Identification Of Direct Target Genes Research Articles

Identification of direct target genes of miR-7, miR-9, miR-96, and miR-182 in the human breast cancer cell lines MCF-7 and MDA-MB-231

Some microRNAs have carcinogenic or tumor suppressive effects in breast cancer, which is the most common cancer in women worldwide. MiR-7 and miR-9 are tumor suppressor microRNAs, which induce apoptosis and inhibit proliferation in breast cancer cells. Moreover, miR-96 and miR-182 are onco-microRNAs that increase proliferation, migration, and tumorigenesis in breast cancer cells. This study aimed to identify the direct target genes of these four microRNAs in the human breast cancer cell lines MCF-7 and MDA-MB-231. Initially, bioinformatics tools were used to identify the target genes that have binding sites for miR-7, MiR-9, MiR-96, and miR-182 and are also associated with breast cancer. Subsequently, the findings of the bioinformatics analysis relating to the effects of these four microRNAs on the 3′-UTR activity of the potential target genes were confirmed using the dual luciferase assay in MCF-7 and MDA-MB-231 cells co-transfected with the vectors containing 3′-UTR segments of the target genes downstream of a luciferase coding gene and each of the microRNAs. Finally, the effects of microRNAs on the endogenous expression of potential target genes were assessed by the overexpression of each of the four microRNAs in MCF-7 and MDA-MB-231 cells. Respectively, three, three, three, and seven genes were found to have binding sites for miR-7, miR-9, miR-96, and miR-182 and were associated with breast cancer. The results of empirical studies including dual luciferase assays and real-time PCR confirmed that miR-7 regulates the expression of BRCA1 and LASP1; MiR-9 regulates the expression of AR; miR-96 regulates the expression of ABCA1; and miR-182 regulates the expression of NBN, TOX3, and LASP1. Taken together, our results suggest that the tumor suppressive effects of miR-7 may be mediated partly by regulating the expression of BRCA1 as a tumor suppressor gene in breast cancer. In addition, this microRNA and miR-182 may have effects on the nodal-positivity and tumor size of breast carcinoma through the regulation of LASP1. The tumor suppressive functions of miR-9 may be mediated partly by suppressing the expression of AR—an oncogene in breast cancer. Moreover, miR-96 may play an oncogenic role in breast cancer by suppressing the apoptosis through the regulation of ABCA1.

Identification of direct targets of plant transcription factors using the GR fusion technique.

The glucocorticoid receptor-dependent activation of plant transcription factors has proven to be a powerful tool for the identification of their direct target genes. In the absence of the synthetic steroid hormone dexamethasone (dex), transcription factors fused to the hormone-binding domain of the glucocorticoid receptor (TF-GR) are held in an inactive state, due to their cytoplasmic localization. This requires physical interaction with the heat shock protein 90 (HSP90) complex. Hormone binding leads to disruption of the interaction between GR and HSP90 and allows TF-GR fusion proteins to enter the nucleus. Once inside the nucleus, they bind to specific DNA sequences and immediately activate or repress expression of their targets. This system is well suited for the identification of direct target genes of transcription factors in plants, as (A) there is little basal protein activity in the absence of dex, (B) steroid application leads to rapid transcription factor activation, (C) no side effects of dex treatment are observed on the physiology of the plant, and (D) secondary effects of transcription factor activity can be eliminated by simultaneous application of an inhibitor of protein biosynthesis, cycloheximide (cyc). In this chapter, we describe detailed protocols for the preparation of plant material, for dex and cyc treatment, for RNA extraction, and for the PCR-based or genome-wide identification of direct targets of transcription factors fused to GR.

MP23-12 GENOME-WIDE MAPPING OF WT1 TARGET GENES REVEALS MOLECULAR MECHANISMS OF WT1 IN NEPHROGENESIS AND WILMS’ TUMOR

You have accessJournal of UrologyKidney Cancer: Basic Research I1 Apr 2014MP23-12 GENOME-WIDE MAPPING OF WT1 TARGET GENES REVEALS MOLECULAR MECHANISMS OF WT1 IN NEPHROGENESIS AND WILMS’ TUMOR Eunah Chung and Joo-Seop Park Eunah ChungEunah Chung More articles by this author and Joo-Seop ParkJoo-Seop Park More articles by this author View All Author Informationhttps://doi.org/10.1016/j.juro.2014.02.880AboutPDF ToolsAdd to favoritesDownload CitationsTrack CitationsPermissionsReprints ShareFacebookTwitterLinked InEmail INTRODUCTION AND OBJECTIVES The Wt1 gene encodes a transcription factor that not only prevents Wilms’ tumor formation but also plays an important role in nephrogenesis. At an early step in nephrogenesis, mesenchymal nephron progenitors become epithelialized to form a renal vesicle, which then undergoes complex morphogenesis and develops into a mature nephron. Wt1 is expressed in both undifferentiated mesenchymal nephron progenitors and differentiating epithelialized nephron progenitors. It was previously reported that conditional deletion of Wt1 arrests differentiation of nephron progenitors. Expression of Wnt4 and Lhx1, two pivotal genes required for differentiation of nephron progenitors, is downregulated in the Wt1 mutant kidney. Loss-of-function mutations of Wt1 are often found in Wilms’ tumor. It was previously reported that, in Wilms’ tumor, phosphorylation of Erk1/2 (MAP kinases) and IRS (insulin receptor substrate), both of which are downstream of IGF signaling pathway, is increased. Identification of direct target genes regulated by Wt1 is essential in understanding how Wt1 regulates differentiation of nephron progenitors and prevents Wilms’ tumor formation. METHODS We performed Wt1 chromatin immunoprecipitation coupled with next-generation sequencing (ChIP-seq) in embryonic kidneys at E16.5. Sequencing libraries were generated by ligation-mediated PCR following a standard protocol and sequencing was performed on Illumina HiSeq 2000. The sequences were aligned to mouse genome (mm9) by using Bowtie and the data were analyzed by using HOMER and GREAT software. RESULTS We identified over 14,000 genomic target sites of Wt1. Our data show that both Wnt4 and Lhx1 are direct targets of Wt1. In addition, our data show that multiple components of IGF-MAP kinase pathways, such as Irs1, Irs2, Igf1r, Mapk1 (Erk2), and Mapk6 (Erk3), are among the top ranked direct targets of Wt1. De novo motif analysis of our Wt1 ChIP-seq data suggest that Wt1 may interact with Foxc2 and Hox proteins to regulate its target genes. CONCLUSIONS Given that expression of Wnt4 and Lhx1 is downregulated in the Wt1 mutant kidney, our Wt1 ChIP-seq data suggest that Wt1 acts as a transcriptional activator for Wnt4 and Lhx1. Considering that a mouse model of Wilms’ tumor requires both the loss of Wt1 and upregulation of Igf2, our data suggest that Wt1 represses expression of multiple components of IGF-MAP kinase pathways. Loss of Wt1 may cause nephron progenitors to become hypersensitive to IGF signaling. Our results provide new insights into how Wt1 regulates nephrogenesis and prevents Wilms’ tumor formation. © 2014FiguresReferencesRelatedDetails Volume 191Issue 4SApril 2014Page: e247-e248 Advertisement Copyright & Permissions© 2014MetricsAuthor Information Eunah Chung More articles by this author Joo-Seop Park More articles by this author Expand All Advertisement Advertisement PDF downloadLoading ...

Mechanism of Cross-Talk between Auxin and Brassinosteroids

Plant development is a complex and fine-controlled process, in which plant hormones play important roles. Two kinds of plant hormones, auxin and brassinosteroids(BRs), show many similar physiological effects (e.g., promoting elongation) and synergistically regulate some biological processes. Recent studies reveal the molecular mechanisms of cross-talk between auxin and BRs and identification of direct target genes of BRs and auxin signaling provide informative clues for the relevant mechanisms. The interactions between auxin and BRs include the regulation of downstream gene expressions, interaction of components of signal transduction pathways, biosynthesis and polar auxin transport, which are summarized in this review.

Identification of target genes of transcription factor CEBPB in acute promyelocytic leukemia cells induced by all-trans retinoic acid

ObjectiveTo indentify target genes of transcription factor CCAAT enhancer-binding protein β (CEBPB) in acute promyelocytic leukemia cells induced by all-trans retinoic acid. MethodsA new strategy for high-throughput identification of direct target genes was established by combining chromatin immunoprecipitation (ChIP) with in vitro selection. Then, 106 potential CEBPB binding fragments from the genome of the all-trans retinoic acid (ATRA)-treated NB4 cells were identified. ResultsOf them, 82 were mapped in proximity to known or previously predicted genes; 7 were randomly picked up for further confirmation by ChIP-PCR and 3 genes (GALM, ITPR2 and ORM2) were found to be specifically up-regulated in the ATRA-treated NB4 cells, indicating that they might be the down-stream target genes of ATRA. ConclusionsOur results provided new insight into the mechanisms of ATRA-induced granulocytic differentiation.

Genome-Wide Identification of Direct Target Genes Implicates Estrogen-Related Receptor α as a Determinant of Breast Cancer Heterogeneity

Estrogen-related receptor alpha (ERRalpha) is an orphan nuclear receptor, the expression of which correlates with negative prognosis in breast cancer. ERRalpha shares functional features with the estrogen receptor alpha (ERalpha) and its activity is modulated by the ERBB2 signaling pathway. Using genome-wide binding sites location analyses in ERalpha-positive and ERalpha-negative breast cancer cell lines, we show that ERRalpha and ERalpha display strict binding site specificity and maintain independent mechanisms of transcriptional activation. Nonetheless, ERRalpha and ERalpha coregulate a small subset of common target genes via binding either to a dual-specificity binding site or to distinct cognate binding sites located within the extended promoter region of the gene. Although ERRalpha signaling in breast cancer cells is mostly independent of ERalpha, the small fraction of common ERRalpha/ERalpha targets comprises genes with high relevance to breast tumor biology, including genes located within the ERBB2 amplicon and GATA3. Finally, unsupervised hierarchical clustering based on the expression profiling of ERRalpha direct target genes in human breast tumors revealed four main clusters that recapitulate established tumor subtypes. Taken together, the identification and functional characterization of the ERRalpha transcriptional network implicate ERRalpha signaling as a determinant of breast cancer heterogeneity.

Identification of Direct Target Genes Using Joint Sequence and Expression Likelihood with Application to DAF-16

A major challenge in the post-genome era is to reconstruct regulatory networks from the biological knowledge accumulated up to date. The development of tools for identifying direct target genes of transcription factors (TFs) is critical to this endeavor. Given a set of microarray experiments, a probabilistic model called TRANSMODIS has been developed which can infer the direct targets of a TF by integrating sequence motif, gene expression and ChIP-chip data. The performance of TRANSMODIS was first validated on a set of transcription factor perturbation experiments (TFPEs) involving Pho4p, a well studied TF in Saccharomyces cerevisiae. TRANSMODIS removed elements of arbitrariness in manual target gene selection process and produced results that concur with one's intuition. TRANSMODIS was further validated on a genome-wide scale by comparing it with two other methods in Saccharomyces cerevisiae. The usefulness of TRANSMODIS was then demonstrated by applying it to the identification of direct targets of DAF-16, a critical TF regulating ageing in Caenorhabditis elegans. We found that 189 genes were tightly regulated by DAF-16. In addition, DAF-16 has differential preference for motifs when acting as an activator or repressor, which awaits experimental verification. TRANSMODIS is computationally efficient and robust, making it a useful probabilistic framework for finding immediate targets.

Regulation of Body Pigmentation by the Abdominal-B Hox Protein and Its Gain and Loss in Drosophila Evolution

Hox genes have been implicated in the evolution of many animal body patterns, but the molecular events underlying trait modification have not been elucidated. Pigmentation of the posterior male abdomen is a recently acquired trait in the Drosophila melanogaster lineage. Here, we show that the Abdominal-B (ABD-B) Hox protein directly activates expression of the yellow pigmentation gene in posterior segments. ABD-B regulation of pigmentation evolved through the gain of ABD-B binding sites in a specific cis-regulatory element of the yellow gene of a common ancestor of sexually dimorphic species. Within the melanogaster species group, male-specific pigmentation has subsequently been lost by at least three different mechanisms, including the mutational inactivation of a key ABD-B binding site in one lineage. These results demonstrate how Hox regulation of traits and target genes is gained and lost at the species level and have general implications for the evolution of body form at higher taxonomic levels.

AGRIS and AtRegNet. A Platform to Link cis-Regulatory Elements and Transcription Factors into Regulatory Networks

Gene regulatory pathways converge at the level of transcription, where interactions among regulatory genes and between regulators and target genes result in the establishment of spatiotemporal patterns of gene expression. The growing identification of direct target genes for key transcription factors (TFs) through traditional and high-throughput experimental approaches has facilitated the elucidation of regulatory networks at the genome level. To integrate this information into a Web-based knowledgebase, we have developed the Arabidopsis Gene Regulatory Information Server (AGRIS). AGRIS, which contains all Arabidopsis (Arabidopsis thaliana) promoter sequences, TFs, and their target genes and functions, provides the scientific community with a platform to establish regulatory networks. AGRIS currently houses three linked databases: AtcisDB (Arabidopsis thaliana cis-regulatory database), AtTFDB (Arabidopsis thaliana transcription factor database), and AtRegNet (Arabidopsis thaliana regulatory network). AtTFDB contains 1,690 Arabidopsis TFs and their sequences (protein and DNA) grouped into 50 (October 2005) families with information on available mutants in the corresponding genes. AtcisDB consists of 25,806 (September 2005) promoter sequences of annotated Arabidopsis genes with a description of putative cis-regulatory elements. AtRegNet links, in direct interactions, several hundred genes with the TFs that control their expression. The current release of AtRegNet contains a total of 187 (September 2005) direct targets for 66 TFs. AGRIS can be accessed at http://Arabidopsis.med.ohio-state.edu.

Genomic identification of direct target genes of LEAFY.

The switch from vegetative to reproductive development in plants necessitates a switch in the developmental program of the descendents of the stem cells in the shoot apical meristem. Genetic and molecular investigations have demonstrated that the plant-specific transcription factor and meristem identity regulator LEAFY (LFY) controls this developmental transition by inducing expression of a second transcription factor, APETALA1, and by regulating the expression of additional, as yet unknown, genes. Here we show that the additional LFY targets include the APETALA1-related factor, CAULIFLOWER, as well as three transcription factors and two putative signal transduction pathway components. These genes are up-regulated by LFY even when protein synthesis is inhibited and, hence, appear to be direct targets of LFY. Supporting this conclusion, cis-regulatory regions upstream of these genes are bound by LFY in vivo. The newly identified LFY targets likely initiate the transcriptional changes that are required for the switch from vegetative to reproductive development in Arabidopsis.

Identification of Novel TGF-β/Smad Gene Targets in Dermal Fibroblasts using a Combined cDNA Microarray/Promoter Transactivation Approach

Despite major advances in the understanding of the intimate mechanisms of transforming growth factor-beta (TGF-beta) signaling through the Smad pathway, little progress has been made in the identification of direct target genes. In this report, using cDNA microarrays, we have focussed our attention on the characterization of extracellular matrix-related genes rapidly induced by TGF-beta in human dermal fibroblasts and attempted to identify the ones whose up-regulation by TGF-beta is Smad-mediated. For a gene to qualify as a direct Smad target, we postulated that it had to meet the following criteria: (1) rapid (30 min) and significant (at least 2-fold) elevation of steady-state mRNA levels upon TGF-beta stimulation, (2) activation of the promoter by both exogenous TGF-beta and co-transfected Smad3 expression vector, (3) up-regulation of promoter activity by TGF-beta blocked by both dominant-negative Smad3 and inhibitory Smad7 expression vectors, and (4) promoter transactivation by TGF-beta not possible in Smad3(-/-) mouse embryo fibroblasts. Using this stringent approach, we have identified COL1A2, COL3A1, COL6A1, COL6A3, and tissue inhibitor of metalloproteases-1 as definite TGF-beta/Smad3 targets. Extrapolation of this approach to other extracellular matrix-related gene promoters also identified COL1A1 and COL5A2, but not COL6A2, as novel Smad targets. Together, these results represent a significant step toward the identification of novel, early-induced Smad-dependent TGF-beta target genes in fibroblasts.