Transcription regulation through chromatin structure and nuclear compartmentalization

Transcription is a very complex multi-step process presenting different levels of regulation. A large amount of general transcription factors and cofactors recruited on the promoters participate, together with the polymerases, in driving RNA production. The formation of chromatin loops allows their interaction with specific transcription factors bound to distant regulatory sequences and the fine tuning of the gene activity. A further level of complexity is provided by the structural and functional compartmentalization of the nucleus. In fact gene transcription takes place in a strongly localized fashion and nuclear architecture can influence genome regulation. One of the most intriguing findings is that an acto-myosin network plays a role in gene transcription. However, the in vivo role of such proteins in gene expression is still largely unclear. During my PhD I developed a technical approach coupling MNase digestion to ChIP by which I showed that the enhancer and minimal promoter of the human uPA gene function as a single transcription control unit forming a stable structure, that is required to sustain the early elongation step of RNAP-II. I next studied the uPA gene in an inducible cell system showing that it is associated with an inactive RNAP-II transcription factory before the onset of its expression while transcriptional induction promotes its association with an active transcription factory. This finding indicates inactive factories as distinct entities from the active ones supporting the notion of specialized transcription factories. I also studied the involvement of MyosinVI in transcription, characterizing its role in regulating RNAP-II activity. MyosinVI is required for phosphorylation of RNAP-II CTD at level of Serin-2 that, in turn, is required for the enzyme to proceed in the elongating phase. Finally, I showed that the transcription factor Prep1 and MyosinVI are associated in a complex and that the recruitment of MyosinVI on the Prep1-target genes is mediated by the transcription factor itself

Loading, Release, Biodegradation, and Biocompatibility of a Nanovector Delivery System

A nanovector multistage system has been created to overcome or bypass sequential barriers within the human body, in order to deliver a therapeutic or imaging agent to a specific location. This innovation consists of a composition that includes two or more stages of particles, such that smaller, later-stage particles are contained in the larger, early-stage particles. An active agent, such as a therapeutic agent or imaging agent, is preferentially delivered and/or localized to a particular target site in the body of a subject. The multistage composition overcomes multiple biological barriers in the body. The multistage composition also allows for simultaneous delivery and localization at the same or different target sites of multiple active agents

Inter-comparison of MARS and FLUKA: Predictions on Energy Deposition in LHC IR Quadrupoles

Detailed modellings of the LHC insertion regions (IR) have earlier been performed to evaluate energy deposition in the IR superconducting magnets [1-4]. Proton-proton collisions at 14 TeV in the centre of mass lead to debris, depositing energy in the IR components. To evaluate uncertainties in those simulations and gain further confidence in the tools and approaches used, inter-comparison calculations have been performed with the latest versions of the FLUKA (2006.3b) [5, 6] and MARS15 [7, 8] Monte Carlo codes. These two codes, used worldwide for multi particle interaction and transport in accelerator, detector and shielding components, have been thoroughly benchmarked by the code authors and the user community (see, for example, recent [9, 10]). In the study described below, a better than 5% agreement was obtained for energy deposition calculated with these two codes - based on different independent physics models - for the identical geometry and initial conditions of a simple model representing the IR5 and its first quadrupole

Perspectives of stack and environmental monitoring in the surroundings of a waste-to-energy plant

Waste-to-Energy (WtE) processes respond both to the emerging need for reducing the flow of waste into the environment and, at the same time, to the increasing demand for energy. Despite, these evident advantages, WtE plants still present some critical issues regarding the emissions of heavy metals into the atmosphere, which are regulated by the environmental legislations but, concerning the European Union, are regarded as groups of metals with no consideration of the different carcinogenic potential of each metal. In addition, there are uncertainties on the estimation of the balance of carbon dioxide, which depends on several factors like transportation, type of incoming waste, processes in use and secondary emissions. Despite great improvements in air pollution control from this sector, persistent organic pollutants are still emitted by WtE plants. Therefore, the implementation of adequate environmental monitoring (EM) plans is essential to monitor the impact of WtE plants in their surroundings, especially in the presence of the population, fields and pastures. In view of these considerations, this paper aims to provide guidance on basic and novel approaches that are necessary for a comprehensive monitoring of the impacts of a new WtE plant in terms of air quality and public health. A case study regarding the EM plan proposed for a new WtE plant will also be reported as an example

The Rest Repression of the Neurosecretory Phenotype Is Negatively Modulated by BHC80, a Protein of the BRAF/HDAC Complex

Expression of neurosecretion by nerve cells requires the levels of the transcription repressor element-1 silencing transcription factor (REST) to be very low. However, when high-REST clones of PC12 cells, defective of neurosecretion, were fused to other high-REST, non-neurosecretory cells, some neurosecretion was recovered. To clarify the mechanism of this recovery, we fused defective PC12 cells with human lymphocytes. A cytogenetic analysis revealed all hybrid clones that recovered neurosecretion to contain a fragment of chromosome 11 including the gene encoding BHC80, a protein of one of the complexes that mediate REST repression. In these clones, REST levels were as high as in defective PC12, whereas BHC80, localized in the nucleus, was 4- to 5-fold higher. Transient transfection of defective PC12 with various amounts of BHC80 cDNA induced (1) in defective PC12, the reexpression of only neurosecretion mRNAs; (2) in defective PC12 cotransfected with the REST negative construct DNA-binding domain (to attenuate gene repression), the recovery of a weak, but complete neurosecretory phenotype, including dense-core granules and their regulated exocytosis. Chromatin immunoprecipitation and immunodepletion analyses revealed the extensive BHC80 association with REST at the genes of two neurosecretion proteins, chromograninB and SNAP25, however only in the low-REST PC12, whereas in high-REST defective PC12 no association was appreciable. In defective PC12 transfected with BHC80 some association was reestablished. Therefore, the recovery of neurosecretion observed after fusion/transfection of defective PC12 depends on the reciprocal level of BHC80 and REST, with BHC80 working as a negative modulator of REST repression. This role appears of possible cell physiological and pathological importance

Methylation of RNA polymerase II non-consensus Lysine residues marks early transcription in mammalian cells

Dynamic post-translational modification of RNA polymerase II (RNAPII) coordinates the co-transcriptional recruitment of enzymatic complexes that regulate chromatin states and processing of nascent RNA. Extensive phosphorylation of serine residues at the largest RNAPII subunit occurs at its structurally-disordered C-terminal domain (CTD), which is composed of multiple heptapeptide repeats with consensus sequence Y1-S2-P3-T4-S5-P6-S7. Serine-5 and Serine-7 phosphorylation mark transcription initiation, whereas Serine-2 phosphorylation coincides with productive elongation. In vertebrates, the CTD has eight non-canonical substitutions of Serine-7 into Lysine-7, which can be acetylated (K7ac). Here, we describe mono- and di-methylation of CTD Lysine-7 residues (K7me1 and K7me2). K7me1 and K7me2 are observed during the earliest transcription stages and precede or accompany Serine-5 and Serine-7 phosphorylation. In contrast, K7ac is associated with RNAPII elongation, Serine-2 phosphorylation and mRNA expression. We identify an unexpected balance between RNAPII K7 methylation and acetylation at gene promoters, which fine-tunes gene expression levels

Psychological Well-Being in Italian Families : An Exploratory Approach to the Study of Mental Health Across the Adult Life Span in the Blue Zone

Self-reported measures of psychological well-being and depressive symptoms were examined across differently aged family members, while controlling for the impact of marital status and personal satisfaction about family and non-family relations. Twenty-one grandchildren (i.e., ages 21-36 years) were recruited with their parents (i.e., 48-66 years old) and grandparents (i.e., 75-101 years of age) in the 'blue zone' of Ogliastra, an Italian area known for the longevity of its inhabitants. Each participant was individually presented a battery of questionnaires assessing their lifestyle and several perceived mental health indices, including the Warwick-Edinburgh Mental Well-Being Scale (WEMWBS, Tennant et al., 2007), and the Center for Epidemiologic Studies Depression Scale (i.e., CES-D, Radloff, 1977). After assessing the level of concordance among adults sharing the same context, the Hierarchical Linear Modeling (HLM) approach was used to assess the nested dataset. It was found that family membership (i.e., grandchildren versus parents and grandparents) predicted the WEMWBS score but not the CES-D when the impact of marital status and personal satisfaction about social (i.e., family and non-family) ties was controlled for. Moreover, two separate repeated-measure Analyses of Variance (ANOVAs) documented similar level of personal satisfaction about social relationships across the three family groups. In conclusions, satisfying social ties with friends and family members together with an active socially oriented life style seems to contribute to the promotion of mental health in adult span

RNA polymerase II primes Polycomb-repressed developmental genes throughout terminal neuronal differentiation

Polycomb repression in mouse embryonic stem cells (ESCs) is tightly associated with promoter co-occupancy of RNA polymerase II (RNAPII) which is thought to prime genes for activation during early development. However, it is unknown whether RNAPII poising is a general feature of Polycomb repression, or is lost during differentiation. Here, we map the genome-wide occupancy of RNAPII and Polycomb from pluripotent ESCs to non-dividing functional dopaminergic neurons. We find that poised RNAPII complexes are ubiquitously present at Polycomb-repressed genes at all stages of neuronal differentiation. We observe both loss and acquisition of RNAPII and Polycomb at specific groups of genes reflecting their silencing or activation. Strikingly, RNAPII remains poised at transcription factor genes which are silenced in neurons through Polycomb repression, and have major roles in specifying other, non-neuronal lineages. We conclude that RNAPII poising is intrinsically associated with Polycomb repression throughout differentiation. Our work suggests that the tight interplay between RNAPII poising and Polycomb repression not only instructs promoter state transitions, but also may enable promoter plasticity in differentiated cells