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The three-dimensional (3D) organization of the genome is highly dynamic, changing during development and varying across different tissues and cell types. Recent studies indicate that these changes alter regulatory interactions, leading to changes in gene expression. Despite its importance, the mechanisms that influence genomic organization remain poorly understood. We have previously identified a network of chromatin boundary elements, or insulators, in the Drosophila Antennapedia homeotic complex (ANT-C). These genomic elements interact with one another to tether chromatin loops that could block or promote enhancer-promoter interactions. To understand the function of these insulators, we assessed their interactions by measuring their 3D nuclear distance in developing animal tissues. Our data suggest that the ANT-C Hox complex might be in a folded or looped configuration rather than in a random or extended form. The architecture of the ANT-C complex, as read out by the pair-wise distance between insulators, undergoes a strong compression during late embryogenesis, coinciding with the reduction of cell and nuclear diameters due to continued cell divisions in post-cleavage cells. Our results suggest that genomic architecture and gene regulation may be influenced by cellular morphology and movement during development.

Cell density regulates many aspects of cell properties and behaviors including metabolism, growth, cytoskeletal structure and locomotion. Importantly, the responses by cultured cells to density signals also uncover key mechanisms that govern animal development and diseases in vivo. Here we characterized a density-responsive reporter system in transgenic Drosophila S2 cells. We show that the reporter genes are strongly induced in a cell density-dependent and reporter-independent fashion. The rapid and reversible induction occurs at the level of mRNA accumulation. We show that multiple DNA elements within the transgene sequences, including a metal response element from the metallothionein gene, contribute to the reporter induction. The reporter induction correlates with changes in multiple cell density and growth regulatory pathways including hypoxia, apoptosis, cell cycle and cytoskeletal organization. Potential applications of such a density-responsive reporter will be discussed.

Pairing of food aroma with selected taste can lead to enhanced food flavor and eating euphoria, but how cross-modal sensory combinations are integrated to increase food reward value remains largely unclear. Here we report that combined stimulation by food aroma and taste drastically increased appetite in well-nourished Drosophila larvae, and the appetizing effect involves a previously uncharacterized smell-taste integration process at axon terminals of two Gr43a gustatory neurons. Molecular genetic analyses of the smell-taste integration reveal a G protein-mediated tuning mechanism in two central neuropeptide F (NPF) neurons. This mechanism converts selected odor stimuli to NPF-encoded appetizing signals that potentiate Gr43a neuronal response to otherwise non-stimulating glucose or oleic acid. Further, NPF-potentiated responses to glucose and oleic acid require a Gr43a-independent and Gr43a-dependent pathway, respectively. Our finding of adaptive expansion of taste neuron response profiles by congruent aroma reveals a previously uncharacterized layer of neural complexity in food flavor perception.

The three-dimensional organization of the eukaryotic genome is important for its structure and function. Recent studies indicate that hierarchies of chromatin loops underlie important aspects of both genomic organization and gene regulation. Looping between insulator or boundary elements interferes with enhancer-promoter communications and limits the spread active or repressive organized chromatin. We have used the SF1 insulator in the Drosophila Antennapedia homeotic gene complex (ANT-C) as a model to study the mechanism and regulation of chromatin looping events. We reported previously that SF1 tethers a transient chromatin loop in the early embryo that insulates the Hox gene Sex comb reduce from the neighbor non-Hox gene fushi tarazu for their independent regulation. To further probe the functional range and connectivity of SF1, we used high-resolution chromosomal conformation capture (3C) to search for SF1 looping partners across ANT-C. We report here the identification of three distal SF1 Tether Elements (STEs) located in the labial, Deformed and Antennapedia Hox gene regions, extending the range of SF1 looping network to the entire complex. These novel STEs are bound by four different combinations of insulator proteins and exhibit distinct behaviors in enhancer block, enhancer-bypass and boundary functions. Significantly, the six STEs we identified so far map to all but one of the major boundaries between repressive and active histone domains, underlining the functional relevance of these long-range chromatin loops in organizing the Hox complex. Importantly, SF1 selectively captured with only 5 STEs out of ~20 sites that display similar insulator binding profiles, indicating that presence of insulator proteins alone is not sufficient to determine looping events. These findings suggest that selective interaction among diverse STE insulators organize the Drosophila Hox genes in the 3D nuclear space.

Our previous studies have demonstrated a critical role of a VEGFR-like signaling pathway in hunger-driven overeating of sugar-rich food in Drosophila larvae. In the current study, we investigate whether the VEGFR signaling mechanism plays a similar role in the feeding behavior of vertebrates using male Sprague Dawley rats. Young rats were treated intracerebroventrically (i.c.v.) with a single dose (2 μg) of VEGFR2 Tyrosine Kinase Inhibitor V (VTKI-V), an N-cyclopropylnaphthamide compound that selectively inhibits the kinase activity of VEGFR2 at subnanomolar concentrations. We find that animals treated with VTKI-V showed markedly attenuated overconsumption of palatable food that is sweet and fatty. The subsequent meal pattern analysis reveals that is achieved by consumption of smaller, shorter meals. Furthermore, i.c.v. injection of VTKI-V decreased body weight gain in animals fed with CHOW or palatable food. These inhibitory effects of the drug were detectable within 24h and persisted for at least five days. Given that body weight was affected by the drug regardless of diet while food intake was selectively altered in palatable diet fed animals, these results raise the possibility that i.c.v. injection of VTKI-V may interfere the functions of two separate VEGFR-mediated mechanisms: one promotes overconsumption of palatable food, and the other mediates body weight gain.

The three-dimensional (3D) organization of the eukaryotic genome is critical for its proper function. Evidence suggests that extensive chromatin loops form the building blocks of the genomic architecture, separating genes and gene clusters into distinct functional domains. These loops are anchored in part by a special type of DNA elements called chromatin boundary elements (CBEs). CBEs were originally found to insulate neighboring genes by blocking influences of transcriptional enhancers or the spread of silent chromatin. However, recent results show that chromatin loops can also play a positive role in gene regulation by looping out intervening DNA and "delivering" remote enhancers to gene promoters. In addition, studies from human and model organisms indicate that the configuration of chromatin loops, many of which are tethered by CBEs, is dynamically regulated during cell differentiation. In particular, a recent work by Li et al has shown that the SF1 boundary, located in the Drosophila Hox cluster, regulates local genes by tethering different subsets of chromatin loops: One subset enclose a neighboring gene ftz, limiting its access by the surrounding Scr enhancers and restrict the spread of repressive histones during early embryogenesis; and the other loops subdivide the Scr regulatory region into independent domains of enhancer accessibility. The enhancer-blocking activity of these CBE elements varies greatly in strength and tissue distribution. Further, tandem pairing of SF1 and SF2 facilitate the bypass of distal enhancers in transgenic flies, providing a mechanism for endogenous enhancers to circumvent genomic interruptions resulting from chromosomal rearrangement. This study demonstrates how a network of chromatin boundaries, centrally organized by SF1, can remodel the 3D genome to facilitate gene regulation during development.

Chromatin boundaries regulate gene expression by modulating enhancer–promoter interactions and insulating transcriptional influences from organized chromatin. However, mechanistic distinctions between these two aspects of boundary function are not well understood. Here we show that SF1, a chromatin boundary located in the Drosophila Antennapedia complex (ANT-C), can insulate the transgenic miniwhite reporter from both enhancing and silencing effects of surrounding genome, a phenomenon known as chromosomal position effect or CPE. We found that the CPE-blocking activity associates with different SF1 sub-regions from a previously characterized insulator that blocks enhancers in transgenic embryos, and is independent of GAF-binding sites essential for the embryonic insulator activity. We further provide evidence that the CPE-blocking activity cannot be attributed to an enhancer-blocking activity in the developing eye. Our results suggest that SF1 contains multiple non-overlapping activities that block diverse transcriptional influences from embryonic or adult enhancers, and from positive and negative chromatin structure. Such diverse insulating capabilities are consistent with the proposed roles of SF1 to functionally separate fushi tarazu (ftz), a non-Hox gene, from the enhancers and the organized chromatin of the neighboring Hox genes.

Chromatin boundary elements (CBEs) are widely distributed in the genome and mediate formation of chromatin loops, but their roles in gene regulation remain poorly understood. The complex expression pattern of the Drosophila homeotic gene Sex combs reduced (Scr) is directed by an unusually long regulatory sequence harboring diverse cis elements and an intervening neighbor gene fushi tarazu (ftz). Here we report the presence of a multitude of CBEs in the Scr regulatory region. Selective and dynamic pairing among these CBEs mediates developmentally regulated chromatin loops. In particular, the SF1 boundary plays a central role in organizing two subsets of chromatin loops: one subset encloses ftz, limiting its access by the surrounding Scr enhancers and compartmentalizing distinct histone modifications, and the other subset subdivides the Scr regulatory sequences into independent enhancer access domains. We show that these CBEs exhibit diverse enhancer-blocking activities that vary in strength and tissue distribution. Tandem pairing of SF1 and SF2, two strong CBEs that flank the ftz domain, allows the distal enhancers to bypass their block in transgenic Drosophila, providing a mechanism for the endogenous Scr enhancer to circumvent the ftz domain. Our study demonstrates how an endogenous CBE network, centrally orchestrated by SF1, could remodel the genomic environment to facilitate gene regulation during development.

Chromatin boundaries or insulators modulate enhancer–promoter interactions in complex genetic loci. However, the mechanism underlying insulator activity is not known. Previous studies showed that the activity of the Drosophila suHw insulator is abolished by the tandem arrangement (pairing) of the insulator elements, suggesting that interactions between insulators or like elements may be involved in their enhancer-blocking mechanism. To test whether such phenomenon reflects a general property of chromatin insulators, we tested the effect of pairing on enhancer-blocking activity of 11 homologous and heterologous insulator combinations using suHw, scs, or SF1 insulators. We found that, unlike the homologous pairing of suHw, the heterologous combinations of suHw with other insulators do not reduce their enhancer-blocking activity. Rather, paired insulators exhibit a higher level of enhancer-blocking activity than either single insulator alone, suggesting that they can function independently or additively. Furthermore, the analyses of two additional chromatin boundaries, scs and SF1, in homologous or heterologous pairing with other boundary elements, also showed no reduction but rather enhancement of insulator activity. We propose that diverse mechanisms may underlie insulator activity, and selective interactions among insulators could influence their function as well as the formations of independent chromatin domains.

The conserved neuropeptide Y (NPY) signaling pathway has been strongly implicated in the stimulation of food uptake in vertebrates as well as in the regulation of food conditioned foraging behaviors of Caenorhabditis elegans. Using in situ RNA hybridization and immunocytochemistry, we report the neuronal network of Drosophila neuropeptide F (dNPF), a human NPY homologue, in the larval central nervous system and its food-dependent modifications. We provide indications that gustatory stimulation by sugar, but not its ingestion or metabolism, is sufficient to trigger long-term, dose-dependent alterations of the dNPF neuronal circuit through both dnpf activation and increased synaptic transmission. Our results strongly suggest that the dNPF neuronal circuit is an integral part of the sensory system that mediates food signaling, providing the neural basis for understanding how invertebrate NPY regulates food response. © 2001 John Wiley & Sons, Inc. J Neurobiol 47: 16–25, 2001

A neuropeptide F (NPF) was isolated from the fruit fly, Drosophila mellanogaster, based on a radioimmunoassay for a gut peptide from the corn earworm, Helicoverpa zea. A partial sequence was obtained from the fly peptide, and a genomic sequence coding for NPF was cloned after inverse polymerase chain reaction and shown to exist as a single genomic copy. The encoded, putative prepropeptide can be processed into an amidated NPF with 36 residues that is related to invertebrate NPF's and the neuropeptide Y family of vertebrates. In situ hybridization and immunocytochemistry showed that Drosophila NPF was expressed in the brain and midgut of fly larvae and adults.

Chromatin boundaries facilitate independent gene regulation by insulating genes from the effects of enhancers or organized chromatin. However, the mechanisms of boundary action are not well understood. To investigate whether boundary function depends on a higher order of chromatin organization, we examined the function of several Drosophila melanogaster insulators in cells with reduced chromatin-remodeling activities. We found that knockdown of NURF301 and ISWI, key components of the nucleosome-remodeling factor (NURF), synergistically disrupted the enhancer-blocking function of Fab7 and SF1 and augmented the function of Fab8. Mutations in Nurf301/Ebx and Iswi also affected the function of these boundaries in vivo. We further show that ISWI was localized on the endogenous Fab7 and Fab8 insulators and that NURF knockdown resulted in a marked increase in the nucleosome occupancy at these insulator sites. In contrast to the effect of NURF knockdown, reduction in dMi-2, the ATPase component of the Drosophila nucleosome-remodeling and deacetylation (NuRD) complex, augmented Fab7 and suppressed Fab8. Our results provide the first evidence that higher-order chromatin organization influences the enhancer-blocking activity of chromatin boundaries. In particular, the NURF and NuRD nucleosome-remodeling complexes may regulate Hox expression by modulating the function of boundaries in these complexes. The unique responses by different classes of boundaries to changes in the chromatin environment may be indicative of their distinct mechanisms of action, which may influence their placement in the genome and selection during evolution.

Background Chromatin boundaries, also known as insulators, regulate gene activity by organizing active and repressive chromatin domains and modulate enhancer-promoter interactions. However, the mechanisms of boundary action are poorly understood, in part due to our limited knowledge about insulator proteins, and a shortage of standard assays by which diverse boundaries could be compared. Results We report here the development of an enhancer-blocking assay for studying insulator activity in Drosophila cultured cells. We show that the activities of diverse Drosophila insulators including suHw, SF1, SF1b, Fab7 and Fab8 are supported in these cells. We further show that double stranded RNA (dsRNA)-mediated knockdown of SuHw and dCTCF factors disrupts the enhancer-blocking function of suHw and Fab8, respectively, thereby establishing the effectiveness of using RNA interference in our cell-based assay for probing insulator function. Conclusion The novel boundary assay provides a quantitative and efficient method for analyzing insulator mechanism and can be further exploited in genome-wide RNAi screens for insulator components. It provides a useful tool that complements the transgenic and genetic approaches for studying this important class of regulatory elements.

In recent years, a novel class of DNA elements called chromatin boundaries or insulators has drawn special attention in the field of gene regulation and chromatin organization. Chromatin boundaries were first identified in Drosophila as genomic regions that separate neighboring chromatin domains of distinctive structure and function. Boundary elements are experimentally defined by two activities: they block the regulatory effects of enhancers from promoters when positioned interveningly, and when placed on both sides of integrated transgenes, they insulate these transgenes from the transcriptional influences of the surrounding genome. Elements with similar properties have been reported in a variety of species from yeast to humans. Molecular genetic and cell biological studies suggest that boundary activity may represent a general mechanism of gene regulation, genome organization, as well as chromatin and nuclear organization. However, the biological functions and the protein components of many boundary elements remain to be characterized. More importantly, the mechanism through which boundaries affect various forms of transcription regulation is still poorly understood.

Chromatin-related functions are associated with spatial organization in the nucleus. We have investigated the relationship between the enhancer-blocking activity and subnuclear localization of the Drosophila melanogaster suHw insulator. Using fluorescent in situ hybridization, we observed that genomic loci containing the gypsy retrotransposon were distributed closer to the nuclear periphery than regions without the gypsy retrotransposon. However, transgenes containing a functional 340 bp suHw insulator did not exhibit such biased distribution towards the nuclear periphery, which suggests that the suHw insulator sequence is not responsible for the peripheral localization of the gypsy retrotransposon. Antibody stains showed that the two proteins essential for the suHw insulator activity, SUHW and MOD(MDG4), are not restricted to the nuclear periphery. The enhancer-blocking activity of suHw remained intact under the heat shock conditions, which was shown to disrupt the association of gypsy, SUHW and MOD(MDG4) with the nuclear periphery. Our results indicate that the suHw insulator can function in the nuclear interior, possibly through local interactions with chromatin components or other nuclear structures.

The intrinsic enhancer–promoter specificity and chromatin boundary/insulator function are two general mechanisms that govern enhancer trafficking in complex genetic loci. They have been shown to contribute to gene regulation in the homeotic gene complexes from fly to mouse. The regulatory region of the Scr gene in the Drosophila Antennapedia complex is interrupted by the neighboring ftz transcription unit, yet both genes are specifically activated by their respective enhancers from such juxtaposed positions. We identified a novel insulator, SF1, in the Scr–ftz intergenic region that restricts promoter selection by the ftz-distal enhancer in transgenic embryos. The enhancer-blocking activity of the full-length SF1, observed in both embryo and adult, is orientation- and enhancer-independent. The core region of the insulator, which contains a cluster of GAGA sites essential for its activity, is highly conserved among other Drosophila species. SF1 may be a member of a conserved family of chromatin boundaries/insulators in the HOM/Hox complexes and may facilitate the independent regulation of the neighboring Scr and ftz genes, by insulating the evolutionarily mobile ftz transcription unit.

Chromatin insulators regulate gene expression by preventing inappropriate enhancer-promoter interactions. Our previous study showed that insulators do not merely function as rigid blockers, rather their activities are quantitative and selective. We have investigated the factors and mechanisms that determine the effectiveness of the suHw insulator in transgenic Drosophila. We show that the suHw-mediated blockage of the AE1 enhancer from a downstream promoter depends on the ability of the promoter to compete for AE1. Promoters that are highly competitive for the enhancer are blocked less effectively. Moreover, blockage of AE1 from its cognate ftz promoter can range from virtually complete to non-detectable, depending on the property of the neighboring upstream promoter. A highly competitive neighboring promoter enhances the suHw-mediated blockage, whereas a less competitive promoter reduces the insulator effectiveness. The influence on insulator effectiveness by both the interacting and the neighboring competing promoters correlates with their ability to compete for the enhancer, which was previously shown to depend on core promoter sequences. Our findings suggest a mechanism at the level of gene organization that modulates insulator effectiveness through promoter competition. The dependence of insulator function on its cis contexts may provide it with more regulatory flexibility while imposing organizational restraints on eukaryotic gene complexes.

Chromatin boundary elements or insulators are believed to regulate gene activity in complex genetic loci by organizing specialized chromatin structures . Here, we report that the enhancer-blocking activity of the Drosophila suHw insulator is sensitive to insulator copy number and position. Two tandem copies of suHw were ineffective in blocking various enhancers from a downstream promoter . Moreover, an enhancer was blocked more effectively from a promoter by two flanking suHw insulators than by a single intervening one. Thus, insulators may modulate enhancer-promoter interactions by interacting with each other and facilitating the formation of chromatin loop domains.

There are numerous examples of shared enhancers interacting with just a subset of target promoters. In some cases, specific enhancer-promoter interactions depend on promoter competition, whereby the activation of a preferred target promoter precludes expression of linked genes. Here, we employ a transgenic embryo assay to obtain evidence that promoter selection is influenced by the TATA element. Both the AE1 enhancer from the Drosophila Antennapedia gene complex (ANT-C) and the IAB5 enhancer from the Bithorax complex (BX-C) preferentially activate TATA-containing promoters when challenged with linked TATA-less promoters. In contrast, the rho neuroectoderm enhancer (NEE) does not discriminate between these two classes of promoters. Thus, certain upstream activators, such as Ftz, prefer TATA-containing promoters, whereas other activators, including Dorsal, work equally well on both classes of promoters. These results provide in vivo evidence that different core promoters possess distinct regulatory activities. We discuss the possibility that an invariant TFIID complex can adopt different conformations on the core promoter.

The Drosophila gypsy retrotransposon disrupts gene activity by blocking the interactions of distal enhancers with target promoters. This enhancer-blocking activity is mediated by a 340 bp insulator DNA within gypsy. The insulator contains a cluster of binding sites for a zinc finger protein, suppressor of Hairy wing [su(Hw)]. Recent studies have shown that a second protein, mod(mdg4), is also important for normal insulator function. Mutations in mod(mdg4) exert paradoxical effects on different gypsy-induced phenotypes. For example, it enhances yellow2 but suppresses cut6. Here, we employ a stripe expression assay in transgenic embryos to investigate the role of mod(mdg4) in gypsy insulator activity. The insulator was inserted between defined enhancers and placed among divergently transcribed reporter genes (white and lacZ) containing distinct core promoter sequences. These assays indicate that mod(mdg4) is essential for the enhancer-blocking activity of the insulator DNA. Moreover, reductions in mod(mdg4)+ activity cause the insulator to function as a promoter-specific silencer that selectively represses white, but not lacZ. The repression of white does not affect the expression of the closely linked lacZ gene, suggesting that the insulator does not propagate changes in chromatin structure. These results provide an explanation for why mod(mdg4) exerts differential effects on different gypsy-induced mutations.