Systematic annotation of gene regulatory elements is a major challenge in genome science. Direct mapping of chromatin modification marks and transcriptional factor binding sites genome-wide 1,2 has successfully identified specific subtypes of regulatory elements 3. In Drosophila several pioneering studies have provided genome-wide identification of Polycomb-Response Elements 4, chromatin states 5, transcription factor binding sites (TFBS) 6–9, PolII regulation 8, and insulator elements 10; however, comprehensive annotation of the regulatory genome remains a significant challenge. Here we describe results from the modENCODE cis-regulatory annotation project. We produced a map of the Drosophila melanogaster regulatory genome based on more than 300 chromatin immuno-precipitation (ChIP) datasets for eight chromatin features, five histone deacetylases (HDACs) and thirty-eight site-specific transcription factors (TFs) at different stages of development. Using these data we inferred more than 20,000 candidate regulatory elements and we validated a subset of predictions for promoters, enhancers, and insulators in vivo. We also identified nearly 2,000 genomic regions of dense TF binding associated with chromatin activity and accessibility. We discovered hundreds of new TF co-binding relationships and defined a TF network with over 800 potential regulatory relationships.

We have analyzed a set of new and existing strong mutations in the myospheroid gene, which encodes the betaPS integrin subunit of Drosophila. In addition to missense and other null mutations, three mutants behave as antimorphic alleles, indicative of dominant negative properties. Unlike null alleles, the three antimorphic mutants are synthetically lethal in double heterozygotes with an inflated (alphaPS2) null allele, and they fail to complement very weak, otherwise viable alleles of myospheroid. Two of the antimorphs result from identical splice site lesions, which create a frameshift in the C-terminal half of the cytoplasmic domain of betaPS. The third antimorphic mutation is caused by a stop codon just before the cytoplasmic splice site. These mutant betaPS proteins can support cell spreading in culture, especially under conditions that appear to promote integrin activation. Analyses of developing animals indicate that the dominant negative properties are not a result of inefficient surface expression, or simple competition between functional and nonfunctional proteins. These data indicate that mutations disrupting the C-terminal cytoplasmic domain of integrin beta subunits can have dominant negative effects in situ, at normal levels of expression, and that this property does not necessarily depend on a specific new protein sequence or structure. The results are discussed with respect to similar vertebrate beta subunit cytoplasmic mutations.

We have investigated the expression and function of the Sox15 transcription factor during the development of the external mechanosensory organs of Drosophila. We find that Sox15 is expressed specifically in the socket cell, and have identified the transcriptional cis-regulatory module that controls this activity. We show that Suppressor of Hairless [Su(H)] and the POU-domain factor Ventral veins lacking (Vvl) bind conserved sites in this enhancer and provide critical regulatory input. In particular, we find that Vvl contributes to the activation of the enhancer following relief of Su(H)-mediated default repression by the Notch signaling event that specifies the socket cell fate. Loss of Sox15 gene activity was found to severely impair the electrophysiological function of mechanosensory organs, due to both cell-autonomous and cell-non-autonomous effects on the differentiation of post-mitotic cells in the bristle lineage. Lastly, we find that simultaneous loss of both Sox15 and the autoregulatory activity of Su(H) reveals an important role for these factors in inhibiting transcription of the Pax family gene shaven in the socket cell, which serves to prevent inappropriate expression of the shaft differentiation program. Our results indicate that the later phases of socket cell differentiation are controlled by multiple transcription factors in a collaborative, and not hierarchical, manner.

The identification and functional studies of DM domain-containing proteins Doublesex, MAB-3, and DMRT1 indicated that flies, nematodes, and humans share at least some of the molecular mechanisms of sex determination. We identified a gene, AmDM1, from the coral Acropora millepora that encodes a homologous DM domain-containing protein. Molecular analyses show that the AmDM1 primary transcript is processed to generate four different messenger RNAs. Alternative use of two polyadenylation sites produces transcripts that vary only in the 3' untranslated regions, whereas alternative splicing generates transcripts with and without the region coding for the DM domain. All the transcripts include a second motif, the DMA domain, which is found in a number of other proteins containing a DM domain. Hermaphroditic A. millepora differentiates sexual cells seasonally before the spring spawn, and Northern blot analysis shows that the AmDM1 transcripts are present at higher levels during sexual differentiation. The non-DM domain-containing messages are also present at significant levels in late embryos, but DM domain transcripts are extremely rare at this stage. These data suggest that the association of DM domain proteins and sexual determination or differentiation predates the separation of the Cnidaria from the rest of the Metazoa.

Transcriptional cis-regulatory modules (CRMs), or enhancers, are responsible for directing gene expression in specific territories and cell types during development. In some instances, the same gene may be served by two or more enhancers with similar specificities. Here we show that the utilization of dual, or "shadow", enhancers is a common feature of genes that are active specifically in neural precursor (NP) cells in Drosophila. By genome-wide computational discovery of statistically significant clusters of binding motifs for both proneural activator (P) proteins and basic helix-loop-helix (bHLH) repressor (R) factors (a "P+R" regulatory code), we have identified NP-specific enhancer modules associated with multiple genes expressed in this cell type. These CRMs are distinct from those previously identified for the corresponding gene, establishing the existence of a dual-enhancer arrangement in which both modules reside close to the gene they serve. Using wild-type and mutant reporter gene constructs in vivo, we show that P sites in these modules mediate activation by proneural factors in "proneural cluster" territories, whereas R sites mediate repression by bHLH repressors, which serves to restrict expression specifically to NP cells. To our knowledge, our results identify the first direct targets of these bHLH repressors. Finally, using genomic rescue constructs for neuralized (neur), we demonstrate that each of the gene's two NP-specific enhancers is sufficient to rescue neur function in the lateral inhibition process by which adult sensory organ precursor (SOP) cells are specified, but that deletion of both enhancers results in failure of this event.neural precursors | dual enhancer modules | cis-regulatory code | proneural proteins | bHLH repressors S pecification of neural precursor (NP) cell fates is a core step in the process of neural development, and there has long been intense interest in its mechanistic basis. Among the most heavily investigated questions in this arena is how NP-specific expression of key regulatory factors associated with the NP fate is achieved. For example, multiple prior studies have used computational methods to identify NP-specific cis-regulatory modules (CRMs) genome-wide, in an attempt to define common transcription factor inputs that might underlie NP-specific gene expression (1-3).Proneural transcriptional activators of the basic helix-loophelix (bHLH) family are the top-level regulators of NP specification. They confer on cells in ectodermal tissue the potential to adopt the NP fate; loss of proneural gene function results in loss of all NPs and thus loss of expression of all NP-specific regulatory factors. However, proneural factors are not expressed only in NPs; rather, they are initially expressed in small groups of cells called proneural clusters (PNCs). At this stage of the process, most or all cells in the cluster have the potential to become an NP. This is prevented by "lateral inhibition", in which the single NP that will ultimately arise from the PNC inhibits al...

SUMMARYThe Notch cell-cell signaling pathway is used extensively in cell fate specification during metazoan development. In many cell lineages, the conditional role of Notch signaling is integrated with the autonomous action of the Numb protein, a Notch pathway antagonist. During Drosophila sensory bristle development, precursor cells segregate Numb asymmetrically to one of their progeny cells, rendering it unresponsive to reciprocal Notch signaling between the two daughters. This ensures that one daughter adopts a Notch-independent, and the other a Notch-dependent, cell fate. In a genome-wide survey for potential Notch pathway targets, the second intron of the numb gene was found to contain a statistically significant cluster of binding sites for Suppressor of Hairless, the transducing transcription factor for the pathway. We show that this region contains a Notchresponsive cis-regulatory module that directs numb transcription in the pIIa and pIIIb cells of the bristle lineage. These are the two precursor cells that do not inherit Numb, yet must make Numb to segregate to one daughter during their own division. Our findings reveal a new mechanism by which conditional and autonomous modes of fate specification are integrated within cell lineages.

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Developmental patterning involves the progressive subdivision of tissue into different cell types by invoking different genetic programs. In particular, cell-cell signaling is a universally deployed means of specifying distinct cell fates in adjacent cells. For this mechanism to be effective, it is essential that an asymmetry be established in the signaling and responding capacities of the participating cells. Here we focus on the regulatory mechanisms underlying the role of the neuralized gene and its protein product in establishing and maintaining asymmetry of signaling through the Notch pathway. The context is the classical process of “lateral inhibition” within Drosophila proneural clusters, which is responsible for distinguishing the sensory organ precursor (SOP) and non-SOP fates among adjacent cells. We find that neur is directly regulated in proneural clusters by both proneural transcriptional activators and Enhancer of split basic helix-loop-helix repressors (bHLH-Rs), via two separate cis-regulatory modules within the neur locus. We show that this bHLH-R regulation is required to prevent the early, pre-SOP expression of neur from being maintained in a subset of non-SOPs following SOP specification. Lastly, we demonstrate that Neur activity in the SOP is required to inhibit, in a cell non-autonomous manner, both neur expression and Neur function in non-SOPs, thus helping to secure the robust establishment of distinct cell identities within the developing proneural cluster.