Hormone receptor-like in 46


Transcriptional regulation

The steroid hormone ecdysone induces a precise sequence of gene activity in Drosophila melanogaster salivary glands in late third larval instar larvae. The acquisition of competence for this response does not result from a single event or pathway but requires factors that accumulate throughout the instar. Individual transcripts become competent to respond at different times and their expression is differentially affected in ecd1, dor22 and BR-C mutants. ecd1 mutants are deficient in ecdysone. dor encodes a protein with a zinc-finger like motif. The induction of early-late transcripts, originally assumed to necessarily follow early transcripts, is partially independent of early transcript activation. Attempts to inhibit the synthesis of regulatory proteins reveal transcript-specific superinduction effects. Furthermore these inhibitors lead to the induction of betaFTZ-F1 and E93 transcripts at levels normally found in prepupal glands. These studies reveal the complexity of the processes underlying the establishment of a hormonal response (Richards, 1999).

In 90-hour salivary glands, all intermolt transcripts, especially EcR and E74B are higher in the presence of cycloheximide. In 100-h glands the effect is less markded although expression ratios were between 2.5 and 3.5 for both cycloheximide and anisomycin. Unexpectedly, E75C transcripts, normally difficult to detect, are dramatically increased in the presence of both inhibitors at 100-h. There is no effect of cyclohexaminde for the early transcript E74A in 90-h glands, while E75A transcripts in the same glands show a 5-fold superinduction. At 100 h, both E74A and E75A ratios are close to 1, as would be expected for an induction that is independent of protein synthesis. In contrast, the inhibitors cause a striking superinduction of E75B transcripts both at 90 and 100 h. DHR3 also undergoes superinduction. A number of mechansims have been suggested to explain the superinduction phenomenon. These include: (1) the existence of a labile repressor, whose rapid turnover is sensitive to inhibitors; (2) the protection of transcripts from degradation either by physical association with inhibitors or by the inhibition of labile mRNases; (3) the inhibition of early protein synthesis to prevent the negative feed-back loop postulated to repress early transcript synthesis and (4) the inhibitor acting as an inducer at concentrations below those necessary to block protein synthesis. Since effects are transcript specific, different combinations of mechanisms may function at each locus (Richards, 1999).

Forward and feedback regulation of cyclic steroid production in Drosophila melanogaster

In most animals, steroid hormones are crucial regulators of physiology and developmental life transitions. Steroid synthesis depends on extrinsic parameters and autoregulatory processes to fine-tune the dynamics of hormone production. In Drosophila, transient increases of the steroid prohormone ecdysone, produced at each larval stage, are necessary to trigger moulting and metamorphosis. Binding of the active ecdysone (20-hydroxyecdysone) to its receptor (EcR) is followed by the sequential expression of the nuclear receptors E75, DHR3 and βFtz-f1, representing a model for steroid hormone signalling. This study has combined genetic and imaging approaches to investigate the precise role of this signalling cascade within the prothoracic gland (PG), where ecdysone synthesis takes place. These receptors operate through an apparent unconventional hierarchy in the PG to control ecdysone biosynthesis. At metamorphosis onset, DHR3 emerges as the downstream component that represses steroidogenic enzymes and requires an early effect of EcR for this repression. To avoid premature repression of steroidogenesis, E75 counteracts DHR3 activity, whereas EcR and βFtz-f1 act early in development through a forward process to moderate DHR3 levels. These findings suggest that within the steroidogenic tissue, a given 20-hydroxyecdysone peak induces autoregulatory processes to sharpen ecdysone production and to confer competence for ecdysteroid biosynthesis at the next developmental phase, providing novel insights into steroid hormone kinetics (Parvy, 2014).

During the past few years, the Drosophila steroidogenic tissue has been extensively used to investigate how extrinsic and intrinsic parameters integrate to coordinate growth with developmental progression. This study shows that EcR, E75, DHR3 and βFtz-f1, which mediate ecdysone signalling, are expressed in the steroidogenic cells and are required for ecdysone synthesis during Drosophila larval development. These findings are consistent with previous studies showing that E75A mutation and βFtz-f1 disruption in the PG induce developmental arrest as a consequence of steroid deficiency. During the past few years, the Drosophila steroidogenic tissue has been extensively used to investigate how extrinsic and intrinsic parameters integrate to coordinate growth with developmental progression. This study shows that EcR, E75, DHR3 and βFtz-f1, which mediate ecdysone signalling, are expressed in the steroidogenic cells and are required for ecdysone synthesis during Drosophila larval development. These findings are consistent with previous studies showing that E75A mutation and βFtz-f1 disruption in the PG induce developmental arrest as a consequence of steroid deficiency signal, while other cells do not respond to this signal. In the case of 20E responsiveness, the gap for acquisition of competence is associated with low ecdysone titres and high βFtz-f1 levels. These findings support the notion that βFtz-f1 also acts as a competent factor for ecdysone biogenesis through a step-forward moderation of DHR3 expression. In addition, as both EcR and DHR3 knockdown also act through an early induced event and affect βFtz-f1 expression, it is conceivable that they participate in the competence acquisition through βFtz-f1. Although the molecular mechanisms that link βFtz-f1 and EcR to DHR3 must still be elucidated, this study reveals that the response following the L2 ecdysone peak is necessary to confer competence for ecdysone biogenesis at the late L3 stage by delaying the DHR3-mediated repression of steroidogenic enzymes (Parvy, 2014).

In summary, this study unravels an autoregulatory mechanism in cyclic ecdysone production. This autoregulation is likely to be coordinated with the processes that adjust ecdysone biogenesis at the L3 stage in response to environmental cues. These include nutrition, insulin signalling and the circadian rhythm that integrates through the prothoracicotropic hormone (PTTH). Interestingly, a downstream effector of PTTH is the NR DHR4, also shown to modulate the ecdysone response at the onset of metamorphosis. However, the fact that DHR4 mutants are not arrested earlier than the prepupal stage, while each RNAi tested in this study provokes a significant arrest at larval stages, suggests that DHR4 acts through an independent mechanism. Moreover, as intermediates of the nutrient, insulin and PTTH signalling interact with NRs that respond to 20E, this study provides a framework to further investigate how environmental parameters integrate with the autoregulation of cyclic ecdysone production (Parvy, 2014).

Targets of Activity

In an initial effort to understand how two orphan receptors, Hr46 and DHR39, might function during development, their DNA binding properties were examined and compared with the known Drosophila nuclear receptor superfamily members that are involved in the ecdysteroid response: Ecdysone receptor, Ultraspiracle, E75A, E78A, and beta FTZ-F1. Upon testing all pairwise combinations of these seven proteins on a panel of seven oligonucleotides, only EcR and Usp bind DNA as a heterodimer, indicating that this interaction is highly specific. With the exception of E78A, which does not bind any sequence tested, each of the remaining proteins, including Hr46 and DHR39, is able to bind to a single consensus AGGTCA half-site; however, each displays different specificities depending on the flanking nucleotide sequence. These observations suggest that the 20E-regulated orphan receptors function as monomers to control the expression of their target genes (Horner, 1995).

The Hr46/DHR3 orphan receptor gene is induced directly by the steroid hormone ecdysone at the onset of Drosophila metamorphosis. Hr46 expression peaks in early prepupae, as the early puff genes are repressed and betaFTZ-F1 is induced. Hr46 directly contributes to both of these regulatory responses. Hr46 protein binds to many ecdysone-induced puffs in the polytene chromosomes, including the early puffs that encode the BR-C and E74 regulatory proteins, as well as the E75, E78 and betaFTZ-F1 orphan receptor loci. Hr46 represses E74A, and to a lesser extent E74B, and it also represses BR-C, E75A, and E78B. Hr46 activates betaFTZ-F1. Three Hr46 binding sites are present downstream from the start site of betaFTZ-F1 transcription, further indicating that this gene is a direct target of Hr46 regulation. Ectopic expression of Hr46 reveals that the polytene chromosome binding pattern is of functional significance. Hr46 is sufficient to repress BR-C, E74A, E75A and E78B transcription as well as induce betaFTZ-F1. Hr46 thus appears to function as a switch that defines the larval-prepupal transition by arresting the early regulatory response to ecdysone at puparium formation and facilitating the induction of the betaFTZ-F1 competence factor in mid-prepupae. This study also provides evidence for direct cross-regulation among orphan members of the nuclear receptor superfamily and further implicates these genes as critical transducers of the hormonal signal during the onset of Drosophila metamorphosis (Lam, 1997).

Hr46 induces ßFTZF1, an orphan nuclear receptor that is essential for the appropriate response to the subsequent prepupal pulse of ecdysone. The DNA binding domain of HR46, and perhaps sequences NH2-terminal to it, are necessary for the activating function of Hr46. Hr46 has two high-affinity binding sites approximately 300 base pairs apart, that lie downstream of the transcription start site of ßFTZF1. Hr46 appears to bind as a monomer to these sites, since sequencing and footprinting analysis have uncovered single consensus Hr46 sites at each of these DNA sites (White, 1997).

Various ecdysteroid responsive genes play important roles in insect moulting and metamorphosis. Late FTZ-F1, a member of the nuclear receptor superfamily, is a unique transcription factor that is induced by a pulse exposure of 20-hydroxyecdysone. Elucidation of the regulation mechanism of this gene during the prepupal period will further understanding of metamorphosis at a molecular level. Using transgenic fly lines carrying various transcription regulatory regions of the FTZ-F1 gene fused to the LacZ gene, cis-regulatory elements in the late FTZ-F1 transcription unit were investigated. The region that governs the stage-specific expression during prepupal period was narrowed down to 1.2kb, from -0.7 to +0.5kb, relative to the transcription start site. Electrophoresis mobility shift assays using staged extracts and various probes within the stage-specific region allowed the identification of binding sites for DHR3, an early late gene product, around 170 and 450bp downstream of the transcription initiation site. Mutations disrupting these binding sites reduce the reporter gene expression without affecting the stage specificity. These deletion and mutation studies of the cis-regulatory element of the FTZ-F1 gene suggest that the DHR3 binding sites located in the 5' non-coding region are involved in the prepupal expression of the gene. These DHR3 binding sites confer high level expression while other elements are also involved in stage-specific expression (Kageyama, 1997).

Protein Interactions

In the early stages of Drosophila metamorphosis Hr46 represses the ecdysone induction of early genes turned on by the pulse of ecdysone that triggers metamorphosis. Hr46 is shown to interact directly with the Ecdysone receptor. The mechanism of Hr46 repression may involve an interaction between the Hr46 and Ecdysone receptor ligand binding domains (White, 1997).

The E75B receptor, which lacks a complete DNA binding domain, inhibits the inductive function of Hr46 on ßFTZF1 by forming a complex with DHR3 on the ßFTZF1 promoter, thereby providing a timing mechanism for ßFTZF1 induction that is dependent on the disappearance of E75B. Thus the repressive function of Hr46 does not involve binding to DNA but instead involves physical interaction with the Ecdysone receptor. E75B fails to bind DNA in the absence of HR46. Thus E75B acts like a co-repressor with HR46, rather than as a competitor with Hr46 for DNA binding; the restricted temporal expression of E75B apparently acts as a precise timer for the onset of ßFTZF1 expression (White, 1997).

Bonus interacts with hormone receptors and inhibits transcription

The Drosophila bonus (bon) gene encodes a homolog of the vertebrate TIF1 transcriptional cofactors. bon is required for male viability, molting, and numerous events in metamorphosis including leg elongation, bristle development, and pigmentation. Most of these processes are associated with genes that have been implicated in the ecdysone pathway, a nuclear hormone receptor pathway required throughout Drosophila development. Bon is associated with sites on the polytene chromosomes and can interact with numerous Drosophila nuclear receptor proteins. Bon binds via an LxxLL motif to the activator function AF-2 domain present in the ligand binding domain of betaFTZ-F1 and behaves as a transcriptional inhibitor in vivo (Beckstead, 2001).

Database searches have revealed that bon encodes the only Drosophila homolog of mammalian TIF1s. Bon exhibits 29% identity with mouse TIF1alpha and mouse TIF1beta, and 26% identity with human TIF1gamma. The overall identity between Bon and TIF1s is similar to the identity observed between the TIF1 members. A higher degree of identity is seen in the N- and C-terminal regions spanning the conserved domains. At the N terminus, a C3HC4 zinc-finger motif or RING finger is followed by two cysteine-rich zinc binding regions (B-boxes) and a coiled coil domain forming a tripartite motif designated RBCC. At the C terminus, a bromodomain is preceded by a C4HC3 zinc-finger motif or PHD finger (Beckstead, 2001 and references therein).

Northern analysis demonstrates that bon produces one predominant 6 kb transcript and two 4 kb transcripts, which each encode a protein of ~140 kDa. The two 4 kb transcripts are only present in 0-3 hr embryos and adult females. It is therefore possible that the 4kb mRNAs are maternal components. bon is expressed throughout embryogenesis and in first instars. Its levels increase in 9-12 hr embryos and are low during the second instar stage. bon is upregulated in late third instar larvae. The upregulation of bon during midembryogenesis and prior to pupariation correlates well with known high titer pulses of ecdysone (Beckstead, 2001).

Immunohistochemical staining of numerous tissues show that Bon is a nuclear protein expressed in most and possibly all cells during embryogenesis, in fat body, imaginal discs, salivary glands, brain, gut, Malpighian tubules, and trachea. Bon is a chromatin-associated protein that localizes to ~10%-15% of the polytene chromosome bands. This pattern is highly reproducible (Beckstead, 2001).

To determine whether the defects seen in bon mutants are due to disruptions in the ecdysone-regulated pathway, the expression of several ecdysone-regulated genes were examined in y w and bon241/bon241 larvae, prepupae, and pupae. In bon241/bon241 animals, levels of betaFTZ-F1, EcR-A, EcR-B, E74A, E74B, and BR-C are reduced. It appears that each gene is upregulated in response to the ecdysone pulse, but is unable to maintain expression in the bon mutants. However, DHR3 transcripts are prematurely expressed and the overall level of expression is elevated in bon241/bon241 animals when compared to y w control animals. In addition, the EcR-A transcript levels appear slightly reduced in bon241/bon241 animals, while the EcR-B transcript levels are severely reduced when compared to controls. Similar observations were made for all of the above genes in bon21B/bon487 animals, except that DHR3 transcript levels are also reduced. Based on these effects on gene expression, defects in larval molting and metamorphosis, and the temporal expression pattern of Bon, it is proposed that Bon plays an important role in the regulation of genes in the ecdysone response pathway (Beckstead, 2001).

To test whether Bon interacts with betaFTZ-F1 as well as other Drosophila nuclear receptors in vitro, binding assays were performed using purified recombinant proteins. Glutathione-S transferase (GST)-fused betaFTZ-F1, alphaFTZ-F1 (amino acids 154-1029), Seven-up (SVP), DHR3, USP, and EcR were immobilized on glutathione-Sepharose and incubated with purified N-terminally His-tagged Bon (His-Bon). His-Bon binds to GST-betaFTZ-F1, GST-alphaFTZ-F1, GST-DHR3, GST-SVP, GST-USP, and GST-EcR, but not to GST alone. Thus, Bon can bind directly to many members of the nuclear receptor family in vitro (Beckstead, 2001).

To determine whether Bon is able to repress transcription, the coding sequence of Bon was fused to the yeast GAL4 DNA binding domain. The resulting fusion protein was tested for its ability to repress transcription activated by ER(C)-VP16, a chimeric activator containing the DBD of ERalpha fused to VP16. GAL4-Bon and ER(C)-VP16 were transiently transfected into S2 cells with a reporter containing a GAL4 binding site (17M) and an estrogen response element (ERE) in front of a thymidine kinase (tk) promoter-CAT fusion (17M-ERE-tk-CAT). GAL4-Bon efficiently represses transcription in a dose-dependent manner. In contrast, coexpression of Bon without the GAL4 DNA binding domain causes a reproducible increase in CAT activity, indicating that repression by Bon is entirely dependent on DNA binding (Beckstead, 2001).

To map the domain of Bon responsible for transcriptional repression, a set of N- and C-terminally truncated derivatives were assayed for their ability to repress VP16-activated transcription in S2 cells. In the absence of the RBCC motif, the GAL4-Bon fusion protein, GAL4-Bon [471-1133]) fails to repress transcription, indicating that the N-terminal region of Bon is required for repression. However, this region is not sufficient for full repression. Consistent with this, a C-terminal truncation, GAL4-Bon (1-890), is a less potent repressor, indicating that the C-terminal residues of the protein including the PHD finger and the bromodomain also contribute to the repression potential of Bon. However, this domain on its own exhibits little repression. A 3- to 4-fold increase in CAT activity is observed with the central region between the coiled-coil and the PHD finger, suggesting that Bon may also contain a 'masked' activation domain. Note, however, that no significant activation was observed with GAL4-Bon (471-890) tested in the absence of ER(C)-VP16. Taken together, these results indicate that most of the repression activity of Bon resides within the N-terminal RBCC domain (Beckstead, 2001).

Bon and TIF1s contain an N-terminal RBCC (RING finger/B boxes/coiled coil) motif. In the absence of the RBCC motif, the GAL4-Bon protein, unlike the full-length protein, fails to repress transcription. The TIF1beta RBCC domain has been shown to be necessary for the oligomerization of TIF1beta and KRAB binding. Because Bon is able to homodimerize, this domain may be involved in formation of protein complexes (Beckstead, 2001).

The PHD finger and bromodomain are characteristic features of nuclear proteins known to be associated with chromatin and/or to function at the chromatin level. For instance, the chromosomal proteins Trithorax and Polycomb-like contain multiple PHD fingers, while the histone acetyltransferases CBP and GCN5 as well as the chromatin-remodeling factor SWI2/SNF2 are also bromodomain containing proteins. Bromodomains have been shown to bind to acetyl-lysine and specifically interact with the amino-terminal tails of histones H3 and H4, suggesting a chromatin-targeting function for this highly evolutionarily conserved domain. Because Bon is localized to hundreds of chromatin bands on Drosophila polytene chromosomes, it is probably involved in chromatin-mediated regulation of transcription of numerous genes (Beckstead, 2001).

Bon can repress both basal and activated transcription when recruited to the promoter region of a target gene, similar to TIF1alpha, -beta, and -gamma. For TIF1alpha and TIF1beta, a link between silencing and histone modification has been established, and TIF1beta is part of a large multiprotein complex that possesses histone deacetylase activity. Moreover, TIF1beta was also reported to colocalize and interact directly with members of the heterochromatin protein 1 (HP1) family. Similar to TIF1beta, TIF1alpha can bind the HP1 proteins in vitro. However, TIF1alpha-mediated repression in transfected cells does not require the integrity of the HP1 interaction domain, nor is there any significant subnuclear colocalization of HP1alpha and TIF1alpha. No interactions were observed between Bon and HP1 in a yeast two-hybrid assay, nor was any evidence found for genetic interactions. However, in a yeast two-hybrid screen, Bon interacted with members of the Polycomb group, suggesting that Bon may also be part of heterochromatin-like complexes and/or may require some of the members of the Polycomb group genes to repress transcription. This would imply that Bon has a dual role, similar to some members of the Polycomb group family: transcriptional repression and heterochromatin formation. Both of these roles may be required in transcriptional repression (Beckstead, 2001).

Upon ecdysone binding, the EcR/USP complex upregulates the expression of a group of transcription factors, many of which are nuclear receptors. During this ecdysone regulatory cascade, both induction and repression of transcription are required to regulate the timing and the response to the ecdysone signal. Bon is able to interact with many members of the nuclear receptor family, suggesting it may have a role in multiple steps during metamorphosis and affect expression of many ecdysone regulated genes. For example, DHR3, a key component of the ecdysone response, is required for patterning and integrity of the adult cuticle, and DHR3 mutant clones exhibit a loss of pigmentation, cuticle defects, and missing bristles, similar to a partial loss of Bon. In addition, mutations in betaFTZ-F1, E74B, and BR-C exhibit malformed legs, which are a result of failure in the ecdysone response pathway. Again, very similar defects are observed in bon mutants. Salivary glands in betaFTZ-F1, BR-C, and bon mutant pupae also fail to undergo apoptosis. The ability of bon mutations to cause phenotypes that resemble defects associated with mutations with multiple members of the pathway suggests that Bon is interacting with several members of the pathway at several stages, in agreement with the biochemical observations (Beckstead, 2001).

The interaction of Bon with nuclear receptors is similar to TIF1alpha but unlike TIF1beta and TIF1gamma. This interaction requires the integrity of the nuclear receptor AF-2 activation domain and is mediated by the Bon/TIF1alpha LxxLL motif. These observations suggest that Drosophila nuclear receptors and Bon have co-evolved to maintain their interaction. It is therefore likely that the biological role of this interaction has been conserved in mammals (Beckstead, 2001).

The Drosophila nuclear receptor E75 contains heme and is gas responsive: E75-DHR3 interaction is regulated by binding of NO of CO to the heme center of E75

Nuclear receptors are a family of transcription factors with structurally conserved ligand binding domains that regulate their activity. Despite intensive efforts to identify ligands, most nuclear receptors are still 'orphans'. The ligand binding pocket of the Drosophila nuclear receptor E75 is shown to contain a heme prosthetic group. E75 absorption spectra, resistance to denaturants, and effects of site-directed mutagenesis indicate a single, coordinately bound heme molecule. A correlation between the levels of E75 expression and the levels of available heme suggest a possible role as a heme sensor. The oxidation state of the heme iron also determines whether E75 can interact with its heterodimer partner DHR3, suggesting an additional role as a redox sensor. Further, the E75-DHR3 interaction is also regulated by the binding of NO or CO to the heme center, suggesting that E75 may also function as a diatomic gas sensor. Possible mechanisms and roles for these interactions are discussed (Reinking, 2005).

Using electronic absorption and mass spectrometry, it has been shown that the Drosophila nuclear receptor E75 contains a single tightly associated heme prosthetic group. Thus, nuclear receptor LBDs can now be added to the limited repertoire of known heme binding motifs. All results here are consistent with a conventional heme-protein interaction mediated by a pair of coordinate bonds. Two highly conserved histidine residues are good candidates for the interacting residues (Reinking, 2005).

The results suggest three general ways in which E75-heme may function. (1) The necessity of heme for E75 LBD stability and the changes in expression levels brought about by supplemental heme suggest that E75 may function as a nuclear monitor of cellular heme levels. (2) The ability of E75-heme to switch between oxidized and reduced states and the effects of these states on CO, NO, and cofactor peptide binding suggest a possible role as a cellular redox sensor. (3) The ability of E75-heme to bind NO and CO, and for these gases to modulate cofactor binding and transcriptional activity, suggests a role in mediating NO and/or CO intercellular signaling. This is the first example of a nuclear receptor with a bipartite ligand binding system (Reinking, 2005).

Nuclear receptors were first characterized based on their ability to bind steroid hormones but are now known to bind a fairly diverse set of lipophilic molecules including fatty acids, phospholipids, retinoids, bile acids, farnesoids, and a range of xenobiotics. Like most other nuclear receptor ligands, heme is also a lipophilic molecule with polarized negative charges. Its molecular weight and solvent-excluded volume, although larger than most, are within the range of other known ligands. For example, one of the closest vertebrate homologs to E75, Peroxisome proliferator activator receptor gamma (PPARgamma), has a pocket that can easily accommodate a molecule of this size. Although heme is not dissimilar to other ligands in most physical attributes, an iron atom within a protoporphyrin ring brings an exciting new dimension to the potential molecular and physiological roles of nuclear receptor proteins (Reinking, 2005).

The tight interaction generally formed between heme molecules and their protein partners, also observed with E75, and the inability to detect apo-E75 suggest that E75 and heme are unlikely to associate and dissociate readily. For this to occur, dedicated cofactors would be required to shuttle the heme in and out of the LBD and to stabilize the LBD in the absence of heme. Although no evidence was found for such cofactors, it is possible that endogenous E75 is expressed in certain tissues or stages in which such cofactors exist. If, however, these cofactors are not present, or do not exist, heme would be required as a dedicated structural component. In the latter case, the levels of free heme in the cell would determine the levels of E75 that can accumulate. Either way, heme availability would determine the levels of active E75 in the cell. If E75 target genes include regulators of heme metabolism, these properties would make E75 an ideal regulator of heme homeostasis (Reinking, 2005).

Heme is required by proteins that control an enormous range of cellular and biological processes. Examples of these processes (and proteins) include energy transfer (mitochondrial cytochromes), lipid and drug metabolism (cytochrome P450s), heme metabolism (heme oxygenase [HO]), oxygen radical detection and removal (superoxide dismutases, catalase, NADPH hydroxylase), gas transport (hemoglobin, myoglobin), iron transport (HO-FE-ATP pump), neuronal differentiation, and behavior (guanylate cyclase, nitric-oxide synthase [NOS]) and circadian rhythm (HO, NPAS2). Interestingly, NOS and HO, the enzymes that produce NO and CO in the cell, are also heme-containing proteins (Reinking, 2005 and references therein).

Of the physiological processes listed above, particularly intriguing and relevant ones are lipid and xenobiotic metabolism. In vertebrates, most of the enzymes that regulate these reactions are cytochrome P450s, which typically use heme to transfer oxygen or hydroxyl groups to or from their substrates. Homeostasis between lipid absorption/production and secretion/breakdown is controlled by nuclear receptors, largely through transcriptional regulation of cytochrome P450 (cyp) genes. In turn, the substrates or products of the P450-regulated reactions are often ligands for the nuclear receptors that regulate expression of the respective P450 genes. This triangular relationship between transcriptional regulators, enzymes, and reaction products provides the necessary feedback for homeostasis. Given that a close link between heme and lipid metabolism is also well documented, the ability of a nuclear receptor to monitor levels of heme could provide a general mechanism for coordinating these processes (Reinking, 2005).

The iron center of E75 can reversibly switch between Fe(III) and Fe(II) oxidation states. Furthermore, the interaction with an HR3-derived peptide is selective for the Fe(II) oxidation state of E75-heme. This set of characteristics sets up the intriguing possibility of E75 acting as a direct redox sensor. An interesting example of this behavior is the reported heme-based redox sensor activity of Ec-DOS, a phosphodiesterase in E. coli. Enzymatic activity is modulated by an allosteric change in the heme-containing PAS domain. The heterodimerization and transcriptional activities of the NPAS2 and Bmal1 transcription factors, which regulate circadian rhythm, have also been shown to depend on redox potential in vitro. NPAS2 has two heme-containing PAS domains, which could provide the mechanism for redox detection. Ec-DOS and NPAS2 also function as gas sensors (Reinking, 2005).

In many heme bound proteins, the heme molecule serves as a cofactor or prosthetic group for the binding of diatomic gases. This is also the case for E75-heme, and NO and CO may serve as E75 ligand(s). Although small, these diatomic gases have been shown to bring about significant changes in protein structure. In heme-containing proteins such as hemoglobin, myoglobin, guanylate cyclase, CooA, and FixL, for example, the binding of O2, CO, or NO causes allosteric rearrangements that modulate protein multimerization, enzymatic activity, and/or the ability to interact with cofactors or target molecules (Reinking, 2005).

In the case of E75, peptide binding studies suggest that CO interferes with the ability of E75 to interact with the DHR3 AF2 motif. Although unable to confirm this by direct binding assays, the results with NO on E75 transcriptional activity suggest that NO may act similarly. This may be analogous to the case of soluble guanylate cyclase, where NO and CO binding both lead to functional activation, but to varying degrees. As with other nuclear receptors, proper placement of the DHR3 AF2 helix within its ligand binding domain is most likely required for DHR3 to bind transcriptional coactivators. If E75 were to sequester the DHR3 AF2 helix, then DHR3 activity would be compromised. By reversing the AF2-E75 interaction, NO and/or CO could then restore normal DHR3-coactivator interactions (Reinking, 2005).

NO and CO are being implicated in a rapidly growing list of signalling processes in vertebrates, and insects. Their ability to diffuse readily between and within cells, and their short half-lives, make them ideal intercellular signaling molecules. In many cases, they act together or in opposition. Examples of processes regulated by NO and CO include blood pressure, cell division, cell death, inflammation, metabolism, hypoxia, diurnal cycles, behavior, and memory. The primary source of NO in tissues is the enzyme NOS and for CO, heme oxygenase. In flies, mutation of the dnos gene results in embryonic lethality. Flies also contain a single heme oxygenase gene, but genetic and functional analyses of dHO are yet to be carried out (Reinking, 2005).

The fact that CO production is dependent upon heme as a substrate, that NOS and heme oxygenase have heme centers, and that they have related physiological functions poses an interesting set of coincidences. This and the convergence of heme with lipid synthesis and related processes, NO and CO gas binding and now E75 function, suggests that E75 may provide a unifying role in the regulation of these processes (Reinking, 2005).

Numerous studies have placed E75 genetically, transcriptionally, and functionally in the ecdysone response pathway, both upstream and downstream of the ecdysone receptor. Thus, E75 appears to play roles in both ecdysone production and response. Ecdysone-regulated processes during insect development include cuticle formation, molting, programmed cell death, neurogenesis, imaginal disk development, and oogenesis. The binding of E75 to heme suggests possible connections between these processes and heme metabolism or function. This could occur at a variety of different levels. Some intriguing possibilities include the regulation of hormone-synthesis pathways, oogenesis arrest, metabolism, and the control of circadian rhythm (Reinking, 2005).

An intimate functional triangle exists between nuclear receptor function, cytochrome P450 expression, and cytochrome P450 substrates. This relationship, taken together with the apparent need for E75 in ecdysone production and response, suggests the possibility that E75 could control ecdysteroid metabolism by regulating the transcription of key cytochrome P450 genes, or more globally by sensing the levels of available heme. The possible ability of E75 to regulate hormone synthesis, and its known role in controlling the progression of molts, metamorphosis, and oogenesis, also brings up other intriguing possible functions. One example might be the ability to monitor energy resources such as lipids and to coordinate these levels with developmental progression. More specifically, the close link between heme, NO, and lipid metabolism could be used to block or postpone molting, pupariation, or oogenesis when energy resources are low, and vice versa (Reinking, 2005).

Interestingly, in mosquitoes, oogenesis is halted prior to chorion deposition until the insect obtains a blood meal. Although the blood-meal components that release the oogenesis arrest have yet to be identified, one of the major components of the blood meal is heme removed from the metabolized hemoglobin. Taken together with the observations that one of the responses of the blood meal is a pulse of ecdysone synthesis and action and that in Drosophila, E75 functions in both a feed forward and downstream role in the ecdysone signaling pathway, E75 may play a key role in this response by responding to the ingested heme and inducing ecdysone synthesis (Reinking, 2005).

A number of possible links also exist between E75 function and a role in regulating circadian rhythms. (1) NO, CO, and heme appear to be important regulators of circadian oscillators. In vertebrates, 'light' and 'dark' inputs are thought to be converted to signals of NO and CO, which in turn modulate the phase and period of the circadian cycle. Heme biosynthesis is also reciprocally regulated by the circadian clock. (2) The closest homolog to E75 in vertebrates, Rev-Erbalpha, is a well-established regulator of circadian rhythm in mammals, also acting as a transcriptional repressor and competing with the nuclear receptor RORalpha, which is a transcriptional activator of the circadian regulator Bmal1. The interaction between Rev-Erbalpha and RORalpha is analogous to that of E75 and HR3, their closest insect homologs. (3) Each larval instar molt, as well as the onset of metamorphosis and adult eclosure, are coupled to the circadian clock. Molts between first and third instars occur every 24 hr, and metamorphosis and eclosure generally begin on the mornings of the fifth and ninth days. These events are regulated by rhythmic pulses of juvenile hormone and ecdysone, which are under E75 control. In insects that take longer to develop, such as Rodnius (21 days), ecdysone levels have been shown to oscillate daily, with levels highest at night. E75 may time these oscillating events indirectly by monitoring feeding activity (lipid intake) or through a more direct role in circadian rhythm (Reinking, 2005 and references therein).

An interesting implication of this study is that the orphan receptor, Rev-Erbalpha, may also bind heme and respond to diatomic gases. If it does not, compensating evolutionary steps may have been adopted to maintain the circuitry of E75-heme-regulated processes. It is also possible that other nuclear receptors may have adopted or conserved the capacity to bind heme and diatomic gases and to regulate corresponding developmental and physiological functions (Reinking, 2005).

Dynamic regulation of Drosophila nuclear receptor activity in vivo

Nuclear receptors are a large family of transcription factors that play major roles in development, metamorphosis, metabolism and disease. To determine how, where and when nuclear receptors are regulated by small chemical ligands and/or protein partners, a `ligand sensor' system was used to visualize spatial activity patterns for each of the 18 Drosophila nuclear receptors in live developing animals. Transgenic lines were established that express the ligand binding domain of each nuclear receptor fused to the DNA-binding domain of yeast GAL4. When combined with a GAL4-responsive reporter gene, the fusion proteins show tissue- and stage-specific patterns of activation. These responses accurately reflect the presence of endogenous and exogenously added hormone, and that they can be modulated by nuclear receptor partner proteins. The amnioserosa, yolk, midgut and fat body, which play major roles in lipid storage, metabolism and developmental timing, were identified as frequent sites of nuclear receptor activity. Dynamic changes in activation were seen that are indicative of sweeping changes in ligand and/or co-factor production. The screening of a small compound library using this system identified the angular psoralen angelicin and the insect growth regulator fenoxycarb as activators of the Ultraspiracle (USP) ligand-binding domain. These results demonstrate the utility of this system for the functional dissection of nuclear receptor pathways and for the development of new receptor agonists and antagonists that can be used to modulate metabolism and disease and to develop more effective means of insect control (Palanker, 2006).

Nine GAL4-LBD ligand sensor lines described in this study show tissue-specific patterns of activity during development: EcR, USP, ERR, FTZ-F1, HNF4, E78, DHR3, DHR38 and DHR96. These transgenic lines will serve as valuable tools for the genetic and molecular dissection of the receptors they represent, the pathways they regulate and the upstream factors and co-factors that modulate their activity. Specifically, the data reported here show that these lines can be used to: (1) indicate tissues and stages in which the corresponding NRs are likely to function; (2) indicate where endogenous ligands and co-factors are likely to be found; (3) suggest NR biological functions; (4) suggest possible NR-NR interactions, cascades and target genes; (5) evaluate putative co-factors and ligands; (6) screen chemical compound libraries for new agonists and antagonists; and (7) screen genetically for new pathway components. The results of these studies will also provide important insights into the ligands, co-factors and functions of their vertebrate NR homologues (Palanker, 2006).

Hormonal regulation of GAL4-LBD activation in the amnioserosa and yolk

Examination of the nine active ligand sensor lines provided a number of insights into possible relationships between their corresponding NRs. For example, although each of these ligand sensors displays unique temporal and spatial patterns of activity, activation in specific tissues and stages is common to many. These common sites of LBD activity may indicate shared functions, hierarchical or physical interactions, or related ligands. Examples of tissues that represent hotspots for GAL4-LBD activation include the amnioserosa, yolk, midgut and fat body (Palanker, 2006).

Each of these tissues, and the stages at which they score positively, correlates well with the presence of putative ligands. The yolk, for example, is believed to act as a storage site for maternally provided ecdysteroids during embryogenesis. Work with other insects has shown that these ecdysteroids are conjugated in an inactive form to vitellin proteins via phosphate bridges. Around mid-embryogenesis, these yolk proteins and phosphate bonds are cleaved, thereby releasing what are presumed to be the earliest biologically active ecdysteroids in the embryo. Interestingly though, GAL4-EcR activation in the amnioserosa depends on the disembodied (dib) gene, which encodes a cytochrome P450 enzyme required in the penultimate step of Ecdysone (E) biosynthesis, suggesting that the final steps in the linear E biosynthetic pathway are required for EcR function in this tissue and contradicting the prediction that this activity would be dependent on maternal ecdysteroids and independent of the zygotic biosynthetic machinery. The mechanisms by which dib exerts this essential role in providing an EcR ligand, however, remain to be determined (Palanker, 2006).

The response of the EcR and USP ligand sensors in the adjacent amnioserosa tissue shows that active ecdysteroids are not present until the hormone reaches the amnioserosa. A recent study of yolk-amnioserosa interactions has revealed dynamic transient projections that emanate from one tissue and contact the other, suggesting that there may be functional interactions between these two cell types. It is possible that these projections mediate the transfer of lipophilic ligand precursors from the yolk to the amnioserosa. This transfer, in turn, could determine the proper timing of EcR activation in the amnioserosa, thus triggering the major morphogenetic movements that establish the body plan of the first instar larva (Palanker, 2006).

Studies of the DHR38 receptor have demonstrated that it can be activated by a distinct set of ecdysteroids from those that activate EcR, through a novel mechanism that does not involve direct ligand binding. The activation of GAL4-DHR38 that was observed in the embryonic amnioserosa is consistent with this model of DHR38 regulation. First, exogenous 20E can only weakly activate GAL4-DHR38, relative to the strong ectopic activation seen with 20E on the EcR ligand sensor. This correlates with the weak ability of 20E to activate DHR38 in cell culture transfection assays relative to the strong 20E activation of EcR. Second, the DHR38 ligand sensor is activated in the amnioserosa earlier than the EcR construct, suggesting that it is responding to a different signal. It is possible that this signal is an ecdysteroid precursor that can act on DHR38 but not EcR - paralleling the ability of DHR38 to be activated by E, the precursor to 20E, which activates EcR. This putative ecdysteroid must be produced in a manner independent of the conventional ecdysteroid biosynthetic pathway, however, since a zygotic dib mutation has no effect on GAL4-DHR38 activation in the amnioserosa. Rather, this early activation may be due to maternal ecdysteroids that are conjugated and inactive in the yolk and transferred to the amnioserosa. These studies highlight the value of combining mutations in hormone biosynthesis with ligand sensor activation as a powerful means of dissecting hormone signaling pathways. Further studies of DHR38 function and regulation in embryos could help clarify the potential significance of this distinct activation response (Palanker, 2006).

DHR3, DHR38 and HNF4 ligand sensors appear to respond to metabolic signals Interestingly, the midgut continues to be a hotspot for ligand sensor activity long after it has engulfed the yolk during embryogenesis. This seems logical, as the midgut is responsible for most lipid absorption and release, and many vertebrate NRs are involved in fatty acid, cholesterol and sterol metabolism and homeostasis. The observed restriction of ligand sensor activity to a narrow group of cells located at the base of the gastric caeca is of particular interest. This is the site where nutrients in a feeding larva are absorbed into the circulatory system. The activation of DHR3, DHR38 and HNF4 ligand sensors in this region of the gastric caeca suggests that these receptors are activated by one or more small nutrient ligands. Moreover, this suggests that the corresponding receptors may exert crucial metabolic functions by acting as nutrient sensors (Palanker, 2006).

Further evidence of metabolic functions for DHR3, DHR38 and HNF4 arises from their ligand sensor activation patterns in the embryonic yolk and larval fat body. The yolk is the main nutrient source for the developing embryo and represents an abundant source of lipids, correlating with specific activation of DHR3, DHR38 and HNF4 ligand sensors in this cell type during embryogenesis. Upon hatching into a larva, the fat body acts as the main metabolic organ of the animal, functionally equivalent to the mammalian liver. Upon absorption by the gastric caeca, nutrients travel through the circulatory system and are absorbed by the fat body, where they are broken down and stored as triglycerides, glycogen and trehalose. Once again, the efficient activation of the DHR3, DHR38 and HNF4 ligand sensors in the fat body of metabolically active third instar larvae, and lack of sensor activity in non-feeding prepupae, supports the model that the corresponding NRs operate as metabolic sensors. This proposed function is consistent with the roles of their vertebrate orthologs. Mammalian ROR, the ortholog of DHR3, binds cholesterol and plays a crucial role in lipid homeostasis. Similarly, mammalian HNF4 can bind C14-18 fatty acids, is required for proper hepatic lipid metabolic gene regulation and lipid homeostasis, and is associated with human Maturity-Onset Diabetes of the Young (MODY1). The studies described here suggest that DHR3 and HNF4 may perform similar metabolic functions in flies, defining a new genetic model system for characterizing these key NRs (Palanker, 2006).

New insights into the regulation of Drosophila xenobiotic responses

Several vertebrate NRs play a central role in xenobiotic responses by directly binding toxic compounds and inducing the expression of key detoxification enzymes such as cytochrome P450s and glutathione transferases. Ligand sensor activation observed in the gut, epidermis, tracheae or fat body could represent xenobiotic responses insofar as toxic compounds could enter the organism through any of these tissues. Directed screens that test xenobiotic compounds for their ability to activate Drosophila NR ligand sensors will provide a means of identifying potential xenobiotic receptors. Understanding these response systems, in turn, could facilitate the production of insect resistant crops and the development of more effective pesticides (Palanker, 2006).

Like its vertebrate orthologs SXR/PXR and CAR, DHR96 has been recently shown to act in insect xenobiotic responses, providing resistance to the sedative effect of phenobarbital and lethality caused by chronic exposure to DDT (King-Jones et al., 2006). DHR96 is also required for the proper transcriptional response of a subset of phenobarbital-regulated genes. DHR96 can be activated by the CAR-selective agonist CITCO, suggesting that it may be regulated in a manner similar to that of the vertebrate xenobiotic receptors. It is also interesting to note that angelicin was found to activate the USP ligand sensor fusion. Angelicin is an angular furanocoumarin that has the furan ring attached at the 7,8 position of the benz-2-pyrone nucleus. Detailed studies have shown that insects have adapted to the presence of furanocoumarins in their host plants by expressing specific cytochrome P450 enzymes that detoxify these compounds. In the black swallowtail butterfly (Papilio polyxenes), furanocoumarins induce the transcription of P450 genes through an unknown regulatory pathway, thereby aiding in xenobiotic detoxification. The observation that angelicin, and not the linear furanocoumarins 8-methoxypsoralen (xanthotoxin) or 5-methoxypsoralen (bergapten), can activate GAL4-USP suggests that NRs may mediate this detoxification response and may be capable of distinguishing between the linear and angular chemical forms. It is possible that USP may mediate this effect on its own or, more likely, as a heterodimer partner with another NR. Similarly, the activation of GAL4-USP by fenoxycarb may represent a xenobiotic response. This activation, however, is weaker and more variable than the activation observed with angelicin. Identifying other factors that mediate xenobiotic responses in Drosophila would provide a new basis for dissecting the control of detoxification pathways in higher organisms (Palanker, 2006).

ERR activity appears to be regulated by a temporally restricted and widespread signal

GAL4-ERR displays a remarkable switch in activity during mid-embryogenesis, from strong activation in the myoblasts to specific and strong activation in the CNS. The ERR ligand sensor also shows widespread transient activation in the mid-third instar, a time when larval ERR gene expression begins, together with a global switch in gene expression that prepares the animal for entry into metamorphosis 1 day later. This so-called mid-third instar transition includes upregulation of EcR, providing sufficient receptor to transduce the high titer late larval 20E hormone pulse, upregulation of the Broad-Complex, which is required for entry into metamorphosis, and induction of the genes that encode a polypeptide glue used to immobilize the puparium for metamorphosis. The signal and receptor that mediate this global reprogramming of gene expression remain undefined. The widespread activation of GAL4-ERR at this stage raises the interesting possibility that it may play a role in this transition. Moreover, given that the only ligand sensors to display widespread transient activation are EcR and USP, in response to 20E, it is possible that this response reflects a systemic mid-third instar pulse of a ERR hormone. Vertebrate members of the ERR family can bind the synthetic estrogen diethylstilbestrol and the selective ER modulator tamoxifen, as well as its metabolite, 4-hydroxytamoxifen, suppressing their otherwise constitutive activity in cell culture. This is notably different from the highly restricted patterns of ERR ligand sensor activity that was detected in Drosophila, which suggests that it does not function as a constitutive activator in vivo. Rather, it is envisioned that the patterns of ERR activation are precisely modulated by protein co-factors and/or one or more ligands to direct the dynamic shifts in activation that are detect during embryogenesis and third instar larval development. Functional studies of the Drosophila homolog of the ERR receptor family may provide a basis for understanding these dynamic shifts in LBD activation, as well as revealing a natural ligand for this NR (Palanker, 2006).

SAYP interacts with DHR3 nuclear receptor and participates in ecdysone-dependent transcription regulation

The role of metazoan coactivator SAYP in nuclear receptor-driven gene activation in the ecdysone cascade of Drosophila is considered. SAYP interacts with DHR3 nuclear receptor and activates the corresponding genes by recruiting the BTFly (Brahma and TFIID) coactivator supercomplex. The knockdown of SAYP leads to a decrease in the level of DHR3-activated transcription. DHR3 and SAYP interact during development and have multiple common targets across the genome (Vorobyeva, 2011).

This study analyzed the role of transcription coactivator SAYP and the SAYP-assembled complex in the transcription activation of several genes activated by ecdysone in Drosophila. The results obtained by different methods show that SAYP interacts with the DHR3 nuclear receptor, a component of the ecdysone cascade. In particular, their direct interaction was demonstrated in the yeast two-hybrid system. In gel filtration and co-IP experiments, a significant proportion of DHR3 proved to be associated with the high-MW SAYP-containing protein complex. The association of these factors is confirmed by data on their colocalization on polytene chromosomes as well as their coexpression and cooperation during development. These data indicate that DHR3 interacts with SAYP both in embryos and in pupae (Vorobyeva, 2011).

DHR3 and SAYP are coordinately recruited onto promoters in pupae and S2 cells. SAYP knockdown has a negative effect on the level of DHR3-driven transcription, which indicates that SAYP mediates the action of DHR3 in transcription activation. Thus, SAYP operates as a classic coactivator, which is recruited by an activator and is important for full-level gene activity. This is in agreement with the previously demonstrated mechanism of SAYP action as a component of the BTFly coactivator complex (Vorobyeva, 2009). As suggested previously, BTFly possesses specific features allowing its employment as a specific and efficient molecular machine for activation of genes in development. DHR3 acts together with other components of the ecdysone pathway to establish a specific pattern of gene activity in a restricted time window. It is suggested that the DHR3-SAYP interaction may be important for such specificity of DHR3 action (Vorobyeva, 2011).

The only known target gene activated by DHR3 is ftz-f1, but the current data indicate that DHR3 together with SAYP may be important for the expression of many other genes, since both these proteins co-occupy multiple sites on the polytene chromosomes. This study has directly shown that DHR3, via the interaction with SAYP, drives the expression of several SAYP-dependent genes in cell culture. These genes have not been recognized as components of the ecdysone cascade, which is evidence that the effect of ecdysone stimulation may be much wider than expected previously (Vorobyeva, 2011).

SAYP and Brahma are important for 'repressive' and 'transient' Pol II pausing

Drosophila SAYP, a homologue of human PHF10/BAF45a, is a metazoan coactivator associated with Brahma and essential for its recruitment on the promoter. The role of SAYP in DHR3 activator-driven transcription of the ftz-f1 gene, a member of the ecdysone cascade was studied. In the repressed state of ftz-f1 in the presence of DHR3, the Pol II complex is pre-recruited on the promoter; Pol II starts transcription but is paused 1.5 kb downstream of the promoter, with SAYP and Brahma forming a 'nucleosomal barrier' (a region of high nucleosome density) ahead of paused Pol II. SAYP depletion leads to the removal of Brahma, thereby eliminating the nucleosomal barrier. During active transcription, Pol II pausing at the same point correlates with Pol II CTD Ser2 phosphorylation. SAYP is essential for Ser2 phosphorylation and transcription elongation. Thus, SAYP as part of the Brahma complex participates in both 'repressive' and 'transient' Pol II pausing (Vorobyeva, 2012).

The mechanism of ftz-f1 transcription activation has been analyzed in S2 cells. Sequential addition and removal of ecdysone allows the DHR3 and ftz-f1 genes in these cells to be activated in accordance with their expression pattern in vivo. This system is of considerable interest, since only a few Drosophila models of activated transcription are available. It also provides the possibility of studying the mechanism of pausing in the active and repressed transcription states of the same gene, whereas previous such studies have been performed with different genes (Vorobyeva, 2012).

Pol II pausing on ftz-f1 occurs at about 1.5 kb downstream of the promoter, i.e. at a much greater distance than that described for other genes (from +30 to +100 nt). Future studies will show how widespread is this mode of pausing. It is of interest in this context that a case of Pol II pausing at 800 bp downstream of the promoter was described for the β-actin gene (Vorobyeva, 2012).

The ftz-f1 activation at the molecular level is a several-stage process. At the first stage, when the ecdysone titer and DHR3 expression are high, DHR3, SAYP, TFIID, Brahma and Pol II accumulate at the promoter. Transcription is initiated, but Pol II is paused 1.5 kb downstream of the promoter; DHR3, SAYP and Brahma are also present at this site, where a nucleosomal barrier is formed. At the next stage, ~1 h after ecdysone removal, promoter-bound factors remain at the same levels, except for SAYP (its level on the promoter decreases). Pol II and associated factors disappear from the site of pausing, and the nucleosomal barrier is eliminated, but the transcription level does not increase. The following stage is characterized by rapid intensification of transcription, which reaches a maximum within several hours; the level of Pol II increases in the body of the gene, and its pausing is observed again, with SAYP and Brahma being present at the corresponding position. In addition, the level of SAYP on the promoter is recovered, indicating that it is highly regulated at different transcription stages. The DHR3 activator is present at the site of pausing, and its level does not change upon SAYP knockdown. This is evidence that DHR3 may participate in SAYP recruitment for subsequent nucleosomal barrier formation and Pol II pausing (Vorobyeva, 2012).

The region of high nucleosome density (nucleosomal barrier) is specific for the repression stage, at which the DHR3 activator induces the assembly of the Pol II preinitiation complex on the promoter and makes paused Pol II competent for transcription initiation. Nucleosomal barrier disruption by SAYP knockdown leads to the full-length transcript synthesis, indicating that the nucleosomal barrier contributes to preventing the entry of Pol II to the transcribed region. The data show that SAYP and Brahma play the crucial role in organization of the nucleosomal barrier: this barrier coincides in location with the peak of these coactivators and disappears after SAYP knockdown, which leads to elimination of Brahma from the gene. Thus, SAYP and Brahma at the stage of repressed transcription have an important role in blocking the synthesis of full-length transcripts. Although the transcription increases upon SAYP depletion and elimination of the nucleosomal barrier, its level remains low, compared with that in the permissive state. This is evidence for the existence of different mechanisms of Pol II pausing regulation, which also correlates with the fact that the depletion of NELF, an important factor of Pol II pausing, causes a 2.5-fold increase in the transcription of hsp70 or hsp26 gene in the repressed state, which, however, does not reaches the level characteristic of a fully activated gene (Vorobyeva, 2012).

The question arises as to the structure of the nucleosomal barrier. As shown previously, the human SWI/SNF complex can not only erase nucleosomes from the template but also produce a stable remodeled dimer of mononucleosome core, with this complex being also needed for converting this product back to the cores. One may suggest that the Drosophila Brahma complex operates in the same way. In the current experiments, the level of histone H3 increased ~2-fold in the region of the nucleosomal barrier, compared with its general level on the gene, which agrees with the assumption concerning the presence of a nucleosome dimer. The fact that the region of nucleosomal barrier is significantly enriched in sequences with a high nucleosome-positioning probability indicates that DNA sequences probably contribute to organization of this barrier (Vorobyeva, 2012).

Previous experiments have revealed a relationship between Pol II pausing and the nucleosomal structure of the template. It has been shown that Pol II stops at the site where the nucleosome density is restored to the average level characteristic of the gene. However, no specific nucleosome-dense regions preventing Pol II transcription have been described as yet (Vorobyeva, 2012).

The transition to the transcription-permissive state correlates with significant rearrangements in the promoter-distal region (disappearance of Brahma, SAYP, Pol II and nucleosomal barrier at the site of Pol II pausing). However, no increase in the ftz-f1 transcription level has been observed within the first 30 min after this transition. As shown in the study on estradiol (ER)-mediated gene expression, productive transcription is preceded by an unproductive cycle (~40 min) that is necessary for promoter preparation to this process. This may be the case for ftz-f1, with a certain period of time being required for rearrangements preceding its active transcription (Vorobyeva, 2012).

At the (+;-) stage, the level of SAYP on the promoter is recovered within 2-3 h after the onset of transcription, with SAYP RNAi influencing the Brahma and TFIID levels on the promoter. Pol II pausing correlating with its Ser2 modification is again observed as the transcription level increases. Although SAYP and Brahma occur again together with paused Pol II, their function appears to be different from that at the repression stage. The nucleosomal barrier is not restored, and SAYP depletion has only a slight effect on chromatin structure (Vorobyeva, 2012).

However, SAYP depletion severely disturbs transient pausing, interfering with Ser2 phosphorylation. This impairs proper transition to productive elongation and leads to a decrease in Pol II level on the body of the gene. Thus, SAYP knockdown not only affects the level of ftz-f1 activation but also shifts the timing of its expression. The slower kinetics of transcription induction together with the slight decrease in the Pol II level on the promoter upon SAYP knockdown are evidence for the retarded Pol II passage in the coding region of the gene and, hence, for disturbances in the elongation mechanisms. Similar consequences are observed for other genes regulating on pausing mechanisms (Vorobyeva, 2012).

The results of this study show that SAYP is important for proper timing of ftz-f1 transcription during Drosophila metamorphosis. The ftz-f1 gene is a major regulator of metamorphosis, that is why its precise activation in time is crucial during development. On the whole, the data provide evidence for the important role of pausing in sequential activation of genes in cascades and indicate that this mechanism may have a general role in development (Vorobyeva, 2012).

In addition, these results also support the idea that Pol II pausing may require not only NELF and DSIF but also other factors, such as nucleosome-remodeling complexes. Interestingly, the depletion of NELF proved to result in an increased nucleosome occupancy at the promoters of some genes (Vorobyeva, 2012).

In summary, this study has found that Pol II pausing is dependent on the interplay of several molecular mechanisms, including the formation of a specific chromatin structure via the action of coactivators. These results indicate that, although Pol II pausing is a genome-wide phenomenon, the specific molecular mechanism controlling paused Pol II activity on individual genes may vary significantly (Vorobyeva, 2012).

Nitric oxide coordinates metabolism, growth, and development via the nuclear receptor E75

Nitric oxide gas acts as a short-range signaling molecule in a vast array of important physiological processes, many of which include major changes in gene expression. How these genomic responses are induced, however, is poorly understood. Previous work showed that ecdysone-induced protein 75 (E75, also known as Eip75B; NR1D3) contains heme constitutively bound to its ligand-binding domain (LBD), and that amino acids coordinately bound to the heme iron can be displaced in vitro by changes in redox state or the presence of nitric oxide (NO) gas (Reinking, 2005; Marvin, 2009). In turn, these structural changes negate the ability of E75 to repress transcription and to reverse the positive transcriptional activity of its heterodimer partner, Drosophila hormone receptor 3 (DHR3; also known as DHR46, NR1F4). This study examined whether these interactions are relevant in vivo and, if so, what their roles are. Using genetic and chemical manipulations, it was shown that nitric oxide is produced in the Drosophila prothoracic gland (PG), where it acts via the nuclear receptor ecdysone-induced protein 75 (E75), reversing its ability to interfere with its heterodimer partner, Drosophila hormone receptor 3 (DHR3). Manipulation of these interactions leads to gross alterations in feeding behavior, fat deposition, and developmental timing. These neuroendocrine interactions and consequences appear to be conserved in vertebrates (Cáceres, 2011).

Although previously documented effects of NO on cells and tissues are numerous, and many of the cytoplasmic mechanisms of action are well documented, this is the first demonstration of a direct effect via a transcription factor in vivo. In the PG, the findings indicate that E75 is the major nuclear mediator of NO function, as evidenced by the similarity and epistatic nature of E75, DHR3, and NOS phenotypes. A review of the literature also shows that these genes tend to be coexpressed in many other Drosophila tissues, where limited analyses also suggest shared functions. Thus, the interactions described in this study are likely to be relevant to numerous other tissues and processes (Cáceres, 2011).

This overlap in expression and functions is also true for the vertebrate NOS and Rev-erb/ROR orthologs. Examples of overlapping functions in vertebrates include their similar roles in lipid metabolism, gluconeogenesis, muscle differentiation, inflammation, circadian rhythm, PGC1α regulation, hypertension, and atherosclerosis. Strikingly, Nos triple-knockout mice that survive gestation are morbidly obese, exhibiting all aspects of metabolic syndrome (e.g., diabetes, hypertension, atherosclerosis), resulting in a maximum life span of 10 mo (Tsutsui, 2006; Tsutsui, 2009). Conversely, NO up-regulation via arginine supplementation yields a reciprocal phenotype, which includes increased lipolysis, fatty acid oxidation, mitochondrial biogenesis, glucose metabolism, and life span. These effects are similar to those seen upon genetic manipulation of the Rev-erbs and RORs (Duez, 2009a; Duez, 2009; Cáceres, 2011 and references therein).

In the case of E75, NO appears to act in two ways. NO blocks the ability of E75 to interfere directly with DHR3-mediated transcriptional activation. However, it also appears to block the ability of E75 to repress target genes independently of DHR3. In vertebrates, no direct interaction has yet been observed between Rev-erbs and RORs. NO does, however, block the ability of the Rev-erbs to repress target genes by preventing the recruitment of coactivators (Pardee, 2009). It is expected that this aspect of NO action is conserved in flies (Cáceres, 2011).

The achievement of critical weight at the end of the third larval instar coincides with a reduction in circulating levels of insulin-like peptides (ILPs) and consequential production of prothoracicotropic hormone (PTTH) peptide by neurons located within the larval brain. The axons of these PTTH-producing neurons extend to the surface of the PG, where binding of the secreted PTTH peptide to Torso receptor results in intracellular signaling (Rewitz, 2009). Intracellular outcomes include activation of Ras/Raf, MEK/Erk, and PKA, and, notably, the production of Calmodulin, NADH, and cytoplasmic Ca2+ influx. The latter three molecules are required cofactors for dNOS enzymatic activity. Hence, it is proposed that PTTH acts, in large part, through NOS activation (Cáceres, 2011).

The remarkable endoreduplication and growth of the PG due to loss of NOS expression is consistent with previous studies showing that NO has negative effects on cell growth. This activity has important implications and potential use in the control of oncogenic growth (Cáceres, 2011).

Interestingly, these effects of NO on PG size and the timing of metamorphosis contradict previous studies that had suggested that PG size is a key determinant of metamorphosis timing. In these studies, metamorphosis appeared to occur when full PG size was achieved. Here, PG size was up to six times larger than normal, with no metamorphosis. Similarly, Colombani (2005) found that altering PG size by manipulation of DMyc or Cyclin D expression could also increase PG size without triggering premature ecdysone production. Furthermore, metamorphosis can be induced prematurely by down-regulation of DHR4 without affecting PG size. These seemingly contradictory results are likely due to cross-talk within the insulin and ecdysone signaling pathways (Cáceres, 2011).

The bright-red color of these enlarged PGs is consistent with previous studies suggesting that E75 and the Rev-erbs also function as heme sensors (Cáceres, 2011).

NOS manipulation in the PG had major effects on lipid uptake and storage, leading to a nearly 20-fold increase in larval lipid content in the case of knockdown, or a nearly fivefold decrease when expressed prematurely. As disruption of EcR activity within fat body cells can also result in lipid overaccumulation (Colombani, 2005), the nonautonomous effects of PG NOS manipulation appear to be ecdysone-mediated. However, it is quite possible that the nearly 100-fold overall changes observed in lipid content may also be due to additional effects on feeding behavior, nutrient uptake, and other aspects of lipid metabolism (Cáceres, 2011).

The extended eating and fat deposition phenotype, caused by failure to produce ecdysone, may equate on many levels to processes underlying obesity and diabetes. Indeed, much as seen with Nos up-regulation, ecdysone supplementation in vertebrates results in decreased appetite, cholesterol synthesis, and weight gain, while at the same time increasing muscle mass and endurance. Given these apparent abilities to switch fat cell activity from lipid storage to lipid mobilization, a better understanding of the signals and mechanisms underlying NO and ecdysone-mediated effects should provide new insights and possible uses in metabolism based disorders (Cáceres, 2011).

Given the previously demonstrated roles for NO, Rev-erbs, and RORs in the control of circadian clocks, and those shown in this study for developmental timing, it is quite possible that NO, Rev-erbs, and RORs also serve general roles in the matching of diurnal, lunar, and seasonal inputs (i.e., light, temperature, and food availability) with appropriate feeding behaviors, metabolism, and developmental progression. Disruption of these functions leads to metabolic, sleep, stress, immune, and hypertension disorders. Hence, further elucidation of ROR, Rev-erb, and NOS interactions in neuroendocrine tissues should provide new insights into how these temporal and metabolic processes are linked and coordinated, and new ways to prevent or treat their related disorders (Cáceres, 2011).

Hormone receptor-like in 46: Biological Overview | Evolutionary Homologs | Developmental Biology | Effects of Mutation | References

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