The importance of DNA sequence for nucleosome positioning in transcriptional regulation.

Nucleosome positioning is a key factor for transcriptional regulation. Nucleosomes regulate the dynamic accessibility of chromatin and interact with the transcription machinery at every stage. Influences to steer nucleosome positioning are diverse, and the according importance of the DNA sequence in contrast to active chromatin remodeling has been the subject of long discussion. In this study, we evaluate the functional role of DNA sequence for all major elements along the process of transcription. We developed a random forest classifier based on local DNA structure that assesses the sequence-intrinsic support for nucleosome positioning. On this basis, we created a simple data resource that we applied genome-wide to the human genome. In our comprehensive analysis, we found a special role of DNA in mediating the competition of nucleosomes with cis-regulatory elements, in enabling steady transcription, for positioning of stable nucleosomes in exons, and for repelling nucleosomes during transcription termination. In contrast, we relate these findings to concurrent processes that generate strongly positioned nucleosomes in vivo that are not mediated by sequence, such as energy-dependent remodeling of chromatin.

Comprehensive Wet-Bench and Bioinformatics Workflow for Complex Microbiota Using Oxford Nanopore Technologies.

The advent of high-throughput sequencing techniques has recently provided an astonishing insight into the composition and function of the human microbiome. Next-generation sequencing (NGS) has become the gold standard for advanced microbiome analysis; however, 3rd generation real-time sequencing, such as Oxford Nanopore Technologies (ONT), enables rapid sequencing from several kilobases to >2 Mb with high resolution. Despite the wide availability and the enormous potential for clinical and translational applications, ONT is poorly standardized in terms of sampling and storage conditions, DNA extraction, library creation, and bioinformatic classification. Here, we present a comprehensive analysis pipeline with sampling, storage, DNA extraction, library preparation, and bioinformatic evaluation for complex microbiomes sequenced with ONT. Our findings from buccal and rectal swabs and DNA extraction experiments indicate that methods that were approved for NGS microbiome analysis cannot be simply adapted to ONT. We recommend using swabs and DNA extractions protocols with extended washing steps. Both 16S rRNA and metagenomic sequencing achieved reliable and reproducible results. Our benchmarking experiments reveal thresholds for analysis parameters that achieved excellent precision, recall, and area under the precision recall values and is superior to existing classifiers (Kraken2, Kaiju, and MetaMaps). Hence, our workflow provides an experimental and bioinformatic pipeline to perform a highly accurate analysis of complex microbial structures from buccal and rectal swabs. IMPORTANCE Advanced microbiome analysis relies on sequencing of short DNA fragments from microorganisms like bacteria, fungi, and viruses. More recently, long fragment DNA sequencing of 3rd generation sequencing has gained increasing importance and can be rapidly conducted within a few hours due to its potential real-time sequencing. However, the analysis and correct identification of the microbiome relies on a multitude of factors, such as the method of sampling, DNA extraction, sequencing, and bioinformatic analysis. Scientists have used different protocols in the past that do not allow us to compare results across different studies and research fields. Here, we provide a comprehensive workflow from DNA extraction, sequencing, and bioinformatic workflow that allows rapid and accurate analysis of human buccal and rectal swabs with reproducible protocols. This workflow can be readily applied by many scientists from various research fields that aim to use long-fragment microbiome sequencing.

Effects of the Novel PFKFB3 Inhibitor KAN0438757 on Colorectal Cancer Cells and Its Systemic Toxicity Evaluation In Vivo.

BACKGROUND: Despite substantial progress made in the last decades in colorectal cancer (CRC) research, new treatment approaches are still needed to improve patients' long-term survival. To date, the promising strategy to target tumor angiogenesis metabolically together with a sensitization of CRC to chemo- and/or radiotherapy by PFKFB3 (6-phosphofructo-2-kinase/fructose-2,6-biphosphatase-3) inhibition has never been tested. Therefore, initial evaluation and validation of newly developed compounds such as KAN0438757 and their effects on CRC cells are crucial steps preceding to in vivo preclinical studies, which in turn may consolidate new therapeutic targets. MATERIALS AND METHODS: The efficiency of KAN0438757 to block PFKFB3 expression and translation in human CRC cells was evaluated by immunoblotting and real-time PCR. Functional in vitro assays assessed the effects of KAN0438757 on cell viability, proliferation, survival, adhesion, migration and invasion. Additionally, we evaluated the effects of KAN0438757 on matched patient-derived normal and tumor organoids and its systemic toxicity in vivo in C57BL6/N mice. RESULTS: High PFKFB3 expression is correlated with a worse survival in CRC patients. KAN0438757 reduces PFKFB3 protein expression without affecting its transcriptional regulation. Additionally, a concentration-dependent anti-proliferative effect was observed. The migration and invasion capacity of cancer cells were significantly reduced, independent of the anti-proliferative effect. When treating colonic patient-derived organoids with KAN0438757 an impressive effect on tumor organoids growth was apparent, surprisingly sparing normal colonic organoids. No high-grade toxicity was observed in vivo. CONCLUSION: The PFKFB3 inhibitor KAN0438757 significantly reduced CRC cell migration, invasion and survival. Moreover, on patient-derived cancer organoids KAN0438757 showed significant effects on growth, without being overly toxic in normal colon organoids and healthy mice. Our findings strongly encourage further translational studies to evaluate KAN0438757 in CRC therapy.

NOTCH Activation via gp130/STAT3 Signaling Confers Resistance to Chemoradiotherapy.

Resistance of tumor cells to chemoradiotherapy represents a fundamental problem in clinical oncology. The underlying mechanisms are actively debated. Here we show that blocking inflammatory cytokine receptor signaling via STAT3 re-sensitized treatment-refractory cancer cells and abolished tumor growth in a xenograft mouse model when applied together with chemoradiotherapy. STAT3 executed treatment resistance by triggering the expression of RBPJ, the key transcriptional regulator of the NOTCH pathway. The mandatory RBPJ interaction partner, NOTCH intracellular domain, was provided by tumor cell-intrinsic expression of NOTCH ligands that caused tonic NOTCH proteolysis. In fact, NOTCH inhibition phenocopied the effect of blocking STAT3 signaling. Moreover, genetic profiling of rectal cancer patients revealed the importance of the STAT3/NOTCH axis as NOTCH expression correlated with clinical outcome. Our data uncovered an unprecedented signal alliance between inflammation and cellular development that orchestrated resistance to chemoradiotherapy. Clinically, our findings allow for biomarker-driven patient stratification and offer novel treatment options.

Constructing temporal regulatory cascades in the context of development and cell differentiation.

Cell differentiation is a complex process orchestrated by sets of regulators precisely appearing at certain time points, resulting in regulatory cascades that affect the expression of broader sets of genes, ending up in the formation of different tissues and organ parts. The identification of stage-specific master regulators and the mechanism by which they activate each other is a key to understanding and controlling differentiation, particularly in the fields of tissue regeneration and organoid engineering. Here we present a workflow that combines a comprehensive general regulatory network based on binding site predictions with user-provided temporal gene expression data, to generate a a temporally connected series of stage-specific regulatory networks, which we call a temporal regulatory cascade (TRC). A TRC identifies those regulators that are unique for each time point, resulting in a cascade that shows the emergence of these regulators and regulatory interactions across time. The model was implemented in the form of a user-friendly, visual web-tool, that requires no expert knowledge in programming or statistics, making it directly usable for life scientists. In addition to generating TRCs the tool links multiple interactive visual workflows, in which a user can track and investigate further different regulators, target genes, and interactions, directing the tool along the way into biologically sensible results based on the given dataset. We applied the TRC model on two different expression datasets, one based on experiments conducted on human induced pluripotent stem cells (hiPSCs) undergoing differentiation into mature cardiomyocytes and the other based on the differentiation of H1-derived human neuronal precursor cells. The model was successful in identifying previously known and new potential key regulators, in addition to the particular time points with which these regulators are associated, in cardiac and neural development.

TFClass: expanding the classification of human transcription factors to their mammalian orthologs.

TFClass is a resource that classifies eukaryotic transcription factors (TFs) according to their DNA-binding domains (DBDs), available online at http://tfclass.bioinf.med.uni-goettingen.de. The classification scheme of TFClass was originally derived for human TFs and is expanded here to the whole taxonomic class of mammalia. Combining information from different resources, checking manually the retrieved mammalian TFs sequences and applying extensive phylogenetic analyses, >39 000 TFs from up to 41 mammalian species were assigned to the Superclasses, Classes, Families and Subfamilies of TFClass. As a result, TFClass now provides the corresponding sequence collection in FASTA format, sequence logos and phylogenetic trees at different classification levels, predicted TF binding sites for human, mouse, dog and cow genomes as well as links to several external databases. In particular, all those TFs that are also documented in the TRANSFAC® database (FACTOR table) have been linked and can be freely accessed. TRANSFAC® FACTOR can also be queried through an own search interface.

NF-Y Binding Site Architecture Defines a C-Fos Targeted Promoter Class.

ChIP-seq experiments detect the chromatin occupancy of known transcription factors in a genome-wide fashion. The comparisons of several species-specific ChIP-seq libraries done for different transcription factors have revealed a complex combinatorial and context-specific co-localization behavior for the identified binding regions. In this study we have investigated human derived ChIP-seq data to identify common cis-regulatory principles for the human transcription factor c-Fos. We found that in four different cell lines, c-Fos targeted proximal and distal genomic intervals show prevalences for either AP-1 motifs or CCAAT boxes as known binding motifs for the transcription factor NF-Y, and thereby act in a mutually exclusive manner. For proximal regions of co-localized c-Fos and NF-YB binding, we gathered evidence that a characteristic configuration of repeating CCAAT motifs may be responsible for attracting c-Fos, probably provided by a nearby AP-1 bound enhancer. Our results suggest a novel regulatory function of NF-Y in gene-proximal regions. Specific CCAAT dimer repeats bound by the transcription factor NF-Y define this novel cis-regulatory module. Based on this behavior we propose a new enhancer promoter interaction model based on AP-1 motif defined enhancers which interact with CCAAT-box characterized promoter regions.

Computational Identification of Key Regulators in Two Different Colorectal Cancer Cell Lines.

Transcription factors (TFs) are gene regulatory proteins that are essential for an effective regulation of the transcriptional machinery. Today, it is known that their expression plays an important role in several types of cancer. Computational identification of key players in specific cancer cell lines is still an open challenge in cancer research. In this study, we present a systematic approach which combines colorectal cancer (CRC) cell lines, namely 1638N-T1 and CMT-93, and well-established computational methods in order to compare these cell lines on the level of transcriptional regulation as well as on a pathway level, i.e., the cancer cell-intrinsic pathway repertoire. For this purpose, we firstly applied the Trinity platform to detect signature genes, and then applied analyses of the geneXplain platform to these for detection of upstream transcriptional regulators and their regulatory networks. We created a CRC-specific position weight matrix (PWM) library based on the TRANSFAC database (release 2014.1) to minimize the rate of false predictions in the promoter analyses. Using our proposed workflow, we specifically focused on revealing the similarities and differences in transcriptional regulation between the two CRC cell lines, and report a number of well-known, cancer-associated TFs with significantly enriched binding sites in the promoter regions of the signature genes. We show that, although the signature genes of both cell lines show no overlap, they may still be regulated by common TFs in CRC. Based on our findings, we suggest that canonical Wnt signaling is activated in 1638N-T1, but inhibited in CMT-93 through cross-talks of Wnt signaling with the VDR signaling pathway and/or LXR-related pathways. Furthermore, our findings provide indication of several master regulators being present such as MLK3 and Mapk1 (ERK2) which might be important in cell proliferation, migration, and invasion of 1638N-T1 and CMT-93, respectively. Taken together, we provide new insights into the invasive potential of these cell lines, which can be used for development of effective cancer therapy.

Relevance of Sp Binding Site Polymorphism in WWOX for Treatment Outcome in Pancreatic Cancer.

BACKGROUND: A genome-wide association study (GWAS) suggested inherited genetic single-nucleotide polymorphisms (SNPs) affecting overall survival (OS) in advanced pancreatic cancer. To identify robust clinical biomarkers, we tested the strongest reported candidate loci in an independent patient cohort, assessed cellular drug sensitivity, and evaluated molecular effects. METHODS: This study comprised 381 patients with histologically verified pancreatic ductal adenocarcinoma treated with gemcitabine-based chemotherapy. The primary outcome was the relationship between germline polymorphisms and OS. Functional assays addressed pharmacological dose-response effects in lymphoblastoid cell lines (LCLs) and pancreatic cancer cell lines (including upon RNAi), gene expression analyses, and allele-specific transcription factor binding. All statistical tests were two-sided. RESULTS: The A allele (26% in Caucasians) at SNP rs11644322 in the putative tumor suppressor gene WWOX conferred worse prognosis. Median OS was 14 months (95% confidence interval [CI] = 12 to 15 months), 13 months (95% CI = 11 to 15 months), and nine months (95% CI = 7 to 12 months) for the GG, GA, and AA genotypes, respectively (P trend < .001 for trend in univariate log-rank assuming a codominant mode of inheritance; advanced disease subgroup P trend < .001). Mean OS was 25 months (95% CI = 21 to 29 months), 19 months (95% CI = 15 to 22 months), and 13 months (95% CI = 10 to 16 months), respectively. This effect held true after adjustment for age, performance status according to Eastern Cooperative Oncology Group classification, TNM, grading, and resection status and was comparable with the strongest established prognostic factors in multivariable analysis. Consistently, reduced responsiveness to gemcitabine, but not 5-fluorouracil, along with lower WWOX expression was demonstrated in LCLs harboring the AA genotype. Likewise, RNAi-mediated WWOX knockdown in pancreatic cancer cells confirmed differential cytostatic drug sensitivity. In electrophoretic mobility shift assays, the A allele exhibited weaker binding of Sp family members Sp1/Sp3. CONCLUSIONS: WWOX rs11644322 represents a major predictive factor in gemcitabine-treated pancreatic cancer. Decreased WWOX expression may interfere with gemcitabine sensitivity, and allele-specific binding at rs11644332 might be a causative molecular mechanism behind the observed clinical associations.

Multiobjective triclustering of time-series transcriptome data reveals key genes of biological processes.

BACKGROUND: Exploratory analysis of multi-dimensional high-throughput datasets, such as microarray gene expression time series, may be instrumental in understanding the genetic programs underlying numerous biological processes. In such datasets, variations in the gene expression profiles are usually observed across replicates and time points. Thus mining the temporal expression patterns in such multi-dimensional datasets may not only provide insights into the key biological processes governing organs to grow and develop but also facilitate the understanding of the underlying complex gene regulatory circuits. RESULTS: In this work we have developed an evolutionary multi-objective optimization for our previously introduced triclustering algorithm δ-TRIMAX. Its aim is to make optimal use of δ-TRIMAX in extracting groups of co-expressed genes from time series gene expression data, or from any 3D gene expression dataset, by adding the powerful capabilities of an evolutionary algorithm to retrieve overlapping triclusters. We have compared the performance of our newly developed algorithm, EMOA- δ-TRIMAX, with that of other existing triclustering approaches using four artificial dataset and three real-life datasets. Moreover, we have analyzed the results of our algorithm on one of these real-life datasets monitoring the differentiation of human induced pluripotent stem cells (hiPSC) into mature cardiomyocytes. For each group of co-expressed genes belonging to one tricluster, we identified key genes by computing their membership values within the tricluster. It turned out that to a very high percentage, these key genes were significantly enriched in Gene Ontology categories or KEGG pathways that fitted very well to the biological context of cardiomyocytes differentiation. CONCLUSIONS: EMOA- δ-TRIMAX has proven instrumental in identifying groups of genes in transcriptomic data sets that represent the functional categories constituting the biological process under study. The executable file can be found at http://www.bioinf.med.uni-goettingen.de/fileadmin/download/EMOA-delta-TRIMAX.tar.gz .

Impact of mineralocorticoid receptor polymorphisms on urinary electrolyte excretion with and without diuretic drugs.

AIM: Polymorphisms in the mineralocorticoid receptor may affect urinary sodium and potassium excretion. We investigated polymorphisms in the MR gene in relation to urinary electrolyte excretion in two separate studies. PATIENTS & METHODS: The genotype-phenotype association was studied in healthy volunteers after single doses of bumetanide, furosemide, torsemide, hydrochlorothiazide, triamterene and after NaCl restriction. RESULTS: High potassium excretion under all conditions except torsemide, and high NaCl excretion after bumetanide and furosemide were associated with the A allele of the intron-3 polymorphism (rs3857080). This polymorphism explained 5-10% of the functional variation and in vitro, rs3857080 affected DNA binding of the transcription factor LHX4. CONCLUSION: rs3857080 may be a promising new candidate for research in cardiac and renal disorders and on antialdosteronergic drugs like spironolactone.

TFClass: a classification of human transcription factors and their rodent orthologs.

TFClass aims at classifying eukaryotic transcription factors (TFs) according to their DNA-binding domains (DBDs). For this, a classification schema comprising four generic levels (superclass, class, family and subfamily) was defined that could accommodate all known DNA-binding human TFs. They were assigned to their (sub-)families as instances at two different levels, the corresponding TF genes and individual gene products (protein isoforms). In the present version, all mouse and rat orthologs have been linked to the human TFs, and the mouse orthologs have been arranged in an independent ontology. Many TFs were assigned with typical DNA-binding patterns and positional weight matrices derived from high-throughput in-vitro binding studies. Predicted TF binding sites from human gene upstream sequences are now also attached to each human TF whenever a PWM was available for this factor or one of his paralogs. TFClass is freely available at http://tfclass.bioinf.med.uni-goettingen.de/ through a web interface and for download in OBO format.

Coexpression and coregulation analysis of time-series gene expression data in estrogen-induced breast cancer cell.

BACKGROUND: Estrogen is a chemical messenger that has an influence on many breast cancers as it helps cells to grow and divide. These cancers are often known as estrogen responsive cancers in which estrogen receptor occupies the surface of the cells. The successful treatment of breast cancers requires understanding gene expression, identifying of tumor markers, acquiring knowledge of cellular pathways, etc. In this paper we introduce our proposed triclustering algorithm δ-TRIMAX that aims to find genes that are coexpressed over subset of samples across a subset of time points. Here we introduce a novel mean-squared residue for such 3D dataset. Our proposed algorithm yields triclusters that have a mean-squared residue score below a threshold δ. RESULTS: We have applied our algorithm on one simulated dataset and one real-life dataset. The real-life dataset is a time-series dataset in estrogen induced breast cancer cell line. To establish the biological significance of genes belonging to resultant triclusters we have performed gene ontology, KEGG pathway and transcription factor binding site enrichment analysis. Additionally, we represent each resultant tricluster by computing its eigengene and verify whether its eigengene is also differentially expressed at early, middle and late estrogen responsive stages. We also identified hub-genes for each resultant triclusters and verified whether the hub-genes are found to be associated with breast cancer. Through our analysis CCL2, CD47, NFIB, BRD4, HPGD, CSNK1E, NPC1L1, PTEN, PTPN2 and ADAM9 are identified as hub-genes which are already known to be associated with breast cancer. The other genes that have also been identified as hub-genes might be associated with breast cancer or estrogen responsive elements. The TFBS enrichment analysis also reveals that transcription factor POU2F1 binds to the promoter region of ESR1 that encodes estrogen receptor α. Transcription factor E2F1 binds to the promoter regions of coexpressed genes MCM7, ANAPC1 and WEE1. CONCLUSIONS: Thus our integrative approach provides insights into breast cancer prognosis.

The architecture of the gene regulatory networks of different tissues.

SUMMARY: The great variety of human cell types in morphology and function is due to the diverse gene expression profiles that are governed by the distinctive regulatory networks in different cell types. It is still a challenging task to explain how the regulatory networks achieve the diversity of different cell types. Here, we report on our studies of the design principles of the tissue regulatory system by constructing the regulatory networks of eight human tissues, which subsume the regulatory interactions between transcription factors (TFs), microRNAs (miRNAs) and non-TF target genes. The results show that there are in-/out-hubs of high in-/out-degrees in tissue networks. Some hubs (strong hubs) maintain the hub status in all the tissues where they are expressed, whereas others (weak hubs), in spite of their ubiquitous expression, are hubs only in some tissues. The network motifs are mostly feed-forward loops. Some of them having no miRNAs are the common motifs shared by all tissues, whereas the others containing miRNAs are the tissue-specific ones owned by one or several tissues, indicating that the transcriptional regulation is more conserved across tissues than the post-transcriptional regulation. In particular, a common bow-tie framework was found that underlies the motif instances and shows diverse patterns in different tissues. Such bow-tie framework reflects the utilization efficiency of the regulatory system as well as its high variability in different tissues, and could serve as the model to further understand the structural adaptation of the regulatory system to the specific requirements of different cell functions. CONTACT: edgar.wingender@bioinf.med.uni-goettingen.de; jwang@nju.edu.cn SUPPLEMENTARY INFORMATION: Supplementary data are available at Bioinformatics online.

Coupled mutation finder: a new entropy-based method quantifying phylogenetic noise for the detection of compensatory mutations.

BACKGROUND: The detection of significant compensatory mutation signals in multiple sequence alignments (MSAs) is often complicated by noise. A challenging problem in bioinformatics is remains the separation of significant signals between two or more non-conserved residue sites from the phylogenetic noise and unrelated pair signals. Determination of these non-conserved residue sites is as important as the recognition of strictly conserved positions for understanding of the structural basis of protein functions and identification of functionally important residue regions. In this study, we developed a new method, the Coupled Mutation Finder (CMF) quantifying the phylogenetic noise for the detection of compensatory mutations. RESULTS: To demonstrate the effectiveness of this method, we analyzed essential sites of two human proteins: epidermal growth factor receptor (EGFR) and glucokinase (GCK). Our results suggest that the CMF is able to separate significant compensatory mutation signals from the phylogenetic noise and unrelated pair signals. The vast majority of compensatory mutation sites found by the CMF are related to essential sites of both proteins and they are likely to affect protein stability or functionality. CONCLUSIONS: The CMF is a new method, which includes an MSA-specific statistical model based on multiple testing procedures that quantify the error made in terms of the false discovery rate and a novel entropy-based metric to upscale BLOSUM62 dissimilar compensatory mutations. Therefore, it is a helpful tool to predict and investigate compensatory mutation sites of structural or functional importance in proteins. We suggest that the CMF could be used as a novel automated function prediction tool that is required for a better understanding of the structural basis of proteins. The CMF server is freely accessible at http://cmf.bioinf.med.uni-goettingen.de.

Using potential master regulator sites and paralogous expansion to construct tissue-specific transcriptional networks.

BACKGROUND: Transcriptional networks of higher eukaryotes are difficult to obtain. Available experimental data from conventional approaches are sporadic, while those generated with modern high-throughput technologies are biased. Computational predictions are generally perceived as being flooded with high rates of false positives. New concepts about the structure of regulatory regions and the function of master regulator sites may provide a way out of this dilemma. METHODS: We combined promoter scanning with positional weight matrices with a 4-genome conservativity analysis to predict high-affinity, highly conserved transcription factor (TF) binding sites and to infer TF-target gene relations. They were expanded to paralogous TFs and filtered for tissue-specific expression patterns to obtain a reference transcriptional network (RTN) as well as tissue-specific transcriptional networks (TTNs). RESULTS: When validated with experimental data sets, the predictions done showed the expected trends of true positive and true negative predictions, resulting in satisfying sensitivity and specificity characteristics. This also proved that confining the network reconstruction to the 1% top-ranking TF-target predictions gives rise to networks with expected degree distributions. Their expansion to paralogous TFs enriches them by tissue-specific regulators, providing a reasonable basis to reconstruct tissue-specific transcriptional networks. CONCLUSIONS: The concept of master regulator or seed sites provides a reasonable starting point to select predicted TF-target relations, which, together with a paralogous expansion, allow for reconstruction of tissue-specific transcriptional networks.

Regulatory coordination of clustered microRNAs based on microRNA-transcription factor regulatory network.

BACKGROUND: MicroRNA (miRNA) is a class of small RNAs of ~22nt which play essential roles in many crucial biological processes and numerous human diseases at post-transcriptional level of gene expression. It has been revealed that miRNA genes tend to be clustered, and the miRNAs organized into one cluster are usually transcribed coordinately. This implies a coordinated regulation mode exerted by clustered miRNAs. However, how the clustered miRNAs coordinate their regulations on large scale gene expression is still unclear. RESULTS: We constructed the miRNA-transcription factor regulatory network that contains the interactions between transcription factors (TFs), miRNAs and non-TF protein-coding genes, and made a genome-wide study on the regulatory coordination of clustered miRNAs. We found that there are two types of miRNA clusters, i.e. homo-clusters that contain miRNAs of the same family and hetero-clusters that contain miRNAs of various families. In general, the homo-clustered as well as the hetero-clustered miRNAs both exhibit coordinated regulation since the miRNAs belonging to one cluster tend to be involved in the same network module, which performs a relatively isolated biological function. However, the homo-clustered miRNAs show a direct regulatory coordination that is realized by one-step regulation (i.e. the direct regulation of the coordinated targets), whereas the hetero-clustered miRNAs show an indirect regulatory coordination that is realized by a regulation comprising at least three steps (e.g. the regulation on the coordinated targets by a miRNA through a sequential action of two TFs). The direct and indirect regulation target different categories of genes, the former predominantly regulating genes involved in emergent responses, the latter targeting genes that imply long-term effects. CONCLUSION: The genomic clustering of miRNAs is closely related to the coordinated regulation in the gene regulatory network. The pattern of regulatory coordination is dependent on the composition of the miRNA cluster. The homo-clustered miRNAs mainly coordinate their regulation rapidly, while the hetero-clustered miRNAs exert control with a delay. The diverse pattern of regulatory coordination suggests distinct roles of the homo-clustered and the hetero-clustered miRNAs in biological processes.

DEEP--a tool for differential expression effector prediction.

High-throughput methods for measuring transcript abundance, like SAGE or microarrays, are widely used for determining differences in gene expression between different tissue types, dignities (normal/malignant) or time points. Further analysis of such data frequently aims at the identification of gene interaction networks that form the causal basis for the observed properties of the systems under examination. To this end, it is usually not sufficient to rely on the measured gene expression levels alone; rather, additional biological knowledge has to be taken into account in order to generate useful hypotheses about the molecular mechanism leading to the realization of a certain phenotype. We present a method that combines gene expression data with biological expert knowledge on molecular interaction networks, as described by the TRANSPATH database on signal transduction, to predict additional--and not necessarily differentially expressed--genes or gene products which might participate in processes specific for either of the examined tissues or conditions. In a first step, significance values for over-expression in tissue/condition A or B are assigned to all genes in the expression data set. Genes with a significance value exceeding a certain threshold are used as starting points for the reconstruction of a graph with signaling components as nodes and signaling events as edges. In a subsequent graph traversal process, again starting from the previously identified differentially expressed genes, all encountered nodes 'inherit' all their starting nodes' significance values. In a final step, the graph is visualized, the nodes being colored according to a weighted average of their inherited significance values. Each node's, or sub-network's, predominant color, ranging from green (significant for tissue/condition A) over yellow (not significant for either tissue/condition) to red (significant for tissue/condition B), thus gives an immediate visual clue on which molecules--differentially expressed or not--may play pivotal roles in the tissues or conditions under examination. The described method has been implemented in Java as a client/server application and a web interface called DEEP (Differential Expression Effector Prediction). The client, which features an easy-to-use graphical interface, can freely be downloaded from the following URL: http://deep.bioinf.med.uni-goettingen.de.

Deriving an ontology for human gene expression sources from the CYTOMER database on human organs and cell types.

CYTOMER is a relational database of organs/tissues, cell types, physiological systems and developmental stages that currently focuses on the human system. From this database, we have derived an ontology for anatomical and morphological structures for the human organism which includes all embryonal stages and the cell types constituting these structures. The ontology has been transferred to the OWL format and is freely available for download at http://cytomer/bioinf.med.uni-goettingen.de.

PathFinder: reconstruction and dynamic visualization of metabolic pathways.

MOTIVATION: Beyond methods for a gene-wise annotation and analysis of sequenced genomes new automated methods for functional analysis on a higher level are needed. The identification of realized metabolic pathways provides valuable information on gene expression and regulation. Detection of incomplete pathways helps to improve a constantly evolving genome annotation or discover alternative biochemical pathways. To utilize automated genome analysis on the level of metabolic pathways new methods for the dynamic representation and visualization of pathways are needed. RESULTS: PathFinder is a tool for the dynamic visualization of metabolic pathways based on annotation data. Pathways are represented as directed acyclic graphs, graph layout algorithms accomplish the dynamic drawing and visualization of the metabolic maps. A more detailed analysis of the input data on the level of biochemical pathways helps to identify genes and detect improper parts of annotations. As an Relational Database Management System (RDBMS) based internet application PathFinder reads a list of EC-numbers or a given annotation in EMBL- or Genbank-format and dynamically generates pathway graphs.