Genes In Mouse Embryonic Fibroblasts Research Articles

Quantification of oxidative stress in live mouse embryonic fibroblasts by monitoring the responses of polyubiquitin genes

Stress-regulated polyubiquitin genes in mammals are expected to be upregulated under oxidative stress conditions. In order to assess gene regulation via the conventional method, the isolation of RNA molecules or the transfection of reporter constructs into cells is frequently required. If the stress response within cells can be monitored in a reversible manner with minimal manipulation, the study of the stress response pathways will become much easier. Herein, we have developed a simple fluorescence plate reader-based assay to monitor the stress responses of polyubiquitin genes in mouse embryonic fibroblasts, in which one allele of the ubiquitin-coding region of the polyubiquitin gene Ubb or Ubc was replaced by the eGFP-puro cassette, thereby placing GFP expression under the control of the endogenous polyubiquitin gene promoter. Using this simple assay, we established that both mammalian polyubiquitin genes are upregulated upon oxidative stress with slightly higher responses from the Ubb promoter. The principal advantage of this assay is that it allows for the monitoring of stress responses of polyubiquitin genes without disrupting cellular growth; this assay can therefore be applied repeatedly to the same cells. Furthermore, by calculating the increase in fluorescence deriving from newly synthesized GFP upon stress, which can be regarded as a bona fide polyubiquitin gene stress response, we were able to determine and directly compare the concentrations of various oxidative stressors that induce the similar cellular stress levels. Therefore, this simple assay may also be employed in the screening of potentially toxic reagents that induce the stress response pathways.

Cell-Specific Interaction of Retinoic Acid Receptors with Target Genes in Mouse Embryonic Fibroblasts and Embryonic Stem Cells

All-trans retinoic acid (RA) induces transforming growth factor beta (TGF-beta)-dependent autocrine growth of mouse embryonic fibroblasts (MEFs). We have used chromatin immunoprecipitation to map 354 RA receptor (RAR) binding loci in MEFs, most of which were similarly occupied by the RAR alpha and RAR gamma receptors. Only a subset of the genes associated with these loci are regulated by RA, among which are several critical components of the TGF-beta pathway. We also show RAR binding to a novel series of target genes involved in cell cycle regulation, transformation, and metastasis, suggesting new pathways by which RA may regulate proliferation and cancer. Few of the RAR binding loci contained consensus direct-repeat (DR)-type elements. The majority comprised either degenerate DRs or no identifiable DRs but anomalously spaced half sites. Furthermore, we identify 462 RAR target loci in embryonic stem (ES) cells and show that their occupancy is cell type specific. Our results also show that differences in the chromatin landscape regulate the accessibility of a subset of more than 700 identified loci to RARs, thus modulating the repertoire of target genes that can be regulated and the biological effects of RA.

Indirect Regulation of Presenilins in CREB-mediated Transcription

Presenilins are essential for synaptic function, memory formation, and neuronal survival. Previously, we reported that expression of cAMP response element-binding protein (CREB) target genes is reduced in the cerebral cortex of presenilin (PS) conditional double knock-out (cDKO) mice. To determine whether the reduced expression of the CREB target genes in these mutant mice is due to loss of presenilin directly or secondary to the impaired neuronal activity, we established a sensitive luciferase reporter system to assess direct transcriptional regulation in cultured cells. We first used immortalized PS-deficient mouse embryonic fibroblasts (MEFs), and found that both CREB-mediated transcription and Notch-mediated HES1 transcription are decreased. However, the ubiquitin-C promoter-mediated transcription is also reduced, and among these three reporters, transfection of exogenous PS1 can rescue only the Notch-mediated HES1 transcription. Further Northern analysis revealed transcriptional alterations of Creb, ubiquitin-C, and other housekeeping genes in PS-deficient MEFs, indicating transcriptional dysregulation in these cells. We then used the Cre/loxP system to develop a postnatal PS-deficient cortical neuronal culture. Surprisingly, in these PS-null neurons, CREB-mediated transcription is not significantly decreased, and levels of total and phosphorylated CREB proteins are unchanged as well. Notch-mediated HES1 transcription is markedly reduced, and this reduction can be rescued by exogenous PS1. Together, our findings suggest that CREB-mediated transcription is regulated indirectly by PS in the adult cerebral cortex, and that attenuation of CREB target gene expression in PS cDKO mice is likely due to reduced neuronal activity in these mutant brains.

Involvement of Transcriptional Mediator Subunit TRAP220/MED1 in Action of Niche Cells to Support Hematopoietic Stem/Progenitor Cells

The mammalian TRAP/Mediator complex is a master transcriptional regulatory complex that integrates signals of diverse activators and recruits RNA polymerase II and other general factors to activate transcription. The TRAP220/MED1 subunit was originally identified as a ligand-dependent coactivator specific for nuclear receptors. We have previously shown through biochemical and mouse genetic studies that MED1 is essential for embryogenesis, cell growth/differentiation and homeostasis, and that it stimulates nuclear receptor-mediated myelomonopoiesis. MED1 also integrates other activators such as GATA-1 and C/EBPβ and appears to mediate erythropoiesis as well. The niche cells in the bone marrow plays a pivotal role in the maintenance of hematopoietic stem/progenitor cells (HSPCs). In this study, we employed mouse embryonic fibroblasts (MEFs) as a model to analyze the role of MED1 in the niche, since MEFs have a mesenchymal feature with the osteoblastic precursor lineage and are known to support HSPCs. To establish an experimental system, we crossed Med1 and p53 double knockouts to obtain Med1+/+/p53−/− and Med1−/−/p53−/− E10.0 embryos from a single female and prepared stable MEF lines. Then the Med1−/−/p53−/− MEFs were stably transfected with a MED1 expression vector (Rev-Med1−/− MEFs) or a control empty vector. When normal mouse bone marrow cells were cocultured with these MEFs treated with mitomycin C for a short period of 2 weeks, cell counts, live cells (MTT assay) and a DNA synthesis (BrdU incorporation) of marrow cells were measured. The number of live cells as well as DNA synthesis on Med1−/− MEFs was significantly decreased during this period, but those on Rev-Med1−/− MEFs recovered to the control levels. Thus the growth stress on MEFs appears to be attenuated on Med1−/− MEFs. When apoptosis of the marrow cells was measured, both the FITC-dUTP incorporation by TdT and annexin V/PI double positive cells were lower for Med1−/− MEFs, indicating that apoptosis was also attenuated. We next assessed the role of MED1 in MEFs to support long-term bone marrow culture. After bone marrow cells were cultured on mitomycin C-treated MEFs for 8 weeks in Myelocult M5300 (StemCell Technologies) or IMDM supplemented with BIT9500 (StemCell Technologies) and LDL, progenitor cells (adherent and nonadherent) were collected and cultured in complete methylcellulose (Methocult M3434; StemCell Technologies), and colonies were counted. The number of both myeloid and erythroid colonies were significantly attenuated (0 to 40% depending on experimental conditions) for cells on Med1−/− MEFs, but colonies for cells cultured on Rev-Med1−/− MEFs recovered to the control level. In order to exclude the possibility that lot differences among MEFs or p53 depletion might have affected the results, we next prepared primary Med1+/+ and Med1−/− MEFs by crossing Med1+/− mice and conducted the long-term culture experiments using these MEFs. The attenuated number of colonies for cells cocultured with Med1−/− MEFs (circa 10% of the control) was reproduced repeatedly, indicating that the observed role of MED1 in MEFs to support HSPCs is intrinsic. Since MED1 converges signals from a series of activators on specific promoters and activates transcription, one or some products of the downstream target genes in MEFs may be responsible for the observed activity to maintain HSPCs. In search for candidate MED1 target gene products among a series of known molecules that possess an activity on HSPCs, only the expression of osteopontin was found to be attenuated in Med1−/− MEFs and reverted in Rev-Med1−/− MEFs. Other factors including Angiopoietin-1 and Jagged-1 were comparable. This fact contrasts with the previous observation of osteopontin knockouts where the null niche cells that restricted the size of HSPC number overexpressed these factors. We next assessed the role of MED1 on the osteopontin promoter. We focused on vitamin D receptor (VDR) and Runx2 among the activators and tested MEFs by luciferase reporter assays. The basal level of transcription without any activators in Med1−/− MEFs was about half of the control. Moreover, both the activation by Runx2 and the liganddependent VDR function were significantly attenuated in Med1−/− MEFs. These results indicate that transcriptional coactivator MED1 in niche cells plays an important role in HSPCs support, and that osteopontin may be one of the downstream candidate target genes for MED1.

Review of: Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

Citation of original article:C. V. Hojilla, I. Kim, Z. Kassiri, J. E. Fat, H. Fang, R. Khokha.Journal of Cell Science2007;120(6): 1050–1060.Abstract of the original article:Multiple cancers exhibit mutations in β-catenin that lead to increased stability, altered localization or amplified activity. β-Catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter β-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient inTimp3have increased β-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, withTimp3−/−MEFs being more sensitive andTimp3−/−MECs more resistant to EGTA-induced cell detachment than the wild type.Timp3−/−MECs had higher dephosphorylated β-catenin levels and increased β-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of β-catenin target genes in MEFs and MECs showed no alteration in Myc, decreasedCcnd1(cyclin D1) and increasedMmp7mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated β-catenin, decrease inCcnd1gene expression and increase inMmp7gene expression. Physiologically,Timp3−/−mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

Metalloproteinase axes increase β-catenin signaling in primary mouse mammary epithelial cells lacking TIMP3

Multiple cancers exhibit mutations in beta-catenin that lead to increased stability, altered localization or amplified activity. beta-catenin is situated at the junction between the cadherin-mediated cell adhesion and Wnt signaling pathways, and TIMP3 functions to alter beta-catenin signaling. Here we demonstrate that primary mouse embryonic fibroblasts (MEFs) and mammary epithelial cells (MECs) deficient in Timp3 have increased beta-catenin signaling. Functionally, the loss of TIMP3 exerted cell-type-specific effects, with Timp3(-/-) MEFs being more sensitive and Timp3(-/-) MECs more resistant to EGTA-induced cell detachment than the wild type. Timp3(-/-) MECs had higher dephosphorylated beta-catenin levels and increased beta-catenin transcriptional activity as measured by TCF/LEF-responsive reporter assays. Real-time PCR analysis of beta-catenin target genes in MEFs and MECs showed no alteration in Myc, decreased Ccnd1 (cyclin D1) and increased Mmp7 mRNA levels upon loss of TIMP3, with the latter occurring only in epithelial cells. Recombinant TIMP3 and synthetic metalloproteinase inhibitors reverted the increase in dephosphorylated beta-catenin, decrease in Ccnd1 gene expression and increase in Mmp7 gene expression. Physiologically, Timp3(-/-) mammary glands displayed accelerated mammary ductal elongation during pubertal morphogenesis. Gain-of-function studies using slow-release TIMP-containing pellets revealed distinct effects of individual TIMPs on ductal morphogenesis. Recombinant TIMP1, TIMP3 and TIMP4 inhibited ductal elongation whereas TIMP2 promoted this process.

Gene expression analysis of TFII‐I modulated genes in mouse embryonic fibroblasts

TFII-I is a founding member of a family of helix-loop-helix transcription factors involved in modulation of genes through interaction with various nuclear factors and chromatin remodeling complexes. Recent studies indicate that TFII-I performs important function in cell physiology and mouse embryogenesis. In order to understand its molecular role, TFII-I was overexpressed in primary mouse embryonic fibroblasts (MEFs) and alterations in gene expression were monitored with a mouse 16 K oligonucleotide microarray. These studies allowed us to identify genes that lie downstream of TFII-I-dependent pathways. Among the modulated candidates were genes involved in the immunity response, catalytic activity, signaling pathways and transcriptional regulation. Expression of several candidates including those for the interferon-stimulated protein (G1p2), small inducible cytokine A7 (Ccl7), ubiquitin-conjugating enzyme 8 (Ube2l6), cysteine-rich protein (Csrp2) and Drosophila delta-like 1 homolog (Dlk1) were confirmed by real-time PCR. The obtained results suggest that TFII-I participates in multiple signaling and regulatory pathways in MEFs.

Expression profiling of BEN regulated genes in mouse embryonic fibroblasts

BEN is a member of the TFII-I family of helix-loop-helix transcription factors. Both TFII-I and BEN are involved in gene regulation through interactions with tissue-specific transcription factors and chromatin remodeling complexes. Identification of the downstream target genes of TFII-I proteins is critical in delineating the regulatory effects of these proteins. In this study, we conducted a microarray analysis to determine gene expression alterations following the overexpression of BEN in primary mouse embryonic fibroblasts (MEFs). We found the BEN-dependent modulation in the expression of large groups of genes representing a wide variety of functional categories including genes important in the immune response, cell cycle, transcriptional regulation and cell signaling. A set of genes identified by the microarray analysis was validated by independent real-time PCR analysis. Among upregulated genes were Shrm, Tgfb2, Ube2l6, G1p2, Ccl7 while downregulated genes were Folr1, Tgfbr2, Csrp2, and Dlk1. These results support a versatile function of TFII-I proteins in vertebrate physiology and lead to an increased understanding of the BEN-dependent molecular events.

TGF-β2 allows pluripotent human embryonic stem cell proliferation on E6/E7 immortalized mouse embryonic fibroblasts

In this study we report observations that mouse embryonic fibroblasts (MEF) capable of supporting expansion of pluripotent, human embryonic stem cells (hESC) fail to support after immortalization using E6/E7 oncogenes in serum conditions; however this can be reversed following addition of exogenous TGF-beta2. Microarray analysis of immortalized and non-immortalized MEF revealed differential gene expression of several TGF-beta related genes. By supplementing TGF-beta2 into E6/E7 immortalized MEF cultures, this enabled proliferation of undifferentiated, pluripotent hESC as demonstrated by marker expression (Oct-4, SSEA-4, alkaline phosphatase) and teratoma formation representing three germ layers following hESC injection into immuno-deficient mice. Subsequent investigation using quantitative real-time PCR highlighted differential gene expression of several extracellular matrix related transcripts in primary and immortal (+/-TGF-beta2) feeder cells including the induction of osteopontin following addition of TGF-beta2. Our results demonstrate that TGF-beta2 and its related genes in MEF play a role in the support of pluripotent hESC expansion.

Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6

Silencing of the Meiotic Genes SMC1β and STAG3 in Somatic Cells by E2F6

The transcriptional basis of adipocyte development

Adipogenesis is the developmental process by which a multipotent mesenchymal stem cell differentiates into a mature adipocyte. This process involves a highly regulated and coordinated cascade of transcription factors that together lead to the establishment of the differentiated state. In the presence of the correct hormonal cues, committed pre-adipocytes express the bZIP factors C/EBPb and C/EBPd. These factors in turn induce the expression of C/EBPa and peroxisome proliferator-activated receptor g (PPARg). C/EBPa and PPARg together promote differentiation by activating adipose-specific gene expression and by maintaining each others expression at high levels. We have investigated the relative contributions of PPARg and C/EBPa to adipogenesis by selectively ablating these genes in mouse embryonic fibroblasts (MEFs). MEFs that lack C/EBPa are able to undergo adipogenesis, but only when PPARg is ectopically expressed. Interestingly, these cells are not sensitive to the metabolic actions of insulin. By way of contrast, cells that lack PPARg are utterly incapable of adipogenic conversion, even when supplemented with high levels of C/EBPa. Our current investigations are centered on the identification of novel adipogenic transcription factors, utilizing a variety of techniques, ranging from BAC transgenics to computational approaches. These approaches will be discussed, along with the roles of some new transcriptional players in adipogenesis, including the O/E family of proteins.

IFN-regulatory factor 3-dependent gene expression is defective in Tbk1 -deficient mouse embryonic fibroblasts

Virus infection, double-stranded RNA, and lipopolysaccharide each induce the expression of genes encoding IFN-alpha and -beta and chemokines, such as RANTES (regulated on activation, normal T cell expressed and secreted) and IP-10 (IFN-gamma inducible protein 10). This induction requires the coordinate activation of several transcription factors, including IFN-regulatory factor 3 (IRF3). The signaling pathways leading to IRF3 activation are triggered by the binding of pathogen-specific products to Toll-like receptors and culminate in the phosphorylation of specific serine residues in the C terminus of IRF3. Recent studies of human cell lines in culture have implicated two noncanonical IkappaB kinase (IKK)-related kinases, IKK-epsilon and Traf family member-associated NF-kappaB activator (TANK)-binding kinase 1 (TBK1), in the phosphorylation of IRF3. Here, we show that purified recombinant IKK-epsilon and TBK1 directly phosphorylate the critical serine residues in IRF3. We have also examined the expression of IRF3-dependent genes in mouse embryonic fibroblasts (MEFs) derived from Tbk1(-/-) mice, and we show that TBK1 is required for the activation and nuclear translocation of IRF3 in these cells. Moreover, Tbk1(-/-) MEFs show marked defects in IFN-alpha and -beta, IP-10, and RANTES gene expression after infection with either Sendai or Newcastle disease viruses or after engagement of the Toll-like receptors 3 and 4 by double-stranded RNA and lipopolysaccharide, respectively. Finally, TRIF (TIR domain-containing adapter-inducing IFN-beta), fails to activate IRF3-dependent genes in Tbk1(-/-) MEFs. We conclude that TBK1 is essential for IRF3-dependent antiviral gene expression.

IKKα, IKKβ, and NEMO/IKKγ Are Each Required for the NF-κB-mediated Inflammatory Response Program

The IKKbeta and NEMO/IKKgamma subunits of the NF-kappaB-activating signalsome complex are known to be essential for activating NF-kappaB by inflammatory and other stress-like stimuli. However, the IKKalpha subunit is believed to be dispensable for the latter responses and instead functions as an in vivo mediator of other novel NF-kappaB-dependent and -independent functions. In contrast to this generally accepted view of IKKalpha's physiological functions, we demonstrate in mouse embryonic fibroblasts (MEFs) that, akin to IKKbeta and NEMO/IKKgamma, IKKalpha is also a global regulator of tumor necrosis factor alpha- and IL-1-responsive IKK signalsome-dependent target genes including many known NF-kappaB targets such as serum amyloid A3, C3, interleukin (IL)-6, IL-11, IL-1 receptor antagonist, vascular endothelial growth factor, Ptx3, beta(2)-microglobulin, IL-1alpha, Mcp-1 and -3, RANTES (regulated on activation normal T cell expressed and secreted), Fas antigen, Jun-B, c-Fos, macrophage colony-stimulating factor, and granulocyte-macrophage colony-stimulating factor. Only a small number of NF-kappaB-dependent target genes were preferentially dependent on IKKalpha or IKKbeta. Constitutive expression of a trans-dominant IkappaBalpha superrepressor (IkappaBalphaSR) in wild type MEFs confirmed that these signalsome-dependent target genes were also dependent on NF-kappaB. A subset of NF-kappaB target genes were IKK-dependent in the absence of exogenous stimuli, suggesting that the signalsome was also required to regulate basal levels of activated NF-kappaB in established MEFs. Overall, a sizable number of novel NF-kappaB/IKK-dependent genes were identified including Secreted Frizzled, cadherin 13, protocadherin 7, CCAAT/enhancer-binding protein-beta and -delta, osteoprotegerin, FOXC2 and FOXF2, BMP-2, p75 neurotrophin receptor, caspase-11, guanylate-binding proteins 1 and 2, ApoJ/clusterin, interferon (alpha and beta) receptor 2, decorin, osteoglycin, epiregulin, proliferins 2 and 3, stromal cell-derived factor, and cathepsins B, F, and Z. SOCS-3, a negative effector of STAT3 signaling, was found to be an NF-kappaB/IKK-induced gene, suggesting that IKK-mediated NF-kappaB activation can coordinately illicit negative effects on STAT signaling.