LCoR (ligand-dependent corepressor) is a transcriptional corepressor widely expressed in fetal and adult tissues that is recruited to agonist-bound nuclear receptors through a single LXXLL motif. LCoR binding to estrogen receptor alpha depends in part on residues in the coactivator binding pocket distinct from those bound by TIF-2. Repression by LCoR is abolished by histone deacetylase inhibitor trichostatin A in a receptor-dependent fashion, indicating HDAC-dependent and -independent modes of action. LCoR binds directly to specific HDACs in vitro and in vivo. Moreover, LCoR functions by recruiting C-terminal binding protein corepressors through two consensus binding motifs and colocalizes with CtBPs in the nucleus. LCoR represents a class of corepressor that attenuates agonist-activated nuclear receptor signaling by multiple mechanisms.

The zona pellucida domain (ZPD) defines a conserved family of membrane-anchored matrix proteins that are, as yet, poorly characterized with respect to their functions during development. Using genetic approaches in flies, we show here that a set of eight ZPD proteins is required for the localized reorganization of embryonic epidermal cells during morphogenesis. Despite varying degrees of sequence conservation, these ZPD proteins exert specific and nonredundant functions in the remodeling of epidermal cell shape. Each one accumulates in a restricted subregion of the apical compartment, where it organizes local interactions between the membrane and the extracellular matrix. In addition, ZPD proteins are required to sculpture the actin-rich cell extensions and maintain appropriate organization of the apical compartment. These results on ZPD proteins therefore reveal a functional subcompartmentalization of the apical membrane and its role in the polarized control of epithelial cell shape during development.

It is well established that developmental programs act during embryogenesis to determine animal morphogenesis. How these developmental cues produce specific cell shape during morphogenesis, however, has remained elusive. We addressed this question by studying the morphological differentiation of the Drosophila epidermis, governed by a well-known circuit of regulators leading to a stereotyped pattern of smooth cells and cells forming actin-rich extensions (trichomes). It was shown that the transcription factor Shavenbaby plays a pivotal role in the formation of trichomes and underlies all examined cases of the evolutionary diversification of their pattern. To gain insight into the mechanisms of morphological differentiation, we sought to identify shavenbaby's downstream targets. We show here that Shavenbaby controls epidermal cell shape, through the transcriptional activation of different classes of cellular effectors, directly contributing to the organization of actin filaments, regulation of the extracellular matrix, and modification of the cuticle. Individual inactivation of shavenbaby's targets produces distinct trichome defects and only their simultaneous inactivation prevent trichome formation. Our data show that shavenbaby governs an evolutionarily conserved developmental module consisting of a set of genes collectively responsible for trichome formation, shedding new light on molecular mechanisms acting during morphogenesis and the way they can influence evolution of animal forms.

BackgroundDevelopmental programs are implemented by regulatory interactions between Transcription Factors (TFs) and their target genes, which remain poorly understood. While recent studies have focused on regulatory cascades of TFs that govern early development, little is known about how the ultimate effectors of cell differentiation are selected and controlled. We addressed this question during late Drosophila embryogenesis, when the finely tuned expression of the TF Ovo/Shavenbaby (Svb) triggers the morphological differentiation of epidermal trichomes.ResultsWe defined a sizeable set of genes downstream of Svb and used in vivo assays to delineate 14 enhancers driving their specific expression in trichome cells. Coupling computational modeling to functional dissection, we investigated the regulatory logic of these enhancers. Extending the repertoire of epidermal effectors using genome-wide approaches showed that the regulatory models learned from this first sample are representative of the whole set of trichome enhancers. These enhancers harbor remarkable features with respect to their functional architectures, including a weak or non-existent clustering of Svb binding sites. The in vivo function of each site relies on its intimate context, notably the flanking nucleotides. Two additional cis-regulatory motifs, present in a broad diversity of composition and positioning among trichome enhancers, critically contribute to enhancer activity.ConclusionsOur results show that Svb directly regulates a large set of terminal effectors of the remodeling of epidermal cells. Further, these data reveal that trichome formation is underpinned by unexpectedly diverse modes of regulation, providing fresh insights into the functional architecture of enhancers governing a terminal differentiation program.

Alveolar epithelial cells (AEC) are directly exposed to high alveolar O(2) tension. Many pulmonary disorders are associated with a decrease in alveolar O(2) tension and AEC need to develop adaptative mechanisms to cope with O(2) deprivation. Under hypoxia, because of inhibition of oxidative phosphorylation, adenosine triphosphate supply is dependent on the ability of cells to increase anaerobic glycolysis. In this study we show that under hypoxia, primary rat AEC maintained their energy status close to that of normoxic cells through increasing anaerobic glycolysis. We therefore examined the effect of hypoxia on glucose transport and evaluated the mechanisms of this regulation. Hypoxia induced a stimulation of Na-independent glucose transport, as shown by the increase in 2-deoxy-D-glucose (DG) uptake. This increase was dependent on time and O(2) concentration: maximal at 0% O(2) for 18 h, and reversible after hypoxic cells were allowed to recover in normoxia. Concomitantly, exposure of AEC to hypoxia (18 h 0% O(2)) induced a 3-fold increase of glucose transporter GLUT1 at both protein and messenger RNA (mRNA) levels. To determine whether the increase in GLUT1 mRNA level was dependent on O(2) deprivation per se or resulted from decrease of oxidative phosphorylation, we examined in normoxic cells the effects of cobalt chloride and Na azide, respectively. Cobalt chloride (100 microM) and Na azide (1 mM) increased both mRNA levels and DG uptake, mimicking the effect of hypoxia. Electrophoretic mobility shift assays revealed a hypoxic and a cobalt chloride induction of a hypoxia-inducible factor (HIF) that bound to the sequence of nucleotides, corresponding to a hypoxia-inducible element upstream of the GLUT1 gene. AEC also expressed this factor under nonhypoxic conditions. Together, our results demonstrate that AEC increased glucose transport in response to hypoxia by regulating GLUT1 gene-encoding protein. This regulation likely occurred at the transcriptional level through the activation of an HIF, the nature of which remains to be elucidated.

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Ligand-dependent corepressor LCoR was identified as a protein that interacts with the estrogen receptor ␣ (ER␣) ligand binding domain in a hormone-dependent manner. LCoR also interacts directly with histone deacetylase 3 (HDAC3) and HDAC6. Notably, HDAC6 has emerged as a marker of breast cancer prognosis. However, although HDAC3 is nuclear, HDAC6 is cytoplasmic in many cells. We found that HDAC6 is partially nuclear in estrogen-responsive MCF7 cells, colocalizes with LCoR, represses transactivation of estrogen-inducible reporter genes, and augments corepression by LCoR. In contrast, no repression was observed upon HDAC6 expression in COS7 cells, where it is exclusively cytoplasmic. LCoR binds to HDAC6 in vitro via a central domain, and repression by LCoR mutants lacking this domain was attenuated. Kinetic chromatin immunoprecipitation assays revealed hormone-dependent recruitment of LCoR to promoters of ER␣-induced target genes in synchrony with ER␣. HDAC6 was also recruited to these promoters, and repeat chromatin immunoprecipitation experiments confirmed the corecruitment of LCoR with ER␣ and with HDAC6. Remarkably, however, although we find evidence for corecruitment of LCoR and ER␣ on genes repressed by the receptor, LCoR and HDAC6 failed to coimmunoprecipitate, suggesting that they are part of distinct complexes on these genes. Although small interfering RNA-mediated knockdown of LCoR or HDAC6 augmented expression of an estrogen-sensitive reporter gene in MCF7 cells, unexpectedly their ablation led to reduced expression of some endogenous estrogen target genes. Taken together, these data establish that HDAC6 can function as a cofactor of LCoR but suggest that they may act in enhance expressing some target genes.Nuclear receptors are ligand-regulated transcription factors whose activities are controlled by a variety of lipophilic extracellular signals, including steroid and thyroid hormones, metabolites of vitamins A (retinoids) and D (1, 2). DNA-bound nuclear receptors regulate transcription by recruiting complexes of coregulatory proteins, classified as coactivators or corepressors depending on whether they act to stimulate or repress transcription (2-4). Many coactivators interact with receptors through signature LXXLL motifs, known as NR boxes, which are oriented within a hydrophobic pocket of agonist-bound receptor ligand binding domains (5). Several coactivators or their associated cofactors possess histone acetyltransferase activity, which essentially caps positively charged lysine residues and loosens their association with DNA, facilitating chromatin remodeling and subsequent access of the transcriptional machinery to promoters.Nuclear receptor corepressors NCoR 7 and SMRT were isolated as factors that interacted with hormone-free but not hormone-bound thyroid and retinoid receptors (6, 7). They bind to receptor ligand binding domains through extended LXXX-IXXX(L/I) motifs known as CoRNR boxes (8, 9) and recruit multiprotein complexes implicated in transcriptional repression and histone deacetylatio...

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