Dataset Information

Transcription profiling of mouse PERK knockouts vs. wikd type

ABSTRACT: to study the proliferation of PERK knockout mice islets. The major changes are the proliferation genes Experiment Overall Design: 2days after birth, Experiment Overall Design: Collagenase P digestion Experiment Overall Design: cRNA label with Ambion kit Experiment Overall Design: Hybridization with Affymetrix mouse 430A chip

INSTRUMENT(S): 418 [Affymetrix]

ORGANISM(S): Mus musculus

SUBMITTER: YULIN LI

PROVIDER: E-GEOD-6193 | ArrayExpress| 2012-03-27

SECONDARY ACCESSION(S): GSE6193PRJNA97547

REPOSITORIES: GEO, ArrayExpress

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Publications

PERK EIF2AK3 control of pancreatic beta cell differentiation and proliferation is required for postnatal glucose homeostasis.

Zhang Wei W Feng Daorong D Li Yulin Y Iida Kaori K McGrath Barbara B Cavener Douglas R DR

Cell metabolism 20061201 6

Mutations in PERK (EIF2AK3) result in permanent neonatal diabetes as well as several other anomalies that underlie the human Wolcott-Rallison syndrome, and these anomalies are mirrored in Perk knockout mice. To identify the cause of diabetes in PERK-deficient mice, we generated a series of tissue- and cell-specific knockouts of the Perk gene and performed a developmental analysis of the progression to overt diabetes. We discovered that PERK is specifically required in the insulin-secreting beta ...[more]

PMID: 17141632

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Project description:In eukaryotes, regulation of mRNA translation enables a fast, localized and finely tuned expression of gene products. Within the translation process, the first stage of translation initiation is most rigorously modulated by the actions of eukaryotic initiation factors (eIFs) and their associated proteins. These 11 eIFs catalyze the joining of the tRNA, mRNA and rRNA into a functional translation complex. Their activity is influenced by a wide variety of extra- and intracellular signals, ranging from global, such as hormone signaling and unfolded proteins, to specific, such as single amino acid imbalance and iron deficiency. Their action is correspondingly comprehensive, in increasing or decreasing recruitment and translation of most cellular mRNAs, and specialized, in targeting translation of mRNAs with regulatory features such as a 5’ terminal oligopyrimidine tract (TOP), upstream open reading frames (uORFs), or an internal ribosomal entry site (IRES). In mammals, two major pathways are linked to targeted mRNA translation. The target of rapamycin (TOR) kinase induces translation of TOP and perhaps other subsets of mRNAs, whereas a family of eIF2 kinases does so with mRNAs containing uORFs or an IRES. TOR targets translation of mRNAs that code for proteins involved in translation, an action compatible with its widely accepted role in regulating cellular growth. The four members of the eIF2 kinase family increase translation of mRNAs coding for stress response proteins such as transcription factors and chaperones. Though all four kinases act on one main substrate, eIF2, published literature demonstrates both common and unique effects by each kinase in response to its specific activating stress. This suggests that the activated eIF2 kinases regulate the translation of both a global and a specific set of mRNAs. Up to now, few studies have attempted to test such a hypothesis; none has been done in mammals. We use array analysis to determine the global mRNA shift into polysomes following a stress response, and to compare the translational response following activation of GCN2 versus PERK, two of the four eIF2alpha kinases. Experiment Overall Design: Perk wild-type or knockout mouse liver were perfused without or with 2,4-di-tert-butylhydroquinone (tBuHQ) for RNA extraction and hybridization of Affymetrix microarrays. RNA was extracted from unfractionated liver samples and polysome fraction of samples separated on sucrose density gradient. To minimize biological variations, we pooled RNA from two perfused liver samples to use in each array analysis. The conditions were total and polysome fraction of Perk+/+, -tBuHQ or +tBuHQ; total and polysome fraction of Perk-/-, -tBuHQ or +tBuHQ. Each array analysis was done in duplicate.

Project description:XBP1 is a transcription factor that is induced by unconventional splicing associated with endoplasmic reticulum stress and plays a role in development of liver and plasma cells. We previously reported that brain derived neurotrophic factor (BDNF) leads to splicing of XBP1 mRNA in neurites, and that XBP1 is required for BDNF-induced neurite extension and branching. To search for the molecular mechanisms of how XBP1 plays a role in neural development, comprehensive gene expression analysis was performed in primary telencephalic neurons obtained from Xbp1 knockout mice at embryonic day 12.5. By searching for the genes induced by BDNF in wild type neurons but this induction was reduced in Xbp1 knockout mice, we found that upregulation of three GABAergic markers, somatostatin (Sst), neuropeptide Y (Npy), and calbindin (Calb1), were compromised in Xbp1 knockout neurons. Attenuated induction of Npy and Calb1 was confirmed by quantitative RT-PCR. In neurons lacking in Xbp1, upregulation of GABAergic markers was attenuated. Impaired BDNF-induced neurite extension in Xbp1 knockout neurons might be mediated by disturbed BDNF-induced differentiation of GABAergic interneurons. Experiment Overall Design: Brain derived neurotrophic factor (BDNF) effect examined in telencephalon primary cultures from Xbp1 knockouts and wild-type controls. Experiment Overall Design: Two female Xbp1 +/- mice were mated with male Xbp1 +/- mice, and at embryonic day 12.5 (E12.5) the embryos were dissected. Among the 25 embryos obtained from the 2 pregnant female mice, 6 were genotyped as Xbp1 -/- and 4 were Xbp1 +/+ by a rapid PCR assay using Z-Taq. Telencephalon was dissected from each embryo, and treated with collagenase and trypsin. Six Xbp1 -/- telencephalon samples and 4 Xbp1 +/+ samples were collected together, respectively. Each sample was divided into 15 aliquots, and the cells were subject to low density culture on plastic culture dishes. The neurons were maintained in a serum-free medium (Neurobasal medium supplemented with 0.5 mM glutamine and B27 supplement [Invitrogen]). On the third day in vitro (3 DIV) , neurons in 10 of 15 dishes in each group were stimulated with BDNF (100 ng/ml). On 4 DIV, neurons in 5 dishes stimulated with BDNF (24 hours BDNF treatment) and 5 dishes with no stimulation (0 hour) were lysed to extract total RNA. On 5 DIV, neurons in 5 dishes stimulated with BDNF (48 hours BDNF treatment) were lysed. Experiment Overall Design: Total RNA was extracted using RNAeasy Micro Kit (Qiagen, Hilden, Germany) according to the protocol provided by the manufacturer. The quantity and quality of RNA were measured using NanoDrop ND-1000. Biotin-labeled cRNA for DNA microarray analysis was synthesized using Two Cycle cDNA Synthesis and IVT labeling Kit. The integrity of the cRNA samples was verified using Test3 Array. Each biotin-labeled cRNA sample from one dish was hybridized to a single Affymetrix GeneChip Mouse Genome 430A 2.0 Array, and totally 30 arrays were used. The hybridization signal on the chip was scanned using an HP GeneArray scanner and processed by MAS5. After imported into GeneSpring software, data normalization was performed by dividing each microarray data set by its median value. Probes called as present in at least half of the 30 samples were selected.

Project description:Disruptions of protein homeostasis in the endoplasmic reticulum (ER) elicit activation of the unfolded protein response (UPR), a translation- and transcription-coupled proteostatic stress response pathway. The primary translational control arm of the UPR is initiated by the PERK-dependent phosphorylation of eIF2α, leading to a large-scale reprogramming of translation and subsequent activation of an ATF4-mediated transcriptional program. It has remained challenging, however, to accurately evaluate the contribution of each component of the eIF2α/ATF4 pathway to the remodelling of transcription and translation. Here, we have used mouse embryonic fibroblasts containing either a knock-in of the non-phosphorylatable eIF2α S51A mutant or knock-out for ATF4 by ribosome profiling and mRNA-seq to define the specific contributions of eIF2α phosphoryation and ATF4 in controlling the translational and transcriptional components of the UPR. These studies show that the transcriptional and translational targets of each P-eIF2α, ATF4, and the other UPR gene expression programs overlapped extensively, leading to a core set of UPR genes whose robust expression in response to ER stress is driven by multiple mechanisms. The identification of other, more factor-specific targets illustrated the potential for functional specialization of the UPR. As the UPR progressed temporally, cells employed distinct combinations of transcriptional and translational mechanisms, initiated by different factors, to adapt to ongoing stress. These effects were accompanied by a buffering effect where changes in mRNA levels were coupled to opposing changes in ribosome loading, a property which makes cooperation between transcription and translation essential to confer robust protein expression. Translational analysis by ribosome profiling and mRNA-seq of PERK pathways mutants during the UPR. Mouse embryonic fibroblasts (MEFs) lacking components of the PERK pathway (eIF2a phosphorylation and ATF4) were subjected to ER stress and analyzed by ribosome profiling.