The transcriptional corepressor Groucho (Gro) is required for the function of many developmentally regulated DNA binding repressors, thus helping to define the gene expression profile of each cell during development. The ability of Gro to repress transcription at a distance together with its ability to oligomerize and bind to histones has led to the suggestion that Gro may spread along chromatin. However, much is unknown about the mechanism of Gro-mediated repression and about the dynamics of Gro targeting.Our chromatin immunoprecipitation sequencing analysis of temporally staged Drosophila embryos shows that Gro binds in a highly dynamic manner primarily to clusters of discrete (<1 kb) segments. Consistent with the idea that Gro may facilitate communication between silencers and promoters, Gro binding is enriched at both cis-regulatory modules, as well as within the promotors of potential target genes. While this Gro-recruitment is required for repression, our data show that it is not sufficient for repression. Integration of Gro binding data with transcriptomic analysis suggests that, contrary to what has been observed for another Gro family member, Drosophila Gro is probably a dedicated repressor. This analysis also allows us to define a set of high confidence Gro repression targets. Using publically available data regarding the physical and genetic interactions between these targets, we are able to place them in the regulatory network controlling development. Through analysis of chromatin associated pre-mRNA levels at these targets, we find that genes regulated by Gro in the embryo are enriched for characteristics of promoter proximal paused RNA polymerase II.Our findings are inconsistent with a one-dimensional spreading model for long-range repression and suggest that Gro-mediated repression must be regulated at a post-recruitment step. They also show that Gro is likely a dedicated repressor that sits at a prominent highly interconnected regulatory hub in the developmental network. Furthermore, our findings suggest a role for RNA polymerase II pausing in Gro-mediated repression.

We describe here a system for the expression and purification of small ubiquitin-related modifier (SUMO) fusion proteins, which often exhibit dramatically increased solubility and stability during expression in bacteria relative to unfused proteins. The vector described here allows expression of a His-tagged protein of interest fused at its N-terminus to SUMO. Using this vector, we have produced a polypeptide consisting of SUMO fused to the Q domain of Drosophila Groucho in a concentrated soluble form. Hydrodynamic analysis shows that, consistent with previous studies on full-length Groucho, the fusion protein forms an elongated tetramer, as well as higher order oligomers. After expressing a protein as a fusion to SUMO, it is often desirable to cleave the SUMO off of the fusion protein using a SUMO-specific protease such as Ulp1. To facilitate such processing, we have constructed a dual expression vector encoding two fusion proteins: one consisting of SUMO fused to Ulp1 and a second consisting of SUMO fused to a His-tagged protein of interest. The SUMO–Ulp1 cleaves both itself and the other SUMO fusion protein in the bacterial cells prior to lysis, and the proteins retain solubility after cleavage.

In Drosophila, the eye and antenna originate from a single epithelium termed the eye-antennal imaginal disc. Illumination of the mechanisms that subdivide this epithelium into eye and antenna would enhance our understanding of the mechanisms that restrict stem cell fate. We show here that Dip3, a transcription factor required for eye development, alters fate determination when misexpressed in the early eye-antennal disc, and have taken advantage of this observation to gain new insight into the mechanisms controlling the eye-antennal switch. Dip3 misexpression yields extra antennae by two distinct mechanisms: the splitting of the antennal field into multiple antennal domains (antennal duplication), and the transformation of the eye disc to an antennal fate. Antennal duplication requires Dip3-induced under proliferation of the eye disc and concurrent over proliferation of the antennal disc. While previous studies have shown that overgrowth of the antennal disc can lead to antennal duplication, our results show that overgrowth is not sufficient for antennal duplication, which may require additional signals perhaps from the eye disc. Eye-to-antennal transformation appears to result from the combination of antennal selector gene activation, eye determination gene repression, and cell cycle perturbation in the eye disc. Both antennal duplication and eye-to-antennal transformation are suppressed by the expression of genes that drive the cell cycle providing support for tight coupling of cell fate determination and cell cycle control. The finding that this transformation occurs only in the eye disc, and not in other imaginal discs, suggests a close developmental and therefore evolutionary relationship between eyes and antennae.

Dorsal interacting protein 3 (Dip3) contains a MADF DNA-binding domain and a BESS protein interaction domain. The Dip3 BESS domain was previously shown to bind to the Dorsal Rel homology domain. We show here that Dip3 also binds to the Relish Rel homology domain and enhances Rel family transcription factor function in both dorsoventral patterning and the immune response. While Dip3 is not essential, Dip3 mutations enhance the embryonic patterning defects that result from dorsal haplo-insufficiency, indicating that Dip3 may render dorsoventral patterning more robust. Dip3 is also required for optimal resistance to immune challenge since Dip3 mutant adults and larvae infected with bacteria have shortened lifetimes relative to infected wild-type flies. Furthermore, the mutant larvae exhibit significantly reduced expression of antimicrobial defense genes. Chromatin immunoprecipitation experiments in S2 cells indicate the presence of Dip3 at the promoters of these genes, and this binding requires the presence of Rel proteins at these promoters.

We show here that the Drosophila MADF/BESS domain transcription factor Dip3, which is expressed in differentiating photoreceptors, regulates neuronal differentiation in the compound eye. Loss of Dip3 activity in photoreceptors leads to an extra photoreceptor in many ommatidia, while ectopic expression of Dip3 in non-neuronal cells results in photoreceptor loss. These findings are consistent with the idea that Dip3 is required non-cell autonomously to block extra photoreceptor formation. Dip3 may mediate the spatially restricted potentiation of Notch (N) signaling since the Dip3 misexpression phenotype is suppressed by reducing N signaling and misexpression of Dip3 leads to ectopic activity of a N-responsive enhancer. Analysis of mosaic ommatidia suggests that no specific photoreceptor must be mutant to generate the mutant phenotype. Remarkably, however, mosaic pupal ommatidia with three or fewer Dip3+ photoreceptors always differentiate an extra photoreceptor, while those with four or more Dip3+ photoreceptors never differentiate an extra photoreceptor. These findings are consistent with the notion that Dip3 in photoreceptors activates a heretofore unsuspected diffusible ligand that may work in conjunction with the N pathway to prevent a subpopulation of undifferentiated cells from choosing a neuronal fate.