Dominant and redundant functions of TFIID involved in the regulation of hepatic genes.

To study the in vivo role of TFIID in the transcriptional regulation of hepatic genes, we generated mice with liver-specific disruption of the TAF10 gene. Inactivation of TAF10 in hepatocytes resulted in the dissociation of TFIID into individual components. This correlated with the downregulation of most hepatocyte-specific genes during embryonic life and a defect in liver organogenesis. Unexpectedly, however, the transcription of less than 5% of active genes was affected by TAF10 inactivation and TFIID disassembly in adult liver. The extent of changes in transcription of the affected genes was dependent on the timing of their activation during liver development, relative to that of TAF10 inactivation. Furthermore, TFIID dissociation from promoters leads to the re-expression of several postnatally silenced hepatic genes. Promoter occupancy analyses, combined with expression profiling, demonstrate that TFIID is required for the initial activation or postnatal repression of genes, while it is dispensable for maintaining ongoing transcription.

Characterization of the nicotinic acetylcholine receptor antibodies after an unexpected increase of antibody titer in thymoma associated myasthenia gravis patients.

Two thymoma-associated myasthenia gravis patients with chronic well-controlled disease but an unexpected increase in anti-nAChR autoantibodies titer are reported. The specificity of anti-nAChR autoantibodies directed against extracellular parts of the receptor was studied in order to investigate the discrepancy between clinical and immunological status. Analysis of the anti-nAChR autoantibodies recognizing the extracellular parts of the nAChR revealed that when the concentration of anti-nAChR autoantibodies titer increased both patients had non-anti-α1 autoantibodies. Since the clinical profile of both patients remained unchanged, the increase of non-anti-α1 autoantibodies did not affect the 2 patients' disease progression. Thus, immunotherapy modification due to an increase of anti-nAChR autoantibodies titer could be erroneous and potentially harmful.

Analysis of nAChR Autoantibodies Against Extracellular Epitopes in MG Patients.

Myasthenia gravis (MG) is an autoimmune disorder caused by autoantibodies targeting components of the postsynaptic membrane of the neuromuscular junction (NMJ), leading to neuromuscular transmission deficiency. In the vast majority of patients, these autoantibodies target the nicotinic acetylcholine receptor (nAChR), a heteropentameric ion channel anchored to the postsynaptic membrane of the NMJ. Autoantibodies in patients with MG may target all the subunits of the receptor at both their extracellular and intracellular regions. Here, we combine immunoadsorption with a cell-based assay to examine the specificity of the patients' autoantibodies against the extracellular part of the nAChR. Our results reveal that these autoantibodies can be divided into distinct groups, based on their target, with probably different impacts on disease severity. Although our findings are based on a small sample group of patients, they strongly support that additional analysis of the specificity of the autoantibodies of patients with MG could serve as a valuable tool for the clinicians' decision on the treatment strategy to be followed.

Allosteric regulation of the primase (DnaG) activity by the clamp-loader (tau) in vitro.

During DNA replication the helicase (DnaB) recruits the primase (DnaG) in the replisome to initiate the polymerization of new DNA strands. DnaB is attached to the tau subunit of the clamp-loader that loads the beta clamp and interconnects the core polymerases on the leading and lagging strands. The tau-DnaB-DnaG ternary complex is at the heart of the replisome and its function is likely to be modulated by a complex network of allosteric interactions. Using a stable ternary complex comprising the primase and helicase from Geobacillus stearothermophilus and the tau subunit of the clamp-loader from Bacillus subtilis we show that changes in the DnaB-tau interaction can stimulate allosterically primer synthesis by DnaG in vitro. The A550V tau mutant stimulates the primase activity more efficiently than the native protein. Truncation of the last 18 C-terminal residues of tau elicits a DnaG-stimulatory effect in vitro that appears to be suppressed in the native tau protein. Thus changes in the tau-DnaB interaction allosterically affect primer synthesis. Although these C-terminal residues of tau are not involved directly in the interaction with DnaB, they may act as a functional gateway for regulation of primer synthesis by tau-interacting components of the replisome through the tau-DnaB-DnaG pathway.

Smyd3 Is a Transcriptional Potentiator of Multiple Cancer-Promoting Genes and Required for Liver and Colon Cancer Development.

Smyd3 is a protein methyltransferase implicated in cancer development. Here we show that Smyd3 expression in mice is required for chemically induced liver and colon cancer formation. In these organs Smyd3 functions in the nucleus, stimulating the transcription of several key regulators involved in cell proliferation, epithelial-mesenchymal transition, the JAK/Stat3 oncogenic pathway, as well as the Myc and Ctnnb1 oncogenes. Smyd3 interacts with H3K4Me3-modified histone tails, which facilitates its recruitment to the core promoter regions of most active genes. Smyd3 binding density on target genes positively correlates with increased RNA polymerase-II density and transcriptional outputs. Despite its widespread distribution, the transcription-potentiating function of Smyd3 is restricted to a particular set of genes, whose expression is induced specifically during carcinogenesis.

The clamp-loader-helicase interaction in Bacillus. Atomic force microscopy reveals the structural organisation of the DnaB-tau complex in Bacillus.

The clamp-loader-helicase interaction is an important feature of the replisome. Although significant biochemical and structural work has been carried out on the clamp-loader-clamp-DNA polymerase alpha interactions in Escherichia coli, the clamp-loader-helicase interaction is poorly understood by comparison. The tau subunit of the clamp-loader mediates the interaction with DnaB. We have recently characterised this interaction in the Bacillus system and established a tau(5)-DnaB(6) stoichiometry. Here, we have obtained atomic force microscopy images of the tau-DnaB complex that reveal the first structural insight into its architecture. We show that despite the reported absence of the shorter gamma version in Bacillus, tau has a domain organisation similar to its E.coli counterpart and possesses an equivalent C-terminal domain that interacts with DnaB. The interaction interface of DnaB is also localised in its C-terminal domain. The combined data contribute towards our understanding of the bacterial replisome.

Regulation of anti-atherogenic apolipoprotein M gene expression by the orphan nuclear receptor LRH-1.

The orphan nuclear receptor liver receptor homolog-1 (LRH-1, NR5A2) has been reported to play a crucial role in early development, in the control of the hepatic inflammatory response, in intestinal cell crypt renewal as well as in bile acid biosynthesis and reverse cholesterol transport (RCT). Here, we report the identification of apolipoprotein M (APOM) as a novel target gene for LRH-1. Using gene-silencing experiments, adenovirus-mediated overexpression, transient transfection, and chromatin immunoprecipitation (ChIP) assays, it is shown that LRH-1 directly regulates human and mouse APOM transcription by binding to an LRH-1 response element located in the proximal APOM promoter region. In addition, we demonstrate that bile acids suppress APOM expression in a SHP-dependent manner in vitro and in vivo by inhibiting LRH-1 transcriptional activity on the APOM promoter as demonstrated by in vivo ChIP assay. Taken together, our results demonstrate that LRH-1 is a novel regulator of APOM transcription and further extend the role of this orphan nuclear receptor in lipoprotein metabolism and cholesterol homeostasis.

Involvement of corepressor complex subunit GPS2 in transcriptional pathways governing human bile acid biosynthesis.

Coordinated regulation of bile acid biosynthesis, the predominant pathway for hepatic cholesterol catabolism, is mediated by few key nuclear receptors including the orphan receptors liver receptor homolog 1 (LRH-1), hepatocyte nuclear factor 4alpha (HNF4alpha), small heterodimer partner (SHP), and the bile acid receptor FXR (farnesoid X receptor). Activation of FXR initiates a feedback regulatory loop via induction of SHP, which suppresses LRH-1- and HNF4alpha-dependent expression of cholesterol 7alpha hydroxylase (CYP7A1) and sterol 12alpha hydroxylase (CYP8B1), the two major pathway enzymes. Here we dissect the transcriptional network governing bile acid biosynthesis in human liver by identifying GPS2, a stoichiometric subunit of a conserved corepressor complex, as a differential coregulator of CYP7A1 and CYP8B1 expression. Direct interactions of GPS2 with SHP, LRH-1, HNF4alpha, and FXR indicate alternative coregulator recruitment strategies to cause differential transcriptional outcomes. In addition, species-specific differences in the regulation of bile acid biosynthesis were uncovered by identifying human CYP8B1 as a direct FXR target gene, which has implications for therapeutic approaches in bile acid-related human disorders.