The investigation and modeling of gene regulatory networks requires computational tools specific to the task. We present several locally developed software tools that have been used in support of our ongoing research into the embryogenesis of the sea urchin. These tools are especially well suited to iterative refinement of models through experimental and computational investigation. They include: BioArray, a macroarray spot processing program; SUGAR, a system to display and correlate large-BAC sequence analyses; SeqComp and FamilyRelations, programs for comparative sequence analysis; and NetBuilder, an environment for creating and analyzing models of gene networks. We also present an overview of the process used to build our model of the Strongylocentrotus purpuratus endomesoderm gene network. Several of the tools discussed in this paper are still in active development and some are available as open source.

Myogenin is the dominant transcriptional regulator of embryonic and fetal muscle differentiation and during maturation is profoundly down-regulated. We show that a highly conserved 17-bp DNA cis-acting sequence element located upstream of the myogenin promoter (myogHCE) is essential for postnatal repression of myogenin in transgenic animals. We present multiple lines of evidence supporting the idea that repression is mediated by the Y-box protein MSY-3. Electroporation in vivo shows that myogHCE and MSY-3 are required for postnatal repression. We further show that, in the C2C12 cell culture system, ectopic MSY-3 can repress differentiation, while reduced MSY-3 promotes premature differentiation. MSY-3 binds myogHCE simultaneously with the homeodomain protein Pbx in postnatal innervated muscle. We therefore propose a model in which the myogHCE motif operates as a switch by specifying opposing functions; one that was shown previously is regulated by MyoD and Pbx and it specifies a chromatin opening, gene-activating function at the time myoblasts begin to differentiate; the other includes MYS-3 and Pbx, and it specifies a repression function that operates during and after postnatal muscle maturation in vivo and in myoblasts before they begin to differentiate.[Keywords: CsdA; MSY-3; myogenin; Y-box; innervation; muscle maturation] Supplemental material is available at http://www.genesdev.org.

To learn how well ungapped sequence comparisons of multiple species can predict cis-regulatory elements in Caenorhabditis elegans, we made such predictions across the large, complex ceh-13/lin-39 locus and tested them transgenically. We also examined how prediction quality varied with different genomes and parameters in our comparisons. Specifically, we sequenced ∼0.5% of the C. brenneri and C. sp. 3 PS1010 genomes, and compared five Caenorhabditis genomes (C. elegans, C. briggsae, C. brenneri, C. remanei, and C. sp. 3 PS1010) to find regulatory elements in 22.8 kb of noncoding sequence from the ceh-13/lin-39 Hox subcluster. We developed the MUSSA program to find ungapped DNA sequences with N-way transitive conservation, applied it to the ceh-13/lin-39 locus, and transgenically assayed 21 regions with both high and low degrees of conservation. This identified 10 functional regulatory elements whose activities matched known ceh-13/lin-39 expression, with 100% specificity and a 77% recovery rate. One element was so well conserved that a similar mouse Hox cluster sequence recapitulated the native nematode expression pattern when tested in worms. Our findings suggest that ungapped sequence comparisons can predict regulatory elements genome-wide.[Supplemental material is available online at www.genome.org. The sequence data from this study have been submitted to GenBank (http://www.ncbi.nlm.nih.gov/Genbank/) under accession nos. FJ362353-FJ36238.] Despite knowledge of entire genome sequences, discovering cisregulatory DNA elements remains surprisingly inefficient. In animal genomes, cis-regulatory elements are located unpredictably around or within the genes they regulate (Woolfe et al. 2005;Davidson 2006;Pennacchio et al. 2006;Engström et al. 2007). These elements, when dissected further, often prove to be composed of individual transcription factor binding sites that are often very loosely defined (Sandelin et al. 2004). Transgenic analysis in vivo is the most definitive way to show that a sequence is regulatory, but it is also the most time consuming and expensive. It is therefore desirable to use other criteria, such as preferential sequence conservation, to identify regions most likely to be functional. To evaluate a strategy for phylogenetic footprinting using four other Caenorhabditis species, we dissected the cis-regulatory structure of a Hox cluster in the nematode Caenorhabditis elegans (Fig. 1A).If two or more species are evolutionarily close enough to show common development and physiology, their genomes are expected to share an underlying gene regulatory network driven by cis-regulatory elements with conserved sequences of several hundred base pairs (Tagle et al. 1988;Davidson 2006;Brown et al. 2007;Li et al. 2007). Within a functional cis-regulatory element, individual transcription-factor binding sites are generally short (∼6-20 bp) with statistical preferences, not strict requirements, for specific bases (Sandelin et al. 2004). Statistical overrepresentation of such motifs has been useful for ide...

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