 day growth back to monolayers.
Activated stress response pathways within multicellular aggregates utilize an autocrine component.
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View SamplesMammalian cells were grown as multicellular aggregates (spheroids) in an effort to determine the signaling events required for two cellular transformations states; primary foreskin fibroblasts (HFF-2) and glioblastoma cancer (T98G) cells, to survive at room temperature under oxygen and nutrient-deprived conditions for extended periods of time (2 weeks) and subsequently grown out from the arrested state as adherent monolayers. HFF-2 cells were cultured in DMEM supplemented with 15% fetal bovine serum and 5% carbon dioxide humidified air at 37 degrees C. T98G cells were cultured in EMEM with 10% FBS, 5% non-essential amino acids and 5% carbon dioxide humidified air at 37 degreesC. Monolayers were grown in T-185 flasks to 60% confluency then split into T-185 flasks coated with a 1% agarose mix in a 2:1 media/water ratio. Cells were suspended in 30 ml of supplemented media and grown for 4 days in order to form multicellular spheroids as described previously by our group (J. Cell. Physiol., 206 [2006] 526-536; see GSE1364 and GSE1455 for similar experiments with HEK293 cells). The suspension was removed from the flasks and centrifuged (1500 x g, 2 min) and the media removed. The pellet was returned to the flasks and then placed in vacuum bags (Dri-shield 2000 moisture barrier bag from Surmount Inc., USA; Cat. number 70068), which were sealed immediately under vacuum (Deni Magic Vac, Champion model; Keystone Manufacturing, USA). Vacuum-sealed flasks were stored for 2 weeks (in the dark) at room temperature. Recovery was initiated by removing the flask from the bag and resuspending the spheroids in supplemented media and placing the flasks in a 5% CO2/humidified air incubator maintained at 37 degreesC. Timepoints for transcriptional analysis were monolayer (control), 4 day growth spheroids, 2 week stored spheroids and 7 day growth back to monolayers.
Activated stress response pathways within multicellular aggregates utilize an autocrine component.
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View SamplesThe Mediator complex is an evolutionary conserved multiprotein complex that plays an essential role in initiating and regulating transcription. Its function is to act as a universal adaptor between RNA Polymerase II and DNA-bound transcription factors to translate regulatory information from activators and repressors to the transcriptional machinery. We have found that the PFT1 gene (which encodes the MED25 subunit of the Mediator complex) is required for the uncompromised expression of both salicylic acid- and jasmonate-dependent defense genes as well as resistance to the leaf-infecting fungal pathogens, Alternaria brassicicola and Botrytis cinerea in Arabidopsis. Surprisingly, we found that the pft1/med25 mutant showed increased resistance to the root infecting pathogen Fusarium oxysporum and that this resistance was independent of classical defense genes. In addition, the over-expression of PFT1 led to increased susceptibility to F. oxysporum. Therefore, to explore this phenomenon further, we wished to use whole genome transcript profiling to identify which genes may be playing a role in pft1/med25-mediated resistance to F. oxysporum.
The mediator complex subunit PFT1 is a key regulator of jasmonate-dependent defense in Arabidopsis.
Specimen part, Treatment
View SamplesJasmonate (JA) signaling plays a key role in mediating both resistance and susceptibility to the root-infecting fungal pathogen Fusarium oxysporum. Within this system, the roles of the JA-signaling repressor gene family of JASMONATE ZIM-domain (JAZ) genes had not been investigated. By screening JAZ T DNA insertion lines for altered resistance or susceptibility to F. oxysporum, we identified a JAZ7 mutant (jaz7-1D) highly susceptible to F. oxysporum infection. Further analyses revealed jaz7-1D exhibits constitutively active JAZ7 expression, enhanced expression of JA-defense marker genes, and increased sensitivity to JA-inhibition of root elongation. To further explore altered JA-signaling and JA-responses in this mutant, we use whole transcriptome profiling of jaz7-1D versus wild-type (Col-0) plants after mock/control and JA treatment.
Characterization of a JAZ7 activation-tagged Arabidopsis mutant with increased susceptibility to the fungal pathogen Fusarium oxysporum.
Specimen part, Treatment
View SamplesAMPK (AAK-2) and calcineurin (TAX-6) mediate longevity exclusively through post-translational modification of CRTC-1, the sole C. elegans CRTC (CREB regulated transcriptional coactivator).
Lifespan extension induced by AMPK and calcineurin is mediated by CRTC-1 and CREB.
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View SamplesThe Brakeless protein performs many important functions during Drosophila development, but how it controls gene expression is not understood. We previously showed that Brakeless can function as a transcriptional co-repressor. Here, we report transcriptional profiling of brakeless mutant embryos to identify additional target genes.
The Brakeless co-regulator can directly activate and repress transcription in early Drosophila embryos.
Specimen part
View SamplesMenopausal estrogen (E2) replacement therapy increases the risk of estrogen receptor (ER)-positive epithelial ovarian cancers (EOC). Whether E2 is tumorigenic or promotes expansion of undiagnosed pre-existing disease is unknown. To determine E2 effects on tumor promotion, we developed an intraperitoneal mouse xenograft model using ZsGreen fluorescent ER- 2008 and ER+ PEO4 human EOC cells. Tumor growth was quantified by in vivo fluorescent imaging. In ER+ tumors, E2 significantly increased size, induced progesterone receptors, and promoted lymph node metastasis, confirming that ER are functional and foster aggressiveness. Laser captured human EOC cells from ER- and ER+ xenografted tumors were profiled for expression of E2-regulated genes. Three classes of E-regulated EOC genes were defined, but less than 10% were shared with E-regulated breast cancer genes. Since breast cancer selective ER modulators (SERM) are therapeutically ineffective in EOC, we suggest that our EOC-specific E-regulated genes can assist pharmacologic discovery of ovarian targeted SERM.
Tissue-specific pathways for estrogen regulation of ovarian cancer growth and metastasis.
Specimen part
View Samplesp63 mutations have been associated with several human hereditary disorders characterized by ectodermal dysplasia such as EEC syndrome, ADULT syndrome and AEC syndrome . The location and functional effects of the mutations that underlie these syndromes reveal a striking genotype-phenotype correlation. Unlike EEC and ADULT that result from missense mutations in the DNA-binding domain of p63, AEC is solely caused by missense mutations in the SAM domain of p63. We report a study on the TAp63a isoform, the first to be expressed during development of the embryonic epithelia, and on its naturally occurring Q540L mutant derived from an AEC patient. To assess the effects of the Q540L mutation, we generated stable cell lines expressing TAp63a wt, DeltaNp63 alpha or the TAp63 alpha-Q540L mutant protein and used them to systematically compare the cell growth regulatory activity of the mutant and wt p63 proteins and to generate, by microarray analysis, a comprehensive profile of differential gene expression. We found that the Q540L substitution impairs the transcriptional activity of TAp63a and causes misregulation of genes involved in the control of cell growth and epidermal differentiation.
The Hay Wells syndrome-derived TAp63alphaQ540L mutant has impaired transcriptional and cell growth regulatory activity.
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