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

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

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

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

KRAS status and risk of venous thromboembolic events in patients with metastatic colorectal cancer: a case-control study.

No abstract availabl

Characterization of the Regulatory Region of the Zebrafish Prep1.1 Gene: Analogies to the Promoter of the Human PREP1

Prep1 is a developmentally essential TALE class homeodomain transcription factor. In zebrafish and mouse, Prep1 is already ubiquitously expressed at the earliest stages of development, with important tissue-specific peculiarities. The Prep1 gene in mouse is developmentally essential and has haploinsufficient tumor suppressor activity [1]. We have determined the human Prep1 transcription start site (TSS) by primer extension analysis and identified, within 20 bp, the transcription start region (TSR) of the zebrafish Prep1.1 promoter. The functions of the zebrafish 5′ upstream sequences were analyzed both by transient transfections in Hela Cells and by injection in zebrafish embryos. This analysis revealed a complex promoter with regulatory sequences extending up to −1.8, possibly −5.0 Kb, responsible for tissue specific expression. Moreover, the first intron contains a conserved tissue-specific enhancer both in zebrafish and in human cells. Finally, a two nucleotides mutation of an EGR-1 site, conserved in all species including human and zebrafish and located at a short distance from the TSS, destroyed the promoter activity of the −5.0 Kb promoter. A transgenic fish expressing GFP under the −1.8 Kb zebrafish promoter/enhancer co-expressed GFP and endogenous Prep1.1 during embryonic development. In the adult fish, GFP was expressed in hematopoietic regions like the kidney, in agreement with the essential function of Prep1 in mouse hematopoiesis. Sequence comparison showed conservation from man to fish of the sequences around the TSS, within the first intron enhancer. Moreover, about 40% of the sequences spread throughout the 5 Kbof the zebrafish promoter are concentrated in the −3 to −5 Kb of the human upstream region