Functional interaction of the retinoblastoma and Ini1/Snf5 tumor suppressors in cell growth and pituitary tumorigenesis

The Ini1 subunit of the SWI/SNF chromatin remodeling complex suppresses formation of malignant rhabdoid tumors in humans and mice. Transduction of Ini1 into Ini1-deficient tumor-derived cell lines has indicated that Ini1 arrests cell growth, controls chromosomal ploidy, and suppresses tumorigenesis by regulating components of the retinoblastoma (Rb) signaling pathway. Furthermore, conditional inactivation of Ini1 in mouse fibroblasts alters the expression of various Rb-E2F-regulated genes, indicating that endogenous Ini1 levels may control Rb signaling in cells. We have reported previously that loss of one allele of Ini1 in mouse fibroblasts results only in a 15% to 20% reduction in total Ini1 mRNA levels due to transcriptional compensation by the remaining Ini1 allele. Here, we examine the effects of Ini1 haploinsufficiency on cell growth and immortalization in mouse embryonic fibroblasts. In addition, we examine pituitary tumorigenesis in Rb-Ini1 compound heterozygous mice. Our results reveal that heterozygosity for Ini1 up-regulates cell growth and immortalization and that exogenous Ini1 down-regulates the growth of primary cells in a Rb-dependent manner. Furthermore, loss of Ini1 is redundant with loss of Rb function in the formation of pituitary tumors in Rb heterozygous mice and leads to the formation of large, atypical Rb(+/-) tumor cells lacking adrenocorticotropic hormone expression. These results confirm in vivo the relationship between Rb and Ini1 in tumor suppression and indicate that Ini1 plays a role in maintaining the morphologic and functional differentiation of corticotrophic cells

Loss of miRNA biogenesis induces p19Arf-p53 signaling and senescence in primary cells

Dicer, an enzyme involved in microRNA (miRNA) maturation, is required for proper cell differentiation and embryogenesis in mammals. Recent evidence indicates that Dicer and miRNA may also regulate tumorigenesis. To better characterize the role of miRNA in primary cell growth, we generated Dicer-conditional mice. Ablation of Dicer and loss of mature miRNAs in embryonic fibroblasts up-regulated p19Arf and p53 levels, inhibited cell proliferation, and induced a premature senescence phenotype that was also observed in vivo after Dicer ablation in the developing limb and in adult skin. Furthermore, deletion of the Ink4a/Arf or p53 locus could rescue fibroblasts from premature senescence induced by Dicer ablation. Although levels of Ras and Myc oncoproteins appeared unaltered, loss of Dicer resulted in increased DNA damage and p53 activity in these cells. These results reveal that loss of miRNA biogenesis activates a DNA damage checkpoint, up-regulates p19Arf-p53 signaling, and induces senescence in primary cells

The Class II Transactivator Requires brahma-Related Gene 1 To Activate Transcription of Major Histocompatibility Complex Class II Genes

The class II transactivator (CIITA) is the key regulator of major histocompatibility complex (MHC) class II gene transcription. We demonstrate here that CIITA requires the ATPase subunit of an hSWI/SNF complex, brahma-related gene 1 (BRG-1), to activate transcription. When introduced into a cell line lacking BRG-1, CIITA was unable to activate cellular MHC class II genes. Reexpression of the wild-type but not an ATP-binding-deficient BRG-1 protein in this cell line restored the ability of CIITA to transactivate transcription of MHC class II genes. Interestingly, when the activity of CIITA was assayed in the BRG-1-deficient cell line by using a plasmid-based reporter assay, BRG-1 was not required for transcriptional activation, suggesting that the chromatin structure on the plasmid is such that BRG-1 is not necessary. Coimmunoprecipitation experiments were performed to determine if BRG-1 and CIITA proteins associate with each other in cells. We found that the two proteins coimmunoprecipitate and that amino acids 1 to 140 of CIITA are sufficient for binding. Taken together, these data suggest that BRG-1 and, very likely, an hSWI/SNF complex are required for transcription of MHC class II genes. The complex is likely recruited to MHC class II promoters, at least in part, by interaction with CIITA

An essential role for Dicer in adipocyte differentiation

Dicer is a cellular enzyme required for the processing of pre-miRNA molecules into mature miRNA, and Dicer and miRNA biogenesis have been found to play important roles in a variety of physiologic processes. Recently, reports of alterations in miRNA expression levels in cultured pre-adipogenic cell lines during differentiation and findings of differences between the miRNA expression signatures of white and brown adipose have suggested that miRNA molecules might regulate adipocyte differentiation and the formation of adipose tissue. However, direct evidence that miRNAs regulate adipogenesis is lacking. To determine if Dicer and mature miRNA govern adipocyte differentiation, we utilized primary cells isolated from mice bearing Dicer-conditional alleles to study adipogenesis in the presence or absence of miRNA biogenesis. Our results reveal that Dicer is required for adipogenic differentiation of mouse embryonic fibroblasts and primary cultures of pre-adipocytes. Furthermore, the requirement for Dicer in adipocyte differentiation is not due to miRNA-mediated alterations in cell proliferation, as deletion of the Ink4a locus and the prevention of premature cellular senescence normally induced in primary cells upon Dicer ablation fails to rescue adipogenic differentiation in fibroblasts and pre-adipocytes

High content image-based screening of a protease inhibitor library reveals compounds broadly active against Rift Valley fever virus and other highly pathogenic RNA viruses.

High content image-based screening was developed as an approach to test a protease inhibitor small molecule library for antiviral activity against Rift Valley fever virus (RVFV) and to determine their mechanism of action. RVFV is the causative agent of severe disease of humans and animals throughout Africa and the Arabian Peninsula. Of the 849 compounds screened, 34 compounds exhibited ≥ 50% inhibition against RVFV. All of the hit compounds could be classified into 4 distinct groups based on their unique chemical backbone. Some of the compounds also showed broad antiviral activity against several highly pathogenic RNA viruses including Ebola, Marburg, Venezuela equine encephalitis, and Lassa viruses. Four hit compounds (C795-0925, D011-2120, F694-1532 and G202-0362), which were most active against RVFV and showed broad-spectrum antiviral activity, were selected for further evaluation for their cytotoxicity, dose response profile, and mode of action using classical virological methods and high-content imaging analysis. Time-of-addition assays in RVFV infections suggested that D011-2120 and G202-0362 targeted virus egress, while C795-0925 and F694-1532 inhibited virus replication. We showed that D011-2120 exhibited its antiviral effects by blocking microtubule polymerization, thereby disrupting the Golgi complex and inhibiting viral trafficking to the plasma membrane during virus egress. While G202-0362 also affected virus egress, it appears to do so by a different mechanism, namely by blocking virus budding from the trans Golgi. F694-1532 inhibited viral replication, but also appeared to inhibit overall cellular gene expression. However, G202-0362 and C795-0925 did not alter any of the morphological features that we examined and thus may prove to be good candidates for antiviral drug development. Overall this work demonstrates that high-content image analysis can be used to screen chemical libraries for new antivirals and to determine their mechanism of action and any possible deleterious effects on host cellular biology

Dicer is required for the formation of white but not brown adipose tissue

Dicer, an enzyme involved in microRNA maturation, is required for proper embryo gastrulation and tissue morphogenesis during mammalian development. Using primary cultures of fibroblasts and pre-adipocytes, we have previously shown that Dicer is essential for early stages of adipogenic cell differentiation. In this present study, we have utilized Dicer-conditional mice to explore a role for Dicer and microRNA biogenesis in the terminal differentiation of adipocytes in vivo and in the formation of white and brown adipose tissue. Deletion of Dicer in differentiated adipocytes in Dicer-conditional, aP2-Cre transgenic mice reduced the level of various adipogenic-associated transcripts and inhibited lipogenesis in white adipocytes, resulting in a severe depletion of white adipose tissue in mice. In contrast, Dicer was not required in vivo for lipogenesis in brown adipose or for brown fat formation. However, Dicer deletion in brown adipose did decrease the expression of genes involved in thermoregulation. The results of our study provide genetic evidence of a role for microRNA molecules in regulating adipogenesis and reveal distinct requirements for Dicer in the formation of white and brown adipose tissue. (c) 2010 Wiley-Liss, Inc

High Content Image Based Analysis Identifies Cell Cycle Inhibitors as Regulators of Ebola Virus Infection

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Development of a HCI-based assay to assess phenotypic changes to the Golgi.

<p>(<b>A</b>) IFA showing co-localization of RVFV Gn (Red) and a GFP-fused Golgi marker (Green) in a HeLa-G cell line that stably expressed RVFV Gn protein. (<b>B</b>) Software development for HCI-based analysis of Golgi. Acquired confocal images of nucleus (I), cell (II) and the Golgi complex (III) in mock-treated HeLa cells. The juxtanuclear Golgi is qualified as “intact Golgi” by image analysis and is marked by the red boundary around the clustered Golgi stacks (insert, IV) in mock treated cells. In contrast, image analysis disqualified many of the numerous scattered spots in the cytoplasm of D011-2120-treated cells as “intact Golgi” (insert, V). (<b>C</b>) Heat map of Golgi number and Golgi area by HCI-based analysis with increasing concentrations of the hit compounds and reference compounds. (<b>D</b>) Dose response curve analysis of lead compounds showing an increased reduction in Golgi as a function of compound concentration. Percent Golgi number is shown in blue and percent infection inhibition is shown in green.</p

Protein Kinase R Degradation Is Essential for Rift Valley Fever Virus Infection and Is Regulated by SKP1-CUL1-F-box (SCF)^FBXW11-NSs E3 Ligase

<div><p>Activated protein kinase R (PKR) plays a vital role in antiviral defense primarily by inhibiting protein synthesis and augmenting interferon responses. Many viral proteins have adopted unique strategies to counteract the deleterious effects of PKR. The NSs (Non-structural s) protein which is encoded by Rift Valley fever virus (RVFV) promotes early PKR proteasomal degradation through a previously undefined mechanism. In this study, we demonstrate that NSs carries out this activity by assembling the SCF (SKP1-CUL1-F-box)^FBXW11 E3 ligase. NSs binds to the F-box protein, FBXW11, <i>via</i> the six amino acid sequence DDGFVE called the degron sequence and recruits PKR through an alternate binding site to the SCF^FBXW11 E3 ligase. We further show that disrupting the assembly of the SCF^FBXW11-NSs E3 ligase with MLN4924 (a small molecule inhibitor of SCF E3 ligase activity) or NSs degron viral mutants or siRNA knockdown of FBXW11 can block PKR degradation. Surprisingly, under these conditions when PKR degradation was blocked, NSs was essential and sufficient to activate PKR causing potent inhibition of RVFV infection by suppressing viral protein synthesis. These antiviral effects were antagonized by the loss of PKR expression or with a NSs deleted mutant virus. Therefore, early PKR activation by disassembly of SCF^FBXW11-NSs E3 ligase is sufficient to inhibit RVFV infection. Furthermore, <i>FBXW11</i> and <i>BTRC</i> are the two homologues of the <i>βTrCP</i> (Beta-transducin repeat containing protein) gene that were previously described to be functionally redundant. However, in RVFV infection, among the two homologues of βTrCP, FBXW11 plays a dominant role in PKR degradation and is the limiting factor in the assembly of the SCF^FBXW11 complex. Thus, FBXW11 serves as a master regulator of RVFV infection by promoting PKR degradation. Overall these findings provide new insights into NSs regulation of PKR activity and offer potential opportunities for therapeutic intervention of RVFV infection.</p></div