Background: Molecular and genetic analyses conducted in model organisms such as Drosophila and vertebrates, have provided a wealth of information about how networks of transcription factors control the proper development of these species. Much less is known, however, about the evolutionary origin of these elaborated networks and their large-scale evolution. Here we report the first evolutionary analysis of a whole superfamily of transcription factors, the basic helix-loop-helix (bHLH) proteins, at the scale of the whole metazoan kingdom.Results: We identified in silico the putative full complement of bHLH genes in the sequenced genomes of 12 different species representative of the main metazoan lineages, including three non-bilaterian metazoans, the cnidarians Nematostella vectensis and Hydra magnipapillata and the demosponge Amphimedon queenslandica. We have performed extensive phylogenetic analyses of the 695 identified bHLHs, which has allowed us to allocate most of these bHLHs to defined evolutionary conserved groups of orthology.Conclusion: Three main features in the history of the bHLH gene superfamily can be inferred from these analyses: (i) an initial diversification of the bHLHs has occurred in the pre-Cambrian, prior to metazoan cladogenesis; (ii) a second expansion of the bHLH superfamily occurred early in metazoan evolution before bilaterians and cnidarians diverged; and (iii) the bHLH complement during the evolution of the bilaterians has been remarkably stable. We suggest that these features may be extended to other developmental gene families and reflect a general trend in the evolution of the developmental gene repertoires of metazoans.

BACKGROUND:DNA methylation plays an important role in development and tumorigenesis by epigenetic modification and silencing of critical genes. The development of PCR-based methylation assays on bisulphite modified DNA heralded a breakthrough in speed and sensitivity for gene methylation analysis. Despite this technological advancement, these approaches require a cumbersome gene by gene primer design and experimental validation. Bisulphite DNA modification results in sequence alterations (all unmethylated cytosines are converted into uracils) and a general sequence complexity reduction as cytosines become underrepresented. Consequently, standard BLAST sequence homology searches cannot be applied to search for specific methylation primers.RESULTS:To address this problem we developed methBLAST, a sequence similarity search program, based on the original BLAST algorithm but querying in silico bisulphite modified genome sequences to evaluate oligonucleotide sequence similarities. Apart from the primer specificity analysis tool, we have also developed a public database termed methPrimerDB for the storage and retrieval of validated PCR based methylation assays. The web interface allows free public access to perform methBLAST searches or database queries and to submit user based information. Database records can be searched by gene symbol, nucleotide sequence, analytical method used, Entrez Gene or methPrimerDB identifier, and submitter's name. Each record contains a link to Entrez Gene and PubMed to retrieve additional information on the gene, its genomic context and the article in which the methylation assay was described. To assure and maintain data integrity and accuracy, the database is linked to other reference databases. Currently, the database contains primer records for the most popular PCR-based methylation analysis methods to study human, mouse and rat epigenetic modifications. methPrimerDB and methBLAST are available at http://medgen.ugent.be/methprimerdb and http://medgen.ugent.be/methblast.CONCLUSION:We have developed two integrated and freely available web-tools for PCR based methylation analysis. methBLAST allows in silico assessment of primer specificity in PCR based methylation assays that can be stored in the methPrimerDB database, which provides a search portal for validated methylation assays.

The S.cerevisiae Uga43(Dal80) protein down-regulates the expression of multiple nitrogen pathway genes. It contains a zinc-finger motif similar to the DNA-binding domain of the vertebrate GATA family of transcription factors; this domain is known to direct binding to 5'-GATA-3' core sequences, The inducible UGA4 gene, which encodes the specific gamma-aminobutyrate permease, undergoes strong repression by Uga43p, This study shows that the 5' region of UGA4 contains a UAS element made of four directly repeated 5'-CGAT(A/T) AG-3' sequences, This element, called UAS(GATA), can potentially confer to the UGA4 gene high-level expression in the absence of inducer, but this potential activity is inhibited by two distinct repression systems. One system is Uga43p-dependent; it operates in cells grown on a poor nitrogen source, The other is the nitrogen repression system, which relies on Ure2p and glutamine and operates when a good nitrogen source is present, Nitrogen repression also blocks the synthesis of Uga43p, making the two repression systems mutually exclusive, Previous studies have shown that expression supported by 5'-GATA-3'-containing UAS elements requires Gln3p, another global nitrogen regulatory factor containing a GATA zinc-finger domain. Although Gln3p contributes to UAS(GATA) activity, evidence suggests that a second factor can potentially direct expression through UAS(GATA) Expression conferred by this putative factor is subject to both Uga43p- and Ure2p-mediated repression, The role of UAS(GATA) in the expression of the UGA4 gene is discussed in relation to its sensitivity to the two distinct repression systems.

The S.cerevisiae Uga43(Dai80) protein down-regulates the expression of muftipie nitrogen pathway genes. It contains a zinc-finger motif similar to the DNA-binding domain of the vertebrate GATA family of transcription factors; this domain is known to direct binding to 5'-GATA-3' core sequences. The inducible UGA4 gene, which encodes the specific γ-amlnobutyrate permease, undergoes strong repression by Uga43p. This study shows that the 5' region of UGA4 contains a UAS element made of four directly repeated 5'-CGAT(A/T) AG-3' sequences. This element, called UAS GATA , can potentially confer to the UGA4 gene high-level ex pression In the absence of inducer, but this potential activity Is Inhibited by two distinct repression systems. One system is Uga43p-dependent; it operates in cells grown on a poor nitrogen source. The other is the nitrogen repression system, which relies on Ure2p and glutamine and operates when a good nitrogen source is present. Nitrogen repression also blocks the synthesis of Uga43p, making the two repression systems mutually exclusive. Previous studies have shown that expression supported by 5′-GATA-3′- containing UAS elements requires Gln3p, another global nitrogen regulatory factor containing a GATA zinc-finger domain. Although Gln3p contributes to UAS activity, evidence suggests that a second factor can potentially direct expression through UAS1 Expression conferred by this putative factor is subject to both Uga43p- and Ure2p-medlated re pression. The role of UAS GATA in the expression of the UGA4 gene is discussed in relation to its sensitivity to the two distinct repression systems.

The UGA43 gene of Saccharomyces cerevisiae is required for repression of inducible genes involved in the utilization of 4-aminobutyric acid (GABA) or urea as nitrogen sources. The UGA43 gene has been cloned by complementation of a uga43 mutation. The N-terminal region of the UGA43 protein is very similar to the DNA-binding zinc-finger region typical of the GATA regulatory factor family in vertebrates. UGA43 is the first reported instance of a GATA protein with a negative regulatory function. The C-terminal region of the predicted UGA43 protein contains a putative leucine zipper. Sequencing of three uga43 mutant alleles suggests that the GATA and putative leucine-zipper regions are both required for the repressive activity of UGA43. UGA43 appears to be a highly regulated gene. On "poor" nitrogen sources, UGA43 transcripts are measured at high levels whereas they are nearly undetectable in conditions of nitrogen catabolite repression. The levels measured on "poor" nitrogen sources are further increased in uga43 mutant cells, suggesting that UGA43 exerts negative autoregulation.