Target Gene Screening Research Articles

A Genome-Wide Screening of Potential Target Genes to Enhance the Antifungal Activity of Micafungin in Schizosaccharomyces pombe

Micafungin is a non-reversible inhibitor of 1, 3-β-D-glucan synthase and interferes with fungal cell wall synthesis. Clinically, micafungin has been shown to be efficacious for the treatment of invasive candidiasis and invasive aspergillosis. However, considering its relatively restricted antifungal spectrum, combination therapy with micafungin plus other agents should be considered in critically ill patients. To identify potential therapeutic targets for syncretic drug combinations that potentiate micafungin action, we carried out a genome-wide screen for altered sensitivity to micafungin by using the model yeast Schizosaccharomyces pombe mutant library. We confirmed that 159 deletion strains in the library are micafungin sensitive and classified them into various functional categories, including cell wall biosynthesis, gene expression and chromatin remodeling, membrane trafficking, signaling transduction, ubiquitination, ergosterol biosynthetic process and a variety of other known functions or still unknown functions. On the other hand, we also investigated the growth inhibitory activities of some well-known drugs in combination with micafungin including antifungal drug amphotericin B, fluconazole and immunosuppressive drug FK506. We found that amphotericin B in combination with micafungin showed a more potent inhibitory activity against wild-type cells than that of micafungin alone, whereas fluconazole in combination with micafungin did not. Also, the immunosuppressive drug FK506 showed synergistic inhibitory effect with micafungin on the growth of wild-type cells, whereas it decreased the inhibitory effect of micafungin in Δpmk1 cells, a deletion mutant of the cell wall integrity mitogen-activated protein kinase (MAPK) Pmk1. Altogether, our findings provide useful information for new potential drug combinations in the treatment of fungal infections.

MeCP2 modulates gene expression pathways in astrocytes

BackgroundMutations in MECP2 encoding methyl-CpG-binding protein 2 (MeCP2) cause the X-linked neurodevelopmental disorder Rett syndrome. Rett syndrome patients exhibit neurological symptoms that include irregular breathing, impaired mobility, stereotypic hand movements, and loss of speech. MeCP2 protein epigenetically modulates gene expression through genome-wide binding to methylated CpG dinucleotides. While neurons have the highest level of MeCP2 expression, astrocytes and other cell types also express detectable levels of MeCP2. Recent studies suggest that astrocytes likely control the progression of Rett syndrome. Thus, the object of these studies was to identify gene targets that are affected by loss of MeCP2 binding in astrocytes.MethodsTo identify gene targets of MeCP2 in astrocytes, combined approaches of expression microarray and chromatin immunoprecipitation of MeCP2 followed by sequencing (ChIP-seq) were compared between wild-type and MeCP2-deficient astrocytes. MeCP2 gene targets were compared with genes in the top 10% of MeCP2 binding levels in gene windows either within 2 kb upstream of the transcription start site, or the ‘gene body’ that extended from transcription start to end site, or 2 kb downstream of the transcription end site.ResultsA total of 118 gene transcripts surpassed the highly significant threshold (P < 0.005, fold change > 1.2) in expression microarray analysis from triplicate cultures. The top 10% of genes with the highest levels of MeCP2 binding were identified in two independent ChIP-seq experiments. Together this integrated, genome-wide screen for MeCP2 target genes provided an overlapping list of 19 high-confidence MeCP2-responsive gene transcripts in astrocytes. Validation of candidate target gene transcripts by RT-PCR revealed that expression of Apoc2, Cdon, Csrp and Nrep were consistently responsive to MeCP2 deficiency in astrocytes.ConclusionsThe first MeCP2 ChIP-seq and gene expression microarray analysis in astrocytes reveals a set of potential MeCP2 target genes that may contribute to normal astrocyte signaling, cell division and neuronal support functions, the loss of which may contribute to the Rett syndrome phenotype.

Development of a multiplex PCR assay for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle.

Tropical theileriosis, bovine babesiosis and anaplasmosis are tick-borne protozoan diseases that impose serious constraints on the health and productivity of domestic cattle in tropical and sub-tropical regions of the world. A common feature of these diseases is that, following recovery from primary infection, animals become persistent carriers of the pathogen and continue to play a critical role in disease epidemiology, acting as reservoirs of infection. This study describes development and evaluation of multiplex and single PCR assays for simultaneous detection of Theileria annulata, Babesia bovis and Anaplasma marginale in cattle. Following in silico screening for candidate target genes representing each of the pathogens, an optimised multiplex PCR assay was established using three primer sets, cytob1, MAR1bB2 and bovar2A, for amplification of genomic DNA of T. annulata, A. marginale and B. bovis respectively. The designed primer sets were found to be species-specific, generating amplicons of 312, 265 and 166 base pairs, respectively and were deemed suitable for the development of a multiplex assay. The sensitivity of each primer pair was evaluated using serial dilutions of parasite DNA, while specificity was confirmed by testing for amplification from DNA of different stocks of each pathogen and other Theileria, Babesia and Anaplasma species. Additionally, DNA preparations derived from field samples were used to evaluate the utility of the single and multiplex PCRs for determination of infection status. The multiplex PCR was found to detect each pathogen species with the same level of sensitivity, irrespective of whether its DNA was amplified in isolation or together with DNA representing the other pathogens. Moreover, single and multiplex PCRs were able to detect each species with equal sensitivity in serially diluted DNA representing mixtures of T. annulata, B. bovis and A. marginale, and no evidence of non-specific amplification from non-target species was observed. Validation that the multiplex PCR efficiently detects single and mixed infections from field samples was demonstrated. The developed assay represents a simple and efficient diagnostic for co-detection of tropical theileriosis, bovine babesiosis and anaplasmosis, and may be a valuable tool for epidemiological studies aimed at assessing the burden of multiple infection with tick-borne pathogens and improving control of the associated diseases in endemic regions.

Regulation of Myelopoiesis by Differentiation Stage-Specific Activities of HOXA9 and HOXA10

Abstract SCI-29HOXA9 and HOXA10 are homeodomain (HD) transcription factors that are implicated in control of myelopoiesis and contribute to myeloid leukemogenesis. These proteins are expressed coordinately during hematopoiesis, with maximal expression in granulocyte/monocyte progenitor (GMP) cells. Engineered overexpression of Hoxa9 or Hoxa10 in primary bone marrow cells expands the GMP population in vitro, and results in myeloproliferation in murine bone marrow transplant experiments. Mice transplanted with Hoxa9- or Hoxa10-overexpressing bone marrow develop acute myeloid leukemia (AML) over time. Consistent with this, increased and sustained expression of a set of HD proteins, including HOXA9 and HOXA10, is found in a subset of human AML, including AML with MLL gene translocations (11q23-AML). Since the DNA-binding HDs of HOXA9 and HOXA10 are highly conserved, we hypothesize that they recognize a common set of target genes. However, since HOXA9 and HOXA10 diverge outside the HD, we considered the unexplored possibility that they perform different functions in regulating such genes. To identify molecular mechanisms for HOX-induced GMP expansion and leukemogenesis, we performed a chromatin immunoprecipitation-based screen for HOXA10 target genes. Gene ontology studies determined that the identified set is enriched for genes encoding growth factors and receptors, including fibroblast growth factor 2 (FGF2). We found that production of FGF2 by Hoxa10-overexpressing GMP stabilizes β-catenin and induces proliferation in an autocrine manner. We also found that HOXA9 and HOXA10 activate common FGF2 cis elements. The Hoxa10-target-gene set is also enriched for HD-transcription factors, including CDX4. We determined that Cdx4 transcription is activated by HOXA10 in GMP, but repressed by HOXA9 in differentiating myeloid cells. CDX4 activates transcription of both Hoxa9 and Hoxa10, identifying a HOX-CDX cross-regulatory mechanism. This mechanism may be influenced by Fgf2, since Hoxa10 and Cdx4 are β-catenin target genes, but β-catenin activity decreases Hoxa9 expression. Gene expression profiling studies indicate that HOXA9, HOXA10, CDX4, and FGF2 are increased in 11q23-AML, suggesting clinical relevance. Arih2 (encoding the E3 ligase Triad1) is another common HOXA9 and HOXA10 target gene that may influence Fgf2 activity. We found that Arih2 transcription is repressed by HOXA9 in myeloid progenitors, but activated by HOXA10 in differentiating phagocytes. FGF receptors are destabilized by ubiquitination, and we found increased FGF-R ubiquitination in Hoxa10-overexpressing cells. Therefore, Triad1-dependent regulation of FGF-R stability is another mechanism for control of FGF2 activity and myeloproliferation by HOXA9 and HOXA10. Therefore, HOXA9 and HOXA10 regulate a common set of target genes that control GMP expansion in a manner that is antagonistic for some genes and cooperative for others. Clinical correlative studies suggest that coordinate control of these genes by HOXA9 and HOXA10 is dysregulated in HOX-overexpressing leukemia. Understanding HOX-regulated gene networks may identify therapeutic targets for HOX-overexpressing leukemias. For example, blocking FGF-related signaling pathways may ameliorate cytokine hypersensitivity in such leukemias, and would be a topic of interest for additional studies. Disclosures:No relevant conflicts of interest to declare.

Specific silencing of the REST target genes in insulin-secreting cells uncovers their participation in beta cell survival.

The absence of the transcriptional repressor RE-1 Silencing Transcription Factor (REST) in insulin-secreting beta cells is a major cue for the specific expression of a large number of genes. These REST target genes were largely ascribed to a function of neurotransmission in a neuronal context, whereas their role in pancreatic beta cells has been poorly explored. To identify their functional significance, we have generated transgenic mice expressing REST in beta cells (RIP-REST mice), and previously discovered that REST target genes are essential to insulin exocytosis. Herein we characterized a novel line of RIP-REST mice featuring diabetes. In diabetic RIP-REST mice, high levels of REST were associated with postnatal beta cell apoptosis, which resulted in gradual beta cell loss and sustained hyperglycemia in adults. Moreover, adenoviral REST transduction in INS-1E cells led to increased cell death under control conditions, and sensitized cells to death induced by cytokines. Screening for REST target genes identified several anti-apoptotic genes bearing the binding motif RE-1 that were downregulated upon REST expression in INS-1E cells, including Gjd2, Mapk8ip1, Irs2, Ptprn, and Cdk5r2. Decreased levels of Cdk5r2 in beta cells of RIP-REST mice further confirmed that it is controlled by REST, in vivo. Using siRNA-mediated knock-down in INS-1E cells, we showed that Cdk5r2 protects beta cells against cytokines and palmitate-induced apoptosis. Together, these data document that a set of REST target genes, including Cdk5r2, is important for beta cell survival.

FltpT2AiCre: A new knock-in mouse line for conditional gene targeting in distinct mono- and multiciliated tissues

We recently identified Flattop (Fltp; 1700009p17Rik) in a screen for potential Foxa2 target and novel mouse organizer genes. Besides its expression in the embryonic node, we found that Fltp is active in other monociliated tissues such as the sensory organs of the inner ear, duct and islets of the pancreas as well as in testis. Additionally, Fltp mRNA is expressed in multiciliated epithelial cells of the lung and of the choroid plexi in the brain. To genetically lineage trace these cells during development and injury as well as to conditionally inactivate genes in these tissues, we generated a Cre recombinase knock-in mouse line using the Fltp gene locus. By homologous recombination we have fused the Fltp open-reading frame to a tandem affinity purification (TAP) tag followed by an intervening viral T2A sequence for co-translational cleavage and an improved Cre recombinase (iCre). This strategy allows both the analysis of the tagged Fltp-TAP-T2A protein and the usage of the iCre recombinase for conditional targeting approaches. Using the ROSA26 reporter mouse line we show that FltpT2AiCre is first active in the monociliated cells of the node, notochord, floorplate and prechordal plate, consistent with the Fltp-TAP-T2A protein production in the node progenitor cells. Furthermore iCre recombinase activity is detected in multiciliated tissues such as choroid plexi of the brain and epithelial cells of the lung with the onset at E10.5 and E13.5, respectively. In the pancreas, β-galactosidase activity is seen in the monociliated cells of the pancreatic duct and islet of Langerhans. Intercrossing FltpT2AiCre mice with the CAG-CAT-EGFP reporter mouse line further confirms iCre activity in multiciliated cells of the lung and brain on a cellular level. Thus, the FltpT2AiCre line is a powerful tool to conditionally inactivate genes in distinct mono- and multiciliated tissues and to analyze the tagged Fltp protein in vivo.

Real-time PCR to assess the Leishmania load in Lutzomyia longipalpis sand flies: Screening of target genes and assessment of quantitative methods

Visceral Leishmaniasis is an endemic disease in Brazil caused by Leishmania infantum chagasi and its main vector species is the sand fly Lutzomyia longipalpis. Epidemiological studies have used conventional PCR techniques to measure the rate of infection of sand flies collected in the field. However, real-time PCR can detect lower parasite burdens, reducing the number of false negatives and improving the quantification of Leishmania parasites in the sand fly. This study compared genes with various copy numbers to detect and quantify L. infantum chagasi in L. longipalpis specimens by real-time PCR. We mixed pools of 1, 10 and 30 male sand flies with various amounts of L. infantum chagasi, forming groups with 50, 500, 5000 and 50,000 Leishmania parasites. For the amplification of L. infantum chagasi DNA, primers targeting kDNA, polymerase α and the 18S ribosome subunit were employed. Parasites were measured by absolute and relative quantification. PCR detection using the amplification of kDNA exhibited the greatest sensitivity among the genes tested, showing the capacity to detect the DNA equivalent of 0.004 parasites. Additionally, the relative quantification using these primers was more accurate and precise. In general, the number of sand flies used for DNA extraction did not influence Leishmania quantification. However, for low-copy targets, such as the polymerase α gene, lower parasite numbers in the sample produced inaccurate quantifications. Thus, qPCR measurement of L. infantum chagasi in L. longipalpis was improved by targeting high copy-number genes; amplification of high copy-number targets increased the sensitivity, accuracy and precision of DNA-based parasite enumeration.

Screening of the target genes transactivated by PS1TP1 protein with suppression subtractive hybridization technique

To construct a subtractive cDNA library of genes transactivated by PS1TP1 protein with suppression subtractive hybridization (SSH) technique. Suppression subtractive hybridization technique and bioinformatics technique were used, the mRNA from HepG2 cells transfected with pcDNA3.1 (-) -PS1TP1 and pcDNA3. 1 (-) empty vector was isolated, respectively; cDNA underwent two times of nested PCR, amplified cDNA fragments were subcloned into pGEM-Teasy vectors to set up the subtractive library. The subtractive library of genes transactivated by PS1TP1 was constructed successfully. Sequence analysis was performed in 43 clones randomly, and the full length sequences were obtained with bioinformatics method and searched for homologous DNA sequence from GenBank, altogether 12 coding sequences were gotten, which consisted of 10 known and 2 unknown ones. The obtained sequences may be target genes transactivated by PS1TP1 protein among which some genes coding proteins involved in cell cycle regulation, metabolism, immunity and cell apoptosis. This finding brought some clues for studying the biological functions of PS1T1.

284 MIR-145 FUNCTIONS AS TUMOR SUPPRESSOR AND REGULATES ACTIN-BINDING PROTEINS (FSCN1 AND SWAP70) IN PROSTATE CANCER

INTRODUCTION AND OBJECTIVES: MicroRNAs are small non-coding RNAs that negatively regulate protein-coding genes involved in the development of various malignancies. Down-regulation of miR-145 has been reported in many types of human cancer, including prostate cancer (PCa). In the last meeting, we demonstrated that miR-145 was the most down-regulated microRNA in bladder cancer and had a tumor suppressive function through targeting an actin-binding protein, FSCN1. Increasing evidence indicates that the miR-145 functions as a tumor suppressive miRNA and causes cell viability inhibitions in human cancer cells. However, there is limited knowledge of its targets in PCa. The aim of this study is to identify target genes of miR-145 based on gene expression analysis of miR-145-transfected PCa cells. METHODS: For genome-wide screening for target genes silenced by miR-145 in PCa, we performed oligo-microarray analysis of miR-145 transfectants (PC3, DU145) compared to miR-negative control transfectants. We employed web-based software in order to search for putative target genes of miR-145, and we focused on fascin homolog 1 (FSCN1) and SWAP switching B-cell complex 70kDa subunit (SWAP70), which is also known as F-actin binding protein involved in activating B cell transformation. The luciferase reporter assay was used to confirm the actual binding sites between miR-145 and FSCN1/ SWAP70 mRNA. Cell viability was evaluated by cell proliferation, wound healing, and matrigel invasion assays in si-FSCN1 and siSWAP70 transfectants. A total of 75 clinical prostate specimens were subjected to immunohistochemistry of SWAP70. RESULTS: The mRNA and protein expression of FSCN1 and SWAP70 were markedly repressed in the miR-145 transfectants. The luciferase reporter assay demonstrated that miR-145 was directly bound to the specific sites of FSCN1 and SWAP70 mRNA. Significant inhibitions of cell proliferation, invasion and migration were observed in the si-FSCN1 and the si-SWAP70 transfectants (p 0.0001) as well as the miR-145 transfectants (p 0.0001). The immunohistochemical score of SWAP70 in PCa specimens was significantly higher than that in benign prostate hyperplasia specimens (p 0.0169). CONCLUSIONS: Our data suggest that tumor suppressive miR-145 directory regulates both FSCN1 and SWAP70 that might have oncogenic functions associated with actin-promoting cell motility and malignant transformation in PCa. These pathways may play critical roles in PCa development and serve as a novel therapeutic target in PCa.

Gene Profiling of Mta1 Identifies Novel Gene Targets and Functions

BackgroundMetastasis-associated protein 1 (MTA1), a master dual co-regulatory protein is found to be an integral part of NuRD (Nucleosome Remodeling and Histone Deacetylation) complex, which has indispensable transcriptional regulatory functions via histone deacetylation and chromatin remodeling. Emerging literature establishes MTA1 to be a valid DNA-damage responsive protein with a significant role in maintaining the optimum DNA-repair activity in mammalian cells exposed to genotoxic stress. This DNA-damage responsive function of MTA1 was reported to be a P53-dependent and independent function. Here, we investigate the influence of P53 on gene regulation function of Mta1 to identify novel gene targets and functions of Mta1.MethodsGene expression analysis was performed on five different mouse embryonic fibroblasts (MEFs) samples (i) the Mta1 wild type, (ii) Mta1 knock out (iii) Mta1 knock out in which Mta1 was reintroduced (iv) P53 knock out (v) P53 knock out in which Mta1 was over expressed using Affymetrix Mouse Exon 1.0 ST arrays. Further Hierarchical Clustering, Gene Ontology analysis with GO terms satisfying corrected p-value<0.1, and the Ingenuity Pathway Analysis were performed. Finally, RT-qPCR was carried out on selective candidate genes.Significance/ConclusionThis study represents a complete genome wide screen for possible target genes of a coregulator, Mta1. The comparative gene profiling of Mta1 wild type, Mta1 knockout and Mta1 re-expression in the Mta1 knockout conditions define “bona fide” Mta1 target genes. Further extensive analyses of the data highlights the influence of P53 on Mta1 gene regulation. In the presence of P53 majority of the genes regulated by Mta1 are related to inflammatory and anti-microbial responses whereas in the absence of P53 the predominant target genes are involved in cancer signaling. Thus, the presented data emphasizes the known functions of Mta1 and serves as a rich resource which could help us identify novel Mta1 functions.

Integration of cap analysis of gene expression and chromatin immunoprecipitation analysis on array reveals genome-wide androgen receptor signaling in prostate cancer cells

The androgen receptor (AR) is a critical transcriptional factor that contributes to the development and the progression of prostate cancer (PCa) by regulating the transcription of various target genes. Genome-wide screening of androgen target genes provides useful information to understand a global view of AR-mediated gene network in PCa. In this study, we performed 5'-cap analysis of gene expression (CAGE) to determine androgen-regulated transcription start sites (TSSs) and chromatin immunoprecipitation (ChIP) on array (ChIP-chip) analysis to identify AR binding sites (ARBSs) and histone H3 acetylated (AcH3) sites in the human genome. CAGE determined 13 110 distinct, androgen-regulated TSSs (P<0.01), and ChIP-chip analysis identified 2872 androgen-dependent ARBSs (P<1e-5) and 25 945 AcH3 sites (P<1e-4). Both androgen-regulated coding genes and noncoding RNAs, including microRNAs (miRNAs) were determined as androgen target genes. Besides prototypic androgen-regulated TSSs in annotated gene promoter regions, there are many androgen-dependent TSSs that are widely distributed throughout the genome, including those in antisense (AS) direction of RefSeq genes. Several pairs of sense/antisense promoters were newly identified within single RefSeq gene regions. The integration of CAGE and ChIP-chip analyses successfully identified a cluster of androgen-inducible miRNAs, as exemplified by the miR-125b-2 cluster on chromosome 21. Notably, the number of androgen-upregulated genes was larger in LNCaP cells treated with R1881 for 24 h than for 6 h, and the percentage of androgen-upregulated genes accompanied with adjacent ARBSs was also much higher in cells treated with R1881 for 24 h than 6 h. On the basis of the Oncomine database, the majority of androgen-upregulated genes containing adjacent ARBSs and CAGE tag clusters in our study were previously confirmed as androgen target genes in PCa. The integrated high-throughput genome analyses of CAGE and ChIP-chip provide useful information for elucidating the AR-mediated transcriptional network that contributes to the development and progression of PCa.

Analysis of Gene Mutations in Children With Cholestasis of Undefined Etiology

The discovery of genetic mutations in children with inherited syndromes of intrahepatic cholestasis allows for diagnostic specificity despite similar clinical phenotypes. Here, we aimed to determine whether mutation screening of target genes could assign a molecular diagnosis in children with idiopathic cholestasis. DNA samples were obtained from 51 subjects with cholestasis of undefined etiology and surveyed for mutations in the genes SERPINA1, JAG1, ATP8B1, ABCB11, and ABCB4 by a high-throughput gene chip. Then, the sequence readouts for all 5 genes were analyzed for mutations and correlated with clinical phenotypes. Healthy subjects served as controls. Sequence analysis of the genes identified 14 (or 27%) subjects with missense, nonsense, deletion, and splice site variants associated with disease phenotypes based on the type of mutation and/or biallelic involvement in the JAG1, ATP8B1, ABCB11, or ABCB4 genes. These patients had no syndromic features and could not be differentiated by biochemical markers or histopathology. Among the remaining subjects, 10 (or ∼20%) had sequence variants in ATP8B1 or ABCB11 that involved only 1 allele, 8 had variants not likely to be associated with disease phenotypes, and 19 had no variants that changed amino acid composition. Gene sequence analysis assigned a molecular diagnosis in 27% of subjects with idiopathic cholestasis based on the presence of variants likely to cause disease phenotypes.

60. Screening of target genes of the bZIP transcription factor OsTGAP1 that regulates the inductive production of diterpenoid phytoalexins in rice

60. Screening of target genes of the bZIP transcription factor OsTGAP1 that regulates the inductive production of diterpenoid phytoalexins in rice

Molecular targets for liver cancer therapy: From screening of target genes to clinical trials

Cancer arises from the accumulation of genetic alterations, and the inactivation of oncogenes, or recovery of suppressor genes, are promising strategies for cancer treatment. Genome-based drug research starts with identification of target genes and is accomplished by exploitation of target-based drugs such as monoclonal antibodies, small molecules and antisense drugs. Recently, clinical trials for treatment of advanced hepatocellular carcinoma (HCC) have been performed, and the effectiveness of sorafenib, an oral multikinase inhibitor of the vascular endothelial growth factor receptor and Ras kinase, has been demonstrated. In addition to known target genes, microarray technology has enabled us to constitute novel therapeutic targets, and many researchers have applied this technology in studies of HCC and have identified candidate target genes, validated to affect cell growth. In addition, promoter arrays for whole-genome epigenetic aberration analysis, ChIP-chip analysis using tiling arrays, and high-throughput sequencing systems have been applied to drug discovery. To elucidate the status of therapeutic target genes in vivo, development of diagnostic markers for stratification of patients is a pressing need. Here, we review recent advances in microarray technology for liver cancer, discuss the innovations and approaches to therapeutic target discovery, and present data regarding the outcome of gene target therapy using monoclonal antibodies and molecular diagnostic markers in our laboratory.

A new approach to retrieve full lengths of functional genes from soil by PCR-DGGE and metagenome walking

Metagenomes are a vast genetic resource, and various approaches have been developed to explore them. Here, we present a new approach to retrieve full lengths of functional genes from soil DNA using PCR-denaturing gradient gel electrophoresis (DGGE) followed by metagenome walking. Partial fragments of benzoate 1,2-dioxygenase alpha subunit gene (benA) were detected from a 3-chlorobenzoate (3CB)-dosed soil by PCR-DGGE, and one DGGE band induced by 3CB was used as a target fragment for metagenome walking. The walking retrieved the flanking regions of the target fragment from the soil DNA, resulting in recovery of the full length of benA and also downstream gene (benB). The same strategy retrieved another gene, tfdC, and a complete tfdC and two downstream genes were obtained from the same soil. PCR-DGGE allows screening for target genes based on their potential for degrading contaminants in the environment. This feature provides an advantage over other existing metagenomic approaches.

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

The miR-200 Family: Central Player for Gain and Loss of the Epithelial Phenotype

Comprehensive Genomic Screens Reveal Multiple Modes of Action of the PLZF-RAR-α Oncoprotein

The t(11;17)(q23;q21) translocation is associated with a retinoic acid-insensitive form of acute promyelocytic leukemia (APL) involving the production of reciprocal fusion proteins PLZF-RARα and RARα-PLZF. These proteins mediate malignant transformation by binding to and dysregulating RARα/RXR and PLZF target genes, respectively. In order to investigate the molecular basis of PLZF-RARα induced leukemia, we performed a genome wide screen for PLZF-RARα direct target genes using a gain of function model in which PLZF-RARα was expressed in human U937 leukemia cells. Chromatin from U937/PLZF-RARα cells was immunoprecipitated using PLZF antibodies, amplified by ligation-mediated PCR and biological triplicates were hybridized to NimbleGen 2.7kB promoter arrays, which represent 24,659 human promoters. We identified 4916 genes directly bound by PLZF-RARα (2/3 biological replicates, FDR <0.2). These genes were highly enriched for ontological categories including immunity and defense (p<10-6), apoptosis (p<2×10-5), cell cycle (p<10-3) and oncogenesis (p<10-2). Gene expression profiling of U937/PLZF-RARα cells revealed that 34% of direct targets were also transcriptionally regulated in response to PLZF-RARα induction. Despite the established role of PLZF-RARα as a transcriptional repressor, 56% of genes bound by PLZF-RARα were upregulated and 44% repressed. Bioinformatic analysis of PLZF-RARα bound sequences using the MATRIXReduce algorithm identified the '-AGGTCA-' core sequence as the highest ranked position specific affinity matrix (PSAM). Comparison of this matrix with known transcription factor binding sites from the JASPAR core database revealed high similarity to the recognition sequence for the RAR-related orphan receptor A1 (RORA1) (E value: 5.2×10-3), RORA2 (3.5×10-2) and RXRA-VDR (4.4×10-2). This suggests that the natural binding site of PLZF-RARα is similar to that of other nuclear receptors. The 'GTCA' core sequence is frequently observed in canonical retinoic acid receptor response elements and this motif was only associated with genes repressed with binding by PLZF-RARα. Together these results are consistent with the idea that PLZF-RARα acts in large part as a dominant negative retinoic acid receptor. A comparison of genes bound directly by PLZF-RARα with gene expression profiles from 22 APL (4 PLZF-RARα, 18 PML-RARα) and 99 acute myeloid leukemias (AML) selected at random from the Erasmus University dataset, using gene set enrichment analysis, revealed that direct targets of PLZF-RARα were differentially repressed in APL when compared to other forms of AML. Overexpression of PLZF-RARα in murine hematopoietic progenitors and human CD34+ cord blood, blocked myeloid differentiation, an effect associated with the repression of C/EBP genes (α, β and ε), which were identified as direct targets of PLZF-RARα by ChIP-chip. Treatment of primary CD34+ cells with ATRA led to an increase in CEBPα and β, but repression of CEBPε was not relieved. Overexpression of PLZF-RARα in primary murine bone marrow led to an increase in the more primitive Sca1+ population, coincident with increased serial replating ability. Overexpression of PLZF-RARα in mouse and human progenitors led to increased proliferation with more cells in the S and G2/M phases of cell cycle. Correlating with this effect, genes with defined roles in hematopoietic stem cell self-renewal including HOXA9 and MPL were bound and activated by the induction of PLZF-RARα in U937 cells. Increased proliferation was also coincident with repressed expression of Cdkn2d (p19) a cyclin dependent kinase inhibitor, also directly bound by PLZF-RARα. PLZF-RARα appears to transform cells through three interlinked modes of action, inhibition of differentiation by direct repression of key myeloid transcription factors, stimulation of proliferation by repression of a cyclin dependent kinase inhibitor and activation of genes critical for self renewal.

Screening of target genes in esophageal squamous cell cancer in Kazakh by oligonucleotide microarray

Objective To screen the differentially expressed genes in esophageal squamous cell cancer (ESCC) and normal tissue of esophageal mucosa in Kazakh. Methods RNA was extracted from the ESCC sections in Kazakh patients, and was amplified to obtain cRNA. The gene expression profiles in ESCC and normal tissue of esophageal mucosa were detected by HG-U133 Plus 2.0 gene chip. The results were analyzed by bioinfor-matics. Results One hundred and seventy differentially expressed genes in ESCC and normal tissue of esophageal mucosa were found, with a difference of more than 10 times in expression levels. Of the 170 genes, 39 were up-regulated (signal log ratio > 3 ) and 131 down-regulated (signal log ratio < - 3). These factors such as cell cycle regulation, apoptosis, cytoskeleton; extracellular matrix, intracellular signal transduction, protein translation and synthesis, and immunological functions were correlated with the genes with abnormal expression. Conclusion The use of oligonucleotide microarray accurately and efficiently screen the 170 target genes in ESCC in Kazakh. It is suggested that these genes may be related to the carcinogenesis and development of ESCC in Kazakh. Key words: Esophageal cancer; Kazakh; Gene expression profile

The Selective Alzheimer's Disease Indicator-1 Gene (Seladin-1/DHCR24) Is a Liver X Receptor Target Gene

The nuclear hormone receptors liver X receptor alpha (LXRalpha) and LXRbeta function as physiological receptors for oxidized cholesterol metabolites (oxysterols) and regulate several aspects of cholesterol and lipid metabolism. Seladin-1 was originally identified as a gene whose expression was down-regulated in regions of the brain associated with Alzheimer's disease. Seladin-1 has been demonstrated to be neuroprotective and was later characterized as 3beta-hydroxysterol-Delta24 reductase (DHCR24), a key enzyme in the cholesterologenic pathway. Seladin-1 has also been shown to regulate lipid raft formation. In a whole genome screen for direct LXRalpha target genes, we identified an LXRalpha occupancy site within the second intron of the Seladin-1/DHCR24 gene. We characterized a novel LXR response element within the second intron of this gene that is able to confer LXR-specific ligand responsiveness to reporter gene in both HepG2 and human embryonic kidney 293 cells. Furthermore, we found that Seladin-1/DHCR24 gene expression is significantly decreased in skin isolated from LXRbeta-null mice. Our data suggest that Seladin-1/DHCR24 is an LXR target gene and that LXR may regulate lipid raft formation.

Rcl is a novel ETV1/ER81 target gene upregulated in breast tumors

ETV1 (ER81) is a transcription factor that can be activated by HER2/Neu, a proto-oncoprotein often overexpressed in metastatic breast tumors. Here, we demonstrate that ETV1 downregulation suppresses proliferation of HER2/Neu-positive MDA-MB-231 breast cancer cells in vitro and tumor formation in vivo, proving for the first time the existence of a critical role of ETV1 in breast cancer cell physiology. A screen for novel ETV1 target genes hinted at Rcl, an enzyme involved in nucleotide metabolism. To characterize the human Rcl gene, we cloned its promoter and found that ETV1 and HER2/Neu cooperated in activating the Rcl promoter, whereas a dominant-negative ETV1 molecule suppressed the Rcl promoter. Moreover, ETV1 and HER2/Neu synergized to upregulate the endogenous Rcl gene. ETV1 also bound to the Rcl promoter in vivo, indicating that Rcl is a bona fide target gene of ETV1. Hybridization of Rcl cDNA to a breast cancer array revealed that Rcl is overexpressed in approximately 40% of all breast tumors. Importantly, its expression significantly escalates with increasing tumor grade, strongly implicating that Rcl contributes to breast tumorigenesis. Since joint overexpression of Rcl with vascular endothelial growth factor, another target gene of ETV1, has been shown to induce tumor formation, Rcl may be one crucial effector of ETV1 and HER2/Neu in breast tumors. Furthermore, given its expression pattern and enzymatic function in nucleotide metabolism, Rcl presents itself as a novel target in breast cancer therapy via modulation of its activity by small molecule drugs.