Leslie Pick

Functional dissection of the mouse Hox-a5 gene.

The Hox genes are clustered in evolutionarily conserved complexes and encode DNA binding proteins that determine positional identity. Ubiquitous expression of fly or mammalian Hox proteins in Drosophila embryos provides an assay for gene function, since different Hox genes induce characteristic homeotic transformations. Drosophila Sex combs reduced (Scr) and its murine cognate Hox‐a5 produce identical transformations in transgenic flies. To study the contributions of domains conserved between the two proteins, truncated versions of mouse Hox‐a5 were assayed for their ability to activate transcription in cultured cells and to induce homeotic transformation and activate target gene expression in transgenic embryos. The homeodomain is essential for protein function and/or nuclear targeting; the N‐terminal region contributes to transcription activity and transformation potential in the embryo, but plays no role in determining functional specificity. The YPWM motif is essential for biological specificity, although it does not contribute to transcriptional activation potential. It was recently shown that the Hox‐a5 YPWM motif is necessary for in vitro interactions with the co‐factor Pbx1. Our results suggest that this type of protein‐protein interaction may be essential for the biological activities of Hox‐a5 and Scr.

Cofactor-interaction motifs and the cooption of a homeotic hox protein into the segmentation pathway of Drosophila melanogaster

Some Drosophila Hox-complex members, including the segmentation gene fushi tarazu (Dm-ftz), have nonhomeotic functions. Characteristic expression in other arthropods supports an ancestral homeotic role for ftz, indicating that ftz function changed during arthropod evolution. Dm-Ftz segmentation function depends on interaction with ftz-F1 via an LXXLL motif and homeodomain N-terminal arm. Hox proteins interact with the cofactor Extradenticle (Exd) via their YPWM motif. Previously, we found that Dm-ftz mediates segmentation but not homeosis, whereas orthologs from grasshopper (Sg-ftz) and beetle (Tc-Ftz), both containing a YPWM motif, have homeotic function. Tc-Ftz, which unlike Sg-Ftz contains an LXXLL motif, displays stronger segmentation function than Sg-Ftz. Cofactor-interaction motifs were mutated in Dm-Ftz and Tc-Ftz and effects were evaluated in Drosophila to assess how these motifs contributed to Ftz evolution. Addition of YPWM to Dm-Ftz confers weak homeotic function, which is increased by simultaneous LXXLL mutation. LXXLL is required for strong segmentation function, which is unimpeded by the YPWM, suggesting that acquisition of LXXLL specialized Ftz for segmentation. Strengthening the Ftz/Ftz-F1 interaction led to degeneration of the YPWM and loss of homeotic activity. Thus, small changes in protein sequence can result in a qualitative switch in function during evolution.

NUCLEAR SCAFFOLD ATTACHMENT STIMULATES, BUT IS NOT ESSENTIAL FOR ARS ACTIVITY IN SACCHAROMYCES CEREVISIAE : ANALYSIS OF THE DROSOPHILA FTZ SAR

Nuclei isolated from eukaryotic cells can be depleted of histones and most soluble nuclear proteins to isolate a structural framework called the nuclear scaffold. This structure maintains specific interactions with genomic DNA at sites known as scaffold attached regions (SARs), which are thought to be the bases of DNA loops. In both Saccharomyces cerevisiae and Schizosaccharomyces pombe, genomic ARS elements are recovered as SARs. In addition, SARs from Drosophila melanogaster bind to yeast nuclear scaffolds in vitro and a subclass of these promotes autonomous replication of plasmids in yeast. In the present report, we present fine mapping studies of the Drosophila ftz SAR, which has both SAR and ARS activities in yeast. The data establish a close relationship between the sequences involved in ARS activity and scaffold binding: ARS elements that can bind the nuclear scaffold in vitro promote more efficient plasmid replication in vivo, but scaffold association is not a strict prerequisite for ARS function. Efficient interaction with nuclear scaffolds from both yeast and Drosophila requires a minimal length of SAR DNA that contains reiteration of a narrow minor groove structure of the double helix.

Hox gene evolution: multiple mechanisms contributing to evolutionary novelties

Hox genes, which are important for determining regional identity in organisms as diverse as flies and humans, are typically considered to be under strong evolutionary constraints because large changes in body plan are usually detrimental to survival. Despite this, there is great body plan diversity in nature, and many of the mechanisms underlying this diversity have been attributed to changes in Hox genes. Over the past year, several studies have examined how Hox genes play a role in evolution of body plans and novelties. Here, we examine four distinct evolutionary mechanisms implicated in Hox gene evolution, which include changes in (1) Hox gene expression, (2) downstream Hox target gene regulation without change in Hox expression, (3) protein‐coding sequence, and (4) posttranscriptional regulation of Hox gene function. We discuss how these types of changes in Hox genes—once thought to be evolutionarily static—underlie morphological diversification. We review recent studies that highlight each of these mechanisms and discuss their roles in the evolution of morphology and novelties.

The nuclear receptor Ftz-F1 and homeodomain protein Ftz interact through evolutionarily conserved protein domains

The Drosophila homeodomain protein Fushi Tarazu (Ftz) and its partner, the orphan receptor Ftz-F1, are members of two distinct families of DNA binding transcriptional regulators. Ftz and Ftz-F1 form a novel partnership in vivo as a Hox/orphan receptor heterodimer. Here we show that the murine Ftz-F1 ortholog SF-1 functionally substitutes for Ftz-F1 in vivo, rescuing the defects of ftz-f1 mutants. This finding identified evolutionarily conserved domains of Ftz-F1 as critical for activity of this receptor in vivo. These domains function, at least in part, by mediating direct protein interactions with Ftz. The Ftz-F1 DNA binding domain interacts strongly with Ftz and dramatically facilitates the binding of Ftz to target DNA. This interaction is augmented by a second interaction between the AF-2 domain of Ftz-F1 and the N-terminus of Ftz via an LRALL sequence in Ftz that is reminiscent of LXXLL motifs in nuclear receptor coactivators. We propose that Ftz-F1 serves as a cofactor for Ftz by facilitating the selection of target sites in the genome that contain Ftz/Ftz-F1 composite binding sites. Ftz, on the other hand, influences Ftz-F1 activity by interacting with its AF-2 domain in a manner that mimics a nuclear receptor coactivator.

Non-periodic cues generate seven ftz stripes in the Drosophila embryo

We have examined the expression pattern of the segmentation gene fushi tarazu (ftz) by in situ hybridization to whole mount embryos using digoxygenin labeled probes. This method has revealed previously undetected stages in the development of the ftz RNA pattern. The ftz stripes arise individually in a distinct, non-linear order along the anterior-posterior axis of the embryo. In addition, the stripes develop differentially along the dorsal-ventral axis; most stripes emerge on the ventral side and then gradually spread dorsally until they surround the entire circumference of the embryo. The order of appearance of ftz stripes is not inversely correlated with the order of appearance of hairy (h) stripes as would be expected if ftz stripes were generated by h repression. Furthermore, the seven ftz stripes are correctly established in embryos carrying mutations in h, eve or runt, with normal expression patterns decaying only after cellularization. Thus, the so called primary pair-rule genes are involved in the refinement rather than establishment of the ftz stripes. The contribution of cis-acting regulatory elements to the ftz pattern was examined. The zebra and upstream elements interact to generate seven correctly positioned stripes at the end of cellularization. However, stripe establishment is not correctly mimicked by any ftz/lac fusion gene: stripes arise in an order drastically different from the endogenous ftz gene suggesting the existence of ftz regulatory elements outside the 10-kb region examined to date. These observations suggest that the ftz pattern is directed by at least two independent regulatory systems: first, stripe establishment is directed by regionally distributed factors that act differentially in individual stripes along both anterior-posterior and dorsal-ventral axes of the egg and, second, stripe refinement and maintenance are mediated by pair-rule gene products that interact with previously identified ftz regulatory elements. This multi-level regulation provides a back-up system that ensures the development of seven stripes in the blastoderm.

Conservation and variation in Hox genes: how insect models pioneered the evo-devo field.

Evolutionary developmental biology, or evo-devo, broadly investigates how body plan diversity and morphological novelties have arisen and persisted in nature. The discovery of Hox genes in Drosophila, and their subsequent identification in most other metazoans, led biologists to try to understand how embryonic genes crucial for proper development have changed to promote the vast morphological variation seen in nature. Insects are ideal model systems for studying this diversity and the mechanisms underlying it because phylogenetic relationships are well established, powerful genetic tools have been developed, and there are many examples of evolutionary specializations that have arisen in nature in different insect lineages, such as the jumping leg of orthopterans and the helmet structures of treehoppers. Here, we briefly introduce the field of evo-devo and Hox genes, discuss functional tools available to study early developmental genes in insects, and provide examples in which changes in Hox genes have contributed to changes in body plan or morphology.

Multiple proteins interact with the fushi tarazu proximal enhancer.

The expression of the Drosophila segmentation gene fushi tarazu (ftz) is controlled at the level of transcription. The proximal enhancer, located approximately 3.4 kb upstream of the transcription start site, directs lacZ fusion gene expression in a ftz-like seven-stripe pattern in transgenic fly embryos. We have taken a biochemical approach to identify DNA-binding proteins that regulate ftz gene expression through the proximal enhancer. DNase I footprinting and methylation interference experiments with staged Drosophila embryo nuclear extracts identified nine protein binding sites in the proximal enhancer. Ten different sequence-specific DNA-binding complexes that interact with eight of these sites were identified. Some interact with multiple sites, while others bind to single sites in the enhancer. Two of the complexes that interact with multiple sites appear to contain the previously described ftz regulators, FTZ-F1 and TTK/FTZ-F2. These in vitro studies allowed us to narrow down the proximal enhancer to a 323-bp DNA fragment that contains all of the protein binding sites. Expression directed by this minimal enhancer element in seven ftz-like stripes in transgenic embryos is identical to that directed by the full-length enhancer. Internal deletions of several sites abolish reporter gene expression in vivo. Thus, the ftz proximal enhancer, like other cell-type-specific eukaryotic enhancers, interacts with an array of proteins that are expected to mediate the establishment, maintenance, and repression of transcription of the ftz gene in seven stripes in the developing embryo.

Surprising flexibility in a conserved Hox transcription factor over 550 million years of evolution

Although metazoan body plans are remarkably diverse, the structure and function of many embryonic regulatory genes are conserved because large changes would be detrimental to development. However, the fushi tarazu (ftz) gene has changed dramatically during arthropod evolution from Hox-like to a pair-rule segmentation gene in Drosophila. Changes in both expression and protein sequence contributed to this new function: ftz expression switched from Hox-like to stripes and changes in Ftz cofactor interaction motifs led to loss of homeotic and gain of segmentation potential. Here, we reconstructed ftz changes in a rigorous phylogenetic context. We found that ftz did not simply switch from Hox-like to segmentation function; rather, ftz is remarkably labile, having undergone multiple changes in sequence and expression. The segmentation LXXLL motif was stably acquired in holometabolous insects after the appearance of striped expression in early insect lineages. The homeotic YPWM motif independently degenerated multiple times. These “degen-YPWMs” showed varying degrees of homeotic potential when expressed in Drosophila, suggesting variable loss of Hox function in different arthropods. Finally, the intensity of ftz Hox-like expression decreased to marginal levels in some crustaceans. We propose that decreased expression levels permitted ftz variants to arise and persist in populations without disadvantaging organismal development. This process, in turn, allowed evolutionary transitions in protein function, as weakly expressed “hopeful gene variants” were coopted into alternative developmental pathways. Our findings show that variation of a pleiotropic transcription factor is more extensive than previously imagined, suggesting that evolutionary plasticity may be widespread among regulatory genes.

A double interaction screen identifies positive and negative ftz gene regulators and ftz-interacting proteins.

Regulatory genes directing embryonic development are expressed in complex patterns. The Drosophila homeobox gene fushi tarazu (ftz) is expressed in a striped pattern that is controlled by several discrete and large cis- regulatory elements. One key cis-element is the ftz proximal enhancer which is required for stripe establishment and which mediates autoregulation by direct binding of Ftz protein. To identify the trans-acting factors that regulate ftz expression and autoregulation, we developed a modified yeast two hybrid screen, the Double Interaction Screen (DIS). The DIS was designed to isolate both DNA binding transcriptional regulators that interact with the proximal enhancer and proteins that interact with Ftz itself when it is bound to the enhancer. The screen identified two candidate Ftz protein cofactors as well as activators and repressors of ftz transcription that bind directly to the enhancer. One of these (Tramtrack (Ttk)) was previously shown to bind to at least five sites in the proximal enhancer; genetic studies suggested that Ttk acts as a repressor of ftz in the embryo. Here we show that, in yeast cells, Ttk protein strongly activates transcription, suggesting that yeast may be missing a necessary co-repressor which is present in Drosophila embryos. Further, we have characterized the activity of a second candidate ftz repressor isolated in the screen - the product of the pair-rule gene sloppy paired - a member of the forkhead family. We show that Slp1 is a DNA binding protein. We have identified a high affinity binding site for Slp1 in the ftz proximal enhancer. Slp1 represses transcription via this binding site in yeast cells, consistent with its role as a direct repressor of ftz stripes in interstripe regions during late stages of embryogenesis. The DIS should be a generally useful method to identify DNA binding transcriptional regulators and protein partners of previously characterized DNA binding proteins.