Distinct modes of regulation of transcription of hepatitis B virus by the nuclear receptors HNF4alpha and COUP-TF1.
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To study the effects of the nuclear receptors (NRs) HNF4alpha 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 HNF4alpha 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 HNF4alpha 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, NRRE(preC,) played the major role in activation of pregenomic RNA synthesis by HNF4alpha. 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 NRRE(preC) or NRRE(enhI). HNF4alpha 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 HNF4alpha but not COUP-TF1 in its NRRE(preC) 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 HNF4alpha but not for repression by COUP-TF1. Thus, HNF4alpha and COUP-TF1 function through different mechanisms to regulate expression of the HBV genes. (+info)
Mechanism of a transcriptional cross talk between transforming growth factor-beta-regulated Smad3 and Smad4 proteins and orphan nuclear receptor hepatocyte nuclear factor-4.
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We have shown previously that the transforming growth factor-beta (TGFbeta)-regulated Sma-Mad (Smad) protein 3 and Smad4 proteins transactivate the apolipoprotein C-III promoter in hepatic cells via a hormone response element that binds the nuclear receptor hepatocyte nuclear factor 4 (HNF-4). In the present study, we show that Smad3 and Smad4 but not Smad2 physically interact with HNF-4 via their Mad homology 1 domains both in vitro and in vivo. The synergistic transactivation of target promoters by Smads and HNF-4 was shown to depend on the specific promoter context and did not require an intact beta-hairpin/DNA binding domain of the Smads. Using glutathione S-transferase interaction assays, we established that two regions of HNF-4, the N-terminal activation function 1 (AF-1) domain (aa 1-24) and the C-terminal F domain (aa 388-455) can mediate physical Smad3/HNF-4 interactions in vitro. In vivo, Smad3 and Smad4 proteins enhanced the transactivation function of various GAL4-HNF-4 fusion proteins via the AF-1 and the adjacent DNA binding domain, whereas a single tyrosine to alanine substitution in AF-1 abolished coactivation by Smads. The findings suggest that the transcriptional cross talk between the TGFbeta-regulated Smads and HNF-4 is mediated by specific functional domains in the two types of transcription factors. Furthermore, the specificity of this interaction for certain target promoters may play an important role in various hepatocyte functions, which are regulated by TGFbeta and the Smads. (+info)
Molecular regionalization of the neocortex is disrupted in Fgf8 hypomorphic mutants.
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The neocortex is divided into multiple areas with specific architecture, molecular identity and pattern of connectivity with the dorsal thalamus. Gradients of transcription factor expression in the cortical primordium regulate molecular regionalization and potentially the patterning of thalamic projections. We show that reduction of Fgf8 levels in hypomorphic mouse mutants shifts early gradients of gene expression rostrally, thereby modifying the molecular identity of rostral cortical progenitors. This shift correlates with a reduction in the size of a molecularly defined rostral neocortical domain and a corresponding rostral expansion of more caudal regions. Despite these molecular changes, the topography of projections between the dorsal thalamus and rostral neocortex in mutant neonates appears the same as the topography of wild-type littermates. Overall, our study demonstrates the role of endogenous Fgf8 in regulating early gradients of transcription factors in cortical progenitor cells and in molecular regionalization of the cortical plate. (+info)
Recruitment of Tat to heterochromatin protein HP1 via interaction with CTIP2 inhibits human immunodeficiency virus type 1 replication in microglial cells.
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The Tat protein of human immunodeficiency virus type 1 (HIV-1) plays a key role as inducer of viral gene expression. We report that Tat function can be potently inhibited in human microglial cells by the recently described nuclear receptor cofactor chicken ovalbumin upstream promoter transcription factor-interacting protein 2 (CTIP2). Overexpression of CTIP2 leads to repression of HIV-1 replication, as a result of inhibition of Tat-mediated transactivation. In contrast, the related CTIP1 was unable to affect Tat function and viral replication. Using confocal microscopy to visualize Tat subcellular distribution in the presence of the CTIPs, we found that overexpression of CTIP2, and not of CTIP1, leads to disruption of Tat nuclear localization and recruitment of Tat within CTIP2-induced nuclear ball-like structures. In addition, our studies demonstrate that CTIP2 colocalizes and associates with the heterochromatin-associated protein HP1alpha. The CTIP2 protein harbors two Tat and HP1 interaction interfaces, the 145-434 and the 717-813 domains. CTIP2 and HP1alpha associate with Tat to form a three-protein complex in which the 145-434 CTIP2 domain interacts with the N-terminal region of Tat, while the 717-813 domain binds to HP1. The importance of this Tat binding interface and of Tat subnuclear relocation was confirmed by analysis of CTIP2 deletion mutants. Our findings suggest that inhibition of HIV-1 expression by CTIP2 correlates with recruitment of Tat within CTIP2-induced structures and relocalization within inactive regions of the chromatin via formation of the Tat-CTIP2-HP1alpha complex. These data highlight a new mechanism of Tat inactivation through subnuclear relocalization that may ultimately lead to inhibition of viral pathogenesis. (+info)
Constructing the mammalian neocortex: the role of intrinsic factors.
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The mammalian neocortex is subdivided into regions that are specialised for the processing of particular forms of information. These regions are distinct in terms of their cytoarchitecture, electrophysiology, and connectivity. How this regional diversity is generated through development is currently a topic of considerable interest and has centered upon two main issues. First, to what extent are these regions prespecified by intrinsic genetic mechanisms? Second, what is the influence of extrinsic activity in transmitting signals that ultimately shape functional regions? Historically, experimental evidence has tended to emphasise the role of extrinsic influences, but the identification and analysis of several genes that are expressed asymmetrically in the developing neocortex have tempered this viewpoint. We review current literature from the standpoint that intrinsic influences act early in neocortical development to generate molecular patterning whose main role is the guidance of long-range projections from the dorsal thalamus. Extrinsic influences appear to generate receptive fields for peripheral input, the summation of which determines the areal extent of particular neocortical region. (+info)
Repression of the luteinizing hormone receptor gene promoter by cross talk among EAR3/COUP-TFI, Sp1/Sp3, and TFIIB.
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Transcription of luteinizing hormone receptor (LHR) gene is activated by Sp1/Sp3 at two Sp1 sites and is repressed by nuclear orphan receptors EAR2 and EAR3 through a direct-repeat (DR) motif. To elucidate the mechanism of the orphan receptor-mediated gene repression, we explored the functional connection between the orphan receptors and Sp1/Sp3 complex, and its impact on the basal transcription machinery. The Sp1(I) site was identified as critical for the repression since its mutation reduced the inhibition by EAR2 and abolished the inhibition by EAR3. Cotransfection analyses in SL2 cells showed that both Sp1 and Sp3 were required for this process since EAR3 displayed a complete Sp1/Sp3-dependent inhibitory effect. Functional cooperation between Sp1 and DR domains was further supported by mutual recruitment of EAR3 and Sp1/Sp3 bound to their cognate sites. Deletion of EAR3 N-terminal and DNA-binding domains that reduced its interaction with Sp1 impaired its inhibitory effect on human LHR (hLHR) gene transcription. Furthermore, we demonstrate interaction of TFIIB with Sp1/Sp3 at the Sp1(I) site besides its association with EAR3 and the TATA-less core promoter region. Such interaction relied on Sp1 site-bound Sp1/Sp3 complex and adaptor protein(s) present in the JAR nuclear extracts. We further demonstrated that EAR3 specifically decreased association of TFIIB to the Sp1(I) site without interfering on its interaction with the hLHR core promoter. The C-terminal region of EAR3, which did not participate in its interaction with Sp1, was required for its inhibitory function and may affect the association of TFIIB with Sp1. Moreover, perturbation of the association of TFIIB with Sp1 by EAR3 was reflected in the reduced recruitment of RNA polymerase II to the promoter. Overexpression of TFIIB counteracted the inhibitory effect of EAR3 and activated hLHR gene transcription in an Sp1 site-dependent manner. These findings therefore indicate that TFIIB is a key component in the regulatory control of EAR3 and Sp1/Sp3 on the initiation complex. Such cross talk among EAR3, TFIIB, and Sp1/Sp3 reveals repression of hLHR gene transcription by nuclear orphan receptors is achieved via perturbation of communication between Sp1/Sp3 at the Sp1-1 site and the basal transcription initiator complex. (+info)
Retinoid X receptor-COUP-TF interactions modulate retinoic acid signaling.
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We have recently described the properties of direct repeats (DRs) of the half-site AGGTCA as hormone response elements (HREs). According to our results, spacing the half sites by 3, 4, or 5 nucleotides determines specificity of response for vitamin D3, thyroid hormone, and retinoic acid receptors, respectively. This so-called 3-4-5 rule led to the prediction that remaining spacing options of 0, 1, and 2 might serve as targets for other nuclear receptors. A concurrent prediction is that receptors recognizing common sites might display more complex or combinatorial interactions. In exploring these predictions, we discovered that both the retinoid X receptor (RXR) and COUP-TF bind preferentially to a DR-1 motif. In vivo, RXR and COUP-TF display antagonistic action such that RXR-mediated activation is fully repressed by COUP-TF. In vitro studies reveal that COUP-TF and RXR form heterodimers on DR-1. Thus, these results support a general proposal in which the half-site spacing preferences may be used as a means to decipher potentially complex and interactive regulatory circuits. (+info)
Antagonism between apolipoprotein AI regulatory protein 1, Ear3/COUP-TF, and hepatocyte nuclear factor 4 modulates apolipoprotein CIII gene expression in liver and intestinal cells.
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Apolipoprotein CIII (apoCIII), a lipid-binding protein involved in the transport of triglycerides and cholesterol in the plasma, is synthesized primarily in the liver and the intestine. A cis-acting regulatory element, C3P, located at -90 to -66 upstream from the apoCIII gene transcriptional start site (+1), is necessary for maximal expression of the apoCIII gene in human hepatoma (HepG2) and intestinal carcinoma (Caco2) cells. This report shows that three members of the steroid receptor superfamily of transcription factors, hepatocyte nuclear factor 4 (HNF-4), apolipoprotein AI regulatory protein 1 (ARP-1), and Ear3/COUP-TF, act at the C3P site. HNF-4 activates apoCIII gene expression in HepG2 and Caco2 cells, while ARP-1 and Ear3/COUP-TF repress its expression in the same cells. HNF-4 activation is abolished by increasing amounts of ARP-1 or Ear3/COUP-TF, and repression by ARP-1 or Ear3/COUP-TF is alleviated by increasing amounts of HNF-4. HNF-4 and ARP-1 bind with similar affinities to the C3P site, suggesting that their opposing transcriptional effects may be mediated by direct competition for DNA binding. HNF-4 and ARP-1 mRNAs are present within the same cells in the liver and intestine, and protein extracts from hepatic tissue, HepG2, and Caco2 cells contain significantly more HNF-4 than ARP-1 or Ear3/COUP-TF binding activities. These findings suggest that the transcription of the apoCIII gene in vivo is dependent, at least in part, upon the intracellular balance of these positive and negative regulatory factors. (+info)