Specific mutations in the ligand binding domain selectively abolish the silencing function of human thyroid hormone receptor beta.

Although most nuclear hormone receptors are ligand-dependent transcriptional activators, certain members of this superfamily, such as thyroid hormone receptor (TR) and retinoic acid receptor (RAR), are involved in transcriptional repression. The silencing function of these receptors has been localized to the ligand binding domain (LBD). Previously, we demonstrated that overexpression of either the entire LBD or only the N-terminal region of the LBD (amino acids 168-259) is able to inhibit the silencing activity of TR. From this result we postulated the existence of a limiting factor (corepressor) that is necessary for TR silencing activity. To support this hypothesis, we identified amino acids in the N-terminal region of the LBD of TR that are important for the corepressor interaction and for the silencing function of TR. The silencing activity of TR was unaffected by overexpression of the LBD of mutant TR (V174A/D177A), suggesting that valine at position 174 and/or aspartic acid at position 177 are important for corepressor interaction. This mutant receptor protein, V174/D177, also lost the ability to silence target genes, suggesting that these amino acids are important for silencing function. Control experiments indicate that this mutant TR maintains its wild-type hormone binding and transactivation functions. These findings further strengthen the idea that the N-terminal region of the LBD of TR interacts with a putative corepressor protein(s) to achieve silencing of basal gene transcription.

Retinoid X receptor alpha forms tetramers in solution.

Protein-protein interactions allow the retinoid X receptor (RXR) to bind to cognate DNA as a homo- or a heterodimer and to participate in mediating the effects of a variety of hormones on gene transcription. Here we report a systematic study of the oligomeric state of RXR in the absence of a DNA template. We have used electrophoresis under nondenaturing conditions and chemical crosslinking to show that in solution, RXR alpha forms homodimers as well as homotetramers. The dissociation constants governing dimer and tetramer formation were estimated by fluorescence anisotropy studies. The results indicate that RXR tetramers are formed with a high affinity and that at protein concentrations higher than about 70 nM, tetramers will constitute the predominant species. Tetramer formation may provide an additional level of the regulation of gene transcription mediated by RXRs.

Tyrosine phosphorylation and activation of STAT5, STAT3, and Janus kinases by interleukins 2 and 15.

The cytokines interleukin 2 (IL-2) and IL-15 have similar biological effects on T cells and bind common hematopoietin receptor subunits. Pathways that involve Janus kinases (JAKs) and signal transducers and activators of transcription (STATs) have been shown to be important for hematopoietin receptor signaling. In this study we identify the STAT proteins activated by IL-2 and IL-15 in human T cells. IL-2 and IL-15 rapidly induced the tyrosine phosphorylation of STAT3 and STAT5, and DNA-binding complexes containing STAT3 and STAT5 were rapidly activated by these cytokines in T cells. IL-4 induced tyrosine phosphorylation and activation of STAT3 but not STAT5. JAK1 and JAK3 were tyrosine-phosphorylated in response to IL-2 and IL-15. Hence, the JAK and STAT molecules that are activated in response to IL-2 and IL-15 are similar but differ from those induced by IL-4. These observations identify the STAT proteins activated by IL-2 and IL-15 and therefore define signaling pathways by which these T-cell growth factors may regulate gene transcription.

Identification of human granzyme B promoter regulatory elements interacting with activated T-cell-specific proteins: implication of Ikaros and CBF binding sites in promoter activation.

Granzyme B serine protease is found in the granules of activated cytotoxic T cells and in natural and lymphokine-activated killer cells. This protease plays a critical role in the rapid induction of target cell DNA fragmentation. The DNA regulatory elements that are responsible for the specificity of granzyme B gene transcription in activated T-cells reside between nt -148 and +60 (relative to the transcription start point at +1) of the human granzyme B gene promoter. This region contains binding sites for the transcription factors Ikaros, CBF, Ets, and AP-1. Mutational analysis of the human granzyme B promoter reveals that the Ikaros binding site (-143 to -114) and the AP-1/CBF binding site (-103 to -77) are essential for the activation of transcription in phytohemagglutinin-activated peripheral blood lymphocytes, whereas mutation of the Ets binding site does not affect promoter activity in these cells.

Activation of Stat5 by interleukin 2 requires a carboxyl-terminal region of the interleukin 2 receptor beta chain but is not essential for the proliferative signal transmission.

The high-affinity interleukin 2 (IL-2) receptor (IL-2R) consists of three subunits: the IL-2R alpha, IL-2R beta c, and IL-2R gamma c chains. Two members of the Janus kinase family, Jak1 and Jak3, are associated with IL-2R beta c and IL-2R gamma c, respectively, and they are activated upon IL-2 stimulation. The cytokine-mediated Jak kinase activation usually results in the activation of a family of latent transcription factors termed Stat (signal transducer and activator of transcription) proteins. Recently, the IL-2-induced Stat protein was purified from human lymphocytes and found to be the homologue of sheep Stat5/mammary gland factor. We demonstrate that the human Stat5 is activated by IL-2 and that Jak3 is required for the efficient activation. The cytoplasmic region of the IL-2R beta c chain required for activation of Stat5 is mapped within the carboxyl-terminal 147 amino acids. On the other hand, this region is not essential for IL-2-induced cell proliferation.

Continuous activation of gp130, a signal-transducing receptor component for interleukin 6-related cytokines, causes myocardial hypertrophy in mice.

To investigate the physiological roles of gp130 in detail and to determine the pathological consequence of abnormal activation of gp130, transgenic mice having continuously activated gp130 were created. This was carried out by mating mice from interleukin 6 (IL-6) and IL-6 receptor (IL-6R) transgenic lines. Offspring overexpressing both IL-6 and IL-6R showed constitutive tyrosine phosphorylation of gp130 and a downstream signaling molecule, acute phase response factor/signal transducer and activator of transcription 3. Surprisingly, the distinguishing feature of such offspring was hypertrophy of ventricular myocardium and consequent thickened ventricular walls of the heart, where gp130 is also expressed, in adulthood. Transgenic mice overexpressing either IL-6 or IL-6R alone did not show detectable myocardial abnormalities. Neonatal heart muscle cells from normal mice, when cultured in vitro, enlarged in response to a combination of IL-6 and a soluble form of IL-6R. The results suggest that activation of the gp130 signaling pathways leads to cardiac hypertrophy and that these signals might be involved in physiological regulation of myocardium.

Surface signaling in pathogenesis.

Surface signaling plays a major role in fungal infection. Topographical features of the plant surface and chemicals on the surface can trigger germination of fungal spores and differentiation of the germ tubes into appressoria. Ethylene, the fruit-ripening hormone, triggers germination of conidia, branching of hyphae, and multiple appressoria formation in Colletotrichum, thus allowing fungi to time their infection to coincide with ripening of the host. Genes uniquely expressed during appressoria formation induced by topography and surface chemicals have been isolated. Disruption of some of them has been shown to decrease virulence on the hosts. Penetration of the cuticle by the fungus is assisted by fungal cutinase secreted at the penetration structure of the fungus. Disruption of cutinase gene in Fusarium solani pisi drastically decreased its virulence. Small amounts of cutinase carried by spores of virulent pathogens, upon contact with plant surface, release small amounts of cutin monomers that trigger cutinase gene expression. The promoter elements involved in this process in F. solani pisi were identified, and transcription factors that bind these elements were cloned. One of them, cutinase transcription factor 1, expressed in Escherichia coli, is phosphorylated. Several protein kinases from F. solani pisi were cloned. The kinase involved in phosphorylation of specific transcription factors and the precise role of phosphorylation in regulating cutinase gene transcription remain to be elucidated.

Glycosylation of the c-Myc transactivation domain.

O-linked N-acetylglucosamine (O-GlcNAc) is an abundant and dynamic posttranslational modification composed of a single monosaccharide, GlcNAc, glycosidically composed of a single monosaccharide, GlcNAc, glycosidically linked to the side-chain hydroxyl of serine or threonine residues. Although O-GlcNAc occurs on a myriad of nuclear and cytoplasmic proteins, only a few have thus far been identified. These O-GlcNAc-bearing proteins are also modified by phosphorylation and form reversible multimeric complexes. Here we present evidence for O-GlcNAc glycosylation of the oncoprotein c-Myc, a helix-loop-helix/leucine zipper phosphoprotein that heterodimerizes with Max and participates in the regulation of gene transcription in normal and neoplastic cells. O-GlcNAc modification of c-Myc is shown by three different methods: (i) demonstration of lectin binding to in vitro translated protein using a protein-protein interaction mobility-shift assay; (ii) glycosidase or glycosyltransferase treatment of in vitro translated protein analyzed by lectin affinity chromatography; and (iii) direct characterization of the sugar moieties on purified recombinant protein overexpressed in either insect cells or Chinese hamster ovary cells. Analyses of serial deletion mutants of c-Myc further suggest that the O-GlcNAc site(s) are located within or near the N-terminal transcription activation/malignant transformation domain, a region where mutations of c-Myc that are frequently found in Burkitt and AIDS-related lymphomas cluster.

Investigação de mecanismos genéticos e epigenéticos em distúrbios do crescimento humano

Parcela considerável de pacientes com distúrbios de crescimento não têm a causa de seus quadros clínicos estabelecida, incluindo aproximadamente 50% dos pacientes com diagnóstico clínico de síndrome de Silver−Russell (SRS) e 10-20% dos pacientes com síndrome de Beckwith-Wiedemann (BWS). O objetivo deste estudo foi investigar as causas genéticas e epigenéticas de distúrbios de crescimento, de etiologia desconhecida, numa contribuição para o entendimento de mecanismos que regulam o crescimento. O estudo compreendeu: (1) a investigação de microdesequilíbrios cromossômicos, por aCGH; (2) a análise do perfil de expressão alelo-específica de genes sujeitos a imprinting (IG), por pirossequenciamento (PSQ) ou sequenciamento de Sanger; (3) a investigação do padrão de metilação global em pacientes com restrição de crescimento, utilizando microarray de metilação. A casuística constituiu-se de 41 pacientes não aparentados, com distúrbios de crescimento, de etiologia desconhecida: (1) 25, com hipótese diagnóstica de SRS; (2) seis, com restrição de crescimento intrauterino e peso ao nascimento abaixo do 10º percentil, associados a outros sinais clínicos; (3) sete, com hipótese diagnóstica de BWS; e (4) três, com macrossomia pré-natal ou pós-natal, associada a outros sinais. A investigação de microdesequilíbrios cromossômicos foi realizada em 40 pacientes. Foram detectadas 58 variantes raras em 30/40 pacientes (75%): 40 foram consideradas provavelmente benignas (18 pacientes, 45%), 12, com efeito patogênico desconhecido (11 pacientes, 27,5%), duas, provavelmente patogênicas (um paciente, 2,5%) e quatro, patogênicas (três pacientes, 7,5%). Essas frequências são comparáveis àquelas descritas em estudos que investigaram CNV em grupos de pacientes com distúrbios de crescimento e outras alterações congênitas, incluindo SRS, e mostram a importância da investigação de microdesequilíbrios cromossômicos nesses pacientes. A diversidade dos microdesequilíbrios cromossômicos identificados é reflexo da heterogeneidade clínica das casuísticas. Neste estudo, muitos dos pacientes com hipótese diagnóstica de SRS e BWS apresentavam sinais clínicos atípicos, explicando a ausência neles das alterações (epi)genéticas que causam essas síndromes. A identificação de CNV características de outras síndromes reflete a sobreposição de sinais clínicos com BWS e SRS. A análise do perfil de expressão alelo-específica de IG foi realizada em um subgrupo de 18 pacientes com restrição de crescimento. Trinta IG com função em proliferação celular, crescimento fetal ou neurodesenvolvimento foram inicialmente selecionados. Após seleção de SNP transcritos com alta frequência na população, genotipagem de pacientes, genitores e indivíduos controle, determinação da expressão dos IG em sangue periférico e seu padrão de expressão (mono ou bialélico), 13 IG, expressos no sangue, tiveram a expressão alelo-específica avaliada, sete deles por PSQ e seis por sequenciamento de Sanger. Alterações no perfil de expressão de dois genes, de expressão normalmente paterna, foram detectadas em 4/18 pacientes (22%). Este estudo é o primeiro a utilizar pirossequenciamento e sequenciamento de Sanger na avaliação do perfil de expressão alelo-específica de IG, em pacientes com restrição de crescimento. Apesar de terem limitações, ambas as técnicas mostraram-se robustas e revelaram alterações de expressão alélica interessantes; entretanto, a relação dessas alterações com o quadro clínico dos pacientes permanece por esclarecer. A investigação da metilação global do DNA foi realizada em subgrupo de 21 pacientes com restrição de crescimento e em 24 indivíduos controle. Dois tipos de análise foram realizados: (1) análise diferencial de grupo e (2) análise diferencial individual. Na primeira análise, em que foi comparado o padrão de metilação do grupo de pacientes com quadro clínico sugestivo de SRS (n=16) com o do grupo controle (n=24), não houve indicação de hipo ou hipermetilação global no grupo SRS. Na segunda análise, foi comparado o padrão de metilação de cada um dos 21 pacientes com restrição de crescimento e dos 24 indivíduos controle, com o padrão de metilação do grupo controle. O número médio de CpG hipermetilados e de segmentos diferencialmente metilados (SDM) foi significativamente maior nos pacientes. Foram identificados 82 SDM hipermetilados, estando 57 associados a gene(s) (69,5%), em 16 pacientes, e 51 SDM hipometilados, 41 deles associados a gene(s) (80,4%), em 10 pacientes. A análise de ontologia genética dos 61 genes associados aos SDM hipo ou hipermetilados nos pacientes destacou genes que atuam no desenvolvimento e na morfogênese do sistema esquelético e de órgãos fetais, e na regulação da transcrição gênica e de processos metabólicos. Alterações de metilação em genes que atuam em processos de proliferação e diferenciação celulares e crescimento foram identificadas em 9/20 dos pacientes (45%), sugerindo implicação clínica. Não foi detectada alteração epigenética comum aos pacientes com diagnóstico clínico de SRS, explicável provavelmente pela heterogeneidade clínica. A investigação de metilação global, utilizando microarray, produziu novos dados que podem contribuir para a compreensão de mecanismos moleculares que influenciam o crescimento pré- e pós-natal. Na translocação aparentemente equilibrada - t(5;6)(q35.2;p22.3)dn, detectada em paciente com suspeita clínica de SRS, a interrupção de um gene, pela quebra no cromossomo 6, pode ser a causa do quadro clínico; alternativamente, a translocação pode ter impactado a regulação de genes de desenvolvimento localizados próximos aos pontos de quebra. A análise de expressão em sangue periférico mostrou que os níveis de cDNA do gene, interrompido pelo ponto de quebra da translocação, estavam reduzidos à metade. Além de sinais típicos da SRS, a paciente apresentava algumas características clínicas sugestivas de displasia cleidocraniana. Assim, a translocação t(5;6) pode ter alterado a interação de genes de desenvolvimento e seus elementos reguladores, levando à desregulação de sua expressão espaço-temporal, e resultando num fenótipo atípico, com características sobrepostas de mais de uma síndrome genética

Activity-Regulated microRNAs: Modulators of Synaptic Growth at the Drosophila Neuromuscular Junction

It is well established that long-term changes in synaptic structure and function are mediated by rapid activity-dependent gene transcription and new protein synthesis. A growing body of evidence supports the involvement of the microRNA (miRNA) pathway in these processes. We have used the Drosophila neuromuscular junction (NMJ) as a model synapse to characterize activity-regulated miRNAs and their important mRNA targets. Here, we have identified five neuronal miRNAs (miRs-1, -8, -289, -314, and -958) that are significantly downregulated in response to neuronal activity. Furthermore we have discovered that neuronal misexpression of three of these miRNAs (miR-8, -289, and -958) is capable of suppressing new synaptic growth in response to activity suggesting that these miRNAs control the translation of biologically relevant target mRNAs. Putative targets of the activity-regulated miRNAs-8 and -289 are significantly enriched in clusters mapping to functional processes including axon development, pathfinding, and axon growth. We demonstrate that activity-regulated miR-8 regulates the 3'UTR of wingless, a presynaptic regulatory protein involved in the process of activity-dependent axon terminal growth. Additionally, we show that the 3'UTR of the protein tyrosine phosophatase leukocyte antengen related (lar), a protein required for axon guidance and synaptic growth, is regulated by activity-regulated miRNAs-8, -289, and -958 in vitro. Both wg and lar were identified as relevant putative targets for co-regulation based through our functional cluster analysis. One putative target of miR-289 is the Ca2+/calmodulin-dependent protein kinase II (CamKII). While CamKII is not predicted as a target for co-regulation by multiple activity-regulated miRNAs we identified it as an especially pertinent target for analysis in our system for two reasons. First, CamKII has an extremely well characterized role in postsynaptic plasticity, but its presynaptic role is less well characterized and bears further analysis. Second, local translation of CamKII mRNA is regulated in part by the miRNA pathway in an activity-dependent manner in dendrites. We find that the CamKII 3'UTR is regulated by miR-289 in-vitro and this regulation is alleviated by mutating the `seed region' of the miR-289 binding site within the CamKII 3'UTR. Furthermore, we demonstrate a requirement for local translation of CamKII in motoneurons in the process of activity-regulated axon terminal growth.

Expression and cellular localization of the voltage-gated calcium channel α2δ3 in the rodent retina

High-voltage-activated calcium channels are hetero-oligomeric protein complexes that mediate multiple cellular processes, including the influx of extracellular Ca2+, neurotransmitter release, gene transcription, and synaptic plasticity. These channels consist of a primary α1 pore-forming subunit, which is associated with an extracellular α2δ subunit and an intracellular β auxiliary subunit, which alter the gating properties and trafficking of the calcium channel. The cellular localization of the α2δ3 subunit in the mouse and rat retina is unknown. In this study using RT-PCR, a single band at ∼305 bp corresponding to the predicted size of the α2δ3 subunit fragment was found in mouse and rat retina and brain homogenates. Western blotting of rodent retina and brain homogenates showed a single 123-kDa band. Immunohistochemistry with an affinity-purified antibody to the α2δ3 subunit revealed immunoreactive cell bodies in the ganglion cell layer and inner nuclear layer and immunoreactive processes in the inner plexiform layer and the outer plexiform layer. α2δ3 immunoreactivity was localized to multiple cell types, including ganglion, amacrine, and bipolar cells and photoreceptors, but not horizontal cells. The expression of the α2δ3 calcium channel subunit to multiple cell types suggests that this subunit participates widely in Ca-channel-mediated signaling in the retina.

A macrophage colony-stimulating factor receptor-green fluorescent protein transgene is expressed throughout the mononuclear phagocyte system of the mouse

The c-fms gene encodes the receptor for macrophage colony-stimulating factor (CSF-1). The gene is expressed selectively in the macrophage and trophoblast cell lineages. Previous studies have indicated that sequences in intron 2 control transcript elongation in tissue-specific and regulated expression of c-fms. In humans, an alternative promoter was implicated in expression of the gene in trophoblasts. We show that in mice, c-fms transcripts in trophoblasts initiate from multiple points within the 2-kilobase (kb) region flanking the first coding exon. A reporter gene construct containing 3.5 kb of 5' flanking sequence and the down-stream intron 2 directed expression of enhanced green fluorescent protein (EGFP) to both trophoblasts and macrophages. EGFP was detected in trophoblasts from the earliest stage of implantation examined at embryonic day 7.5. During embryonic development, EGFP highlighted the large numbers of c-fms-positive macrophages, including those that originate from the yolk sac. In adult mice, EGFP location Was consistent with known F4/80-positive macrophage populations, including Langerhans cells of the skin, and permitted convenient sorting of isolated tissue macrophages from disaggregated tissue. Expression of EGFP in transgenic mice was dependent on intron 2 as no lines with detectable EGFP expression were obtained where either all of intron 2 or a conserved enhancer element FIRE (the Fms intronic regulatory element) was removed. We have therefore defined the elements required to generate myeloid- and trophoblast-specific transgenes as well as a model system for the study of mononuclear phagocyte development and function. (C) 2003 by The American Society of Hematology.

Histone-deacetylase inhibitors for the treatment of cancer

Histone deacetylase inhibitors (HDACi) are a promising new class of chemotherapeutic drug currently in early phase clinical trials. A large number of structurally diverse HDACi have been purified or synthesised that mostly inhibit the activity of all eleven class I and II HDACs. While these agents demonstrate many features required for anti-cancer activity such as low toxicity against normal cells and an ability to inhibit tumor cell growth and survival at nanomolar concentrations, their mechanisms of action are largely unknown. Initially, a model was proposed whereby HDACi-mediated transactivation of a specific gene or set of genes was responsible for the inhibition of cell cycle progression or induction of apoptosis. Given that HDACs can regulate the activity of a number of nonhistone proteins and that histone acetylation is important for events such as DNA replication and mitosis that do not directly involve gene transcription, it appears that the initial mechanistic model for HDACi may have been too simple. Herein, we provide an update on the transcription-dependent and - independent events that may be important for the anti-tumor activities of HDACi and discuss the use of these compounds in combination with other chemotherapeutic drugs.

A conformationally sensitive GHR [growth hormone (GH) receptor] antibody: Impact on GH signaling and GHR proteolysis

The GH receptor (GHR) mediates metabolic and somatogenic actions of GH. Its extracellular domain (ECD; residues 1-246) has two subdomains, each with seven beta strands organized into two antiparallel beta sheets, connected by a short hinge region. Most of the ECD residues involved in GH binding reside in subdomain 1, whereas subdomain 2 harbors a dimerization interface between GHR dimers that alters conformation in response to GH. A regulated GHR metalloprotease cleavage site is in the membrane-proximal stem region of subdomain 2. We have identified a monoclonal anti-ECD antibody, anti-GHR(ext-mAb), which recognizes the rabbit and human GHRs by immunoprecipitation, but less so after GH treatment. By immunoblotting and immunoprecipitation, anti-GHR(ext-mAb) recognized a glutathione-S-transferase (GST) fusion incorporating subdomain 2, but not one including subdomain 1. In transient transfection experiments, anti-GHR(ext-mAb) failed to recognize by immunoprecipitation a previously characterized dimerization interface mutant GHR that is incompetent for signaling. In signaling experiments, brief pretreatment of GH-responsive human fibrosarcoma cells with anti-GHR(ext-mAb) dramatically inhibited GH-induced Janus kinase 2 and signal transducer and activator of transcription 5 tyrosine phosphorylation and prevented GH-induced GHR disulfide linkage (a reflection of GH-induced conformational changes). In contrast, anti-GHR(ext-mAb) only partially inhibited radiolabeled GH binding, suggesting its effects on signaling were not simply via inhibition of binding. Furthermore, anti-GHR(ext-mAb) prevented phorbol ester-stimulated GHR proteolysis, but GHR cleavage site mutants were normally recognized by the antibody, indicating that the stem region cleavage site is not a direct epitope. A Fab fragment of anti-GHR(ext-mAb) inhibited GH-induced GHR disulfide linkage and signaling, as well as phorbol ester-induced GHR proteolysis, in a fashion similar to the intact antibody. Thus, our findings suggest that anti-GHR(ext-mAb) has promise as a GH antagonist and as a tool in studies of conformational changes required for GHR activation.

Functional dissection of the Suppressor of UnderReplication protein of Drosophila melanogaster: identification of domains influencing chromosome binding and DNA replication

The Suppressor of UnderReplication (SuUR) gene controls the DNA underreplication in intercalary and pericentric heterochromatin of Drosophila melanogaster salivary gland polytene chromosomes. In the present work, we investigate the functional importance of different regions of the SUUR protein by expressing truncations of the protein in an UAS-GAL4 system. We find that SUUR has at least two separate chromosome-binding regions that are able to recognize intercalary and pericentric heterochromatin specifically. The C-terminal part controls DNA underreplication in intercalary heterochromatin and partially in pericentric heterochromatin regions. The C-terminal half of SUUR suppresses endoreplication when ectopically expressed in the salivary gland. Ectopic expression of the N-terminal fragments of SUUR depletes endogenous SUUR from polytene chromosomes, causes the SuUR(-) stopphenotype and induces specific swellings in heterochromatin.