Pausing for thought: disrupting the early transcription elongation checkpoint leads to developmental defects and tumourigenesis

Factors affecting transcriptional elongation have been characterized extensively in in vitro, single cell (yeast) and cell culture systems; however, data from the context of multicellular organisms has been relatively scarce. While studies in homogeneous cell populations have been highly informative about the underlying molecular mechanisms and prevalence of polymerase pausing, they do not reveal the biological impact of perturbing this regulation in an animal. The core components regulating pausing are expressed in all animal cells and are recruited to the majority of genes, however, disrupting their function often results in discrete phenotypic effects. Mutations in genes encoding key regulators of transcriptional pausing have been recovered from several genetic screens for specific phenotypes or interactions with specific factors in mice, zebrafish and flies. Analysis of these mutations has revealed that control of transcriptional pausing is critical for a diverse range of biological pathways essential for animal development and survival

The Groucho Co-repressor Is Primarily Recruited to Local Target Sites in Active Chromatin to Attenuate Transcription

Gene expression is regulated by the complex interaction between transcriptional activators and repressors, which function in part by recruiting histone-modifying enzymes to control accessibility of DNA to RNA polymerase. The evolutionarily conserved family of Groucho/Transducin-Like Enhancer of split (Gro/TLE) proteins act as co-repressors for numerous transcription factors. Gro/TLE proteins act in several key pathways during development (including Notch and Wnt signaling), and are implicated in the pathogenesis of several human cancers. Gro/TLE proteins form oligomers and it has been proposed that their ability to exert long-range repression on target genes involves oligomerization over broad regions of chromatin. However, analysis of an endogenous gro mutation in Drosophila revealed that oligomerization of Gro is not always obligatory for repression in vivo. We have used chromatin immunoprecipitation followed by DNA sequencing (ChIP-seq) to profile Gro recruitment in two Drosophila cell lines. We find that Gro predominantly binds at discrete peaks (<1 kilobase). We also demonstrate that blocking Gro oligomerization does not reduce peak width as would be expected if Gro oligomerization induced spreading along the chromatin from the site of recruitment. Gro recruitment is enriched in "active" chromatin containing developmentally regulated genes. However, Gro binding is associated with local regions containing hypoacetylated histones H3 and H4, which is indicative of chromatin that is not fully open for efficient transcription. We also find that peaks of Gro binding frequently overlap the transcription start sites of expressed genes that exhibit strong RNA polymerase pausing and that depletion of Gro leads to release of polymerase pausing and increased transcription at a bona fide target gene. Our results demonstrate that Gro is recruited to local sites by transcription factors to attenuate rather than silence gene expression by promoting histone deacetylation and polymerase pausing

Allergic bronchopulmonary aspergillosis with coexistant aspergilloma: a case report

<p>Abstract</p> <p>Introduction</p> <p>The coexistence of allergic bronchopulmonary aspergillosis and aspergilloma is rare.</p> <p>Case presentation</p> <p>We present the case of a 56-year-old Caucasian man who worked as a farmer, with infiltrates in the right lower and middle lung lobes, partial consolidation of the middle lobe and with previous diagnosis of chronic obstructive bronchitis. Evaluation of our patient led to the diagnosis of allergic bronchopulmonary aspergillosis with coexistent aspergilloma in the right lower lobe. He was treated with oral methylprednisolone and itraconazole. At the five-year follow-up he is without any sign of recurrence.</p> <p>Conclusion</p> <p>Aspergillus infection after the inhalation of spores in the form of a hypersensitivity reaction and saprophytic colonization can be coexistent.</p

Cell-cell adhesion regulates Merlin/NF2 interaction with the PAF complex

The PAF complex (PAFC) coordinates transcription elongation and mRNA processing and its CDC73/parafibromin subunit functions as a tumour suppressor. The NF2/Merlin tumour suppressor functions both at the cell cortex and nucleus and is a key mediator of contact inhibition but the molecular mechanisms remain unclear. In this study we have used affinity proteomics to identify novel Merlin interacting proteins and show that Merlin forms a complex with multiple proteins involved in RNA processing including the PAFC and the CHD1 chromatin remodeller. Tumour-derived inactivating mutations in both Merlin and the CDC73 PAFC subunit mutually disrupt their interaction and growth suppression by Merlin requires CDC73. Merlin interacts with the PAFC in a cell density-dependent manner and we identify a role for FAT cadherins in regulating the Merlin-PAFC interaction. Our results suggest that in addition to its function within the Hippo pathway, Merlin is part of a tumour suppressor network regulated by cell-cell adhesion which coordinates post-initiation steps of the transcription cycle of genes mediating contact inhibition

Renal artery stenosis-when to screen, what to stent?

Renal artery stensosis (RAS) continues to be a problem for clinicians, with no clear consensus on how to investigate and assess the clinical significance of stenotic lesions and manage the findings. RAS caused by fibromuscular dysplasia is probably commoner than previously appreciated, should be actively looked for in younger hypertensive patients and can be managed successfully with angioplasty. Atheromatous RAS is associated with increased incidence of cardiovascular events and increased cardiovascular mortality, and is likely to be seen with increasing frequency. Evidence from large clinical trials has led clinicians away from recommending interventional revascularisation towards aggressive medical management. There is now interest in looking more closely at patient selection for intervention, with focus on intervening only in patients with the highest-risk presentations such as flash pulmonary oedema, rapidly declining renal function and severe resistant hypertension. The potential benefits in terms of improving hard cardiovascular outcomes may outweigh the risks of intervention in this group, and further research is needed

Vaccine Potential of Nipah Virus-Like Particles

Nipah virus (NiV) was first recognized in 1998 in a zoonotic disease outbreak associated with highly lethal febrile encephalitis in humans and a predominantly respiratory disease in pigs. Periodic deadly outbreaks, documentation of person-to-person transmission, and the potential of this virus as an agent of agroterror reinforce the need for effective means of therapy and prevention. In this report, we describe the vaccine potential of NiV virus-like particles (NiV VLPs) composed of three NiV proteins G, F and M. Co-expression of these proteins under optimized conditions resulted in quantifiable amounts of VLPs with many virus-like/vaccine desirable properties including some not previously described for VLPs of any paramyxovirus: The particles were fusogenic, inducing syncytia formation; PCR array analysis showed NiV VLP-induced activation of innate immune defense pathways; the surface structure of NiV VLPs imaged by cryoelectron microscopy was dense, ordered, and repetitive, and consistent with similarly derived structure of paramyxovirus measles virus. The VLPs were composed of all the three viral proteins as designed, and their intracellular processing also appeared similar to NiV virions. The size, morphology and surface composition of the VLPs were consistent with the parental virus, and importantly, they retained their antigenic potential. Finally, these particles, formulated without adjuvant, were able to induce neutralizing antibody response in Balb/c mice. These findings indicate vaccine potential of these particles and will be the basis for undertaking future protective efficacy studies in animal models of NiV disease

The Unconserved Groucho Central Region Is Essential for Viability and Modulates Target Gene Specificity

Groucho (Gro) is a Drosophila corepressor required by numerous DNA-binding repressors, many of which are distributed in gradients and provide positional information during development. Gro contains well-conserved domains at its N- and C-termini, and a poorly conserved central region that includes the GP, CcN, and SP domains. All lethal point mutations in gro map to the conserved regions, leading to speculation that the unconserved central domains are dispensable. However, our sequence analysis suggests that the central domains are disordered leading us to suspect that the lack of lethal mutations in this region reflects a lack of order rather than an absence of essential functions. In support of this conclusion, genomic rescue experiments with Gro deletion variants demonstrate that the GP and CcN domains are required for viability. Misexpression assays using these same deletion variants show that the SP domain prevents unrestrained and promiscuous repression by Gro, while the GP and CcN domains are indispensable for repression. Deletion of the GP domain leads to loss of nuclear import, while deletion of the CcN domain leads to complete loss of repression. Changes in Gro activity levels reset the threshold concentrations at which graded repressors silence target gene expression. We conclude that co-regulators such as Gro are not simply permissive components of the repression machinery, but cooperate with graded DNA-binding factors in setting borders of gene expression. We suspect that disorder in the Gro central domains may provide the flexibility that allows this region to mediate multiple interactions required for repression

Establishment of Motor Neuron-V3 Interneuron Progenitor Domain Boundary in Ventral Spinal Cord Requires Groucho-Mediated Transcriptional Corepression

Background: Dorsoventral patterning of the developing spinal cord is important for the correct generation of spinal neuronal types. This process relies in part on cross-repressive interactions between specific transcription factors whose expression is regulated by Sonic hedgehog. Groucho/transducin-like Enhancer of split (TLE) proteins are transcriptional corepressors suggested to be recruited by at least certain Sonic hedgehog-controlled transcription factors to mediate the formation of spatially distinct progenitor domains within the ventral spinal cord. The aim of this study was to characterize the involvement of TLE in mechanisms regulating the establishment of the boundary between the most ventral spinal cord progenitor domains, termed pMN and p3. Because the pMN domain gives rise to somatic motor neurons while the p3 domain generates V3 interneurons, we also examined the involvement of TLE in the acquisition of these neuronal fates. Methodology and Principal Findings: A combination of in vivo loss- and gain-of-function studies in the developing chick spinal cord was performed to characterize the role of TLE in ventral progenitor domain formation. It is shown here that TLE overexpression causes increased numbers of p3 progenitors and promotes the V3 interneuron fate while suppressing the motor neuron fate. Conversely, dominant-inhibition of TLE increases the numbers of pMN progenitors and postmitotic motor neurons. Conclusion: Based on these results, we propose that TLE is important to promote the formation of the p3 domain an

Groucho binds two conserved regions of LEF-1 for HDAC-dependent repression

<p>Abstract</p> <p>Background</p> <p><it>Drosophila </it>Groucho and its human Transducin-like-Enhancer of Split orthologs (TLEs) function as transcription co-repressors within the context of Wnt signaling, a pathway with strong links to cancer. The current model for how Groucho/TLE's modify Wnt signaling is by direct competition with β-catenin for LEF/TCF binding. The molecular events involved in this competitive interaction are not defined and the actions of Groucho/TLEs within the context of Wnt-linked cancer are unknown.</p> <p>Methods</p> <p>We used <it>in vitro </it>protein interaction assays with the LEF/TCF family member LEF-1, and <it>in vivo </it>assays with Wnt reporter plasmids to define Groucho/TLE interaction and repressor function.</p> <p>Results</p> <p>Mapping studies reveal that Groucho/TLE binds two regions in LEF-1. The primary site of recognition is a 20 amino acid region in the Context Dependent Regulatory domain. An auxiliary site is in the High Mobility Group DNA binding domain. Mutation of an eight amino acid sequence within the primary region (RFSHHMIP) results in a loss of Groucho action in a transient reporter assay. <it>Drosophila </it>Groucho, human TLE-1, and a truncated human TLE isoform Amino-enhancer-of-split (AES), work equivalently to repress LEF-1•β-catenin transcription in transient reporter assays, and these actions are sensitive to the HDAC inhibitor Trichostatin A. A survey of Groucho/TLE action in a panel of six colon cancer cell lines with elevated β-catenin shows that Groucho is not able to repress transcription in a subset of these cell lines.</p> <p>Conclusion</p> <p>Our data shows that Groucho/TLE repression requires two sites of interaction in LEF-1 and that a central, conserved amino acid sequence within the primary region (F S/T/P/xx y I/L/V) is critical. Our data also reveals that AES opposes LEF-1 transcription activation and that both Groucho and AES repression require histone deacetylase activity suggesting multiple steps in Groucho competition with β-catenin. The variable ability of Groucho/TLE to oppose Wnt signaling in colon cancer cells suggests there may be defects in one or more of these steps.</p

Do obese but metabolically normal women differ in intra-abdominal fat and physical activity levels from those with the expected metabolic abnormalities? A cross-sectional study

<p>Abstract</p> <p>Background</p> <p>Obesity remains a major public health problem, associated with a cluster of metabolic abnormalities. However, individuals exist who are very obese but have normal metabolic parameters. The aim of this study was to determine to what extent differences in metabolic health in very obese women are explained by differences in body fat distribution, insulin resistance and level of physical activity.</p> <p>Methods</p> <p>This was a cross-sectional pilot study of 39 obese women (age: 28-64 yrs, BMI: 31-67 kg/m²) recruited from community settings. Women were defined as 'metabolically normal' on the basis of blood glucose, lipids and blood pressure. Magnetic Resonance Imaging was used to determine body fat distribution. Detailed lifestyle and metabolic profiles of participants were obtained.</p> <p>Results</p> <p>Women with a healthy metabolic profile had lower intra-abdominal fat volume (geometric mean 4.78 l [95% CIs 3.99-5.73] vs 6.96 l [5.82-8.32]) and less insulin resistance (HOMA 3.41 [2.62-4.44] vs 6.67 [5.02-8.86]) than those with an abnormality. The groups did not differ in abdominal subcutaneous fat volume (19.6 l [16.9-22.7] vs 20.6 [17.6-23.9]). A higher proportion of those with a healthy compared to a less healthy metabolic profile met current physical activity guidelines (70% [95% CIs 55.8-84.2] vs 25% [11.6-38.4]). Intra-abdominal fat, insulin resistance and physical activity make independent contributions to metabolic status in very obese women, but explain only around a third of the variance.</p> <p>Conclusion</p> <p>A sub-group of women exists who are metabolically normal despite being very obese. Differences in fat distribution, insulin resistance, and physical activity level are associated with metabolic differences in these women, but account only partially for these differences. Future work should focus on strategies to identify those obese individuals most at risk of the negative metabolic consequences of obesity and on identifying other factors that contribute to metabolic status in obese individuals.</p