DEAF-1, a novel protein that binds an essential region in a Deformed response element.

A 120 bp homeotic response element that is regulated specifically by Deformed in Drosophila embryos contains a single binding site for Deformed protein. However, a 24 bp sub-element containing this site does not constitute a Deformed response element. Specific activation requires a second region in the 120 bp element, which presumably contains one or more binding sites for Deformed cofactors. We have isolated a novel protein from Drosophila nuclear extracts which binds specifically to a site in this second region. This protein, which we call DEAF-1 (Deformed epidermal autoregulatory factor-1), contains three conserved domains. One of these includes a cysteine repeat motif that is similar to a motif found in Drosophila Nervy and the human MTG8 proto-oncoprotein, and another matches a region of Drosophila Trithorax. Mutations in the response element designed to improve binding to DEAF-1 in vitro resulted in increased embryonic expression. Conversely, small mutations designed to diminish binding to DEAF-1 resulted in reduced expression of the element. Thus, DEAF-1 is likely to contribute to the functional activity, and perhaps to the homeotic specificity, of this response element. Consistent with this hypothesis, we have discovered DEAF-1 binding sites in other Deformed response elements.     

DEAF-1 function is essential for the early embryonic development of Drosophila

The Drosophila protein DEAF-1 is a sequence-specific DNA binding protein that was isolated as a putative cofactor of the Hox protein Deformed (Dfd). In this study, we analyze the effects of loss or gain of DEAF-1 function on Drosophila development. Maternal/zygotic mutations of DEAF-1 largely result in early embryonic arrest prior to the expression of zygotic segmentation genes, although a few embryos develop into larvae with segmentation defects of variable severity. Overexpression of DEAF-1 protein in embryos can induce defects in migration/closure of the dorsal epidermis, and overexpression in adult primordia can strongly disrupt the development of eye or wing. The DEAF-1 protein associates with many discrete sites on polytene chromosomes, suggesting that DEAF-1 is a rather general regulator of gene expression.     

Control of the spineless antennal enhancer: Direct repression of antennal target genes by Antennapedia

It is currently thought that antennal target genes are activated in Drosophila by the combined action of Distal-less, homothorax, and extradenticle, and that the Hox gene Antennapedia prevents activation of antennal genes in the leg by repressing homothorax. To test these ideas, we analyze a 62 bp enhancer from the antennal gene spineless that is specific for the third antennal segment. This enhancer is activated by a tripartite complex of Distal-less, Homothorax, and Extradenticle. Surprisingly, Antennapedia represses the enhancer directly, at least in part by competing with Distal-less for binding. We show that Antennapedia is required in the leg only within a proximal ring that coexpresses Distal-less, Homothorax and Extradenticle. We conclude that the function of Antennapedia in the leg is not to repress homothorax, as has been suggested, but to directly repress spineless and other antennal genes that would otherwise be activated within this ring.     

Enhancer sequences influence the role of the amino-terminal domain of bicoid in transcription

Bicoid (Bcd) is a Drosophila melanogaster morphogenetic gradient that controls embryonic patterning by activating target gene expression in a concentration-dependent manner. In this study we describe experiments to determine how different enhancers respond to Bcd distinctively, focusing on two natural Bcd-responsive enhancer elements, hunchback (hb) and knirps (kni). Our results show that, on the hb enhancer element, the amino-terminal domain of Bcd (residues 1 to 91) plays primarily an inhibitory role, whereas on the kni enhancer element this same Bcd domain plays a positive role at low protein concentrations. We further demonstrate that while the amino-terminal domain is largely dispensable for cooperative binding to the hb enhancer element, it is preferentially required for cooperative binding to the kni enhancer element. Alteration of the arrangement of Bcd binding sites in the kni enhancer element reduces the role of the amino-terminal domain in cooperative DNA binding but increases the effectiveness of the self-inhibitory function. In addition, elimination of symmetric pairs of Bcd binding sites in the kni enhancer element reduces both DNA binding and activation by Bcd. We propose that the amino-terminal domain of Bcd is an enhancer-specific switch that contributes to the protein's ability to activate different target genes in distinct manners.     

Synergistic activation of a Drosophila enhancer by HOM/EXD and DPP signaling.

The homeotic proteins encoded by the genes of the Drosophila HOM and the vertebrate HOX complexes do not bind divergent DNA sequences with a high selectivity. In vitro, HOM (HOX) specificity can be increased by the formation of heterodimers with Extradenticle (EXD) or PBX homeodomain proteins. We have identified a single essential Labial (LAB)/EXD-binding site in a Decapentaplegic (DPP)-responsive enhancer of the homeotic gene lab which drives expression in the developing midgut. We show that LAB and EXD bind cooperatively to the site in vitro, and that the expression of the enhancer in vivo requires exd and lab function. In addition, point mutations in either the EXD or the LAB subsite compromise enhancer function, strongly suggesting that EXD and LAB bind to this site in vivo. Interestingly, we found that the activity of the enhancer is only stimulated by DPP signaling significantly upon binding of LAB and EXD. Thus, the enhancer appears to integrate positional information via the homeotic gene lab, and spatiotemporal information via DPP signaling; only when these inputs act in concert in an endodermal cell is the enhancer fully active. Our results illustrate how a tissue-specific response to DPP can be generated through synergistic effects on an enhancer carrying both DPP- and HOX-responsive sequences.     

Exploring Functional Redundancy in the Immunoglobulin μ Heavy-Chain Gene Enhancer

Immunoglobulin (Ig) μ heavy-chain gene enhancer activity is mediated by multiple DNA binding proteins. Mutations of several protein binding sites in the enhancer do not affect enhancer activity significantly. This feature, termed redundancy, is thought to be due to functional compensation of the mutated sites by other elements within the enhancer. In this study, we identified the elements that make the basic helix-loop-helix (bHLH) protein binding sites, μE2 and μE3, redundant. The major compensatory element is a binding site for interferon regulatory factors (IRFs) and not one of several other bHLH protein binding sites. These studies also provide the first evidence for a role of IRF proteins in Ig heavy-chain gene expression. In addition, we reconstituted the activity of a monomeric μ enhancer in nonlymphoid cells and defined the domains of the ETS gene required for function.     

The protein kinase C pathway acts through multiple transcription factors to repress gonadotropin-releasing hormone gene expression in hypothalamic GT1-7 neuronal cells

The GnRH gene uses two well-defined regions to target expression to a small population of hypothalamic GnRH neurons: a 173-bp proximal promoter and a 300-bp enhancer localized at approximately -1800 to -1500 bp from the start site. Interaction of multiple factors with the GnRH enhancer and promoter is required to confer neuron-specific expression in vivo and in cells in culture. In addition, the expression of the GnRH gene is regulated by numerous neurotransmitters and hormones. Several of these effectors act through membrane receptors to trigger the protein kinase C pathway, and 12-O-tetradecanoyl phorbol-13-acetate (TPA), a modulator of this pathway, has been shown to suppress GnRH gene expression through the promoter. We find that TPA suppresses expression through the GnRH enhancer as well as the promoter. In the enhancer, an Oct-1 binding site, a Pbx/Prep binding site, Msx/Dlx binding sites, and a previously unidentified protein-binding element at -1793, all contribute to TPA suppression. TPA treatment leads to decreased binding of Oct-1 and Pbx1a/Prep to their sites. However, a complex formed by GT1-7 nuclear extracts on the -1793 site is not affected by TPA treatment. It is known that cooperative interaction among multiple factors is necessary for GnRH gene expression; thus, one mechanism by which TPA suppresses GnRH gene expression is to disengage some of these factors from their cis-regulatory elements.     

A Functional Insulator Screen Identifies NURF and dREAM Components to Be Required for Enhancer-Blocking

Chromatin insulators of higher eukaryotes functionally divide the genome into active and inactive domains. Furthermore, insulators regulate enhancer/promoter communication, which is evident from the Drosophila bithorax locus in which a multitude of regulatory elements control segment specific gene activity. Centrosomal protein 190 (CP190) is targeted to insulators by CTCF or other insulator DNA-binding factors. Chromatin analyses revealed that insulators are characterized by open and nucleosome depleted regions. Here, we wanted to identify chromatin modification and remodelling factors required for an enhancer blocking function. We used the well-studied Fab-8 insulator of the bithorax locus to apply a genome-wide RNAi screen for factors that contribute to the enhancer blocking function of CTCF and CP190. Among 78 genes required for optimal Fab-8 mediated enhancer blocking, all four components of the NURF complex as well as several subunits of the dREAM complex were most evident. Mass spectrometric analyses of CTCF or CP190 bound proteins as well as immune precipitation confirmed NURF and dREAM binding. Both co-localise with most CP190 binding sites in the genome and chromatin immune precipitation showed that CP190 recruits NURF and dREAM. Nucleosome occupancy and histone H3 binding analyses revealed that CP190 mediated NURF binding results in nucleosomal depletion at CP190 binding sites. Thus, we conclude that CP190 binding to CTCF or to other DNA binding insulator factors mediates recruitment of NURF and dREAM. Furthermore, the enhancer blocking function of insulators is associated with nucleosomal depletion and requires NURF and dREAM.