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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 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.