CiteULike: emptyhb's even-skipped_enhancer

[duplicate] Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene.

Hilde Janssens — 2008-07-17T19:54:20-00:00

Nat Genet (17 September 2006)

Here we present a quantitative and predictive model of the transcriptional readout of the proximal 1.7 kb of the control region of the Drosophila melanogaster gene even skipped (eve). The model is based on the positions and sequence of individual binding sites on the DNA and quantitative, time-resolved expression data at cellular resolution. These data demonstrated new expression features, first reported here. The model correctly predicts the expression patterns of mutations in trans, as well as point mutations, insertions and deletions in cis. It also shows that the nonclassical expression of stripe 7 driven by this fragment is activated by the protein Caudal (Cad), and repressed by the proteins Tailless (Tll) and Giant (Gt).

Information display by transcriptional enhancers

Meghana Kulkarni — 2008-07-02T21:43:37-00:00

Development, Vol. 130, No. 26. (29 December 2003), pp. 6569-6575.

Transcriptional enhancers integrate positional and temporal information to regulate the complex expression of developmentally controlled genes. Current models suggest that enhancers act as computational devices, receiving multiple inputs from activators and repressors and resolving them into a single positive or a negative signal that is transmitted to the basal transcriptional machinery. We show that a simple, compact enhancer is capable of representing both repressed and activated states at the same time and in the same nucleus. This finding suggests that closely apposed factor binding sites, situated within compact cis-elements, can be independently interpreted by the transcriptional machinery, possibly through successive enhancer-promoter interactions. These results provide clear evidence that the computational functions usually ascribed to the enhancer itself are actually shared with the basal machinery. In contrast to the autonomous computer model of enhancer function, an information-display or `billboard' model of enhancer activity may better describe many developmentally regulated transcriptional enhancers. 10.1242/dev.00890

Sepsid even-skipped Enhancers Are Functionally Conserved in Drosophila Despite Lack of Sequence Conservation

Emily Hare — 2008-06-27T18:46:42-00:00

PLoS Genet, Vol. 4, No. 6. (27 June 2008), e1000106.

The gene expression pattern specified by an animal regulatory sequence is generally viewed as arising from the particular arrangement of transcription factor binding sites it contains. However, we demonstrate here that regulatory sequences whose binding sites have been almost completely rearranged can still produce identical outputs. We sequenced the even-skipped locus from six species of scavenger flies (Sepsidae) that are highly diverged from the model species Drosophila melanogaster, but share its basic patterns of developmental gene expression. Although there is little sequence similarity between the sepsid eve enhancers and their well-characterized D. melanogaster counterparts, the sepsid and Drosophila enhancers drive nearly identical expression patterns in transgenic D. melanogaster embryos. We conclude that the molecular machinery that connects regulatory sequences to the transcription apparatus is more flexible than previously appreciated. In exploring this diverse collection of sequences to identify the shared features that account for their similar functions, we found a small number of short (20–30 bp) sequences nearly perfectly conserved among the species. These highly conserved sequences are strongly enriched for pairs of overlapping or adjacent binding sites. Together, these observations suggest that the local arrangement of binding sites relative to each other is more important than their overall arrangement into larger units of cis-regulatory function.

Regulation of even-skipped stripe 2 in the Drosophila embryo.

S Small — 2008-04-24T09:15:41-00:00

The EMBO journal, Vol. 11, No. 11. (November 1992), pp. 4047-4057.

In an effort to determine how crude gradients of transcriptional activators and repressors specify sharp stripes of gene expression in the early embryo, we have conducted a detailed study of even-skipped (eve) stripe 2. A combination of promoter fusions and P-transformation assays were used to show that a 480 bp region of the eve promoter is both necessary and sufficient to direct a stripe of LacZ expression within the limits of the endogenous eve stripe 2. The maternal morphogen bicoid (bcd) and the gap proteins hunchback (hb), Kruppel (Kr) and giant (gt) all bind with high affinity to closely linked sites within this small promoter element. Activation appears to depend on cooperative interactions among bcd and hb proteins, since disrupting single binding sites cause catastrophic reductions in expression. gt is directly involved in the formation of the anterior border, although additional repressors may participate in this process. Forming the posterior border of the stripe involves a delicate balance between limiting amounts of the bcd activator and the Kr repressor. We propose that the clustering of activator and repressor binding sites in the stripe 2 element is required to bring these weakly interacting regulatory factors into close apposition so that they can function both cooperatively and synergistically to control transcription.

Functional Evolution of a cis-Regulatory Module

Michael Ludwig — 2007-09-23T23:02:44-00:00

PLoS Biology, Vol. 3, No. 4. (1 April 2005), e93.

Lack of knowledge about how regulatory regions evolve in relation to their structure–function may limit the utility of comparative sequence analysis in deciphering cis-regulatory sequences. To address this we applied reverse genetics to carry out a functional genetic complementation analysis of a eukaryotic cis-regulatory module—the even-skipped stripe 2 enhancer—from four Drosophila species. The evolution of this enhancer is non-clock-like, with important functional differences between closely related species and functional convergence between distantly related species. Functional divergence is attributable to differences in activation levels rather than spatiotemporal control of gene expression. Our findings have implications for understanding enhancer structure–function, mechanisms of speciation and computational identification of regulatory modules.

Functional analysis of eve stripe 2 enhancer evolution in Drosophila: rules governing conservation and change.

MZ Ludwig — 2006-12-03T23:29:52-00:00

Development, Vol. 125, No. 5. (March 1998), pp. 949-958.

Experimental investigations of eukaryotic enhancers suggest that multiple binding sites and trans-acting regulatory factors are often required for wild-type enhancer function. Genetic analysis of the stripe 2 enhancer of even-skipped (eve), an important developmental gene in Drosophila, provides support for this view. Given the importance of even-skipped expression in early Drosophila development, it might be predicted that many structural features of the stripe 2 enhancer will be evolutionarily conserved, including the DNA sequences of protein binding sites and the spacing between them. To test this hypothesis, we compared sequences of the stripe 2 enhancer between four species of Drosophila: D. melanogaster, D. yakuba, D. erecta and D. pseudoobscura. Our analysis revealed a large number of nucleotide substitutions in regulatory protein binding sites for bicoid, hunchback, Kruppel and giant, as well as a systematic change in the size of the enhancer. Some of the binding sites in D. melanogaster are either absent or modified in other species. One functionally important bicoid-binding site in D. melanogaster appears to be recently evolved. We, therefore, investigated possible functional consequences of sequence differences among these stripe 2 enhancers by P-element-mediated transformation. This analysis revealed that the eve stripe 2 enhancer from each of the four species drove reporter gene expression at the identical time and location in D. melanogaster embryos. Double staining of native eve protein and transgene mRNA in early embryos showed that the reporter gene mimicked native eve expression and, in every case, produced sharply defined stripes at the blastoderm stage that were coincident with eve stripe 2 protein. We argue that stripe 2 eve expression in Drosophila evolution can be viewed as being under constant stabilizing selection with respect to the location of the anterior and posterior borders of the stripe. We further hypothesize that the stripe 2 enhancer is functionally robust, so that its evolution may be governed by the fixation of both slightly deleterious and adaptive mutations in regulatory protein binding sites as well as in the spacing between binding sites. This view allows for a slow but continual turnover of functionally important changes in the stripe 2 enhancer.

Bicoid determines sharp and precise target gene expression in the Drosophila embryo.

O Crauk — 2005-11-14T04:17:39-00:00

Curr Biol, Vol. 15, No. 21. (8 November 2005), pp. 1888-1898.

Background: The activity of the Bicoid (Bcd) transcription factor is a useful example of how quantitative information contained in a smooth morphogen gradient is transformed into discrete and precise patterns of target gene expression. There are two distinct and important aspects to this process: the "sharpening" of the posterior borders of the expression domains and the "precision" of where the target genes are expressed along the length of the embryo as the syncytial embryo begins to cellularize. Although the sharpening phenomenon was observed over a decade ago, it is still poorly understood. Results: Here, we show that a Bcd reporter gene containing binding sites only for Bcd is expressed, like natural targets of Bcd, in a precise domain with a sharp boundary. Analysis of embryos expressing deleted forms of Bcd indicates that the sharpness of the Bcd target gene hunchback's expression involves the glutamine-rich and C-terminal activation domains of Bcd. Furthermore, several artificial Gal4-derived transcription factors expressed as gradients in the embryo share Bcd's ability to drive precise target gene expression with sharp boundaries. Conclusion: Thus, contrary to recent reports proposing that the Bcd gradient is not sufficient to establish precise positional information, we show that Bcd drives precise and sharp expression of its target genes through a process that depends exclusively on its ability to activate transcription.

Evidence for stabilizing selection in a eukaryotic enhancer element

Michael Ludwig — 2006-04-22T18:30:45-00:00

Nature, Vol. 403, No. 6769. (3 February 2000), pp. 564-567.

Evolutionary dynamics of the enhancer region of even-skipped in Drosophila.

MZ Ludwig — 2007-10-16T21:48:54-00:00

Mol Biol Evol, Vol. 12, No. 6. (November 1995), pp. 1002-1011.

We report findings on naturally occurring variation in the regulatory region of even-skipped in Drosophila. This pair-rule gene encodes a homeobox-containing transcription factor, is expressed as a series of seven transverse stripes in developing embryos, and defines parasegmental boundaries. The 5' flanking region of the gene contains a 671-bp enhancer governing stripe 2 expression. The stripe 2 enhancer contains multiple binding sites for four transcription factors that provide positional information in developing blastoderm, the positive regulators bicoid and hunchback and the repressors giant and Kruppel. The study compares polymorphism and divergence in Drosophila melanogaster and Drosophila simulans for the enhancer region, the spacer between the enhancer and the transcription start site, the untranslated leader, the first exon and the intron of eve. Contrary to expectations, we find a relatively high level of variation in the stripe 2 enhancer region, including point substitutions and insertion/deletions in binding sites, and a comparable level of variation in the other noncoding regions. The patterns of variation and divergence within the enhancer region and between regions of the locus fit a model of neutral molecular evolution. We suggest that the multiplicity of binding sites in the enhancer provides a redundancy in function that allows flexibility in the sequence requirements and structural design of the promoter.

Short-range repression permits multiple enhancers to function autonomously within a complex promoter.

S Gray — 2008-02-28T17:41:30-00:00

Genes Dev., Vol. 8, No. 15. (1 August 1994), pp. 1829-1838.

Transcriptional repressors play a key role in establishing localized patterns of gene expression in the early Drosophila embryo. Several different modes of repression have been implicated in previous studies, including competition and direct interference with the transcription complex. Here, we present evidence for "quenching," whereby activators and repressors co-occupy neighboring sites in a target promoter, but the repressor blocks the ability of the activator to contact the transcription complex. This study centers on a zinc finger repressor, snail (sna), which represses the expression of neuroectodermal regulatory genes in the presumptive mesoderm. We show that sna can mediate efficient repression when bound 50-100 bp from upstream activator sites. Repression does not depend on proximity of sna-binding sites to the transcription initiation site. sna is not a dedicated repressor but, instead, appears to block disparate activators. We discuss the importance of quenching as a means of permitting separate enhancers to function autonomously within a complex promoter. 10.1101/gad.8.15.1829

The Drosophila even-skipped promoter is transcribed in a stage-specific manner in vitro and contains multiple, overlapping factor-binding sites.

D Read — 2008-02-28T17:23:52-00:00

Mol. Cell. Biol., Vol. 10, No. 8. (1 August 1990), pp. 4334-4344.

To investigate the factors contributing to regulation of expression of the Drosophila segmentation gene even-skipped (eve), we have analyzed both the in vitro transcription and eve-promoter-binding proteins in embryo extracts. We show that the eve promoter is accurately and efficiently expressed in nuclear extracts derived from Drosophila embryos and that transcription is more efficient in extracts prepared from embryos at early stages of development than in those from older embryos, broadly reproducing the temporal pattern of expression observed in vivo. This stage-specific expression is dependent on sequences upstream of the eve transcription start site which contain multiple binding sites for at least two distinct proteins present in embryo nuclei. One of these proteins, the binding sites for which correspond to the sequences required for stage-specific expression, appears to be the previously described GAGA factor. Although the binding activity of the GAGA factor remains constant, the level of the binding activity of the other protein, which we have called the TCCT factor, changes during the course of embryogenesis. Activity is first detected 3 to 5 h after fertilization and decreases during later stages of embryogenesis. We discuss the possibility that the TCCT factor plays a role in the maintenance or refinement of the eve expression pattern.

Quantitative and predictive model of transcriptional control of the Drosophila melanogaster even skipped gene.

Hilde Janssens — 2006-09-26T20:33:36-00:00

Sequence-specific DNA-binding activities of the gap proteins encoded by hunchback and Kruppel in Drosophila

Dusan Stanojevic — 2007-12-15T22:33:54-00:00

Nature, Vol. 341, No. 6240. (1989), pp. 331-335.