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Background: The recent progress in molecular biology generates an increasing interest in investigating molecular biomarkers as markers of response to treatments. The present work is motivated by a study, where the objective was to explore the potential of the molecular biomarkers of renin-angiotensin-aldosterone system (RAAS) to identify the undertaken antihypertensive treatments in the general population. Population-based studies offer an opportunity to assess the effectiveness of treatments in real-world scenarios. However, lack of quality documentation, especially when electronic health record linkage is unavailable, leads to inaccurate reporting and classification bias., Method: We present a machine learning clustering technique to determine the potential of measured RAAS biomarkers for the identification of undertaken treatments in the general population. The biomarkers were simultaneously determined through a novel mass-spectrometry analysis in 800 participants of the Cooperative Health Research In South Tyrol (CHRIS) study with documented antihypertensive treatments. We assessed the agreement, sensitivity and specificity of the resulting clusters against known treatment types. Through the lasso penalized regression, we identified clinical characteristics associated with the biomarkers, accounting for the effects of cluster and treatment classifications., Results: We identified three well-separated clusters: cluster 1 (n = 444) preferentially including individuals not receiving RAAS-targeting drugs; cluster 2 (n = 235) identifying angiotensin type 1 receptor blockers (ARB) users (weighted kappa κ w  = 74%; sensitivity = 73%; specificity = 83%); and cluster 3 (n = 121) well discriminating angiotensin-converting enzyme inhibitors (ACEi) users (κ w  = 81%; sensitivity = 55%; specificity = 90%). Individuals in clusters 2 and 3 had higher frequency of diabetes as well as higher fasting glucose and BMI levels. Age, sex and kidney function were strong predictors of the RAAS biomarkers independently of the cluster structure., Conclusions: Unsupervised clustering of angiotensin-based biomarkers is a viable technique to identify individuals on specific antihypertensive treatments, pointing to a potential application of the biomarkers as useful clinical diagnostic tools even outside of a controlled clinical setting., (© 2023. The Author(s).)

Developing Future Biologists (DFB) is an inclusive, trainee-run organization that strives to excite and engage the next generation of biologists, regardless of race, gender or socioeconomic status, in the field of developmental biology. DFB offers a week-long course consisting of active lectures, hands-on laboratory sessions, and professional development opportunities through interactions with scientists from a variety of backgrounds and careers. A major goal of DFB is to propel undergraduate students from underserved communities to pursue biomedical research opportunities and advanced degrees in science. To achieve this goal, we provide DFB participants with continuing access to a diverse network of scientists that students can utilize to secure opportunities and foster success throughout multiple stages of their research careers. Here, we describe the flourishing DFB program at the University of Michigan to encourage other institutions to create their own DFB programs., Competing Interests: Competing interests The authors declare no competing or financial interests., (© 2023. Published by The Company of Biologists Ltd.)

The Hedgehog signaling pathway is part of the ancient developmental-evolutionary animal toolkit. Frequently co-opted to pattern new structures, the pathway is conserved among eumetazoans yet flexible and pleiotropic in its effects. The Hedgehog receptor, Patched, is transcriptionally activated by Hedgehog, providing essential negative feedback in all tissues. Our locus-wide dissections of the cis-regulatory landscapes of fly patched and mouse Ptch1 reveal abundant, diverse enhancers with stage- and tissue-specific expression patterns. The seemingly simple, constitutive Hedgehog response of patched/Ptch1 is driven by a complex regulatory architecture, with batteries of context-specific enhancers engaged in promoter-specific interactions to tune signaling individually in each tissue, without disturbing patterning elsewhere. This structure-one of the oldest cis-regulatory features discovered in animal genomes-explains how patched/Ptch1 can drive dramatic adaptations in animal morphology while maintaining its essential core function. It may also suggest a general model for the evolutionary flexibility of conserved regulators and pathways.

The Hedgehog (Hh) signaling pathway directs a multitude of cellular responses during embryogenesis and adult tissue homeostasis. Stimulation of the pathway results in activation of Hh target genes by the transcription factor Ci/Gli, which binds to specific motifs in genomic enhancers. In Drosophila, only a few enhancers (patched, decapentaplegic, wingless, stripe, knot, hairy, orthodenticle) have been shown by in vivo functional assays to depend on direct Ci/Gli regulation. All but one (orthodenticle) contain more than one Ci/Gli site, prompting us to directly test whether homotypic clustering of Ci/Gli binding sites is sufficient to define a Hh-regulated enhancer. We therefore developed a computational algorithm to identify Ci/Gli clusters that are enriched over random expectation, within a given region of the genome. Candidate genomic regions containing Ci/Gli clusters were functionally tested in chicken neural tube electroporation assays and in transgenic flies. Of the 22 Ci/Gli clusters tested, seven novel enhancers (and the previously known patched enhancer) were identified as Hh-responsive and Ci/Gli-dependent in one or both of these assays, including: Cuticular protein 100A (Cpr100A); invected (inv), which encodes an engrailed-related transcription factor expressed at the anterior/posterior wing disc boundary; roadkill (rdx), the fly homolog of vertebrate Spop; the segment polarity gene gooseberry (gsb); and two previously untested regions of the Hh receptor-encoding patched (ptc) gene. We conclude that homotypic Ci/Gli clustering is not sufficient information to ensure Hh-responsiveness; however, it can provide a clue for enhancer recognition within putative Hedgehog target gene loci.

Some transcriptional enhancers work best with one type of promoter, while ignoring others. How widespread is such specificity across the genome? A new study finds that, in a fair fight, most enhancers prefer to activate promoters resembling those of their parent genes., (Copyright © 2015 Elsevier Ltd. All rights reserved.)

As genomes evolve, proteins with novel functions arise primarily from gene duplication and divergence events. A new study identifies several molecular mechanisms by which related transcription factors diverge over time to control new sets of target genes and novel cellular functions., (Copyright © 2014 Elsevier Ltd. All rights reserved.)

Why do some genes seem to respond in a 'digital', on/off manner to a graded signal, while others produce an 'analog', graded response? A new study suggests that the DNA-binding properties of transcription factors can strongly influence the response patterns of gene networks., (Copyright © 2013 Elsevier Ltd. All rights reserved.)

In the era of functional genomics, the role of transcription factor (TF)-DNA binding affinity is of increasing interest: for example, it has recently been proposed that low-affinity genomic binding events, though frequent, are functionally irrelevant. Here, we investigate the role of binding site affinity in the transcriptional interpretation of Hedgehog (Hh) morphogen gradients. We noted that enhancers of several Hh-responsive Drosophila genes have low predicted affinity for Ci, the Gli family TF that transduces Hh signalling in the fly. Contrary to our initial hypothesis, improving the affinity of Ci/Gli sites in enhancers of dpp, wingless and stripe, by transplanting optimal sites from the patched gene, did not result in ectopic responses to Hh signalling. Instead, we found that these enhancers require low-affinity binding sites for normal activation in regions of relatively low signalling. When Ci/Gli sites in these enhancers were altered to improve their binding affinity, we observed patterning defects in the transcriptional response that are consistent with a switch from Ci-mediated activation to Ci-mediated repression. Synthetic transgenic reporters containing isolated Ci/Gli sites confirmed this finding in imaginal discs. We propose that the requirement for gene activation by Ci in the regions of low-to-moderate Hh signalling results in evolutionary pressure favouring weak binding sites in enhancers of certain Hh target genes.

How do gene regulatory networks evolve? A new study in yeasts shows that cis- and trans-regulatory changes resulted in a hybrid state of coexisting ancestral and derived regulatory circuits. This hybrid state then diversified into a variety of modern networks., (Copyright © 2012 Elsevier Ltd. All rights reserved.)

This paper, in the form of a frequently asked questions page (FAQ), addresses outstanding questions about "shadow enhancers", quasi-redundant cis-regulatory elements, and their proposed roles in transcriptional control. Questions include: What exactly are shadow enhancers? How many genes have shadow/redundant/distributed enhancers? How redundant are these elements? What is the function of distributed enhancers? How modular are enhancers? Is it useful to study a single enhancer in isolation? In addition, a revised definition of "shadow enhancers" is proposed, and possible mechanisms of shadow enhancer function and evolution are discussed., (Copyright © 2012 WILEY Periodicals, Inc.)

This review focuses on a single cis-regulatory element: the sparkling eye enhancer of the Drosophila dPax2 gene. sparkling responds to Notch and EGFR signaling, along with other direct regulatory inputs, to drive gene expression that is restricted to cone cells of the developing fly retina. Functional, genetic, biochemical, evolutionary, and bioinformatic analyses have revealed surprising properties of sparkling, which may provide new insights into cis-regulatory logic and mechanisms of transcriptional activation. These properties include: a very high density of regulatory information and a correspondingly low "junk" content; an unexpectedly complex combinatorial code; tight functional constraints on enhancer organization, paradoxically coupled with high turnover of DNA sequence and binding site position; a requirement for weak binding of the transcription factor Su(H) to low-affinity sites in order to maintain a cell-type-specific response to Notch signaling; and multiple specialized regulatory sequences conferring functionally distinct activation activities, all of which are required in concert to achieve proper gene expression in vivo., (Copyright © 2012 Elsevier Inc. All rights reserved.)

Morphogens control patterns of transcription in development, often by establishing concentration gradients of a single transcriptional activator. However, many morphogens, including Hedgehog, create opposing activator and repressor gradients (OARGs). In contrast to single activator gradients, it is not well understood how OARGs control transcriptional patterns. We present a general thermodynamic model that explains how spatial patterns of gene expression are established within OARGs. The model predicts that differences in enhancer binding site affinities for morphogen-responsive transcription factors (TFs) produce discrete transcriptional boundaries, but only when either activators or repressors bind cooperatively. This model quantitatively predicts the boundaries of gene expression within OARGs. When trained on experimental data, our model accounts for the counterintuitive observation that increasing the affinity of binding sites in enhancers of Hedgehog target genes produces more restricted transcription within Hedgehog gradients in Drosophila. Because our model is general, it may explain the role of low-affinity binding sites in many contexts, including mammalian Hedgehog gradients.

Background: Enhancers are genomic cis-regulatory sequences that integrate spatiotemporal signals to control gene expression. Enhancer activity depends on the combination of bound transcription factors as well as-in some cases-the arrangement and spacing of binding sites for these factors. Here, we examine evolutionary changes to the sequence and structure of sparkling, a Notch/EGFR/Runx-regulated enhancer that activates the dPax2 gene in cone cells of the developing Drosophila eye., Results: Despite functional and structural constraints on its sequence, sparkling has undergone major reorganization in its recent evolutionary history. Our data suggest that the relative strengths of the various regulatory inputs into sparkling change rapidly over evolutionary time, such that reduced input from some factors is compensated by increased input from different regulators. These gains and losses are at least partly responsible for the changes in enhancer structure that we observe. Furthermore, stereotypical spatial relationships between certain binding sites ("grammar elements") can be identified in all sparkling orthologs-although the sites themselves are often recently derived. We also find that low binding affinity for the Notch-regulated transcription factor Su(H), a conserved property of sparkling, is required to prevent ectopic responses to Notch in noncone cells., Conclusions: Rapid DNA sequence turnover does not imply either the absence of critical cis-regulatory information or the absence of structural rules. Our findings demonstrate that even a severely constrained cis-regulatory sequence can be significantly rewired over a short evolutionary timescale., (Copyright © 2011 Elsevier Ltd. All rights reserved.)

Gradients of diffusible signaling proteins control precise spatial patterns of gene expression in the developing embryo. Here, we use quantitative expression measurements and thermodynamic modeling to uncover the cis-regulatory logic underlying spatially restricted gene expression in a Hedgehog (Hh) gradient in Drosophila. When Hh signaling is low, the Hh effector Gli, known as Cubitus interruptus (Ci) in Drosophila, acts as a transcriptional repressor; when Hh signaling is high, Gli acts as a transcriptional activator. Counterintuitively and in contrast to previous models of Gli-regulated gene expression, we found that low-affinity binding sites for Ci were required for proper spatial expression of the Hh target gene decapentaplegic (dpp) in regions of low Hh signal. Three low-affinity Ci sites enabled expression of dpp in response to low signal; increasing the affinity of these sites restricted dpp expression to regions of maximal signaling. A model incorporating cooperative repression by Ci correctly predicted the in vivo expression of a reporter gene controlled by a single Ci site. Our work clarifies how transcriptional activators and repressors, competing for common binding sites, can transmit positional information to the genome. It also provides an explanation for the widespread presence of conserved, nonconsensus Gli binding sites in Hh target genes.

Competing Interests: The authors have declared that no competing interests exist.

Enhancers integrate spatiotemporal information to generate precise patterns of gene expression. How complex is the regulatory logic of a typical developmental enhancer, and how important is its internal organization? Here, we examine in detail the structure and function of sparkling, a Notch- and EGFR/MAPK-regulated, cone cell-specific enhancer of the Drosophila Pax2 gene, in vivo. In addition to its 12 previously identified protein-binding sites, sparkling is densely populated with previously unmapped regulatory sequences, which interact in complex ways to control gene expression. One segment is essential for activation at a distance, yet dispensable for other activation functions and for cell type patterning. Unexpectedly, rearranging sparkling's regulatory sites converts it into a robust photoreceptor-specific enhancer. Our results show that a single combination of regulatory inputs can encode multiple outputs, and suggest that the enhancer's organization determines the correct expression pattern by facilitating certain short-range regulatory interactions at the expense of others., (Copyright 2010 Elsevier Inc. All rights reserved.)

The highly conserved Wingless/Wnt signaling pathway controls many developmental processes by regulating the expression of target genes, most often through members of the TCF family of DNA-binding proteins. In the absence of signaling, many of these targets are silenced, by mechanisms involving TCFs that are not fully understood. Here we report that the chromatin remodeling proteins ISWI and ACF1 are required for basal repression of WG target genes in Drosophila. This regulation is not due to global repression by ISWI and ACF1 and is distinct from their previously reported role in chromatin assembly. While ISWI is localized to the same regions of Wingless target gene chromatin as TCF, we find that ACF1 binds much more broadly to target loci. This broad distribution of ACF1 is dependent on ISWI. ISWI and ACF1 are required for TCF binding to chromatin, while a TCF-independent role of ISWI-ACF1 in repression of Wingless targets is also observed. Finally, we show that Wingless signaling reduces ACF1 binding to WG targets, and ISWI and ACF1 regulate repression by antagonizing histone H4 acetylation. Our results argue that WG signaling activates target gene expression partly by overcoming the chromatin barrier maintained by ISWI and ACF1.

Signaling pathways usually activate transcriptional targets in a cell type-specific manner. Notable exceptions are pathway-specific feedback antagonists, which serve to restrict the range or duration of the signal. These factors are often activated by their respective pathways in a broad array of cell types. For example, the Wnt ligand Wingless (Wg) activates the naked cuticle (nkd) gene in all tissues examined throughout Drosophila development. How does the nkd gene respond in such an unrestricted manner to Wg signaling? Analysis in cell culture revealed regions of the nkd locus that contain Wg response elements (WREs) that are directly activated by the pathway via the transcription factor TCF. In flies, Wg signaling activates these WREs in multiple tissues, in distinct but overlapping patterns. These WREs are necessary and largely sufficient for nkd expression in late stage larval tissues, but only contribute to part of the embryonic expression pattern of nkd. These results demonstrate that nkd responsiveness to Wg signaling is achieved by several WREs which are broadly (but not universally) activated by the pathway. The existence of several WREs in the nkd locus may have been necessary to allow the Wg signaling-Nkd feedback circuit to remain intact as Wg expression diversified during animal evolution.

Enhancers, or cis-regulatory elements, are the principal determinants of spatiotemporal patterning of gene expression. For reasons of clinical and research utility, it is desirable to build customized enhancers that drive novel gene expression patterns, but currently, we largely rely on "found" genomic elements. Synthetic enhancers, assembled from transcription factor binding sites taken from natural signal-regulated enhancers, generally fail to behave like their wild-type counterparts when placed in transgenic animals, suggesting that important aspects of enhancer function are still unexplored. As a step toward the creation of a truly synthetic regulatory element, we have undertaken an extensive structure-function study of an enhancer of the Drosophila decapentaplegic (dpp) gene that drives expression in the developing visceral mesoderm (VM). Although considerable past efforts have been made to dissect the dppVM enhancer, transgenic experiments presented here indicate that its activity cannot be explained by the known regulators alone. dppVM contains multiple, previously uncharacterized, regulatory sites, some of which exhibit functional redundancy. The results presented here suggest that even the best-studied enhancers must be further dissected before they can be fully understood, and before faithful synthetic elements based on them can be created. Implications for developmental genetics, mathematical modeling, and therapeutic applications are discussed.

The fruit fly Drosophila is a leading model system for the study of transcriptional control by cis-regulatory elements, or enhancers. Here we present a rapid, high-efficiency system for directionally cloning PCR-amplified, PCR-mutated, or synthetic enhancer sequences into the Ganesh family of P element reporter constructs, which contain reporter genes encoding nuclear-localized eGFP, DsRed, or beta-galactosidase. This system, which is scalable for either small projects or high-throughput approaches, makes use of both TOPO and Gateway cloning technologies for directional, efficient cloning, without the need for restriction digestion or ligation reactions. It should be especially useful for those researchers who wish to test large numbers of putative enhancers, those who are undertaking detailed mutational analyses of enhancer sequences, or those who wish to avoid the difficulties sometimes encountered in traditional cloning strategies.

The initiation of retinal development in Drosophila begins at the posterior center (PC) of the eye disc margin. The front of the differentiation wave, recognized as a morphogenetic furrow (MF), moves from posterior to anterior. What determines MF initiates from the specific PC site is still unclear. The unpaired (upd) gene is expressed at PC at early third instar, just before the time of MF initiation. Therefore, upd is expressed at the appropriate time and location for a specific role in defining the site of MF initiation. upd encodes a ligand for the Jak/STAT signaling pathway. In this report, we showed that the Upd/Jak/STAT signaling is required and sufficient to determine MF initiation. This is primarily achieved by repressing the transcription of wingless (wg), which is known to block MF initiation.

Lateral inhibition, wherein a single cell signals to its neighbors to prevent them from adopting its own fate, is the best-known setting for cell-cell communication via the Notch (N) pathway. During peripheral neurogenesis in Drosophila, sensory organ precursor (SOP) cells arise within proneural clusters (PNCs), small groups of cells endowed with SOP fate potential by their expression of proneural transcriptional activators. SOPs use N signaling to activate in neighboring PNC cells the expression of multiple genes that inhibit the SOP fate. These genes respond transcriptionally to direct regulation by both the proneural proteins and the N pathway transcription factor Suppressor of Hairless [Su(H)], and their activation is generally highly asymmetric; i.e. only in the inhibited (non-SOP) cells of the PNC, and not in SOPs. We show that the substantially higher proneural protein levels in the SOP put this cell at risk of inappropriately activating the SOP-inhibitory genes, even without input from N-activated Su(H). We demonstrate that this is prevented by direct ;default' repression of these genes by Su(H), acting through the same binding sites it uses for activation in non-SOPs. We show that de-repression of even a single N pathway target gene in the SOP can extinguish the SOP cell fate. Finally, we define crucial roles for the adaptor protein Hairless and the co-repressors Groucho and CtBP in conferring repressive activity on Su(H) in the SOP. Our work elucidates the regulatory logic by which N signaling and the proneural proteins cooperate to create the neural precursor/epidermal cell fate distinction during lateral inhibition.

In vivo green fluorescent protein (GFP)/red fluorescent protein (RFP) double-labeling studies have been hampered by several inconvenient properties of DsRed, the first described RFP. These disadvantages include a very slow (> 24 h) maturation time, emission of contaminating green light, and low solubility. A recently developed variant of DsRed, called DsRed.T4, has a much shorter maturation time, no significant green emission, and improved solubility. We have constructed Drosophila P-element transformation vectors encoding DsRed.T4 for promoter/enhancer analysis, labeling of living cells, or RFP tagging of proteins. These new vectors have all of the features of the widely used Pelican/Stinger GFP vectors, including insulator sequences to reduce position effects, an extensive polylinker, and both cytoplasmic and nuclear-localized forms of the reporter. We have also constructed an upstream activating sequence (UAS)-DsRed.T4 vector, for GAL4 activation of the reporter. We find that DsRed.T4 is very easily detected in transgenic flies without contamination of the GFP signal and that it matures to its fluorescent form nearly simultaneously with GFP. This advance in Drosophila reporter technology makes timed double-labeling experiments in developing transgenic animals possible for the first time.

The DNA-binding transcription factor Suppressor of Hairless [Su(H)] functions as an activator during Notch (N) pathway signaling, but can act as a repressor in the absence of signaling. Hairless (H), a novel Drosophila protein, binds to Su(H) and has been proposed to antagonize N signaling by inhibiting DNA binding by Su(H). Here we show that, in vitro, H directly binds two corepressor proteins, Groucho (Gro) and dCtBP. Reduction of gro or dCtBP function enhances H mutant phenotypes and suppresses N phenotypes in the adult mechanosensory bristle. This activity of gro is surprising, because it is directed oppositely to its traditionally defined role as a neurogenic gene. We find that Su(H)-H complexes can bind to DNA with high efficiency in vitro. Furthermore, a H-VP16 fusion protein causes dominant-negative phenotypes in vivo, a result consistent with the proposal that H functions in transcriptional repression. Taken together, our findings indicate that "default repression" of N pathway target genes by an unusual adaptor/corepressor complex is essential for proper cell fate specification during Drosophila peripheral nervous system development.

Suppressor of Hairless [Su(H)]/Lag-1/RBP-Jkappa/CBF1 is the only known transducing transcription factor for Notch receptor signaling. Here, we show that Su(H) has three distinct functions in the development of external mechanosensory organs in Drosophila: Notch-dependent transcriptional activation and a novel auto-repression function, both of which direct cell fate decisions, and a novel auto-activation function required for normal socket cell differentiation. This third phase of activity, the first known Notch-independent activation function for Su(H) in development, depends on a cell type-specific autoregulatory enhancer that is active throughout adult life and is required for proper mechanoreception. These results establish a direct link between a broadly deployed cell signaling pathway and an essential physiological function of the nervous system.

To evaluate the role of angiotensin-converting enzyme (ACE) gene insertion/deletion (I/D) polymorphism in the progression of immunoglobulin A glomerulonephritis (IgA-GN), genotype distribution in 81 biopsy-proven cases of IgA-GN was studied. A logistic regression model showed that the risk for homozygous DD was not significantly elevated in patients with IgA-GN compared with healthy subjects (odds ratio = 1.16; confidence interval [CI], 0.4 to 3.3). However, the 5-year (78% v 90%) and 10-year (52% v 82%) renal survival rates for 47 patients with serum creatine (Cr) levels of 1.5 mg/dL or less at biopsy was significantly less in DD patients (n = 18; chi2 = 5.41; P = 0.02). The hazard ratio (HR) for DD (multivariate analysis from Cox proportional model after adjustment for known factors of progression, such as hypertension [HPT] and proteinuria [PTO]) was 3.07 (CI, 1.1 to 9.4). The HR for heavy PTO was 6.1 (CI, 1.9 to 19). The association of DD genotype with progression was even more striking when patients with other risk factors (heavy proteinuria) were excluded, as shown by DD-related risk in the absence (HR = 3.6; CI, 1.1 to 12) and presence (HR = 2; CI, 0.4 to 10) of PTO. The risk ratio was further increased by the coexistence of DD + PTO (HR = 9.16; CI, 1.8 to 15.7). Furthermore, in a cross-sectional study among patients with IgA-GN, a logistic regression model showed that the risk for homozygous DD was greater, although not at a statistically significant level in the end-stage renal failure subgroup compared with the normal renal function subgroup (odds ratio = 3.16; CI, 0.7 to 13.7) after adjustment by sex, age at biopsy, HPT, PTO, and therapy. Last, DD was significantly more frequent in those patients who started hemodialysis at an earlier age (chi2 for trend = 6.81; P = 0.009). Our study further supports that ACE genotype is a risk factor not for the development, but for the worsening of IgA-GN clinical course. However, on the basis of current knowledge, we cannot exclude that I/D polymorphism may simply serve as a prognostic marker, eventually linked with other discrete loci involved in the progression of renal damage.

The pre-cellular Drosophila embryo contains 10 well characterized sequence-specific transcriptional repressors, which represent a broad spectrum of DNA-binding proteins. Previous studies have shown that two of the repressors, Hairy and Dorsal, recruit a common co-repressor protein, Groucho. Here we present evidence that three different repressors, Knirps, Krüppel and Snail, recruit a different co-repressor, dCtBP. Mutant embryos containing diminished levels of maternal dCtBP products exhibit both segmentation and dorsoventral patterning defects, which can be attributed to loss of Krüppel, Knirps and Snail activity. In contrast, the Dorsal and Hairy repressors retain at least some activity in dCtBP mutant embryos. dCtBP interacts with Krüppel, Knirps and Snail through a related sequence motif, PXDLSXK/H. This motif is essential for the repression activity of these proteins in transgenic embryos. We propose that dCtBP represents a major form of transcriptional repression in development, and that the Groucho and dCtBP co-repressors mediate separate pathways of repression.

Transcriptional repression is essential for the conversion of crude maternal gradients into sharp territories of tissue differentiation in the Drosophila embryo. Evidence will be presented suggesting that some of the embryonic repressors function through a short-range 'quenching' mechanism, whereby a repressor works over short distances (ca. 50 b.p.) to block neighbouring activators within a target enhancer. This type of repression can explain how different enhancers work autonomously within complex modular promoters. However, at least one of the repressors operating in the early embryo works through a long-range, or silencing, mechanism. The binding of a silencer to a given enhancer leads to the inactivation of all enhancers within a complex promoter. The analysis of chromatin boundary elements suggest that silencers and enhancers might work through distinct mechanisms. We speculate that silencers constrain the evolution of complex promoters.