Convergence of Wnt signalling on the HNF4a-driven transcription in controlling liver zonation

BACKGROUND & AIMS: In each hepatocyte, the specific repertoire of gene expression is influenced by its exact location along the portocentrovenular axis of the hepatic lobule and provides a reason for the liver functions compartmentalization defined "metabolic zonation." So far, few molecular players controlling genetic programs of periportal (PP) and perivenular (PV) hepatocytes have been identified; the elucidation of zonation mechanisms remains a challenge for experimental hepatology. Recently, a key role in induction and maintenance of the hepatocyte heterogeneity has been ascribed to Wnt/beta-catenin pathway. We sought to clarify how this wide-ranging stimulus integrates with hepatocyte specificity. METHODS: Reverse transcriptase polymerase chain reaction (RT-PCR) allowed the transcriptional profiling of hepatocytes derived from in vitro differentiation of liver stem cells. The GSK3beta inhibitor 6-bromoindirubin-3'-oxime (BIO) was used for beta-catenin stabilization. Co-immunoprecipitations were used to study biochemical protein interactions while ChIP assays allowed the in vivo inspection of PV and PP genes regulatory regions. RESULTS: We found that spontaneous differentiation of liver stem cells gives rise to PP hepatocytes that, after Wnt pathway activation, switch into PV hepatocytes. Next, we showed that the Wnt downstream player LEF1 interacts with the liver-enriched transcriptional factor HNF4alpha. Finally, we unveiled that the BIO induced activation of PV genes correlates with LEF1 binding to both its own and HNF4alpha consensus, and the repression of PP genes correlates with HNF4alpha displacement from its own consensus. CONCLUSION: Our data show a direct and hitherto unknown convergence of the canonical Wnt signaling on the HNF4alpha-driven transcription providing evidences of a mechanism controlling liver zonated gene expression

Reduced 2D form coherence and 3D structure from motion sensitivity in developmental dyscalculia

Developmental dyscalculia (DD) is a specific learning disability affecting the development of numerical and arithmetical skills. The origin of DD is typically attributed to the suboptimal functioning of key regions within the dorsal visual stream (parietal cortex) which support numerical cognition. While DD individuals are often impaired in visual numerosity perception, the extent to which they also show a wider range of visual dysfunctions is poorly documented. In the current study we measured sensitivity to global motion (translational and flow), 2D static form (Glass patterns) and 3D structure from motion in adults with DD and control subjects. While sensitivity to global motion was comparable across groups, thresholds for static form and structure from motion were higher in the DD compared to the control group, irrespective of associated reading impairments. Glass pattern sensitivity predicted numerical abilities, and this relation could not be explained by recently reported differences in visual crowding. Since global form sensitivity has often been considered an index of ventral stream function, our findings could indicate a cortical dysfunction extending beyond the dorsal visual stream. Alternatively, they would fit with a role of parietal cortex in form perception under challenging conditions requiring multiple element integration

The lncRNA HOTAIR transcription is controlled by HNF4α-induced chromatin topology modulation

The expression of the long noncoding RNA HOTAIR (HOX Transcript Antisense Intergenic RNA) is largely deregulated in epithelial cancers and positively correlates with poor prognosis and progression of hepatocellular carcinoma and gastrointestinal cancers. Furthermore, functional studies revealed a pivotal role for HOTAIR in the epithelial-to-mesenchymal transition, as this RNA is causal for the repressive activity of the master factor SNAIL on epithelial genes. Despite the proven oncogenic role of HOTAIR, its transcriptional regulation is still poorly understood. Here hepatocyte nuclear factor 4-α (HNF4α), as inducer of epithelial differentiation, was demonstrated to directly repress HOTAIR transcription in the mesenchymal-to epithelial transition. Mechanistically, HNF4α was found to cause the release of a chromatin loop on HOTAIR regulatory elements thus exerting an enhancer-blocking activity

The Snail repressor recruits EZH2 to specific genomic sites through the enrollment of the lncRNA HOTAIR in epithelial-to-mesenchymal transition

The transcription factor Snail is a master regulator of cellular identity and epithelial-to-mesenchymal transition (EMT) directly repressing a broad repertoire of epithelial genes. How chromatin modifiers instrumental to its activity are recruited to Snail-specific binding sites is unclear. Here we report that the long non-coding RNA (lncRNA) HOTAIR (for HOX Transcript Antisense Intergenic RNA) mediates a physical interaction between Snail and enhancer of zeste homolog 2 (EZH2), an enzymatic subunit of the polycomb-repressive complex 2 and the main writer of chromatin-repressive marks. The Snail-repressive activity, here monitored on genes with a pivotal function in epithelial and hepatic morphogenesis, differentiation and cell-type identity, depends on the formation of a tripartite Snail/HOTAIR/EZH2 complex. These results demonstrate an lncRNA-mediated mechanism by which a transcriptional factor conveys a general chromatin modifier to specific genes, thereby allowing the execution of hepatocyte transdifferentiation; moreover, they highlight HOTAIR as a crucial player in the Snail-mediated EMT.Oncogene advance online publication, 25 July 2016; doi:10.1038/onc.2016.260

Recombinase-based conditional and reversible gene regulation via XTR alleles

Synthetic biological tools that enable precise regulation of gene function within in vivo systems have enormous potential to discern gene function in diverse physiological settings. Here we report the development and characterization of a synthetic gene switch that, when targeted in the mouse germline, enables conditional inactivation, reports gene expression and allows inducible restoration of the targeted gene. Gene inactivation and reporter expression is achieved through Cre-mediated stable inversion of an integrated gene-trap reporter, whereas inducible gene restoration is afforded by Flp-dependent deletion of the inverted gene trap. We validate our approach by targeting the p53 and Rb genes and establishing cell line and in vivo cancer model systems, to study the impact of p53 or Rb inactivation and restoration. We term this allele system XTR, to denote each of the allelic states and the associated expression patterns of the targeted gene: eXpressed (XTR), Trapped (TR) and Restored (R)

MTHFR C677T polymorphism analysis: A simple, effective restriction enzyme-based method improving previous protocols

Background: 5,10-Methylentetrahydrofolate reductase (MTHFR) C677T polymorphism is one of the most studied genetic variations in the human genome. Polymerase chain reaction-restriction fragment length polymorphism (PCR-RFLP) is one of the most used techniques to characterize the point mutations in genomic sequences because of its suitability and low cost. The most widely used method for the MTHFR C677T polymorphism characterization was developed by Frosst et al. (1995) but appears to have some technical limitations. The aim of this study was to propose a novel PCR-RFLP method for the detection of this polymorphism. Methods: In order to retrieve all published articles possibly describing any PCR-RFLP methods useful to analyze MTHFR C677T polymorphism, we performed systematic queries on PubMed, using a combination of Boolean operators (AND/OR) and MeSH terms. Amplify software was used in order to design a new primer pair following the optimal standard criteria. Primer-BLAST software was used to check primer pair's biological specificity. Results: The analysis of previous literature showed that PCR-RFLP method remains the most used technique. None of the 108 primer pairs described was ideal with regard to main accepted primer pair biochemical technical parameters. The new primer pair amplifies a DNA-fragment of 513 base pair (bp) that, in the presence of the polymorphism, is cut by Hinf I enzyme in two pieces of 146 bp and 367 bp and clearly visible on 2% agarose gel. The level of expertise and the materials required are minimal and the protocol takes one day to carry out. Conclusion: Our original PCR-RFLP strategy, specifically designed to make the analysis optimal with respect to PCR primers and gel analysis, fits the ideal criteria compared to the widely used strategy by Frosst et al (1995) as well as any other PCR-RFLP strategies proposed for MTHFR C677T polymorphism genotyping to date

An epistatic mini-circuitry between the transcription factors Snail and HNF4\uce\ub1 controls liver stem cell and hepatocyte features exhorting opposite regulation on stemness-inhibiting microRNAs

Preservation of the epithelial state involves the stable repression of epithelial-to-mesenchymal transition program, whereas maintenance of the stem compartment requires the inhibition of differentiation processes. A simple and direct molecular mini-circuitry between master elements of these biological processes might provide the best device to keep balanced such complex phenomena. In this work, we show that in hepatic stem cell Snail, a transcriptional repressor of the hepatocyte differentiation master gene HNF4\uce\ub1, directly represses the expression of the epithelial microRNAs (miRs)-200c and-34a, which in turn target several stem cell genes. Notably, in differentiated hepatocytes HNF4\uce\ub1, previously identified as a transcriptional repressor of Snail, induces the miRs-34a and-200a, b, c that, when silenced, causes epithelial dedifferentiation and reacquisition of stem traits. Altogether these data unveiled Snail, HNF4\uce\ub1 and miRs-200a, b, c and-34a as epistatic elements controlling hepatic stem cell maintenance/differentiation. \uc2\ua9 2012 Macmillan Publishers Limited. All rights reserved

Heritability and mechanisms of n-3 long chain polyunsaturated fatty acid deposition in the flesh of Atlantic salmon

N-3 long chain polyunsaturated fatty acids (n-3LC-PUFA) are essential components of vertebrate membrane lipids and are crucially deficient in modern Western diets. The main human dietary source for n-3LC-PUFA is fish and seafood, particularly oily fish and over 50% of global fish production is currently supplied by aquaculture. However, increasing pressure to include vegetable oils, which are devoid of n-3LC-PUFA, in aquaculture feeds reduces the content of these crucial nutrients in farmed fish flesh. The aim of this study was to measure the heritability and infer mechanisms determining flesh n-3LC-PUFA content in Atlantic salmon. This was achieved by analysing flesh lipid parameters in 48 families of Atlantic salmon, and by measuring differences in hepatic mRNA expression in families with high and low flesh n-3LC-PUFA. The results show that flesh n-3LC-PUFA level is a highly heritable trait (h2 = 0.77±0.14) and indicate the involvement of increased lipid transport, most likely in the form of very low density lipoprotein (VLDL) from liver. This increase in lipid transport may be associated with increased activity of a transcription factor, hepatic nuclear factor 4α (HNF4α), possibly as a result of family differences in transforming growth factor β1 (Tgfβ1) signalling. This study paves the way for identification of quantitative trait loci and gene interaction networks that are associated with levels of n-3LC-PUFA in fish flesh. Such markers can be used to assist the sustainable production of Atlantic salmon and provide optimal levels of critical nutrients for human consumers

HNF1α inhibition triggers epithelial-mesenchymal transition in human liver cancer cell lines

<p>Abstract</p> <p>Background</p> <p>Hepatocyte Nuclear Factor 1α (HNF1α) is an atypical homeodomain-containing transcription factor that transactivates liver-specific genes including albumin, α-1-antitrypsin and α- and β-fibrinogen. Biallelic inactivating mutations of <it>HNF1A </it>have been frequently identified in hepatocellular adenomas (HCA), rare benign liver tumors usually developed in women under oral contraceptives, and in rare cases of hepatocellular carcinomas developed in non-cirrhotic liver. HNF1α-mutated HCA (H-HCA) are characterized by a marked steatosis and show activation of glycolysis, lipogenesis, translational machinery and mTOR pathway. We studied the consequences of HNF1α silencing in hepatic cell lines, HepG2 and Hep3B and we reproduced most of the deregulations identified in H-HCA.</p> <p>Methods</p> <p>We transfected hepatoma cell lines HepG2 and Hep3B with siRNA targeting HNF1α and obtained a strong inhibition of HNF1α expression. We then looked at the phenotypic changes by microscopy and studied changes in gene expression using qRT-PCR and Western Blot.</p> <p>Results</p> <p>Hepatocytes transfected with HNF1α siRNA underwent severe phenotypic changes with loss of cell-cell contacts and development of migration structures. In HNF1α-inhibited cells, hepatocyte and epithelial markers were diminished and mesenchymal markers were over-expressed. This epithelial-mesenchymal transition (EMT) was related to the up regulation of several EMT transcription factors, in particular <it>SNAIL </it>and <it>SLUG</it>. We also found an overexpression of TGFβ1, an EMT initiator, in both cells transfected with HNF1α siRNA and H-HCA. Moreover, TGFβ1 expression is strongly correlated to HNF1α expression in cell models, suggesting regulation of TGFβ1 expression by HNF1α.</p> <p>Conclusion</p> <p>Our results suggest that HNF1α is not only important for hepatocyte differentiation, but has also a role in the maintenance of epithelial phenotype in hepatocytes.</p

A Novel Human Cytomegalovirus Locus Modulates Cell Type-Specific Outcomes of Infection

Clinical strains of HCMV encode 20 putative ORFs within a region of the genome termed ULb′ that are postulated to encode functions related to persistence or immune evasion. We have previously identified ULb′-encoded pUL138 as necessary, but not sufficient, for HCMV latency in CD34+ hematopoietic progenitor cells (HPCs) infected in vitro. pUL138 is encoded on polycistronic transcripts that also encode 3 additional proteins, pUL133, pUL135, and pUL136, collectively comprising the UL133-UL138 locus. This work represents the first characterization of these proteins and identifies a role for this locus in infection. Similar to pUL138, pUL133, pUL135, and pUL136 are integral membrane proteins that partially co-localized with pUL138 in the Golgi during productive infection in fibroblasts. As expected of ULb′ sequences, the UL133-UL138 locus was dispensable for replication in cultured fibroblasts. In CD34+ HPCs, this locus suppressed viral replication in HPCs, an activity attributable to both pUL133 and pUL138. Strikingly, the UL133-UL138 locus was required for efficient replication in endothelial cells. The association of this locus with three context-dependent phenotypes suggests an exciting role for the UL133-UL138 locus in modulating the outcome of viral infection in different contexts of infection. Differential profiles of protein expression from the UL133-UL138 locus correlated with the cell-type dependent phenotypes associated with this locus. We extended our in vitro findings to analyze viral replication and dissemination in a NOD-scid IL2Rγcnull-humanized mouse model. The UL133-UL138NULL virus exhibited an increased capacity for replication and/or dissemination following stem cell mobilization relative to the wild-type virus, suggesting an important role in viral persistence and spread in the host. As pUL133, pUL135, pUL136, and pUL138 are conserved in virus strains infecting higher order primates, but not lower order mammals, the functions encoded likely represent host-specific viral adaptations