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Differences in gene expression patterns in mouse embryonic fibroblast Flp-In-3T3 cell lines stably expressing Pax6 or Pax6(5a).

ABSTRACT: Novel target genes for the two transcription factor isoforms Pax6 and Pax6(5a) were identified by generating mouse embryonic fibroblast cell lines stably expressing either Pax6 or Pax6(5a), and comparing their gene expression pattern with the original mouse embryonic fibroblast cell line.

ORGANISM(S): Mus musculus

PROVIDER: GSE31402 | GEO | 2012/03/22

SECONDARY ACCESSION(S): PRJNA145725

REPOSITORIES: GEO

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Differences in gene expression patterns in mouse embryonic fibroblast Flp-In-3T3 cell lines stably expressing Pax6 or Pax6(5a).

Project description:Novel target genes for the two transcription factor isoforms Pax6 and Pax6(5a) were identified by generating mouse embryonic fibroblast cell lines stably expressing either Pax6 or Pax6(5a), and comparing their gene expression pattern with the original mouse embryonic fibroblast cell line. Flp-In-3T3 cell lines were generated according to the manufacturers instruction (Invitrogen), using the mouse Pax6 and Pax6(5a) cDNA as insert. Correct inserts were verified by PCR, sequencing and Westernblot.

2012-03-22 | E-GEOD-31402 | biostudies-arrayexpress

3T3 cell lines stably expressing Pax6 or Pax6(5a)--a new tool used for identification of common and isoform specific target genes.

Project description:Pax6 and Pax6(5a) are two isoforms of the evolutionary conserved Pax6 gene often co-expressed in specific stochiometric relationship in the brain and the eye during development. The Pax6(5a) protein differs from Pax6 by having a 14 amino acid insert in the paired domain, causing the two proteins to have different DNA binding specificities. Difference in functions during development is proven by the fact that mutations in the 14 amino acid insertion for Pax6(5a) give a slightly different eye phenotype than the one described for Pax6. Whereas quite many Pax6 target genes have been published during the last years, few Pax6(5a) specific target genes have been reported on. However, target genes identified by Pax6 knockout studies can probably be Pax6(5a) targets as well, since this isoform also will be affected by the knockout. In order to identify new Pax6 target genes, and to try to distinguish between genes regulated by Pax6 and Pax6(5a), we generated FlpIn-3T3 cell lines stably expressing Pax6 or Pax6(5a). RNA was harvested from these cell lines and used in gene expression microarrays where we identified a number of genes differentially regulated by Pax6 and Pax6(5a). A majority of these were associated with the extracellular region. By qPCR we verified that Ncam1, Ngef, Sphk1, Dkk3 and Crtap are Pax6(5a) specific target genes, while Tgfbi, Vegfa, EphB2, Klk8 and Edn1 were confirmed as Pax6 specific target genes. Nbl1, Ngfb and seven genes encoding different glycosyl transferases appeared to be regulated by both. Direct binding to the promoters of Crtap, Ctgf, Edn1, Dkk3, Pdgfb and Ngef was verified by ChIP. Furthermore, a change in morphology of the stably transfected Pax6 and Pax6(5a) cells was observed, and the Pax6 expressing cells were shown to have increased proliferation and migration capacities.

| S-EPMC3285655 | biostudies-literature

Pax6 regulates the expression of Dkk3 in murine and human cell lines, and altered responses to Wnt signaling are shown in FlpIn-3T3 cells stably expressing either the Pax6 or the Pax6(5a) isoform.

Project description:Pax6 is a transcription factor important for early embryo development. It is expressed in several cancer cell lines and tumors. In glioblastoma, PAX6 has been shown to function as a tumor suppressor. Dickkopf 3 (Dkk3) is well established as a tumor suppressor in several tumor types, but not much is known about the regulation of its expression. We have previously found that Pax6 and Pax6(5a) increase the expression of the Dkk3 gene in two stably transfected mouse fibroblast cell lines. In this study the molecular mechanism behind this regulation is looked at. Western blot and reverse transcriptase quantitative PCR (RT-qPCR) confirmed higher level of Dkk3 expression in both Pax6 and Pax6(5a) expressing cell lines compared to the control cell line. By the use of bioinformatics and electrophoretic mobility shift assay (EMSA) we have mapped a functional Pax6 binding site in the mouse Dkk3 promoter. The minimal Dkk3 promoter fragment required for transcriptional activation by Pax6 and Pax6(5a) was a 200 bp region just upstream of the transcriptional start site. Mutation of the evolutionary conserved binding site in this region abrogated transcriptional activation and binding of Pax6/Pax6(5a) to the mouse Dkk3 promoter. Since the identified Pax6 binding site in this promoter is conserved, RT-qPCR and Western blot were used to look for regulation of Dkk3/REIC expression in human cell lines. Six of eight cell lines tested showed changes in Dkk3/REIC expression after PAX6 siRNA knockdown. Interestingly, we observed that the Pax6/Pax6(5a) expressing mouse fibroblast cell lines were less responsive to canonical Wnt pathway stimulation than the control cell line when TOP/FOP activity and the levels of active β-catenin and GSK3-β Ser9 phosphorylation were measured after LiCl stimulation.

| S-EPMC4100929 | biostudies-literature

Agrobacterium tumefaciens 5A strain:5A

Project description:Agrobacterium tumefaciens 5A strain:5A Genome sequencing and assembly

| PRJNA75079 | ENA

Pax6 mutant neocortex vs. wild type

Project description:Pax6 is a transcription factor with key functional roles in embryonic development. In order to identify downstream effectors of Pax6 in the developing cerebral cortex we performed microarray analysis. We compared gene expression profiles of cortical tissues isolated from wild type and Pax6-/- mouse embryos. In order to identify Pax6 downstream targets we carried out microarray analysis of Pax6-/- mutant mice. Pax6 is highly expressed in the mouse cerebral cortex at embryonic day E14.5, therefore we selected this tissue in order to compare gene expression profiles between wild type and Pax6-/- homozygous cortici. RNA samples were isolated from three mutant and three wild type embryos.

2011-10-31 | E-GEOD-32271 | biostudies-arrayexpress

Pax6 mutant neocortex vs. wild type

2011-10-31 | GSE32271 | GEO

Identification of in vivo DNA-binding mechanisms of Pax6 and reconstruction of Pax6-dependent gene regulatory networks during lens and forebrain development

Project description:The transcription factor Pax6 is comprised of the paired domain (PD) and homeodomain (HD). In the developing forebrain, Pax6 is expressed in ventricular zone precursor cells and in specific subpopulations of neurons; absence of Pax6 results in disrupted cell proliferation and cell fate specification. Pax6 also regulates the entire lens developmental program. To reconstruct Pax6-dependent gene regulatory networks (GRNs), ChIP-seq studies were performed using lens and forebrain chromatin from mice. A total of 3,723 (forebrain) and 3,514 (lens) Pax6-containing peaks were identified, with ~70% of them found in both tissues and thereafter called “common” peaks. Analysis of Pax6-bound peaks identified motifs that closely resemble Pax6-PD, -PD/HD and -HD established binding sequences. Mapping of H3K4me1, H3K4me3, H3K27ac, H3K27me3 and RNA polymerase II revealed distinct types of tissue-specific enhancers bound by Pax6. Pax6 directly regulates cortical neurogenesis through activation (e.g. Dmrta1 and Ngn2) and repression (e.g. Ascl1, Fezf2, and Gsx2) of transcription factors. In lens, Pax6 directly regulates cell cycle exit control via components of FGF (Fgfr2, Ccnd1, and Prox1) and Wnt (Dkk3, Wnt7a, Lrp6, Bcl9l, and Ccnd1) signaling pathways. Collectively, these studies provide genome-wide analysis of Pax6-dependent GRNs in lens and forebrain and establish novel roles of Pax6 in organogenesis. Examination of Pax6 in mouse embryonic forebrain and newborn lens. We performed ChIP-seq on mouse E12.5 embryonic forebrain and newborn lens. Genome-wide binding sites of Pax6, H3K4me1, H3K4me3, H3K27ac, H3K27me3, and Pol2 were generated. We also performed RNA-seq on mouse E12.5 embryonic forebrain and newborn lens epithelial cells and fibers.

2015-07-09 | E-GEOD-66961 | biostudies-arrayexpress

Shigella flexneri 5a

Project description:Shigella flexneri 5a

| PRJNA16047 | ENA

Mus musculus

Project description:Differences in gene expression patterns in mouse embryonic fibroblast Flp-In-3T3 cell lines stably expressing Pax6 or Pax6(5a).

| PRJNA145725 | ENA

Toxoplasma gondii eIF-5A knockdown iTraq

Project description:Proliferative stage (tachyzoite) is a critical factor in the transmission and pathogenesis of Toxoplasma gondii (T. gondii), which is capable of infecting a large variety of host cells. Proto-oncogene eukaryotic translation initiation factor (eIF-5A) is thought to function in regulation of many cellular processes including cell proliferation. Investigation the specific roles of eIF-5A involved in T. gondii is instrumental for the elucidation of tachyzoite replication. Here, eIF-5A knockdown strain was generated and analyzed by isobaric tags for relative and absolute quantification (iTraq), which suggested that proto-oncogene eIF-5A also acts as an essential gene to ensure proper protein synthesis during T. gondii invasion and replication.

2021-09-09 | PXD016810 | Pride

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