Targetfinder.org: a resource for systematic discovery of transcription factor target genes

Targetfinder.org (http://targetfinder.org/) provides a web-based resource for finding genes that show a similar expression pattern to a group of user-selected genes. It is based on a large-scale gene expression compendium (>1200 experiments, >13 000 genes). The primary application of Targetfinder.org is to expand a list of known transcription factor targets by new candidate target genes. The user submits a group of genes (the ‘seed’), and as a result the web site provides a list of other genes ranked by similarity of their expression to the expression of the seed genes. Additionally, the web site provides information on a recovery/cross-validation test to check for consistency of the provided seed and the quality of the ranking. Furthermore, the web site allows to analyse affinities of a selected transcription factor to the promoter regions of the top-ranked genes in order to select the best new candidate target genes for further experimental analysis

TransFind—predicting transcriptional regulators for gene sets

The analysis of putative transcription factor binding sites in promoter regions of coregulated genes allows to infer the transcription factors that underlie observed changes in gene expression. While such analyses constitute a central component of the in-silico characterization of transcriptional regulatory networks, there is still a lack of simple-to-use web servers able to combine state-of-the-art prediction methods with phylogenetic analysis and appropriate multiple testing corrected statistics, which returns the results within a short time. Having these aims in mind we developed TransFind, which is freely available at http://transfind.sys-bio.net/

Inhibition of CDK8 mediator kinase suppresses estrogen dependent transcription and the growth of estrogen receptor positive breast cancer

Hormone therapy targeting estrogen receptor (ER) is the principal treatment for ER-positive breast cancers. However, many cancers develop resistance to hormone therapy while retaining ER expression. Identifying new druggable mediators of ER function can help to increase the efficacy of ER-targeting drugs. Cyclin-dependent kinase 8 (CDK8) is a Mediator complex-associated transcriptional regulator with oncogenic activities. Expression of CDK8, its paralog CDK19 and their binding partner Cyclin C are negative prognostic markers in breast cancer. Meta-analysis of transcriptome databases revealed an inverse correlation between CDK8 and ERalpha expression, suggesting that CDK8 could be functionally associated with ER. We have found that CDK8 inhibition by CDK8/19-selective small-molecule kinase inhibitors, by shRNA knockdown or by CRISPR/CAS9 knockout suppresses estrogen-induced transcription in ER-positive breast cancer cells; this effect was exerted downstream of ER. Estrogen addition stimulated the binding of CDK8 to the ER-responsive GREB1 gene promoter and CDK8/19 inhibition reduced estrogen-stimulated association of an elongation-competent phosphorylated form of RNA Polymerase II with GREB1. CDK8/19 inhibitors abrogated the mitogenic effect of estrogen on ER-positive cells and potentiated the growth-inhibitory effects of ER antagonist fulvestrant. Treatment of estrogen-deprived ER-positive breast cancer cells with CDK8/19 inhibitors strongly impeded the development of estrogen independence. In vivo treatment with a CDK8/19 inhibitor Senexin B suppressed tumor growth and augmented the effects of fulvestrant in ER-positive breast cancer xenografts. These results identify CDK8 as a novel downstream mediator of ER and suggest the utility of CDK8 inhibitors for ER-positive breast cancer therapy

Transcriptional landscape of the human and fly genomes: Nonlinear and multifunctional modular model of transcriptomes

Regions of the genome not coding for proteins or not involved in cis-acting regulatory activities are frequently viewed as lacking in functional value. However, a number of recent large-scale studies have revealed significant regulated transcription of unannotated portions of a variety of plant and animal genomes, allowing a new appreciation of the widespread transcription of large portions of the genome. High-resolution mapping of the sites of transcription of the human and fly genomes has provided an alternative picture of the extent and organization of transcription and has offered insights for biological functions of some of the newly identified unannotated transcripts. Considerable portions of the unannotated transcription observed are developmental or cell-type-specific parts of protein-coding transcripts, often serving as novel, alternative 5′ transcriptional start sites. These distal 5′ portions are often situated at significant distances from the annotated gene and alternatively join with or ignore portions of other intervening genes to comprise novel unannotated protein-coding transcripts. These data support an interlaced model of the genome in which many regions serve multifunctional purposes and are highly modular in their utilization. This model illustrates the underappreciated organizational complexity of the genome and one of the functional roles of transcription from unannotated portions of the genome. Copyright 2006, Cold Spring Harbor Laboratory Press © 2006 Cold Spring Harbor Laboratory Press

Despite WT1 binding sites in the promoter region of human and mouse nucleoporin glycoprotein 210, WT1 does not influence expression of GP210

BACKGROUND: Glycoprotein 210 (GP210) is a transmembrane component of the nuclear pore complex of metazoans, with a short carboxyterminus protruding towards the cytoplasm. Its function is unknown, but it is considered to be a major structural component of metazoan nuclear pores. Yet, our previous findings showed pronounced differences in expression levels in embryonic mouse tissues and cell lines. In order to identify factors regulating GP210, the genomic organization of human GP210 was analyzed in silico. RESULTS: The human gene was mapped to chromosome 3 and consists of 40 exons spread over 102 kb. The deduced 1887 amino acid showed a high degree of alignment homology to previously reported orthologues. Experimentally we defined two transcription initiation sites, 18 and 29 bp upstream of the ATG start codon. The promoter region is characterized by a CpG island and several consensus binding motifs for gene regulatory transcription factors, including clustered sites associated with Sp1 and the Wilms' tumor suppressor gene zinc finger protein (WT1). In addition, distal to the translation start we found a (GT)n repetitive sequence, an element known for its ability to bind WT1. Homologies for these motifs could be identified in the corresponding mouse genomic region. However, experimental tetracycline dependent induction of WT1 in SAOS osteosarcoma cells did not influence GP210 transcription. CONCLUSION: Although mouse GP210 was identified as an early response gene during induced metanephric kidney development, and WT1 binding sites were identified in the promoter region of the human GP210 gene, experimental modulation of WT1 expression did not influence expression of GP210. Therefore, WT1 is probably not regulating GP210 expression. Instead, we suggest that the identified Sp binding sites are involved

Downregulation of ETS Rescues Diabetes-Induced Reduction of Endothelial Progenitor Cells

Transplantation of vasculogenic progenitor cells (VPC) improves neovascularization after ischemia. However, patients with type 2 diabetes mellitus show a reduced VPC number and impaired functional activity. Previously, we demonstrated that p38 kinase inhibition prevents the negative effects of glucose on VPC number by increasing proliferation and differentiation towards the endothelial lineage in vitro. Moreover, the functional capacity of progenitor cells is reduced in a mouse model of metabolic syndrome including type 2 diabetes (Lepr(db)) in vivo.The aim of this study was to elucidate the underlying signalling mechanisms in vitro and in vivo. Therefore, we performed DNA-protein binding arrays in the bone marrow of mice with metabolic syndrome, in blood-derived progenitor cells of diabetic patients as well as in VPC ex vivo treated with high levels of glucose. The transcriptional activation of ETS transcription factors was increased in all samples analyzed. Downregulation of ETS1 expression by siRNA abrogated the reduction of VPC number induced by high-glucose treatment. In addition, we observed a concomitant suppression of the non-endothelial ETS-target genes matrix metalloproteinase 9 (MMP9) and CD115 upon short term lentiviral delivery of ETS-specific shRNAs. Long term inhibition of ETS expression by lentiviral infection increased the number of cells with the endothelial markers CD144 and CD105.These data demonstrate that diabetes leads to dysregulated activation of ETS, which blocks the functional activity of progenitor cells and their commitment towards the endothelial cell lineage

Mutations in the C-terminus of the X protein of hepatitis B virus regulate Wnt-5a expression in hepatoma Huh7 cells: cDNA microarray and proteomic analyses

Background: The hepatitis B virus x gene (HBx) is a promiscuous transactivator implicated in the development of hepatocellular carcinoma (HCC). The present study was designed to investigate the molecular events regulated by HBx. Methods: Genomic and proteomic expression profiling was performed in Huh7 HCC cells transfected with HBx mutants with a C-terminal deletion. The gene and protein expression of wingless-type murine-mammary-tumour virus (MMTV) integration site family, member 5A (Wnt-5a) was validated by analyses of reverse transcription–polymerase chain reaction (RT–PCR), real-time RT–PCR, western blot and immunohistochemistry. Results: Differentially expressed genes and proteins were found in the transfected Huh7 HCC cells; most of them were involved in transcriptional regulation, although others including oncogenes or tumor suppressor genes, and molecules involved in cell junctions, signal transduction pathways, metabolism or the immune response were also observed. The expression of the Wnt-5a gene was elevated >10-fold in Huh7 cells transfected with the HBx3′-30 amino acid deletion mutant. However, the expression was downregulated by the transfection with the HBx3′-40 amino acid deletion mutant. The changes in Wnt-5a expression were also observed in human HCC tissues, compared with corresponding non-cancerous liver tissues. A negative correlation was found between the expression of Wnt-5a and HBx COOH mutations in HCC tissues. Conclusions: HBx mutants may participate in the development and progression of HCC, at least in part through the Wnt-5a pathway

Systematic discovery and characterization of regulatory motifs in ENCODE TF binding experiments

Recent advances in technology have led to a dramatic increase in the number of available transcription factor ChIP-seq and ChIP-chip data sets. Understanding the motif content of these data sets is an important step in understanding the underlying mechanisms of regulation. Here we provide a systematic motif analysis for 427 human ChIP-seq data sets using motifs curated from the literature and also discovered de novo using five established motif discovery tools. We use a systematic pipeline for calculating motif enrichment in each data set, providing a principled way for choosing between motif variants found in the literature and for flagging potentially problematic data sets. Our analysis confirms the known specificity of 41 of the 56 analyzed factor groups and reveals motifs of potential cofactors. We also use cell type-specific binding to find factors active in specific conditions. The resource we provide is accessible both for browsing a small number of factors and for performing large-scale systematic analyses. We provide motif matrices, instances and enrichments in each of the ENCODE data sets. The motifs discovered here have been used in parallel studies to validate the specificity of antibodies, understand cooperativity between data sets and measure the variation of motif binding across individuals and species.National Institutes of Health (U.S.) (HG004037)National Institutes of Health (U.S.) (HG007000)National Institutes of Health (U.S.) (HG006991

Transcription Factors Bind Thousands of Active and Inactive Regions in the Drosophila Blastoderm

Identifying the genomic regions bound by sequence-specific regulatory factors is central both to deciphering the complex DNA cis-regulatory code that controls transcription in metazoans and to determining the range of genes that shape animal morphogenesis. We used whole-genome tiling arrays to map sequences bound in Drosophila melanogaster embryos by the six maternal and gap transcription factors that initiate anterior–posterior patterning. We find that these sequence-specific DNA binding proteins bind with quantitatively different specificities to highly overlapping sets of several thousand genomic regions in blastoderm embryos. Specific high- and moderate-affinity in vitro recognition sequences for each factor are enriched in bound regions. This enrichment, however, is not sufficient to explain the pattern of binding in vivo and varies in a context-dependent manner, demonstrating that higher-order rules must govern targeting of transcription factors. The more highly bound regions include all of the over 40 well-characterized enhancers known to respond to these factors as well as several hundred putative new cis-regulatory modules clustered near developmental regulators and other genes with patterned expression at this stage of embryogenesis. The new targets include most of the microRNAs (miRNAs) transcribed in the blastoderm, as well as all major zygotically transcribed dorsal–ventral patterning genes, whose expression we show to be quantitatively modulated by anterior–posterior factors. In addition to these highly bound regions, there are several thousand regions that are reproducibly bound at lower levels. However, these poorly bound regions are, collectively, far more distant from genes transcribed in the blastoderm than highly bound regions; are preferentially found in protein-coding sequences; and are less conserved than highly bound regions. Together these observations suggest that many of these poorly bound regions are not involved in early-embryonic transcriptional regulation, and a significant proportion may be nonfunctional. Surprisingly, for five of the six factors, their recognition sites are not unambiguously more constrained evolutionarily than the immediate flanking DNA, even in more highly bound and presumably functional regions, indicating that comparative DNA sequence analysis is limited in its ability to identify functional transcription factor targets

The possible functions of duplicated ets (GGAA) motifs located near transcription start sites of various human genes

Transcription is one of the most fundamental nuclear functions and is an enzyme complex-mediated reaction that converts DNA sequences into mRNA. Analyzing DNA sequences of 5′-flanking regions of several human genes that respond to 12-O-tetradecanoyl-phorbol-13-acetate (TPA) in HL-60 cells, we have identified that the ets (GGAA) motifs are duplicated, overlapped, or clustered within a 500-bp distance from the most 5′-upstream region of the cDNA. Multiple protein factors including Ets family proteins are known to recognize and bind to the GGAA containing sequences. In addition, it has been reported that the ets motifs play important roles in regulation of various promoters. Here, we propose a molecular mechanism, defined by the presence of duplication and multiplication of the GGAA motifs, that is responsible for the initiation of transcription of several genes and for the recruitment of binding proteins to the transcription start site (TSS) of TATA-less promoters