A genome-wide computational approach to define microRNA-Polycomb/trithorax gene regulatory circuits in Drosophila.

Characterization of gene regulatory networks is fundamental to understanding homeostatic development. This process can be simplified by analyzing relatively simple genomes such as the genome of Drosophila melanogaster. In this work we have developed a computational framework in Drosophila to explore for the presence of gene regulatory circuits between two large groups of transcriptional regulators: the epigenetic group of the Polycomb/trithorax (PcG/trxG) proteins and the microRNAs (miRNAs). We have searched genome-wide for miRNA targets in PcG/trxG transcripts as well as for Polycomb Response Elements (PREs) in miRNA genes. Our results show that 10% of the analyzed miRNAs could be controlling PcG/trxG gene expression, while 40% of those miRNAs are putatively controlled by the selected set of PcG/trxG proteins. The integration of these analyses has resulted in the predicted existence of 3 classes of miRNA-PcG/trxG crosstalk interactions that define potential regulatory circuits. In the first class, miRNA-PcG circuits are defined by miRNAs that reciprocally crosstalk with PcG. In the second, miRNA-trxG circuits are defined by miRNAs that reciprocally crosstalk with trxG. In the third class, miRNA-PcG/trxG shared circuits are defined by miRNAs that crosstalk with both PcG and trxG regulators. These putative regulatory circuits may uncover a novel mechanism in Drosophila for the control of PcG/trxG and miRNAs levels of expression. The computational framework developed here for Drosophila melanogaster can serve as a model case for similar analyses in other species. Moreover, our work provides, for the first time, a new and useful resource for the Drosophila community to consult prior to experimental studies investigating the epigenetic regulatory networks of miRNA-PcG/trxG mediated gene expression.

Drosophila SCE/dRING E3-ligase inhibits apoptosis in a Dp53 dependent manner.

The Polycomb group (PcG) of proteins control developmental gene silencing and are highly conserved between flies and mammals. PcG proteins function by controlling post-translational modification of histones, such as ubiquitylation, which impacts chromatin compaction and thus gene transcription. Changes in PcG cellular levels have drastic effects on organismal development and are involved in the generation of human pathologies such as cancer. However, the mechanisms controlling their levels of expression and their physiological effects are only partially understood. In this work we describe the effects of modulating levels of SCE/dRING, a conserved E3 ubiquitin ligase and member of the PcG known to mono-ubiquitylate histone H2A. We find that inactivation of Sce induces apoptosis, an effect that is decreased in the absence of Dp53 function. However, over-expression of SCE produce no developmental effects but inhibits DP53-induced apoptosis. Thus, Sce functions as a Dp53-dependent apoptosis inhibitor. The SCE inhibition of DP53-induced apoptosis requires dRYBP, an ubiquitin binding protein and member of the PcG. Moreover, this inhibition of apoptosis involves the reduction of DP53 protein levels. Finally, high levels of SCE inhibit X-ray induced apoptosis as well as the apoptosis associated with tumor growth. We propose that SCE, together with dRYBP, inhibits apoptosis either by epigenetically regulating Dp53 transcription or by controlling the stabilization of DP53 protein levels thus promoting its ubiquitylation for proteaosomal degradation. This function may generate a homeostatic balance between apoptosis and proliferation during development that provides cell survival during the initiation and progression of disease processes.

MicroRNA miR-7 contributes to the control of Drosophila wing growth.

BACKGROUND: The control of organ growth is critical for correct animal development. From flies to mammals, the mechanisms regulating growth are conserved and the role of microRNAs in this process is emerging. The conserved miR-7 has been described to control several aspects of development. RESULTS: Here, we have analyzed the function of miR-7 during Drosophila wing development. We found that loss of miR-7 function results in a reduction of wing size and produces wing cells that are smaller than wild type cells. We also found that loss of miR-7 function interferes with the cell cycle by affecting the G1 to S phase transition. Further, we present evidence that miR-7 is expressed in the wing imaginal discs and that the inactivation of miR-7 increases the expression of Cut and Senseless proteins in wing discs. Finally, our results show that the simultaneous inactivation of miR-7 and either cut, Notch, or dacapo rescues miR-7 loss of function wing size reduction phenotype. CONCLUSIONS: The results from this work reveal, for the first time, that miR-7 functions to regulate Drosophila wing growth by controlling cell cycle phasing and cell mass through its regulation of the expression of dacapo and the Notch signaling pathway.

Polycomb controls gliogenesis by regulating the transient expression of the Gcm/Glide fate determinant.

The Gcm/Glide transcription factor is transiently expressed and required in the Drosophila nervous system. Threshold Gcm/Glide levels control the glial versus neuronal fate choice, and its perdurance triggers excessive gliogenesis, showing that its tight and dynamic regulation ensures the proper balance between neurons and glia. Here, we present a genetic screen for potential gcm/glide interactors and identify genes encoding chromatin factors of the Trithorax and of the Polycomb groups. These proteins maintain the heritable epigenetic state, among others, of HOX genes throughout development, but their regulatory role on transiently expressed genes remains elusive. Here we show that Polycomb negatively affects Gcm/Glide autoregulation, a positive feedback loop that allows timely accumulation of Gcm/Glide threshold levels. Such temporal fine-tuning of gene expression tightly controls gliogenesis. This work performed at the levels of individual cells reveals an undescribed mode of Polycomb action in the modulation of transiently expressed fate determinants and hence in the acquisition of specific cell identity in the nervous system.

Drosophila p53 controls Notch expression and balances apoptosis and proliferation.

A balance between cell proliferation and apoptosis is important for normal development and tissue homeostasis. Under stress conditions, the conserved tumor suppressor and transcription factor Dp53 induces apoptosis to contribute to the maintenance of homeostasis. However, in some cases Dp53-induced apoptosis results in the proliferation of surrounding non-apoptotic cells. To gain insight into the Dp53 function in the control of apoptosis and proliferation, we studied the interaction between the Drosophila Dp53 and Notch genes. We present evidence that simultaneous reduction of Dp53 and Notch function synergistically increases the wing phenotype of Notch heterozygous mutant flies. Further, we found that a Notch cis-regulatory element is responsive to loss and gain of Dp53 function and that over-expression of Dp53 up-regulates Notch mRNA and protein expression. These findings suggest not only that Dp53 and Notch act together to control wing development but also indicate that Dp53 transcriptionally regulates Notch expression. Moreover, using Notch  gain and loss of function mutations we examined the relevance of Dp53 and Notch interactions in the process of Dp53-apoptosis induced proliferation. Results show that proliferation induced by Dp53 over-expression is dependent on Notch, thus identifying Notch as a new player in Dp53-induced proliferation. Interestingly, we found that Dp53-induced Notch activation and proliferation occurs even under conditions where apoptosis was inhibited. Our findings highlight the conservation between flies and vertebrates of the Dp53 and Notch cross-talk and suggest that Dp53 has a dual role regulating cell death and proliferation gene networks to control the homeostatic balance between apoptosis and proliferation.

Lipidomic profiling of Drosophila strains Canton-S and white1118 reveals intraspecific lipid variations in basal metabolic rate.

Drosophila melanogaster is a well-established model system for studies on lipid metabolism and energy homeostasis. In this study, we identified and quantified the main components of the lipid profile of two widely utilized Drosophila strains, namely Canton-S and white1118, under identical experimental conditions. Differences observed between the strains can be attributed to inherent metabolic divergences, thus limiting the influence of confounding factors. Using the comprehensive lipid data acquired, we applied cluster analysis and PLS-DA techniques to ascertain whether the lipidome could effectively differentiate between the strains. Certain lipid features, such as triacylglycerols, polar lipids, and specific sterol components, could be distinguished between flies of both strains regardless of sex. Our results suggest that although Canton-S and white1118 have similar lipid profiles and distributions, a selected subset of lipids demonstrates clear discriminatory potential between strains, thereby bearing significant implications for planning biological studies using these strains as control references.

A novel dRYBP-SCF complex functions to inhibit apoptosis in Drosophila.

A balanced response to intrinsic and extrinsic apoptotic signals is crucial to support homeostatic development and animal survival. Regulation of activation and inhibition of apoptotic pathways involves diverse mechanisms including protein ubiquitylation to control expression levels of apoptotic factors. Here we report that drosophila Ring and YY1 Binding Protein (dRYBP) protein interacts both genetically and biochemically with the E3 ubiquitin ligase SKPA, dCULLIN, F-box (SCF) complex to synergistically inhibit apoptosis in Drosophila. Further, we show that the loss of skpA function activates the intrinsic pathway of apoptosis and down-regulates the levels of expression of the anti-apoptotic DIAP1 protein. Accordingly, the apoptosis induced by inactivation of skpA and dRYBP is rescued by loss of function of the pro-apoptotic gene reaper and overexpression of DIAP1. Of interest, we also find that high levels of SKPA protein rescue the wing phenotype induced by overexpression of Reaper protein. Finally, we demonstrate that overexpression of SKPA inhibits both developmental and radiation-induced apoptosis. We propose that the function of the dRYBP-SCF complex in the inhibition of apoptosis might possibly be to control the levels of the pro-apoptotic and anti-apoptotic proteins most likely by promoting their ubiquitylation and consequently, proteasomal degradation. Given the evolutionary conservation of the dRYBP and the SCF proteins, our results suggest that their mammalian homologs may function in balancing cell survival versus cell death during normal and pathological development.

Functional characterization of the dRYBP gene in Drosophila.

The Drosophila dRYBP gene has been described to function as a Polycomb-dependent transcriptional repressor. To determine the in vivo function of the dRYBP gene, we have generated mutations and analyzed the associated phenotypes. Homozygous null mutants die progressively throughout development and present phenotypes variable both in their penetrance and in their expressivity, including disrupted oogenesis, a disorganized pattern of the syncytial nuclear divisions, defects in pattern formation, and decreased wing size. Although dRYBP mutations do not show the homeotic-like phenotypes typical of mutations in the PcG and trxG genes, they enhance the phenotypes of mutations of either the Sex comb extra gene (PcG) or the trithorax gene (trxG). Finally, the dRYBP protein interacts physically with the Sex comb extra and the Pleiohomeotic proteins, and the homeotic-like phenotypes produced by the high levels of the dRYBP protein are mediated through its C-terminal domain. Our results indicate that the dRYBP gene functions in the control of cell identity together with the PcG/trxG proteins. Furthermore, they also indicate that dRYBP participates in the control of cell proliferation and cell differentiation and we propose that its functional requirement may well depend on the robustness of the animal.

The microRNA-306/abrupt regulatory axis controls wing and haltere growth in Drosophila.

Growth control relies on extrinsic and intrinsic mechanisms that regulate and coordinate the size and pattern of organisms. This control is crucial for a homeostatic development and healthy physiology. The gene networks acting in this process are large and complex: factors involved in growth control are also important in diverse biological processes and these networks include multiple regulators that interact and respond to intra- and extra-cellular inputs that may ultimately converge in the control of the cell cycle. In this work we have studied the function of the Drosophila abrupt gene, coding for a BTB-ZF protein and previously reported to be required for wing vein pattern, in the control of haltere and wing growth. We have found that inactivation of abrupt reduces the size of the wing and haltere. We also found that the microRNA miR-306 controls abrupt expression and that miR-306 and abrupt genetically interact to control wing size. Moreover, the reduced appendage size due to abrupt inactivation is rescued by overexpression of Cyclin-E and by inactivation of dacapo. These findings define a miR-306-abrupt regulatory axis that controls wing and haltere size, whereby miR-306 maintains appropriate levels of abrupt expression which, in turn, regulates the cell cycle. Thus, our results uncover a novel function of abrupt in the regulation of the size of Drosophila appendages during development and contribute to the understanding of the coordination between growth and pattern as well as to the understanding of abrupt oncogenic function in flies.

Epigenetic and non-epigenetic functions of the RYBP protein in development and disease.

Over the last decades significant advances have been made in our understanding of the molecular mechanisms controlling organismal development. Among these mechanisms the knowledge gained on the roles played by epigenetic regulation of gene expression is extensive. Epigenetic control of transcription requires the function of protein complexes whose specific biochemical activities, such as histone mono-ubiquitylation, affect chromatin compaction and, consequently activation or repression of gene expression. Complexes composed of Polycomb Group (PcG) proteins promote transcriptional silencing while those containing trithorax group (trxG) proteins promote transcriptional activation. However, other epigenetic protein factors, such as RYBP, have the ability to interact with both PcG and trxG and thus putatively participate in the reversibility of chromatin compaction, essential to respond to developmental cues and stress signals. This review discusses the developmental and mechanistic functions of RYBP, a ubiquitin binding protein, in epigenetic control mediated by the PcG/trxG proteins to control transcription. Recent experimental evidence indicates that proteins regulating chromatin compaction also participate in other molecular mechanisms controlling development, such as cell death. This review also discusses the role of RYBP in apoptosis through non-epigenetic mechanisms as well as recent investigations linking the role of RYBP to apoptosis and cancer.

The Drosophila RYBP gene functions as a Polycomb-dependent transcriptional repressor.

The Polycomb and trithorax groups of genes control the maintenance of homeotic gene expression in a variety of organisms. A putative participant in the regulation of this process is the murine RYBP (Ring and YY1 Binding Protein) gene. Sequence comparison between different species has identified the homologous gene in Drosophila, the dRYBP gene. We have investigated whether dRYBP participates in the mechanisms of silencing of homeotic genes expression. We first studied its expression by RNA in situ hybridisation and detected dRYBP expression ubiquitously and throughout development. Moreover, we generated a polyclonal anti-dRYBP antibody that recognises the dRYBP protein. dRYBP protein is nuclear and expressed maternally and ubiquitously throughout development. To study the transcriptional activity of dRYBP, we generated a fusion protein containing the entire dRYBP protein and the GAL4 DNA binding domain. This fusion protein functions, in vivo, as a transcriptional repressor throughout development. Importantly, this repression is dependent on the function of the Polycomb group genes. Furthermore, using the GAL4/UAS system, we have over expressed dRYBP in the haltere and the wing imaginal discs. In the haltere discs, high levels of dRYBP repress the expression of the homeotic Ultrabithorax gene. This repression is Polycomb dependent. In the wing discs, dRYBP over expression produces a variety of phenotypes suggesting the overall miss regulation of the many putative genes affected by high levels of dRYBP. Taking together, our results indicate that dRYBP is able to interact with PcG proteins to repress transcription suggesting that the dRYBP gene might belong to the Polycomb group of genes in Drosophila.

Functional dissection of the mouse tyrosinase locus control region identifies a new putative boundary activity.

Locus control regions (LCRs) are complex high-order chromatin structures harbouring several regulatory elements, including enhancers and boundaries. We have analysed the mouse tyrosinase LCR functions, in vitro, in cell lines and, in vivo, in transgenic mice and flies. The LCR-core (2.1 kb), located at -15 kb and carrying a previously described tissue-specific DNase I hypersensitive site, operates as a transcriptional enhancer that efficiently transactivates heterologous promoters in a cell-specific orientation-independent manner. Furthermore, we have investigated the boundary activity of these sequences in transgenic animals and cells. In mice, the LCR fragment (3.7 kb) rescued a weakly expressed reference construct that displays position effects. In Drosophila, the LCR fragment and its core insulated the expression of a white minigene reporter construct from chromosomal position effects. In cells, sequences located 5' from the LCR-core displayed putative boundary activities. We have obtained genomic sequences surrounding the LCR fragment and found a LINE1 repeated element at 5'. In B16 melanoma and L929 fibroblast mouse cells, this element was found heavily methylated, supporting the existence of putative boundary elements that could prevent the spreading of condensed chromatin from the LINE1 sequences into the LCR fragment, experimentally shown to be in an open chromatin structure.

dRYBP counteracts chromatin-dependent activation and repression of transcription.

Chromatin dependent activation and repression of transcription is regulated by the histone modifying enzymatic activities of the trithorax (trxG) and Polycomb (PcG) proteins. To investigate the mechanisms underlying their mutual antagonistic activities we analyzed the function of Drosophila dRYBP, a conserved PcG- and trxG-associated protein. We show that dRYBP is itself ubiquitylated and binds ubiquitylated proteins. Additionally we show that dRYBP maintains H2A monoubiquitylation, H3K4 monomethylation and H3K36 dimethylation levels and does not affect H3K27 trimethylation levels. Further we show that dRYBP interacts with the repressive SCE and dKDM2 proteins as well as the activating dBRE1 protein. Analysis of homeotic phenotypes and post-translationally modified histones levels show that dRYBP antagonizes dKDM2 and dBRE1 functions by respectively preventing H3K36me2 demethylation and H2B monoubiquitylation. Interestingly, our results show that inactivation of dBRE1 produces trithorax-like related homeotic transformations, suggesting that dBRE1 functions in the regulation of homeotic genes expression. Our findings indicate that dRYBP regulates morphogenesis by counteracting transcriptional repression and activation. Thus, they suggest that dRYBP may participate in the epigenetic plasticity important during normal and pathological development.

dRYBP contributes to the negative regulation of the Drosophila Imd pathway.

The Drosophila humoral innate immune response fights infection by producing antimicrobial peptides (AMPs) through the microbe-specific activation of the Toll or the Imd signaling pathway. Upon systemic infection, the production of AMPs is both positively and negatively regulated to reach a balanced immune response required for survival. Here, we report the function of the dRYBP (drosophila Ring and YY1 Binding Protein) protein, which contains a ubiquitin-binding domain, in the Imd pathway. We have found that dRYBP contributes to the negative regulation of AMP production: upon systemic infection with Gram-negative bacteria, Diptericin expression is up-regulated in the absence of dRYBP and down-regulated in the presence of high levels of dRYBP. Epistatic analyses using gain and loss of function alleles of imd, Relish, or skpA and dRYBP suggest that dRYBP functions upstream or together with SKPA, a member of the SCF-E3-ubiquitin ligase complex, to repress the Imd signaling cascade. We propose that the role of dRYBP in the regulation of the Imd signaling pathway is to function as a ubiquitin adaptor protein together with SKPA to promote SCF-dependent proteasomal degradation of Relish. Beyond the identification of dRYBP as a novel component of Imd pathway regulation, our results also suggest that the evolutionarily conserved RYBP protein may be involved in the human innate immune response.

Drosophila melanogaster dHCF interacts with both PcG and TrxG epigenetic regulators.

Repression and activation of gene transcription involves multiprotein complexes that modify chromatin structure. The integration of these complexes at regulatory sites can be assisted by co-factors that link them to DNA-bound transcriptional regulators. In humans, one such co-factor is the herpes simplex virus host-cell factor 1 (HCF-1), which is implicated in both activation and repression of transcription. We show here that disruption of the gene encoding the Drosophila melanogaster homolog of HCF-1, dHCF, leads to a pleiotropic phenotype involving lethality, sterility, small size, apoptosis, and morphological defects. In Drosophila, repressed and activated transcriptional states of cell fate-determining genes are maintained throughout development by Polycomb Group (PcG) and Trithorax Group (TrxG) genes, respectively. dHCF mutant flies display morphological phenotypes typical of TrxG mutants and dHCF interacts genetically with both PcG and TrxG genes. Thus, dHCF inactivation enhances the mutant phenotypes of the Pc PcG as well as brm and mor TrxG genes, suggesting that dHCF possesses Enhancer of TrxG and PcG (ETP) properties. Additionally, dHCF interacts with the previously established ETP gene skd. These pleiotropic phenotypes are consistent with broad roles for dHCF in both activation and repression of transcription during fly development.

High levels of dRYBP induce apoptosis in Drosophila imaginal cells through the activation of reaper and the requirement of trithorax, dredd and dFADD.

Drosophila RYBP (dRYBP; Ring1 and YY1 Binding Protein) is a Polycomb and trithorax interacting protein, whose homologous RYBP/DEDAF mammalian counterparts exhibit tumor cell-specific killing activity. Here we show that although endogenous dRYBP is not involved in developmental apoptosis, high levels of exogenous dRYBP induce apoptosis in all the imaginal discs of the fly, indicating that dRYBP apoptotic activity is not specific to tumor cells. We also show that dRYBP-induced apoptosis is inhibited by high levels of either p35 or DIAP1 (Drosophila Inhibitor of Apoptosis Protein 1), and requires the function of the pro-apoptotic REAPER, HID and GRIM proteins, the apical caspase DREDD, the adaptor dFADD protein as well as TRITHORAX (TRX), an epigenetic transcriptional regulator. Furthermore, we demonstrate that overexpression of TRX also induces apoptosis in the imaginal discs. Finally, we show that the expression of reaper-lacZ is upregulated both upon dRYBP-induced apoptosis and upon TRX-induced apoptosis in imaginal discs and that the reaper gene is a direct target of dRYBP in Drosophila embryos. Our results indicate that dRYBP triggers in a receptor-mediated apoptotic pathway that also includes TRX-dependent epigenetic regulation of gene expression.

The Polyhomeotic protein induces hyperplastic tissue overgrowth through the activation of the JAK/STAT pathway.

Epigenetic mechanisms controlling cellular proliferation are essential to animal development. Moreover, altered levels of expression of the epigenetic regulator proteins are associated with the development and progression of human diseases like cancer. We have studied the effects of high levels of Polyhomeotic (PH) protein, a member of the Polycomb Group (PcG), during the proliferation of the imaginal discs in Drosophila. Overexpression of PH protein causes induction of proliferation, accompanied with induction of JNK-dependent apoptosis. As a result, massive hyperplastic overgrowth is produced and the corresponding differentiated tissues show phenotypes related with mis-regulation of homeotic gene expression. We have found that high levels of PH upregulate the JAK/STAT pathway through the de-repression of Unpaired (UPD), the extracellular ligand of the Drosophila JAK/STAT signalling cascade. Moreover, inactivation of the JAK/STAT pathway in the presence of a large amount of PH protein greatly reduces the tissue overgrowth, demonstrating a functional role of JAK/STAT in PH-induced hyperplasia. Finally, we have observed that decapentaplegic and d-myc, two growth genes and putative targets of the JAK/STAT pathway, are also overexpressed in the PH-induced tumors. We propose that during normal development, the PcG proteins act to maintain inactive the JAK/STAT pathway. Upon cellular stress, changes in the levels of PcG proteins expression are induced and JAK/STAT is activated leading to tumor development. Our results show a functional relationship between the PcG gene expression and the JAK/STAT pathway, both of which are found to be perturbed in tumorigenesis.

Function of the Trithorax-like gene during Drosophila development.

Maintenance of homeotic gene expression during Drosophila development relies on the Polycomb and the trithorax groups of genes. Classically, the Polycomb proteins act as repressors of homeotic gene function, whereas trithorax proteins function as activators. However, recent investigation has indicated that some of these maintenance genes may act both as repressors and activators. One of those is the Drosophila Trithorax-like gene that codes for the GAGA factor. To investigate its dual activator/repressor role, we have studied the function of the Trithorax-like throughout Drosophila development. Embryos lacking both the maternal and the zygotic Trithorax-like function do not develop suggesting that Trithorax-like might be required in oogenesis. Homozygous Trithorax-like null mutant embryos show reduced expression levels of some of the homeotic proteins. Trithorax-like mutant larval clones, however, do not show phenotypes indicative of either activation or repression of homeotic gene function. These results suggest that Trithorax-like is required during embryogenesis but not throughout larval development for the regulation of homeotic gene expression. Moreover, this temporal requirement seems also to regulate MCP-mediated silencing. Finally, lack of Trithorax-like function modulates the gain of function phenotypes caused by over-expression of homeotic genes. To explain Trithorax-like gene function, we propose a model where very early in development, GAGA factor probably establishes a chromatin ground state for transcription. The differential "on/off" transcriptional state of the homeotic genes is then established and propagated by the action of the specific regulatory proteins independently of the GAGA factor. We also suggest that GAGA factor may not have a dual activator/repressor function. Rather, Trithorax-like mutations may produce dual loss of activation and loss of repression effects.

Genetic and molecular characterization of a novel iab-8 regulatory domain in the Abdominal-B gene of Drosophila melanogaster.

Homeotic (or Hox) genes are key determinants in specifying the anteroposterior axis of most animals. The temporal and spatial expression of these genes requires the presence of large and complex cis-regulatory regions. The Abdominal-B Hox gene of the bithorax complex of Drosophila is regulated by several infraabdominal domains, which determine Abdominal-B expression in abdominal segments A5 to A9 (parasegments 10 to 14). Some of the infraabdominal domains have been characterized, including an infraabdominal-8 domain, which has been located 3' to the Abdominal-B transcription unit. We have analyzed the expression and mutant phenotype of a P-lacZ element inserted close to the Abdominal-B m origin of transcription and of derivatives of this transposon. Some of these derivatives represent a particular class of mutations in the bithorax complex, because they transform the eighth and ninth abdominal segments without affecting more anterior metameres. The analysis of these mutations and of transformants carrying sequences upstream the Abdominal-B m transcription unit has allowed us to define a new infraabdominal-8 regulatory region, located 5' to the Abdominal-B transcription unit, and has helped to characterize better the complex regulation of the Abdominal-B gene.