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Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation.

ABSTRACT: Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 growth-regulated genes in parallel in a breast epithelial cell line, MCF10A, and a prostate cancer cell line, PC3. Depletion of an unexpectedly high number of genes (25%) differentially affected proliferation of the two cell lines. Knockdown of many genes that spare PC3 (p53-) but inhibit MCF10A (p53+) proliferation induces p53 in MCF10A cells. EBNA1BP2, involved in ribosome biogenesis, is an example of such a gene, with its depletion arresting MCF10A at G1/S in a p53-dependent manner. TARCOR is thus useful for identifying cell type-specific genes and pathways involved in proliferation and also for exploring the heterogeneity of cell lines. In particular, our data emphasize the importance of considering the genetic status, when performing siRNA screens in mammalian cells.

SUBMITTER: Machida YJ

PROVIDER: S-EPMC1635399 | biostudies-other | 2006 Nov

REPOSITORIES: biostudies-other

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Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation.

Machida Yuichi J YJ Chen Yuefeng Y Machida Yuka Y Malhotra Ankit A Sarkar Sukumar S Dutta Anindya A

Molecular biology of the cell 20060906 11

Differences in the genetic and epigenetic make up of cell lines have been very useful for dissecting the roles of specific genes in the biology of a cell. Targeted comparative RNAi (TARCOR) analysis uses high throughput RNA interference (RNAi) against a targeted gene set and rigorous quantitation of the phenotype to identify genes with a differential requirement for proliferation between cell lines of different genetic backgrounds. To demonstrate the utility of such an analysis, we examined 257 ...[more]

PMID: 16957053

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Project description:BackgroundDengue virus (DENV) is the causative agent of Dengue fever and the life-threatening Dengue Haemorrhagic fever or Dengue shock syndrome. In the absence of anti-viral agents or vaccine, there is an urgent need to develop an effective anti-viral strategy against this medically important viral pathogen. The initial interplay between DENV and the host cells may represent one of the potential anti-viral targeting sites. Currently the involvements of human membrane trafficking host genes or factors that mediate the infectious cellular entry of dengue virus are not well defined.ResultsIn this study, we have used a targeted small interfering RNA (siRNA) library to identify and profile key cellular genes involved in processes of endocytosis, cytoskeletal dynamics and endosome trafficking that are important and essential for DENV infection. The infectious entry of DENV into Huh7 cells was shown to be potently inhibited by siRNAs targeting genes associated with clathrin-mediated endocytosis. The important role of clathrin-mediated endocytosis was confirmed by the expression of well-characterized dominant-negative mutants of genes in this pathway and by using the clathrin endocytosis inhibitor chlorpromazine. Furthermore, DENV infection was shown to be sensitive to the disruption of human genes in regulating the early to late endosomal trafficking as well as the endosomal acidic pH. The importance and involvement of both actin and microtubule dynamics in mediating the infectious entry of DENV was also revealed in this study.ConclusionsTogether, the findings from this study have provided a detail profiling of the human membrane trafficking cellular genes and the mechanistic insight into the interplay of these host genes with DENV to initiate an infection, hence broadening our understanding on the entry pathway of this medically important viral pathogen. These data may also provide a new potential avenue for development of anti-viral strategies and treatment of DENV infection.

Project description:UnlabelledHerpes simplex virus (HSV) utilizes and subverts host chromatin mechanisms to express its lytic gene products in mammalian cells. The host cell attempts to silence the incoming viral genome by epigenetic mechanisms, but the viral VP16 and ICP0 proteins promote active chromatin on the viral genome by recruiting other host epigenetic factors. However, the dependence on VP16 and ICP0 differs in different cell lines, implying cell type-dependent functional contributions of epigenetic factors for HSV gene expression. In this study, we performed a targeted RNA interference (RNAi) screen for cellular chromatin factors that are involved in regulation of herpes simplex virus (HSV) gene expression in U2OS osteosarcoma cells, a cell line that complements ICP0 mutant and VP16 mutant virus replication. In this screen, we found the same general classes of chromatin factors that regulate HSV gene expression in U2OS cells as in other cell types, including histone demethylases (HDMs), histone deacetylases (HDACs), histone acetyltransferases (HATs), and chromatin-remodeling factors, but the specific factors within these classes are different from those identified previously for other cell types. For example, KDM3A and KDM1A (LSD1) both demethylate mono- and dimethylated H3K9, but KDM3A emerged in our screen of U2OS cells. Further, small interfering RNA (siRNA) and inhibitor studies support the idea that KDM1A is more critical in HeLa cells, as observed previously, while KDM3A is more critical in U2OS cells. These results argue that different cellular chromatin factors are critical in different cell lines to carry out the positive and negative epigenetic effects exerted on the HSV genome.ImportanceUpon entry into the host cell nucleus, the herpes simplex virus genome is subjected to host epigenetic silencing mechanisms. Viral proteins recruit cellular epigenetic activator proteins to reverse and counter the cellular silencing mechanisms. Some of the host silencing and activator functions involved in HSV gene expression have been identified, but there have been indications that the host cell factors may vary in different cell types. In this study, we performed a screen of chromatin factors involved in HSV gene regulation in osteosarcoma cells, and we found that the chromatin factors that are critical for HSV gene expression in these cells are different from those for previously studied cell types. These results argue that the specific chromatin factors operative in different cell lines and cell types may differ. This has implications for epigenetic drugs that are under development.

Project description:BackgroundThe identification of genes essential for survival is of theoretical importance in the understanding of the minimal requirements for cellular life, and of practical importance in the identification of potential drug targets in novel pathogens. With the great time and expense required for experimental studies aimed at constructing a catalog of essential genes in a given organism, a computational approach which could identify essential genes with high accuracy would be of great value.ResultsWe gathered numerous features which could be generated automatically from genome sequence data and assessed their relationship to essentiality, and subsequently utilized machine learning to construct an integrated classifier of essential genes in both S. cerevisiae and E. coli. When looking at single features, phyletic retention, a measure of the number of organisms an ortholog is present in, was the most predictive of essentiality. Furthermore, during construction of our phyletic retention feature we for the first time explored the evolutionary relationship among the set of organisms in which the presence of a gene is most predictive of essentiality. We found that in both E. coli and S. cerevisiae the optimal sets always contain host-associated organisms with small genomes which are closely related to the reference. Using five optimally selected organisms, we were able to improve predictive accuracy as compared to using all available sequenced organisms. We hypothesize the predictive power of these genomes is a consequence of the process of reductive evolution, by which many parasites and symbionts evolved their gene content. In addition, essentiality is measured in rich media, a condition which resembles the environments of these organisms in their hosts where many nutrients are provided. Finally, we demonstrate that integration of our most highly predictive features using a probabilistic classifier resulted in accuracies surpassing any individual feature.ConclusionUsing features obtainable directly from sequence data, we were able to construct a classifier which can predict essential genes with high accuracy. Furthermore, our analysis of the set of genomes in which the presence of a gene is most predictive of essentiality may suggest ways in which targeted sequencing can be used in the identification of essential genes. In summary, the methods presented here can aid in the reduction of time and money invested in essential gene identification by targeting those genes for experimentation which are predicted as being essential with a high probability.

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Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation.

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Targeted comparative RNA interference analysis reveals differential requirement of genes essential for cell proliferation.

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