Littérature scientifique sur le sujet « COUP-TF1 »

ABSTRACT To study the effects of the nuclear receptors (NRs) HNF4α and COUP-TF1 on the life cycle of hepatitis B virus (HBV), the human hepatoma cell line Huh7 was transiently cotransfected with plasmids containing the HBV genome and encoding these two NRs. Overexpression of HNF4α and COUP-TF1 led to a 9-fold increase and a 7- to 10-fold decrease, respectively, in viral DNA synthesis. These two NRs also exhibited distinct modes of regulation of viral transcription. Overexpression of HNF4α led to a more-than-10-fold increase in synthesis of the pregenomic RNA but to only a 2- to 3-fold increase in synthesis of the pre-C and S RNAs. Moreover, the NR response element within the pre-C promoter, NRREpreC, played the major role in activation of pregenomic RNA synthesis by HNF4α. On the other hand, overexpression of COUP-TF1 led to an over-10-fold repression of synthesis of both pre-C and pregenomic RNAs mediated through either NRREpreC or NRREenhI. HNF4α and COUP-TF1 antagonized each other's effects on synthesis of pregenomic RNA and viral DNA when they were co-overexpressed. A naturally occurring HBV variant which allows for binding by HNF4α but not COUP-TF1 in its NRREpreC exhibited significantly higher levels of synthesis of pregenomic RNA and viral DNA than wild-type HBV in coexpression experiments. Last, deletion analysis revealed that non-NRRE sequences located within both the C and pre-S1 regions are also essential for maximum activation of the pregenomic promoter by HNF4α but not for repression by COUP-TF1. Thus, HNF4α and COUP-TF1 function through different mechanisms to regulate expression of the HBV genes.

Kallikrein-binding protein (KBP) is a component of the kallikrein-kinin system that mediates vasodilation and inhibits tumor growth by antagonizing vascular endothelial growth factor-mediated angiogenesis. We demonstrate that KBP gene expression is repressed by T3 and modulated by the orphan nuclear receptor, chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1). In hypothyroid mice, KBP mRNA expression in the testis was increased 2.1-fold compared with euthyroid mice. We have identified two negative thyroid hormone response elements (nTREs) in the mouse KBP gene, nTRE1 located in the 5′ flanking region (−53 to −29) and nTRE2, located in the first intron (104–132). We used functional assays, cofactor knockdown, and chromatin immunoprecipitation assays to characterize nTRE1 and nTRE2 in hepatic (HepG2) and testes (GC-1spg) cell lines. Reporter expression directed by both elements was enhanced with addition of thyroid hormone receptor and repressed with the addition of T3. COUP-TF1 enhanced basal expression of both elements but blunted unliganded thyroid hormone receptor enhancement and T3 repression of nTRE1 but not nTRE2. Both nTREs bound nuclear corepressor and binding increased in response to T3. Nuclear corepressor knockdown resulted in loss of T3 repression of both nTRE1 and nTRE2. COUP-TF1, which usually represses T3 induction of positive thyroid hormone response elements, reverses T3 repression mediated by nTRE1 in the mouse KBP gene. Endogenous KBP expression is repressed by T3 and two functional nTREs, both of which are required, have been characterized in the KBP gene. COUP-TF1 may be an important factor to modulate expression of genes that are repressed by T3.

Expression of enhanced green fluorescent protein (EGFP) under control of the promoter-enhancer of chicken infectious anemia virus (CAV) is increased in an oestrogen receptor-enhanced cell line when treated with oestrogen and the promoter-enhancer binds unidentified proteins that recognize a consensus oestrogen response element (ERE). Co-transfection assays with the CAV promoter and the nuclear receptor chicken ovalbumin upstream promoter transcription factor 1 (COUP-TF1) showed that expression of EGFP was decreased by 50 to 60 % in DF-1 and LMH cells. The CAV promoter that included sequences at and downstream of the transcription start point had less expression than a short promoter construct. Mutation of a putative E box at this site restored expression levels. Electromobility shift assays showed that the transcription regulator delta-EF1 (δEF1) binds to this E box region. These findings indicate that the CAV promoter activity can be affected directly or indirectly by COUP-TF1 and δEF1.

vHNF1 (also termed HNF1 beta) is a member of the hepatocyte nuclear fa ctor 1 (HNF1; also termed HNF1 alpha) family of homeodomain-containing transcription factors that interact with a sequence motif found in the regulatory regions of a large number of genes expressed mainly in the liver. It has been suggested that vHNF1 plays a role in early differentiation of specialized epithelia of several endoderm- and mesoderm-derived organs, with HNF1 playing a role in later stages. In support of this idea, expression of vHNF1 but not HNF1 is induced upon treatment of the embryonal carcinoma cell line F9 with retinoic acid. We have cloned and analyzed the vHNF1 promoter to gain a better understanding of the regulation of vHNF1 expression and how it relates to the expression of HNF1. We have identified five sites of DNA-protein interaction within the first 260 bp upstream of the transcription start site, which involve at least three different families of transcription factors. Two sites, a distal DR-1 motif and a proximal octamer motif, are the most important for promoter activity. The DR-1 motif interacts with several members of the steroid hormone receptor superfamily including HNF4, COUP-TFI/Ear3, COUP-TFII/Arp1, and RAR alpha/RXR alpha heterodimers. The vHNF1 promoter is transactivated by COUP-TFI/Ear3 and COUP-TFII/Arp1 and, unlike the HNF1 promoter, is virtually unaffected by HNF4. Interestingly, the proximal octamer site and not the DR-1 site is required for COUP-TFI/Ear3 and COUP-TFII/Arp1 transactivation of the vHNF1 promoter. COUP-TFI/Ear3 does not bind directly to this proximal octamer site. We present evidence of an interaction between COUP-TFI/Ear3 and the octamer-binding proteins in vitro and in the cell, suggesting that COUP-TFI and COUP-TFII activate the vHNF1 promoter via an indirect mechanism.

In hepatocytes, the amount of the Spot 14 (S14) protein is closely related to the full expression of enzymes involved in the glycolytic and lipogenic pathways. In the present study we address the role played by this protein in the control of transcription of the L-type pyruvate kinase (L-PK) gene in primary hepatocytes. We show that human S14, which by itself does not bind to the L-PK promoter, physically interacts with the human chicken ovalbumin upstream promoter-transcription factor 1 (COUP-TF1) and induces the switch of this factor from a repressor to an activator. However, the enhancing activity of S14 and COUP-TF1 depends on the presence of a proximal GC-rich box (the L0 element) that specifically binds nuclear proteins from the livers of rats fed a glucose-rich diet. Moreover, the L0 element, which strongly binds dephosphorylated specificity protein 1 (Sp1), loses all affinity when this factor is phosphorylated by cAMP-dependent protein kinase. Mutations that affect binding of Sp1 and nuclear proteins to the L0 box also decrease basal transcription and impair glucose responsiveness of the promoter. These results therefore shed light on the mechanism by which the S14 protein, whose concentration rapidly rises after glucose intake, contributes to the full activity of the L-PK promoter.

The erythropoietin (Epo) gene is regulated by hypoxia-inducible cis-acting elements in the promoter and in a 3' enhancer, both of which contain consensus hexanucleotide hormone receptor response elements which are important for function. A group of 11 orphan nuclear receptors, transcribed and translated in vitro, were screened by the electrophoretic mobility shift assay. Of these, hepatic nuclear factor 4 (HNF-4), TR2-11, ROR alpha 1, and EAR3/COUP-TF1 bound specifically to the response elements in the Epo promoter and enhancer and, except for ROR alpha 1, formed DNA-protein complexes that had mobilities similar to those observed in nuclear extracts of the Epo-producing cell line Hep3B. Moreover, both anti-HNF-4 and anti-COUP antibodies were able to supershift complexes in Hep3B nuclear extracts. Like Epo, HNF-4 is expressed in kidney, liver, and Hep3B cells but not in HeLa cells. Transfection of a plasmid expressing HNF-4 into HeLa cells enabled an eightfold increase in the hypoxic induction of a luciferase reporter construct which contains the minimal Epo enhancer and Epo promoter, provided that the nuclear hormone receptor consensus DNA elements in both the promoter and the enhancer were intact. The augmentation by HNF-4 in HeLa cells could be abrogated by cotransfection with HNF-4 delta C, which retains the DNA binding domain of HNF-4 but lacks the C-terminal activation domain. Moreover, the hypoxia-induced expression of the endogenous Epo gene was significantly inhibited in Hep3B cells stably transfected with HNF-4 delta C. On the other hand, cotransfection of EAR3/COUP-TF1 and the Epo reporter either with HNF-4 into HeLa cells or alone into Hep3B cells suppressed the hypoxia induction of the Epo reporter. These electrophoretic mobility shift assay and functional experiments indicate that HNF-4 plays a critical positive role in the tissue-specific and hypoxia-inducible expression of the Epo gene, whereas the COUP family has a negative modulatory role.

Il fattore di trascrizione Sox2 è espresso nel sistema nervoso dall’inizio del suo sviluppo dove è richiesto per il mantenimento delle cellule staminali. Nell'uomo, le mutazioni eterozigoti di Sox2 sono collegate a vari difetti del sistema nervoso centrale, inclusi i difetti visivi. Il sistema visivo è composto dall'occhio, dal nucleo talamico genicolato dorsolaterale (dLGN) e dalla corteccia visiva, che sono altamente interconnessi. L'occhio, infatti, invia le afferenze retiniche ad uno specifico nucleo talamico dorsale, il dLGN, i cui neuroni a loro volta proiettano verso l'area corticale visiva. La corteccia visiva elabora input visivi e proietta al dLGN in un circuito complesso. Numerosi geni sono importanti per il corretto sviluppo del sistema visivo e Sox2 è uno di questi. Sox2 è espresso in tutti e tre i componenti del sistema visivo nel topo; mentre il suo ruolo nello sviluppo della retina è ben descritto si sa poco riguardo al suo ruolo nel talamo. Per studiare l’importanza di Sox2 nel talamo per il corretto sviluppo dell'asse visivo, abbiamo generato un knockout condizionale talamico di Sox2 nei neuroni post-mitotici. Abbiamo osservato che la perdita di Sox2 nel dLGN porta a una forte riduzione delle dimensioni del dLGN, all’alterazione delle proiezioni neuronali retino-talamiche, talamo-corticali e cortico-talamiche e, di conseguenza, a una difettiva definizione dell'area visiva corticale. Abbiamo scoperto che nei mutanti talamici di Sox2 il gene Efna5, importante nel guidare gli assoni retinici verso il dLGN, e i geni SERT e vMAT2 che codificano per trasportatori di serotonina, importanti per la corretta formazione di proiezioni talamo-corticali, sono fortemente sottoregolati nel dLGN mutante. Per identificare tutti i potenziali geni che potrebbero mediare la funzione di Sox2 nel talamo, abbiamo eseguito il sequenziamento dell'RNA (RNA-seq) su dLGN di controlli e mutanti di Sox2. Abbiamo scoperto che i geni deregolati sono arricchiti in geni che codificano per molecole importanti per la guida degli assoni e per molecole coinvolte nella neurotrasmissione e nelle sinapsi. È interessante notare che l'ablazione talamica di un altro fattore di trascrizione, COUP-TF1, porta a difetti del sistema visivo simili a quelli descritti per Sox2. Inoltre, le mutazioni eterozigoti nel gene COUP-TF1 nell'uomo portano all'atrofia ottica e a disabilità intellettive. Abbiamo scoperto che Sox2 e COUP-TF1 sono co-espressi negli stessi neuroni post-mitotici del dLGN. Sorprendentemente, l'espressione di COUP-TF1 non varia nei mutanti talamici di Sox2, facendo nascere la possibilità che Sox2 e COUP-TF abbiano target comuni nel talamo. Pertanto, abbiamo esaminato l'espressione, nei mutanti COUP-TF1, di geni sottoregolati nei mutanti talamici di Sox2 e sorprendentemente abbiamo scoperto che sembrano sovraregolati, suggerendo che i due fattori di trascrizione potrebbero agire sugli stessi geni ma in modo opposto. Per capire meglio se i due fattori di trascrizione regolano geni comuni, stiamo eseguendo l'analisi dell'espressione genica mediante RNA-seq anche sui mutanti talamici COUP-TF1. Inoltre, stiamo generando topi doppi mutanti per Sox2 e COUP-TF1 per scoprire come questi geni regolano espressione genica; è plausibile che regolino geni comuni per bilanciare la loro espressione nei neuroni talamici.
The transcription factor Sox2 is expressed in the nervous system from the beginning of its development where it is required for stem cells maintenance. In humans, Sox2 heterozygous mutations are linked to various central nervous system defects, including visual defects. The visual system is composed of the eye, the dorsolateral geniculate thalamic nucleus (dLGN) and the visual cortex, which are highly interconnected. The eye, in fact, sends retinal afferent to a specific dorsal thalamic nucleus, the dLGN, whose neurons in turn project to the visual cortical area. The visual cortex elaborates visual inputs and projects back to the dLGN in a complex circuit. Several genes are important for the correct development of the visual system and Sox2 is one of them. Sox2 is expressed in all the three components of the visual system in mouse; while its role in the development of the retina is well characterized little is known about its role in the thalamus. To investigate Sox2 requirement in the thalamus for the correct establishment of the visual axis, we generated a thalamic Sox2 conditional knock-out in post-mitotic neurons. We observed that Sox2 loss in the dLGN leads to a strong reduction in size of the dLGN, aberrant retino-geniculate, thalamo-cortical and cortico-thalamic neural projections and, consequently, to a defective patterning of the cortical visual area. We found that in Sox2 thalamic mutants the Efna5 gene, important in guiding retinal axons towards the dLGN, and the serotonin transporters encoding genes SERT and vMAT2, involved in the establishment of thalamo-cortical projections, are strongly downregulated in the mutant dLGN. To identify all the potential genes that could mediate Sox2 function in the thalamus, we performed RNA sequencing (RNA-seq) on control and Sox2 mutant dLGNs. We noticed that misregulated genes are enriched in genes encoding axon guidance molecules and molecules involved in neurotransmission and synapses. Interestingly, thalamic ablation of another transcription factor, COUP-TF1, leads to defects of the visual system similar to the ones described for Sox2. In addition, heterozygous mutations in the COUP-TF1 gene in human lead to optic atrophy and intellectual disabilities. Interestingly, we found that Sox2 and COUP-TF1 are co-expressed in the same post-mitotic neurons of the dLGN. Surprisingly, COUP-TF1 expression does not vary in Sox2 thalamic mutants, arising the possibility that Sox2 and COUP-TF have common target in the thalamus. Therefore, we looked at the expression, in COUP-TF1 mutants, of genes downregulated in Sox2 thalamic mutants and we surprisingly found that they appear upregulated, suggesting that the two transcription factors could act on the same genes but in an opposite way. To better understand if the two transcription factors regulate common genes, we are performing gene expression analyses by RNA-seq also on COUP-TF1 thalamic mutants, with the aim to identify an overlap with Sox2 regulated genes. Moreover, we are generating Sox2 and COUP-TF1 double mutant mice to unveil how these genes regulate gene expression; it is plausible that they regulate common genes to balance their expression in thalamic neurons.