The role of SUMOylation during development.

During the development of multicellular organisms, transcriptional regulation plays an important role in the control of cell growth, differentiation and morphogenesis. SUMOylation is a reversible post-translational process involved in transcriptional regulation through the modification of transcription factors and through chromatin remodelling (either modifying chromatin remodelers or acting as a 'molecular glue' by promoting recruitment of chromatin regulators). SUMO modification results in changes in the activity, stability, interactions or localization of its substrates, which affects cellular processes such as cell cycle progression, DNA maintenance and repair or nucleocytoplasmic transport. This review focuses on the role of SUMO machinery and the modification of target proteins during embryonic development and organogenesis of animals, from invertebrates to mammals.

Scavenger receptors mediate the role of SUMO and Ftz-f1 in Drosophila steroidogenesis.

SUMOylation participates in ecdysteroid biosynthesis at the onset of metamorphosis in Drosophila melanogaster. Silencing the Drosophila SUMO homologue smt3 in the prothoracic gland leads to reduced lipid content, low ecdysone titers, and a block in the larval-pupal transition. Here we show that the SR-BI family of Scavenger Receptors mediates SUMO functions. Reduced levels of Snmp1 compromise lipid uptake in the prothoracic gland. In addition, overexpression of Snmp1 is able to recover lipid droplet levels in the smt3 knockdown prothoracic gland cells. Snmp1 expression depends on Ftz-f1 (an NR5A-type orphan nuclear receptor), the expression of which, in turn, depends on SUMO. Furthermore, we show by in vitro and in vivo experiments that Ftz-f1 is SUMOylated. RNAi-mediated knockdown of ftz-f1 phenocopies that of smt3 at the larval to pupal transition, thus Ftz-f1 is an interesting candidate to mediate some of the functions of SUMO at the onset of metamorphosis. Additionally, we demonstrate that the role of SUMOylation, Ftz-f1, and the Scavenger Receptors in lipid capture and mobilization is conserved in other steroidogenic tissues such as the follicle cells of the ovary. smt3 knockdown, as well as ftz-f1 or Scavenger knockdown, depleted the lipid content of the follicle cells, which could be rescued by Snmp1 overexpression. Therefore, our data provide new insights into the regulation of metamorphosis via lipid homeostasis, showing that Drosophila Smt3, Ftz-f1, and SR-BIs are part of a general mechanism for uptake of lipids such as cholesterol, required during development in steroidogenic tissues.

Drosophila Sal and Salr are transcriptional repressors.

The SALL (Spalt-like) family of zinc-finger transcription factors is conserved in metazoans. In Drosophila Sal (Spalt) and Salr (Spalt-related) control the expression of genes involved in wing and central nervous system development, including cell adhesion and cytoskeletal proteins. In humans, SALL mutations associate with congenital disorders such as the Townes-Brocks and Okihiro syndromes. Human and Drosophila SALL proteins are modified by SUMO (small ubiquitin-related modifier), which influences their subnuclear localization. In the present study, we have analysed the transcriptional activity of Drosophila Sall proteins in cultured cells. We show that both Sal and Salr act as transcriptional repressors in Drosophila cells where they repress transcription through an AT-rich sequence. Furthermore, using the UAS/Gal4 heterologous system, Drosophila Sal and Salr repress transcription in human cells. Under our experimental conditions, only in the case of Salr is the repression activity dependent on the HDAC (histone deacetylase) complex. This complex might interact with the C-terminal zinc fingers of Salr. We describe the differential subcellular localizations of Sal and Salr fragments and identify their repression domains. Surprisingly, both repressors also contain transcription activation domains. In addition, under our experimental conditions SUMOylation has differential effects on Sal and Salr repressor activity. Phylogenetic comparison between nematodes, insects and vertebrates identifies conserved peptide sequences that are presumably critical for SALL protein function.

Role of Epiprofin, a zinc-finger transcription factor, in limb development.

The formation and maintenance of the apical ectodermal ridge (AER) is critical for the outgrowth and patterning of the vertebrate limb. In the present work, we have investigated the role of Epiprofin (Epfn/Sp6), a member of the SP/KLF transcription factor family that is expressed in the limb ectoderm and the AER, during limb development. Epfn mutant mice have a defective autopod that shows mesoaxial syndactyly in the forelimb and synostosis (bony fusion) in the hindlimb and partial bidorsal digital tips. Epfn mutants also show a defect in the maturation of the AER that appears flat and broad, with a double ridge phenotype. By genetic analysis, we also show that Epfn is controlled by WNT/b-CATENIN signaling in the limb ectoderm. Since the less severe phenotypes of the conditional removal of b-catenin in the limb ectoderm strongly resemble the limb phenotype of Epfn mutants, we propose that EPFN very likely functions as a modulator of WNT signaling in the limb ectoderm.

Sumoylation modulates the activity of Spalt-like proteins during wing development in Drosophila.

The Spalt-like family of zinc finger transcription factors is conserved throughout evolution and is involved in fundamental processes during development and during embryonic stem cell maintenance. Although human SALL1 is modified by SUMO-1 in vitro, it is not known whether this post-translational modification plays a role in regulating the activity of this family of transcription factors. Here, we show that the Drosophila Spalt transcription factors are modified by sumoylation. This modification influences their nuclear localization and capacity to induce vein formation through the regulation of target genes during wing development. Furthermore, spalt genes interact genetically with the sumoylation machinery to repress vein formation in intervein regions and to attain the wing final size. Our results suggest a new level of regulation of Sall activity in vivo during animal development through post-translational modification by sumoylation. The evolutionary conservation of this family of transcription factors suggests a functional role for sumoylation in vertebrate Sall members.

Origin and Development of the Adipose Tissue, a Key Organ in Physiology and Disease.

Adipose tissue is a dynamic organ, well known for its function in energy storage and mobilization according to nutrient availability and body needs, in charge of keeping the energetic balance of the organism. During the last decades, adipose tissue has emerged as the largest endocrine organ in the human body, being able to secrete hormones as well as inflammatory molecules and having an important impact in multiple processes such as adipogenesis, metabolism and chronic inflammation. However, the cellular progenitors, development, homeostasis and metabolism of the different types of adipose tissue are not fully known. During the last decade, Drosophila melanogaster has demonstrated to be an excellent model to tackle some of the open questions in the field of metabolism and development of endocrine/metabolic organs. Discoveries ranged from new hormones regulating obesity to subcellular mechanisms that regulate lipogenesis and lipolysis. Here, we review the available evidences on the development, types and functions of adipose tissue in Drosophila and identify some gaps for future research. This may help to understand the cellular and molecular mechanism underlying the pathophysiology of this fascinating key tissue, contributing to establish this organ as a therapeutic target.

Glioblastoma invasion factor ODZ1 is induced by microenvironmental signals through activation of a Stat3-dependent transcriptional pathway.

We have previously shown that the transmembrane protein ODZ1 serves for glioblastoma (GBM) cells to invade the surrounding tissue through activation of RhoA/ROCK pathway. However, the transcriptional machinery used by GBM cells to regulate the expression of ODZ1 is unknown. Here we show that interaction with tumor microenvironment elements, mainly activated monocytes through IL-6 secretion, and the extracellular matrix protein fibronectin, induces the Stat3 transcriptional pathway and upregulates ODZ1 which results in GBM cell migration. This signaling route is abrogated by blocking the IL-6 receptor, inhibiting Jak kinases or knocking down Stat3. Furthermore, we have identified a Stat3 responsive element in the ODZ1 gene promoter, about 1 kb from the transcription start site. Luciferase-reporter assays confirmed that the promoter responds to the presence of monocytic cells and this activation is greatly reduced when the Stat3 site is mutated or following treatment with a neutralizing anti-IL-6 receptor antibody or transfecting GBM cells with a dominant negative variant of Stat3. Overall, we show that monocyte-secreted IL-6 and the extracellular matrix protein fibronectin activate the axis Stat3-ODZ1 and promote migration of GBM cells. This is the first described transcriptional mechanism used by tumor cells to promote the expression of the invasion factor ODZ1.

SUMO and ubiquitin modifications during steroid hormone synthesis and function.

Steroid hormones control many aspects of animal physiology and behaviour. They are highly regulated, among other mechanisms, by post-translational modifications of the transcription factors involved in their synthesis and response. In the present review, we will focus on the influence of SUMO (small ubiquitin-related modifier) and ubiquitin modifications on the function of transcription factors involved in adrenal cortex formation, steroidogenesis and the hormonal response.

SUMOylation in the control of cholesterol homeostasis.

SUMOylation-protein modification by the small ubiquitin-related modifier (SUMO)-affects several cellular processes by modulating the activity, stability, interactions or subcellular localization of a variety of substrates. SUMO modification is involved in most cellular processes required for the maintenance of metabolic homeostasis. Cholesterol is one of the main lipids required to preserve the correct cellular function, contributing to the composition of the plasma membrane and participating in transmembrane receptor signalling. Besides these functions, cholesterol is required for the synthesis of steroid hormones, bile acids, oxysterols and vitamin D. Cholesterol levels need to be tightly regulated: in excess, it is toxic to the cell, and the disruption of its homeostasis is associated with various disorders like atherosclerosis and cardiovascular diseases. This review focuses on the role of SUMO in the regulation of proteins involved in the metabolism of cholesterol.

Genetic manipulation of LKB1 elicits lethal metastatic prostate cancer.

Gene dosage is a key defining factor to understand cancer pathogenesis and progression, which requires the development of experimental models that aid better deconstruction of the disease. Here, we model an aggressive form of prostate cancer and show the unconventional association of LKB1 dosage to prostate tumorigenesis. Whereas loss of Lkb1 alone in the murine prostate epithelium was inconsequential for tumorigenesis, its combination with an oncogenic insult, illustrated by Pten heterozygosity, elicited lethal metastatic prostate cancer. Despite the low frequency of LKB1 deletion in patients, this event was significantly enriched in lung metastasis. Modeling the role of LKB1 in cellular systems revealed that the residual activity retained in a reported kinase-dead form, LKB1K78I, was sufficient to hamper tumor aggressiveness and metastatic dissemination. Our data suggest that prostate cells can function normally with low activity of LKB1, whereas its complete absence influences prostate cancer pathogenesis and dissemination.

Ecdysone promotes growth of imaginal discs through the regulation of Thor in D. melanogaster.

Animals have a determined species-specific body size that results from the combined action of hormones and signaling pathways regulating growth rate and duration. In Drosophila, the steroid hormone ecdysone controls developmental transitions, thereby regulating the duration of the growth period. Here we show that ecdysone promotes the growth of imaginal discs in mid-third instar larvae, since imaginal discs from larvae with reduced or no ecdysone synthesis are smaller than wild type due to smaller and fewer cells. We show that insulin-like peptides are produced and secreted normally in larvae with reduced ecdysone synthesis, and upstream components of insulin/insulin-like signaling are activated in their discs. Instead, ecdysone appears to regulate the growth of imaginal discs via Thor/4E-BP, a negative growth regulator downstream of the insulin/insulin-like growth factor/Tor pathways. Discs from larvae with reduced ecdysone synthesis have elevated levels of Thor, while mutations in Thor partially rescue their growth. The regulation of organ growth by ecdysone is evolutionarily conserved in hemimetabolous insects, as shown by our results obtained using Blattella germanica. In summary, our data provide new insights into the relationship between components of the insulin/insulin-like/Tor and ecdysone pathways in the control of organ growth.

Expression of the Scavenger Receptor Class B type I (SR-BI) family in Drosophila melanogaster.

In mammals, cholesterol is transformed into steroid hormones in the adrenal gland, the ovaries or the testes. The Scavenger Receptors Class B Type I (SR-BI) are membrane proteins that belong to the CD36 family and participate in the selective uptake of high density lipoprotein cholesteryl ester in the mammalian steroidogenic tissues. Fourteen members of the CD36 family have been identified in Diptera, although their expression patterns remain uncharacterized. Using in situ hybridization we have characterized the expression patterns of the fourteen SR-BIs in Drosophila melanogaster. We analyzed three different developmental larval stages prior to and during the peak of the insect steroid hormone ecdysone, which triggers the larval to pupal transition. We focused on the steroidogenic tissues, such as the prothoracic gland, the ovaries and the testes, and extended our analysis to non-steroidogenic tissues, such as the fat body, salivary glands, the gut, the gastric caeca or the central nervous system. Our results show highly regulated expression patterns, with three genes crq, pes and Snmp being upregulated in steroidogenic tissues at the onset of pupariation when steroidogenesis is crucial. This study underlines the importance of the transport of cholesterol and steroids in the process of ecdysone synthesis.

Functional analysis of the SUMOylation pathway in Drosophila.

SUMOylation, a reversible process used as a 'fine-tuning' mechanism to regulate the role of multiple proteins, is conserved throughout evolution. This post-translational modification affects several cellular processes by the modulation of subcellular localization, activity or stability of a variety of substrates. A growing number of proteins have been identified as targets for SUMOylation, although, for many of them, the role of SUMO conjugation on their function is unknown. The use of model systems might facilitate the study of SUMOylation implications in vivo. In the present paper, we have compiled what is known about SUMOylation in Drosophila melanogaster, where the use of genetics provides new insights on SUMOylation's biological roles.

Smt3 is required for Drosophila melanogaster metamorphosis.

Sumoylation, the covalent attachment of the small ubiquitin-related modifier SUMO to target proteins, regulates different cellular processes, although its role in the control of development remains unclear. We studied the role of sumoylation during Drosophila development by using RNAi to reduce smt3 mRNA levels in specific tissues. smt3 knockdown in the prothoracic gland, which controls key developmental processes through the synthesis and release of ecdysteroids, caused a 4-fold prolongation of larval life and completely blocked the transition from larval to pupal stages. The reduced ecdysteroid titer of smt3 knockdown compared with wild-type larvae explains this phenotype. In fact, after dietary administration of exogenous 20-hydroxyecdysone, knockdown larvae formed pupal cases. The phenotype is not due to massive cell death or degeneration of the prothoracic glands at the time when puparium formation should occur. Knockdown cells show alterations in expression levels and/or the subcellular localisation of enzymes and transcription factors involved in the regulation of ecdysteroid synthesis. In addition, they present reduced intracellular channels and a reduced content of lipid droplets and cholesterol, which could explain the deficit in steroidogenesis. In summary, our study indicates that Smt3 is required for the ecdysteroid synthesis pathway at the time of puparium formation.

Expression of the Drosophila melanogaster ATP synthase alpha subunit gene is regulated by a transcriptional element containing GAF and Adf-1 binding sites.

Mitochondrial biogenesis is a complex and highly regulated process that requires the controlled expression of hundreds of genes encoded in two separated genomes, namely the nuclear and mitochondrial genomes. To identify regulatory proteins involved in the transcriptional control of key nuclear-encoded mitochondrial genes, we have performed a detailed analysis of the promoter region of the alpha subunit of the Drosophila melanogaster F1F0 ATP synthase complex. Using transient transfection assays, we have identified a 56 bp cis-acting proximal regulatory region that contains binding sites for the GAGA factor and the alcohol dehydrogenase distal factor 1. In vitro mutagenesis revealed that both sites are functional, and phylogenetic footprinting showed that they are conserved in other Drosophila species and in Anopheles gambiae. The 56 bp region has regulatory enhancer properties and strongly activates heterologous promoters in an orientation-independent manner. In addition, Northern blot and RT-PCR analysis identified two alpha-F1-ATPase mRNAs that differ in the length of the 3' untranslated region due to the selection of alternative polyadenylation sites.

Expression and regulation of eHAND during limb development.

eHAND is a bHLH transcription factor with important functions during embryogenesis. Here, we report that eHAND has a dynamic pattern of expression during limb development. In chick embryos, eHAND expression is first observed in the ventral mesoderm of the emerging limb. Its expression is then restricted to an anteroventral area of mesoderm at mid-level in the proximodistal axis. At later stages, expression is observed in the autopod encompassing the ventral tendons of the digits. In mouse embryos, only the anteroventral domain of expression is conserved, the early ventral expression not being detectable and the late pattern of expression differing clearly from that in the chick. A constant feature of all areas of expression is their ventral and anterior localization. Respecification of the anterior mesoderm as occurs secondarily to Sonic hedgehog (SHH) or retinoic acid application to the anterior border leads to down-regulation of eHAND expression. Accordingly, eHAND expression is not detectable in talpid(2) mutant limbs, which are considered to be posteriorized limbs. However, eHAND expression is little modified in oligozeugodactyly, a chick mutant that lacks Shh signaling in the limb but retains certain anteroposterior polarity. Interestingly, eHAND expression is also linked to the ventral identity of the mesoderm and is repressed by the dorsal ectoderm. It is also positively regulated by bone morphogenetic protein signaling, which is also known to participate in dorsoventral patterning. We suggest that eHAND expression may be related to the anteroventral identity of the mesoderm. However, in overexpression experiments using retroviral vectors, only a low percentage of cases (5%) showed phenotypic alterations, consisting of a duplication of digit 2.

Pax6 regulates regional development and neuronal migration in the cerebral cortex.

Mutations in the Pax6 gene disrupt telencephalic development, resulting in a thin cortical plate, expansion of proliferative layers, and the absence of the olfactory bulb. The primary defect in the neuronal cell population of the developing cerebral cortex was analysed by using mouse chimeras containing a mixture of wild-type and Pax6-deficient cells. The chimeric analysis shows that Pax6 influences cellular activity throughout corticogenesis. At early stages, Pax6-deficient and wildtype cells segregate into exclusive patches, indicating an inability of different cell genotypes to interact. At later stages, cells are sorted further based on telencephalic domains. Pax6-deficient cells are specifically reduced in the mediocaudal domain of the dorsal telencephalon, indicating a role in regionalization. In addition, Pax6 regulates the process of radial migration of neuronal precursors. Loss of Pax6 particularly affects movement of neuronal precursors at the subventricular zone/intermediate zone boundary at a transitional migratory phase essential for entry into the intermediate zone. We suggest that the primary role of Pax6 is the continual regulation of cell surface properties responsible for both cellular identity and radial migration, defects of which cause regional cell sorting and abnormalities of migration in chimeras.