Lubica Supekova

Genomic analysis of the host response to hepatitis C virus infection

We have examined the progression of hepatitis C virus (HCV) infections by gene expression analysis of liver biopsies in acutely infected chimpanzees that developed persistent infection, transient viral clearance, or sustained clearance. Both common responses and outcome-specific changes in expression were observed. All chimpanzees showed gene expression patterns consistent with an IFN-α response that correlated with the magnitude and duration of infection. Transient and sustained viral clearance were uniquely associated with induction of IFN-γ-induced genes and other genes involved in antigen processing and presentation and the adaptive immune response. During the early stages of infection, host genes involved in lipid metabolism were also differentially regulated. We also show that drugs that affect these biosynthetic pathways can regulate HCV replication in HCV replicon systems. Our results reveal genome-wide transcriptional changes that reflect the establishment, spread, and control of infection, and they reveal potentially unique antiviral programs associated with clearance of HCV infection.

Genome-wide functional analysis of human cell-cycle regulators

Human cells have evolved complex signaling networks to coordinate the cell cycle. A detailed understanding of the global regulation of this fundamental process requires comprehensive identification of the genes and pathways involved in the various stages of cell-cycle progression. To this end, we report a genome-wide analysis of the human cell cycle, cell size, and proliferation by targeting >95% of the protein-coding genes in the human genome using small interfering RNAs (siRNAs). Analysis of >2 million images, acquired by quantitative fluorescence microscopy, showed that depletion of 1,152 genes strongly affected cell-cycle progression. These genes clustered into eight distinct phenotypic categories based on phase of arrest, nuclear area, and nuclear morphology. Phase-specific networks were built by interrogating knowledge-based and physical interaction databases with identified genes. Genome-wide analysis of cell-cycle regulators revealed a number of kinase, phosphatase, and proteolytic proteins and also suggests that processes thought to regulate G1-S phase progression like receptor-mediated signaling, nutrient status, and translation also play important roles in the regulation of G2/M phase transition. Moreover, 15 genes that are integral to TNF/NF-κB signaling were found to regulate G2/M, a previously unanticipated role for this pathway. These analyses provide systems-level insight into both known and novel genes as well as pathways that regulate cell-cycle progression, a number of which may provide new therapeutic approaches for the treatment of cancer.

Identification of Human Kinases Involved in Hepatitis C Virus Replication by Small Interference RNA Library Screening

The propagation of the hepatitis C virus (HCV) is a complex process that requires both host and viral proteins. To facilitate identification of host cell factors that are required for HCV replication, we screened a panel of small interference RNAs that preferentially target human protein kinases using an HCV replicon expressing the firefly luciferase gene as a genetic reporter. Small interference RNAs specific for three human kinases, Csk, Jak1, and Vrk1, were identified that reproducibly reduce viral RNA and viral protein levels in HCV replicon-bearing cells. Treatment of replicon cells with a small molecule inhibitor of Csk also resulted in a significant reduction in HCV RNA and proteins, further supporting a role for Csk in HCV replication. The effects of siRNAs targeting eight kinases known to be negatively regulated by Csk were then examined; knock down of one of these kinases, Fyn, resulted in up-regulation of the HCV replicon, suggesting that Csk mediates its effect on HCV replication through Fyn. This conclusion was further corroborated by demonstration that replicon cells treated with Csk inhibitor contained lower levels of the phosphorylated form of Fyn than control cells.

siRNA in human cells selectively localizes to target RNA sites

Recent observations of RNA interference (RNAi) in the nuclei of human cells raise key questions about the extent to which nuclear and cytoplasmic RNAi pathways are shared. By directly visualizing the localization of small interfering RNA (siRNA) in live human cells, we show here that siRNA either selectively localizes in the cytoplasm or translocates into the nucleus, depending on where the silencing target RNA resides. Two siRNAs that target the small nuclear 7SK and U6 RNAs localize into the nucleus as duplexes. In contrast, an siRNA targeting the cytoplasmic hepatitis C virus replicon RNA dissociates, and only antisense strand distributes in the cytoplasm of the cells harboring the target RNA, whereas sense strand gets degraded. At the same time, both strands of the latter siRNA are distributed throughout the cytoplasm and nucleus in cells lacking the silencing target RNA. These results suggest the existence of a mechanism by which the RNAi machinery orchestrates a target-determined localization of the siRNA and the corresponding RNAi activity, and also provide evidence for formation of nuclear-programmed active RNA induced silencing complexes directly in the nucleus.

Pan-Src Family Kinase Inhibitors Replace Sox2 during the Direct Reprogramming of Somatic Cells **

Ectopic expression of the four transcription factors Oct4, Klf4, Sox2 and c-Myc reprograms adult somatic cells to induced pluripotent stem (iPS) cells.[1] Although iPS cells hold considerable promise as tools in research and drug discovery, the clinical application of iPS cells is hindered by the use of viruses that deliver the exogenous factors and modify the host genome. It is therefore of great interest to replace virally transduced factors with either proteins or small molecules. To date a number of compounds have been identified that facilitate reprogramming of somatic cells. Among these are kenpaullone[2], valproic acid[3] and inhibitors of TGFβ-signaling.[4] Here we have exploited a reporter based screen[2] to identify a new class of compounds that functionally replace Sox2: inhibitors of the Src family of kinases. These molecules provide novel tools to study the molecular mechanism of Sox2 in reprogramming. n nTo screen for small molecule replacements of Sox2, mouse embryonic fibroblasts (MEFs) harboring the firefly luciferase (Fluc) gene in the Nanog locus[2] (NL-MEFs) were transduced with Oct4, Klf4 and c-Myc (OKM), seeded into 1536-well plates in standard growth media and assayed against a large chemical library[5] (750,000 compounds; 2.2 μM). Compounds that reproducibly and dose-dependently activated the NL reporter >2.5-fold over vehicle-treated controls (Figure 1a) were then counter-screened in a cell based SV40-driven Fluc assay to rule out false positives that directly and non-specifically induce luciferase signal.[2, 6] n n n nFigure 1 n nChemical complementation of Sox2 n n n nTo confirm that filtered hit compounds which activate Nanog gene expression also replace Sox2, iPS cell colony formation was used as a secondary assay. Specifically, Klf4 and c-Myc were delivered retrovirally to O4NR-MEFs[1b] (cells harboring a Doxycycline (Dox)-inducible Oct4 cDNA in the collagen locus and the neomycin-resistance gene in the Oct4 locus), and Oct4 expression was induced by addition of Dox to the culture media (day 0). Two days later, positive screen hits (1-10 μM) were added to OKM-expressing MEFs in place of Sox2. After 10 days of compound treatment, growth media was supplemented with neomycin to select for colonies that reactivated the endogenous Oct4 locus. The reactivation of epigenetically silenced pluripotency-associated genes is required for somatic cells to transition to the iPS cell state.[7] n nDox-independent, neomycin resistant colonies were not observed in DMSO-treated (0.1%, v/v) controls, indicating that vehicle-treated cells had not removed the epigenetic silencing marks from the Oct4 promoter (which drives NeoR) and were thus not pluripotent. Among the compounds tested, one compound, iPYrazine (iPY; 10 μM), promoted the formation of neomycin-resistant iPS cell colonies (Figure 1b, blue bars) that survived and could be cultured in the absence of Dox. Transgenic Oct4 independent (minus Dox) growth of the iPY-treated iPS cells demonstrated that they had reactivated and relied on endogenous Oct4 to maintain the pluripotent state. In addition, OKM transduction combined with iPY treatment of MEFs carrying a GFP reporter under control of the endogenous Oct4 locus[8] also gave rise to stable, GFP-positive iPS cell lines (Figure S1, Supporting Information). n niPS cells derived from O4NR-MEFs with iPY, Dox and KM-transduction grew as pluripotent stem cell colonies in the absence of Dox and iPY. Moreover, these cells were indistinguishable from ES cells by morphological criteria and expressed the pluripotency-associated markers Oct4 and SSEA1 (Figure 1c). We next tested the differentiation potential of the iPY-derived iPS cells in a teratoma assay by injecting 106 cells subcutaneously into NOD-SCID mice. Tumors were isolated 3 weeks later and histological analyses demonstrated that cell types of all three germ layers were present; these included neural tissues, bone, cartilage and ciliated epithelium (Figure 1d). Furthermore, iPY-derived iPS cells contributed to live chimeras, as shown in Figure 1d. The results from this series of analyses indicate that the iPY-derived, Sox2-free iPS cells are pluripotent. n nIn order to identify the biological target of iPY, we profiled the compound against a biochemical panel of tyrosine kinases (51 kinases; Table S1). From this analysis, we found that iPY potently inhibited a number of tyrosine kinases at 5 μM. Commercially available inhibitors (Figures 2a-b and Table S2) of these candidate kinase targets were then assayed for their ability to replace Sox2 in the iPS cell colony formation assay. As shown in Figure 2b, the pan-Src family kinase (SFK) inhibitors Dasatinib[9] and PP1[10] (Figure 2b) were able to recapitulate the activity of iPY. Interestingly, both Dasatinib and PP1 were >2-fold more active than iPY and efficiently replaced Sox2 (Figure 2b). Moreover, the pan-SFK inhibitors gave rise to colonies with a similar efficiency to TGFβ inhibitors (SB-431542 and LY-364947). The latter have been reported to replace Sox2 and served as a positive control in this study.[4] In addition to TGFβ inhibitors, Ichida et al. have also reported that the SFK inhibitor PP1 is able to replace Sox2.[4a] Together with our work, these results indicate that iPY is likely playing a role in reprogramming by inhibiting Src kinases, although additional mechanisms cannot be excluded. n n n nFigure 2 n nSrc family kinase and TGFβ-inhibitors recapitulate the Sox2 replacement activity of iPY n n n nSFKs are a class of proto-oncogene tyrosine kinases that include nine mammalian members (i.e., c-Src, Yes, Fyn, Fgr, Lck, Hck, Blk, Lyn and Frk).[11] Several members of the SFK family have been reported to influence embryonic stem (ES) cell self-renewal and differentiation.[12] For example, activation of c-Src signaling promotes ES cell differentiation.[13] Consistent with this observation we find that the activation of Src signaling in MEFs with JK239[14] potently inhibits 4-factor reprogramming (Figure 2c). Together, our results suggest that SFK signaling is an important mediator of somatic cell reprogramming, where activation of the SFK pathway prevents reprogramming and inhibition allows for reprogramming in the absence of exogenous Sox2. n nPreviously, Ichida et al. demonstrated that small molecule mediated inhibition of TGFβ-signaling with LY-364947 or E-616452 can replace Sox2 through the activation of Nanog expression.[4a] The results from our screen, which rely on Nanog activation as a surrogate for the replacement of Sox2, suggest that the inhibition of SFK- and TGFβ-signaling may converge on a similar mechanism; that is, the function of Sox2 can be replaced during direct reprogramming by activating Nanog expression. Another potential scenario comes from the observation that both Nanog[15] and SFK inhibition[13] are capable of maintaining the self-renewing pluripotent state in ES cells. Thus, TGFβ inhibitor-mediated Nanog activation and pan-SFK inhibition may instead converge on a common mechanism in which the differentiation of newly formed iPS cells is prevented, thereby assisting in the transition to an undifferentiated state. In either case, it is interesting to note that inhibition of distinct signaling responses converge on a common end point. n nIn summary, we applied a cell-based, high-throughput chemical screen to identify small molecules that replace Sox2 during somatic cell reprogramming. The identification of novel SFK inhibitors provides new chemical tools to study the mechanisms underlying direct reprogramming and may ultimately help to bring iPS cell technology one step closer to clinical application.

Transcriptional profiling of the effects of 25-hydroxycholesterol on human hepatocyte metabolism and the antiviral state it conveys against the hepatitis C virus

Background Hepatitis C virus (HCV) infection is a global health problem. A number of studies have implicated a direct role of cellular lipid metabolism in the HCV life cycle and inhibitors of the mevalonate pathway have been demonstrated to result in an antiviral state within the host cell. Transcriptome profiling was conducted on Huh-7 human hepatoma cells bearing subgenomic HCV replicons with and without treatment with 25-hydroxycholesterol (25-HC), an inhibitor of the mevalonate pathway that alters lipid metabolism, to assess metabolic determinants of pro- and antiviral states within the host cell. These data were compared with gene expression profiles from HCV-infected chimpanzees. Results Transcriptome profiling of Huh-7 cells treated with 25-HC gave 47 downregulated genes, 16 of which are clearly related to the mevalonate pathway. Fewer genes were observed to be upregulated (22) in the presence of 25-HC and 5 genes were uniquely upregulated in the HCV replicon bearing cells. Comparison of these gene expression profiles with data collected during the initial rise in viremia in 4 previously characterized HCV-infected chimpanzees yielded 54 overlapping genes, 4 of which showed interesting differential regulation at the mRNA level in both systems. These genes are PROX1, INSIG-1, NK4, and UBD. The expression of these genes was perturbed with siRNAs and with overexpression vectors in HCV replicon cells, and the effect on HCV replication and translation was assessed. Both PROX1 and NK4 regulated HCV replication in conjunction with an antiviral state induced by 25-hydroxycholesterol. Conclusion Treatment of Huh-7 cells bearing HCV replicons with 25-HC leads to the downregulation of many key genes involved in the mevalonate pathway leading to an antiviral state within the host cell. Furthermore, dysregulation of a larger subset of genes not directly related to the mevalonate pathway occurs both in 25-HC-treated HCV replicon harbouring cells as well as during the initial rise in viremia in infected chimpanzees. Functional studies of 3 of these genes demonstrates that they do not directly act as antiviral gene products but that they indirectly contribute to the antiviral state in the host cell. These genes may also represent novel biomarkers for HCV infection, since they demonstrate an outcome-specific expression profile.

Genomic Effects of Polyamide/DNA Interactions on mRNA Expression

Here we characterize the biological activity of a hairpin polyamide 1 that inhibits binding of the minor-groove transcription factor LEF-1, constitutively expressed in colon cancers. Genome-wide analysis of mRNA expression in DLD1 colon cancer cells treated with 1 reveals that a limited number of genes are affected; the most significant changes correspond to genes related to cell cycle, signaling, and proteolysis rather than the anticipated WNT signaling pathway. Treated cells display increased doubling time and hypersensitivity to DNA damage that most likely results from downregulation of DNA-damage checkpoint genes, including YWAE (14-3-3epsilon protein) and DDIT3. Promoter analyses on a genomic level revealed numerous potential polyamide binding sites and multiple possible mechanisms for transcriptional antagonism, underscoring the utility of gene expression profiling in understanding the effects of polyamides on transcription at the cellular level.

Dual regulation of hepatitis C viral RNA by cellular RNAi requires partitioning of Ago2 to lipid droplets and P-bodies

The antiviral role of RNA interference (RNAi) in humans remains to be better understood. In RNAi, Ago2 proteins and microRNAs (miRNAs) or small interfering RNAs (siRNAs) form endonucleolytically active complexes which down-regulate expression of target mRNAs. P-bodies, cytoplasmic centers of mRNA decay, are involved in these pathways. Evidence exists that hepatitis C virus (HCV) utilizes host cellular RNAi machinery, including miRNA-122, Ago1-4, and Dicer proteins for replication and viral genome translation in Huh7 cells by, so far, nebulous mechanisms. Conversely, synthetic siRNAs have been used to suppress HCV replication. Here, using a combination of biochemical, transfection, confocal imaging, and digital image analysis approaches, we reveal that replication of HCV RNA depends on recruitment of Ago2 and miRNA-122 to lipid droplets, while suppression of HCV RNA by siRNA and Ago2 involves interaction with P-bodies. Such partitioning of Ago2 proteins into different complexes and separate subcellular domains likely results in modulation of their activity by different reaction partners. We propose a model in which partitioning of host RNAi and viral factors into physically and functionally distinct subcellular compartments emerges as a mechanism regulating the dual interaction of cellular RNAi with HCV RNA.

A single mutation in the first transmembrane domain of yeast COX2 enables its allotopic expression

During the course of evolution, a massive reduction of the mitochondrial genome content occurred that was associated with transfer of a large number of genes to the nucleus. To further characterize factors that control the mitochondrial gene transfer/retention process, we have investigated the barriers to transfer of yeast COX2, a mitochondrial gene coding for a subunit of cytochrome c oxidase complex. Nuclear-recoded Saccharomyces cerevisiae COX2 fused at the amino terminus to various alternative mitochondrial targeting sequences (MTS) fails to complement the growth defect of a yeast strain with an inactivated mitochondrial COX2 gene, even though it is expressed in cells. Through random mutagenesis of one such hybrid MTS-COX2, we identified a single mutation in the first Cox2 transmembrane domain (W56 → R) that (i) results in the cellular expression of a Cox2 variant with a molecular mass indicative of MTS cleavage, which (ii) supports growth of a cox2 mutant on a nonfermentable carbon source, and that (iii) partially restores cytochrome c oxidase-specific respiration by the mutant mitochondria. COX2W56R can be allotopically expressed with an MTS derived from S. cerevisiae OXA1 or Neurospora crassa SU9, both coding for hydrophobic mitochondrial proteins, but not with an MTS derived from the hydrophilic protein Cox4. In contrast to some other previously transferred genes, allotopic COX2 expression is not enabled or enhanced by a 3′-UTR that localizes mRNA translation to the mitochondria, such as yeast ATP23′-UTR. Application of in vitro evolution strategies to other mitochondrial genes might ultimately lead to yeast entirely lacking the mitochondrial genome, but still possessing functional respiratory capacity.

Transcriptional Effects of the Potent Enediyne Anti-Cancer Agent Calicheamicin γ1I

Abstract We have investigated the mode of action of calicheamicin in living cells by using oligonucleotide microarrays to monitor its effects on gene expression across the entire yeast genome. Transcriptional effects were observed as early as 2 min into drug exposure. Among these effects were the upregulation of two nuclear proteins encoding a Y′-helicase (a subtelomerically encoded protein whose function is to maintain telomeres) and a suppressor of rpc10 and rpb40 mutations (both rpc10 and rpb40 encode RNA polymerase subunits). With longer calicheamicin exposure, genes involved in chromatin arrangement, DNA repair and/or oxidative damage, DNA synthesis and cell cycle checkpoint control as well as other nuclear proteins were all differentially expressed. Additionally, ribosomal proteins and a variety of metabolic, biosynthetic, and stress response genes were also altered in their expression.