ChIPping away at gene regulation

The coordinated regulation of gene expression in higher eukaryotes is complex and poorly understood. Recent technological advances have allowed the first insights into these networks on a genome-wide scale. These investigations have identified transcription factor target sites in the genome and successfully predicted cooperative interactions with other factors. However, a detailed understanding of the processes that coordinate gene expression remains elusive. Here, we highlight the advances that have been made using current methods, and the need for new technologies to address the gaps in our knowledge and to map these complex pathways further.

Regulation of endosome fusion

Homotypic fusion between early endosomes can be reconstituted in vitro. By using wortmannin and LY294002, inhibitors of phosphatidylinositol (Pl) 3-kinase, a requirement for this activity has been established in order for fusion to proceed efficiently. It has been shown that Pl 3-kinase activity is required downstream of rab5 activation, although a large excess of activated rab5 can overcome wortmannin inhibition. A series of experiments have also been performed which indicate a role for early endosomal autoantigen 1 (EEA1) in determining fusion efficiency. EEA1 dissociates from membranes following wortmannin treatment. It is proposed that the requirement of endosome fusion for Pl 3-kinase activity is to promote the association of EEA1 with endosomes.

Regulation of human dUTPase gene expression and p53-mediated transcriptional repression in response to oxaliplatin-induced DNA damage

Deoxyuridine triphosphate nucleotidohydrolase (dUTPase) catalyzes the hydrolysis of dUTP to dUMP and PPi. Although dUTP is a normal intermediate in DNA synthesis, its accumulation and misincorporation into DNA is lethal. Importantly, uracil misincorporation is a mechanism of cytotoxicity induced by fluoropyrimidine chemotherapeutic agents including 5-fluorouracil (5-FU) and elevated expression of dUTPase is negatively correlated with clinical response to 5-FU-therapy. In this study we performed the first functional characterization of the dUTPase promoter and demonstrate a role for E2F-1 and Sp1 in driving dUTPase expression. We establish a direct role for both mutant and wild-type forms of p53 in modulating dUTPase promoter activity. Treatment of HCT116 p53(+/+) cells with the DNA-damaging agent oxaliplatin induced a p53-dependent transcriptional downregulation of dUTPase not observed in the isogenic null cell line. Oxaliplatin treatment induced enrichment of p53 at the dUTPase promoter with a concomitant reduction in Sp1. The suppression of dUTPase by oxaliplatin promoted increased levels of dUTP that was enhanced by subsequent addition of fluoropyrimidines. The novel observation that oxaliplatin downregulates dUTPase expression may provide a mechanistic basis contributing to the synergy observed between 5-FU and oxaliplatin in the clinic. Furthermore, these studies provide the first evidence of a direct transcriptional link between the essential enzyme dUTPase and the tumor suppressor p53.

What is being taught in Religious Education classes in Northern Ireland and to what extent should states be concerned about regulation, quality and control of the subject?

This paper reports on a research project designed to discover what schools are teaching in Religious Education in Northern Ireland and what procedures are in place to maintain standards in the delivery of the subject. A search through literature shows that little research has been carried out to determine what is being taught in Religious Education in Northern Ireland. It also indicates that there are very weak systems of control to measure the effectiveness or quality of what is delivered. A survey of the websites of all Post-Primary schools in the region was used to provide some answers to the basic question of what is being taught in RE. Using content and discourse analysis of these alongside supporting documentary sources (textbooks and exam specifications), it is possible to get a clearer picture of how the Northern Ireland Core Syllabus for Religious Education and any additional curricular elements are delivered in schools. The findings show that a significant minority of schools do not publicly articulate what pupils do in religious education. In situations where the content of religious education is made clear, some definite trends are evident. Despite the existence of a statutory core syllabus, there is significant variation in what is taught in schools. The content is most divergent from the syllabus in relation to the teaching of World Religions at Key Stage 3 and at Key Stage 4 whole elements of the syllabus are neglected due to limited conformity between the syllabus and exam specifications. These results raise important questions about the systems of regulation and control of the subject in the region. In law the subject is exempt from formal inspection by the local inspection authority; instead, a form of inspection is allowed for by the Christian churches who design the syllabus, but it is a process that is either entirely neglected or entirely unreported in situations where it does occur. It is argued that these findings raise questions of more general concern for this and other regions in Europe where the teaching of religious education is largely unregulated. For example, to what extent should states take an interest in what is taught in religious education, how it is delivered, what values it promotes and how standards of teaching and learning in the subject are upheld?

Regulation of TLR2, TLR4 and CD14 mRNA in gingival fibroblasts.

Gingival fibroblasts constitutively express pattern recognition molecules including the Toll-like receptors (TLRs) and produce various cytokines following interaction with bacterial ligands including LPS. Hence gingival fibroblasts are thought to play an important role in the pathogenesis of chronic inflammatory periodontal disease.
Objectives: The aim of this study was to investigate the regulation of expression of TLRs and CD-14 mRNA by gingival fibroblasts, and subsequently the responsiveness of these cells to bacterial stimulation Methods: Gingival fibroblasts were stimulated with IL-1ß (10ng/ml), IFN-g (1000IU/ml), P. gingivalis LPS (1µg/ml), E. coli LPS (1µg/ml) or P. gingivalis sonicate (10µg/ml) for 6 and 24 hr. TLR2, TLR4 and CD14 mRNA expression was subsequently determined by Q-PCR utilising Taqman chemistry. The effects of each factor on mRNA expression was analysed by ANOVA. Cells were pre-incubated with IFN-g (1000IU/ml) for 48hr followed by stimulation with E. coli LPS over the concentration range 0 - 10.0 µg/ml for a further 48 hr. IL-8 production by fibroblasts was subsequently determined by ELISA. Results: After 24 hr IFN-g induced a statistically significant increase in TLR2, TLR4 and CD14 mRNA expression. In contrast, IL-1ß, P. gingivalis LPS, E. coli LPS and P. gingivalis sonicate had no significant effect on mRNA expression at either timepoint. Following pre-stimulation with IFN-g, E. coli LPS increased IL-8 production by gingival fibroblasts in a concentration-dependent manner. Conclusion: IFN-g stimulates mRNA expression levels of TLR2, TLR4 and CD14 in gingival fibroblasts, which may subsequently lead to an increased responsiveness of fibroblasts to bacterial stimulation.

Quantitative regulation of FLC via coordinated transcriptional initiation and elongation

The basis of quantitative regulation of gene expression is still poorly understood. In Arabidopsis thaliana, quantitative variation in expression of FLOWERING LOCUS C (FLC) influences the timing of flowering. In ambient temperatures, FLC expression is quantitatively modulated by a chromatin silencing mechanism involving alternative polyadenylation of antisense transcripts. Investigation of this mechanism unexpectedly showed that RNA polymerase II (Pol II) occupancy changes at FLC did not reflect RNA fold changes. Mathematical modeling of these transcriptional dynamics predicted a tight coordination of transcriptional initiation and elongation. This prediction was validated by detailed measurements of total and chromatin-bound FLC intronic RNA, a methodology appropriate for analyzing elongation rate changes in a range of organisms. Transcription initiation was found to vary ∼ 25-fold with elongation rate varying ∼ 8- to 12-fold. Premature sense transcript termination contributed very little to expression differences. This quantitative variation in transcription was coincident with variation in H3K36me3 and H3K4me2 over the FLC gene body. We propose different chromatin states coordinately influence transcriptional initiation and elongation rates and that this coordination is likely to be a general feature of quantitative gene regulation in a chromatin context.

DED or alive: assembly and regulation of the death effector domain complexes.

Death effector domains (DEDs) are protein-protein interaction domains initially identified in proteins such as FADD, FLIP and caspase-8 involved in regulating apoptosis. Subsequently, these proteins have been shown to have important roles in regulating other forms of cell death, including necroptosis, and in regulating other important cellular processes, including autophagy and inflammation. Moreover, these proteins also have prominent roles in innate and adaptive immunity and during embryonic development. In this article, we review the various roles of DED-containing proteins and discuss recent developments in our understanding of DED complex formation and regulation. We also briefly discuss opportunities to therapeutically target DED complex formation in diseases such as cancer.

Thanking the Greeks: The Crisis of the Rule of Law in EU Enlargement Regulation

This paper showcases the weaknesses of EU enlargement law and demonstrates how one Member State – namely, Greece – is notable for abusing this weakness, for harming the candidate countries, the EU, and the institutions alike, for stripping the EU position of its predictability, and for undermining the EU Commission’s efforts. Accordingly, Greece has severely incapacitated the key procedural rule of law component of the EU’s enlargement regulation, turning it into a randomised political game and ignoring any long-term goals of stability, prosperity, and peace that the process is to stand for. Following a walk through Greece’s engagement throughout a number of enlargement rounds, the paper concludes that the duty of loyalty – which is presumably able to discipline Member States that undermine the common effort – should find a new meaning in the context of EU enlargement.

The EU Clinical Trials Regulation: Key Priorities, Purposes and Aims and the Implications for Public Health

The replacement of the European Union (EU) Clinical Trials Directive by the new Clinical Trials Regulation (CTR), which entered into force on 16 June 2014 but will not apply before 28 May 2016, provides an opportunity to review the legal and political context within which this important aspect of research law and policy sits and to reflect on the implications for public health. My aim in this article is to relate the context to the key purposes and aims of EU law and policy on clinical trials in order to explain and clarify its orientation. On that basis, I argue that the CTR and the changes it introduces to the law on clinical trials are part of the EU's continued focus on market optimisation. It is this focus that orients and directs the wider pharmaceutical development pipeline, but that undermines the achievement of key public health objectives.

Employment regulation, game theory, and the lacuna in employee participation in liberal economies

Employee participation is a vital ingredient of what the International Labour Organization (ILO) calls ‘representation security’. This article provides theoretical and empirical insights relating to social policy impact of worker participation, specifically the European Information and Consultation Directive (ICD) for employee voice rights. While existing research on the ICD offers important empirical insights, there is a need for further theoretical analysis to examine the potential effectiveness of the regulations in liberal market economies (LMEs). Drawing on data from 16 case studies, the article uses game theory and the prisoner's dilemma framework to explain why national implementing legislation is largely ineffective in diffusing mutual gains cooperation in two LMEs: UK and the Republic of Ireland. Three theoretical (metaphorical) propositions advance understanding of the policy impact of national information & consultation regulations in LMEs.

Regulation of single exocytotic events by the cAMP signaling pathway

Permanece por esclarecer como a via de sinalização do cAMP modula a exocitose regulada. Os principais objetivos deste trabalho foram: i) avaliar o efeito do cAMP nos eventos exocitóticos, nas propriedades dos poros de fusão e na secreção hormonal; ii) perceber o impacto da sinalização por cAMP-HCN na exocitose e nas propriedades do poro de fusão; e iii) estudar as propriedades do poro de fusão na presença de um agente neurotóxico comum, como o alumínio. Lactotrofos, isolados a partir da hipófise anterior de ratos Wistar machos, foram usados como modelo celular. Os eventos unitários de fusão exocitótica e a prolactina (PRL) libertada foram avaliados, respetivamente, em ensaios eletrofisiológicos efectuados segundo a técnica de contacto hermético no modo sobre a célula aderida à pipeta porta-elétrodo e com recurso a métodos imunológicos de deteção. Os níveis intracelulares de cAMP foram aumentados por 3-isobutil-1-metilxantina (IMBX), forscolina e N6,2'-O-dibutiril adenosina- 3',5'-monofosfato cíclico (dbcAMP). A expressão dos canais HCN foi determinada por Western-blot, qRT-PCR e imunocitoquímica em combinação com microscopia confocal. Culturas primárias de lactotrofos foram também transfetadas com DNA plasmídico que codifica HCN2 juntamente com a proteína-verde-fluorescente e um agente farmacológico foi usado para avaliar o efeito de cAMP-HCN na exocitose. Observou-se que os lactotrofos responderam à forscolina e ao dbcAMP libertando PRL de um modo bifásico e dependente da concentração, uma vez que a secreção aumentou e diminuiu, respectivamente, na gama de baixas e altas concentrações. Os compostos que elevaram os níveis de cAMP aumentaram os eventos transientes e impediram a fusão completa. Além disso, o dbcAMP promoveu o aparecimento de eventos exocitóticos transientes de elevada periodicidade, cujos poros de fusão, de maior diâmetro, se mativeram abertos durante mais tempo. A expressão das quatro isoformas de HCN foi confirmada nos lactotrofos ao nível do mRNA e, tal como no coração, rim e hipófise, o mais abundante codifica a isoforma HCN2. Nos lactotrofos com sobre-expressão desta isoforma, o dbcAMP não só aumentou a frequência dos eventos transientes e a condutância dos poros, mas também a frequência dos eventos de fusão completa. Enquanto o bloqueador dos canais HCN, ZD7288, reduziu a frequência dos eventos transientes e de fusão completa desencadeados por dbcAMP e diminuiu o diâmetro dos poros de fusão. A simultânea diminuição da libertação de PRL, da frequência dos eventos transientes e do diâmetro dos poros de fusão representaram as principais alterações observados após pré-tratamento dos lactotrofos com concentração micromolar de alumínio. Em conclusão, os resultados demonstram que elevados níveis de cAMP reduzem a secreção de PRL devido à estabilização dos poros de fusão no estado de maior abertura. Além disso, a via de sinalização cAMP-HCN afecta a actividade exocitótica e modifica as propriedades dos poros de fusão, que parecem ser igualmente importantes na citotoxicidade induzida por alumínio.

Molecular regulation of cork development: the QsMYB1 gene

Cork is a natural and renewable material obtained as a sustainable product from cork oak (Quercus suber L.) during the tree’s life. Cork formation is a secondary growth derived process resulting from the activity of cork cambium. However, despite its economic importance, only very limited knowledge is available about the molecular mechanisms underlying the regulation of cork biosynthesis and differentiation. The work of this PhD thesis was focused on the characterization of an R2R3-MYB transcription factor, the QsMYB1, previously identified as being putatively involved in the regulatory network of cork development. The first chapter introduces cork oak and secondary growth, with special emphasis on cork biosynthesis. Some findings concerning transcriptional regulation of secondary growth are also described. The MYB superfamily and the R2R3-MYB family (in particular) of transcription factors are introduced. Chapter II presents the complete QsMYB1 gene structure with the identification of two alternative splicing variants. Moreover, the results of QsMYB1 expression analysis, done by real-time PCR, in several organs and tissues of cork oak are also reported. Chapter III is dedicate to study the influence of abiotic stresses (drought and high temperature) and recovery on QsMYB1 expression levels. The effects of exogenous application of phytohormones on the expression profile of QsMYB1 gene are evaluated on Chapter IV. Chapter V describes the reverse genetic approach to obtain transgenic lines of Populus tremula L. x tremulóides Michx. overexpressing the QsMYB1 gene. Finally, in Chapter VI the final conclusions of this PhD thesis are presented and some future research directions are pointed based on the obtained results.