Project description:We used a fractionation scheme to isolate protrusions and cell bodies from control or APC knockdown fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). Four replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 7% of detected RNAs were enriched in protrusions of control cells. Knockdown of APC reduced the protrusion enrichement of a subset of these RNAs, indicating that they are dependent on APC for their localization. Localization of the remaining RNAs was not significantly affected upon APC knockdown suggesting that their localization is mediated by an APC-independent mechanism.
Project description:We generated fibroblast lines overexpressing beta-globin constructs under a doxycycline-inducible promoter. These constructs contain the beta-globin coding sequence followed by either a control 3'UTR (HBB) or the Pkp4 3'UTR. The Pkp4 3'UTR carries a protrusion-targeting element and is expected to compete with localization of endogenous protrusion-localized mRNAs. We used a fractionation scheme to isolate protrusions and cell bodies from HBB and Pkp4 UTR-expressing fibroblasts, which were induced to extend protrusions in response to addition of lysophosphatidic acid (LPA). 4 replicate protrusion and cell body samples for each cell type were analyzed by RNA-Seq. To identify RNAs enriched in protrusions, a protrusion/cell body ratio was calculated. We find that ca. 4.5% of detected RNAs were enriched in protrusions of control HBB cells. Overexpression of the Pkp4 UTR reduced the protrusion enrichement of a subset of these RNAs, while the degree of localization of the remaining RNAs was not significantly affected.
Project description:We identified genes expressed in mouse liver that are regulated by Cux2, a highly female-specific liver transcription factor whose expression is regulated by sex-dependent plasma GH patterns. Using siRNA to knockdown Cux2 expression in female liver, we show that female specific genes are predominantly repressed by Cux2 knockdown. In contrast, similar numbers of male-biased genes are repressed as are induced by Cux2 knockdown. A scrambled, non-specific siRNA was used as a control. (Published in: TL Conforto et al 2012, Mol Cell Biol. 2012, 32:4611-4627. PubMed PMID: 22966202; PMCID: PMC3486175)
Project description:PURPOSE: To provide a detailed gene expression profile of the normal postnatal mouse cornea. METHODS: Serial analysis of gene expression (SAGE) was performed on postnatal day (PN)9 and adult mouse (6 week) total corneas. The expression of selected genes was analyzed by in situ hybridization. RESULTS: A total of 64,272 PN9 and 62,206 adult tags were sequenced. Mouse corneal transcriptomes are composed of at least 19,544 and 18,509 unique mRNAs, respectively. One third of the unique tags were expressed at both stages, whereas a third was identified exclusively in PN9 or adult corneas. Three hundred thirty-four PN9 and 339 adult tags were enriched more than fivefold over other published nonocular libraries. Abundant transcripts were associated with metabolic functions, redox activities, and barrier integrity. Three members of the Ly-6/uPAR family whose functions are unknown in the cornea constitute more than 1% of the total mRNA. Aquaporin 5, epithelial membrane protein and glutathione-S-transferase (GST) omega-1, and GST alpha-4 mRNAs were preferentially expressed in distinct corneal epithelial layers, providing new markers for stratification. More than 200 tags were differentially expressed, of which 25 mediate transcription. CONCLUSIONS: In addition to providing a detailed profile of expressed genes in the PN9 and mature mouse cornea, the present SAGE data demonstrate dynamic changes in gene expression after eye opening and provide new probes for exploring corneal epithelial cell stratification, development, and function and for exploring the intricate relationship between programmed and environmentally induced gene expression in the cornea. Keywords: other