Maté Ongenaert

The N-myc downstream regulated gene (NDRG) family: diverse functions, multiple applications

The N‐myc downstream regulated gene (NDRG) family of proteins consists of 4 members, NDRG1–4, which are well conserved through evolution. The first member to be discovered and responsible for the family name was NDRG1, because its expression is repressed by the proto‐oncogenes MYCN and MYC. All family members are characterized by an α/β hydrolase‐fold motif; however, the precise molecular and cellular function of these family members has not been fully elucidated. Although the exact function of NDRG family members has not been clearly elucidated, emerging evidence suggests that mutations in these genes are associated with diverse neurological and electrophysiological syndromes. In addition, aberrant expression as well as tumor suppressor and oncogenic functions affecting key hallmarks of carcinogenesis such as cell proliferation, differentiation, migration, invasion, and stress response have been reported for several of the NDRG proteins. In this review, we summarize the current literature on the NDRG family members concerning their structure, origin, and tissue distribution. In addition, we review the current knowledge regarding the regulation and signaling of the NDRG family members in development and normal physiology. Finally, their role in disease and potential clinical applications (their role as detection or prognostic markers) are discussed.—Melotte, V., Qu, X., Ongenaert, M., van Criekinge, W., de BruÏne, A. P., Baldwin, H. S., van Engeland, M. The N‐myc downstream regulated gene (NDRG) family: diverse functions, multiple applications. FASEB J. 24,4153–4166 (2010). www.fasebj.org

PubMeth: a cancer methylation database combining text-mining and expert annotation

Epigenetics, and more specifically DNA methylation is a fast evolving research area. In almost every cancer type, each month new publications confirm the differentiated regulation of specific genes due to methylation and mention the discovery of novel methylation markers. Therefore, it would be extremely useful to have an annotated, reviewed, sorted and summarized overview of all available data. PubMeth is a cancer methylation database that includes genes that are reported to be methylated in various cancer types. A query can be based either on genes (to check in which cancer types the genes are reported as being methylated) or on cancer types (which genes are reported to be methylated in the cancer (sub) types of interest). The database is freely accessible at http://www.pubmeth.org. PubMeth is based on text-mining of Medline/PubMed abstracts, combined with manual reading and annotation of preselected abstracts. The text-mining approach results in increased speed and selectivity (as for instance many different aliases of a gene are searched at once), while the manual screening significantly raises the specificity and quality of the database. The summarized overview of the results is very useful in case more genes or cancer types are searched at the same time.

Phylogenetic and specificity studies of two-domain GNA-related lectins: Generation of multispecificity through domain duplication and divergent evolution

A re-investigation of the occurrence and taxonomic distribution of proteins built up of protomers consisting of two tandem arrayed domains equivalent to the GNA [Galanthus nivalis (snowdrop) agglutinin] revealed that these are widespread among monotyledonous plants. Phylogenetic analysis of the available sequences indicated that these proteins do not represent a monophylogenetic group but most probably result from multiple independent domain duplication/in tandem insertion events. To corroborate the relationship between inter-domain sequence divergence and the widening of specificity range, a detailed comparative analysis was made of the sequences and specificity of a set of two-domain GNA-related lectins. Glycan microarray analyses, frontal affinity chromatography and surface plasmon resonance measurements demonstrated that the two-domain GNA-related lectins acquired a marked diversity in carbohydrate-binding specificity that strikingly contrasts the canonical exclusive specificity of their single domain counterparts towards mannose. Moreover, it appears that most two-domain GNA-related lectins interact with both high mannose and complex N-glycans and that this dual specificity relies on the simultaneous presence of at least two different independently acting binding sites. The combined phylogenetic, specificity and structural data strongly suggest that plants used domain duplication followed by divergent evolution as a mechanism to generate multispecific lectins from a single mannose-binding domain. Taking into account that the shift in specificity of some binding sites from high mannose to complex type N-glycans implies that the two-domain GNA-related lectins are primarily directed against typical animal glycans, it is tempting to speculate that plants developed two-domain GNA-related lectins for defence purposes.

miRBase Tracker: keeping track of microRNA annotation changes

Since 2002, information on individual microRNAs (miRNAs), such as reference names and sequences, has been stored in miRBase, the reference database for miRNA annotation. As a result of progressive insights into the miRNome and its complexity, miRBase underwent addition and deletion of miRNA records, changes in annotated miRNA sequences and adoption of more complex naming schemes over time. Unfortunately, miRBase does not allow straightforward assessment of these ongoing miRNA annotation changes, which has resulted in substantial ambiguity regarding miRNA identity and sequence in public literature, in target prediction databases and in content on various commercially available analytical platforms. As a result, correct interpretation, comparison and integration of miRNA study results are compromised, which we demonstrate here by assessing the impact of ignoring sequence annotation changes. To address this problem, we developed miRBase Tracker (www.mirbasetracker.org), an easy-to-use online database that keeps track of all historical and current miRNA annotation present in the miRBase database. Three basic functionalities allow researchers to keep their miRNA annotation up-to-date, reannotate analytical miRNA platforms and link published results with outdated annotation to the latest miRBase release. We expect miRBase Tracker to increase the transparency and annotation accuracy in the field of miRNA research. Database URL: www.mirbasetracker.org

Neuroblastoma epigenetics: from candidate gene approaches to genome-wide screenings.

Neuroblastoma (NB) is a childhood tumor originating from sympathetic nervous system cells. Although recently new insights into genes involved in NB have emerged, the molecular basis of neuroblastoma development and progression still remains poorly understood. The best-characterized genetic alterations include amplification of the proto-oncogene MYCN, ALK activating mutations or amplification, gain of chromosome arm 17q and losses of 1p, 3p, and 11q. Epigenetic alterations have been described as well: caspase-8 (CASP8) and RAS-association domain family 1 isoform A (RASSF1A) DNA-methylation are important events for the development and progression of neuroblastoma. In total, there are about 75 genes described as epigenetically affected in NB cell lines and/or NB primary samples. These epigenetic alterations were either found using a candidate gene approach or based on the analysis of genome-wide screening techniques. This review gives an extensive overview of all epigenetic changes described in NB as of today, with a main focus on both prognostic use and the potential of genome-wide techniques to find epigenetic prognostic biomarkers in NB. We summarize the key findings so far and the state-of-the-art of the upcoming methods at a unique time frame in the transition towards combined genome wide chromatin immune-precipitation (ChIP) and DNA sequencing techniques.

Methylation-specific digital karyotyping of HPV16E6E7-expressing human keratinocytes identifies novel methylation events in cervical carcinogenesis.

Transformation of epithelial cells by high‐risk human papillomavirus (hrHPV) types can lead to anogenital carcinomas, particularly cervical cancer, and oropharyngeal cancers. This process is associated with DNA methylation alterations, often affecting tumour suppressor gene expression. This study aimed to comprehensively unravel genome‐wide DNA methylation events linked to a transforming hrHPV‐infection, which is driven by deregulated expression of the viral oncogenes E6 and E7 in dividing cells. Primary human keratinocytes transduced with HPV16E6E7 and their untransduced counterparts were subjected to methylation‐specific digital karyotyping (MSDK) to screen for genome‐wide DNA‐methylation changes at different stages of HPV‐induced transformation. Integration of the obtained methylation profiles with genome‐wide gene expression patterns of cervical carcinomas identified 34 genes with increased methylation in HPV‐transformed cells and reduced expression in cervical carcinomas. For 12 genes (CLIC3, CREB3L1, FAM19A4, LFNG, LHX1, MRC2, NKX2‐8, NPTX‐1, PHACTR3, PRDM14, SOST and TNFSF13) specific methylation in HPV‐containing cell lines was confirmed by semi‐quantitative methylation‐specific PCR. Subsequent analysis of FAM19A4, LHX1, NKX2‐8, NPTX‐1, PHACTR3 and PRDM14 in cervical tissue specimens showed increasing methylation levels for all genes with disease progression. All six genes were frequently methylated in cervical carcinomas, with highest frequencies (up to 100%) seen for FAM19A4, PHACTR3 and PRDM14. Analysis of hrHPV‐positive cervical scrapes revealed significantly increased methylation levels of the latter three genes in women with high‐grade cervical disease compared to controls. In conclusion, MSDK analysis of HPV16‐transduced keratinocytes at different stages of HPV‐induced transformation resulted in the identification of novel DNA methylation events, involving FAM19A4, LHX1, NKX2‐8, PHACTR3 and PRDM14 genes in cervical carcinogenesis. These genes may provide promising triage markers to assess the presence of (pre)cancerous cervical lesions in hrHPV‐positive women. Copyright

Critical evaluation of the use of bioinformatics as a theoretical tool to find high-potential sources of ACE inhibitory peptides

A bioinformatics analysis to screen for high-potential sources of angiotensin converting enzyme (ACE) inhibitory peptides was conducted in the area of insect muscle proteins. Vertebrate muscle proteins are reported as good sources of ACE inhibitory peptides, while the research on invertebrate muscle proteins is limited. A phylogenetic tree constructed with actin sequences of both vertebrate and invertebrate species indicated a high homology. Furthermore, a quantitative in silico ACE inhibition analysis suggested that actin proteins of invertebrates have potentials as new sources of ACE inhibitory peptides. On one insect, Bombyx mori, a more detailed in silico analysis was done followed by a small experimental study. The in silico analysis indicated B. mori as a high-potential source of ACE inhibitory peptides and this was supported by the ACE inhibitory activity of the partially purified actin preparation. In conclusion, in food science, in silico analysis can be used as fast initial screening tool to look for high-potential sources of ACE inhibitory peptides and other peptidic bioactivities.

Discovery of DNA methylation markers in cervical cancer using relaxation ranking

BackgroundTo discover cancer specific DNA methylation markers, large-scale screening methods are widely used. The pharmacological unmasking expression microarray approach is an elegant method to enrich for genes that are silenced and re-expressed during functional reversal of DNA methylation upon treatment with demethylation agents. However, such experiments are performed in in vitro (cancer) cell lines, mostly with poor relevance when extrapolating to primary cancers. To overcome this problem, we incorporated data from primary cancer samples in the experimental design. A strategy to combine and rank data from these different data sources is essential to minimize the experimental work in the validation steps.AimTo apply a new relaxation ranking algorithm to enrich DNA methylation markers in cervical cancer.ResultsThe application of a new sorting methodology allowed us to sort high-throughput microarray data from both cervical cancer cell lines and primary cervical cancer samples. The performance of the sorting was analyzed in silico. Pathway and gene ontology analysis was performed on the top-selection and gives a strong indication that the ranking methodology is able to enrich towards genes that might be methylated. Terms like regulation of progression through cell cycle, positive regulation of programmed cell death as well as organ development and embryonic development are overrepresented. Combined with the highly enriched number of imprinted and X-chromosome located genes, and increased prevalence of known methylation markers selected from cervical (the highest-ranking known gene is CCNA1) as well as from other cancer types, the use of the ranking algorithm seems to be powerful in enriching towards methylated genes.Verification of the DNA methylation state of the 10 highest-ranking genes revealed that 7/9 (78%) gene promoters showed DNA methylation in cervical carcinomas. Of these 7 genes, 3 (SST, HTRA3 and NPTX1) are not methylated in normal cervix tissue.ConclusionThe application of this new relaxation ranking methodology allowed us to significantly enrich towards methylation genes in cancer. This enrichment is both shown in silico and by experimental validation, and revealed novel methylation markers as proof-of-concept that might be useful in early cancer detection in cervical scrapings.

Proteins with an Euonymus lectin-like domain are ubiquitous in Embryophyta

BackgroundCloning of the Euonymus lectin led to the discovery of a novel domain that also occurs in some stress-induced plant proteins. The distribution and the diversity of proteins with an Euonymus lectin (EUL) domain were investigated using detailed analysis of sequences in publicly accessible genome and transcriptome databases.ResultsComprehensive in silico analyses indicate that the recently identified Euonymus europaeus lectin domain represents a conserved structural unit of a novel family of putative carbohydrate-binding proteins, which will further be referred to as the Euonymus lectin (EUL) family. The EUL domain is widespread among plants. Analysis of retrieved sequences revealed that some sequences consist of a single EUL domain linked to an unrelated N-terminal domain whereas others comprise two in tandem arrayed EUL domains. A new classification system for these lectins is proposed based on the overall domain architecture. Evolutionary relationships among the sequences with EUL domains are discussed.ConclusionThe identification of the EUL family provides the first evidence for the occurrence in terrestrial plants of a highly conserved plant specific domain. The widespread distribution of the EUL domain strikingly contrasts the more limited or even narrow distribution of most other lectin domains found in plants. The apparent omnipresence of the EUL domain is indicative for a universal role of this lectin domain in plants. Although there is unambiguous evidence that several EUL domains possess carbohydrate-binding activity further research is required to corroborate the carbohydrate-binding properties of different members of the EUL family.

Novel TAL1 targets beyond protein-coding genes: identification of TAL1-regulated microRNAs in T-cell acute lymphoblastic leukemia

Novel TAL1 targets beyond protein-coding genes: identification of TAL1-regulated microRNAs in T-cell acute lymphoblastic leukemia