Developmental dynamics of transcription and genome architecture

Abstract Regulation of gene expression is necessary for the control of complex developmental processes. The genome has to shape in specific conformations to fit inside the nucleus and to tether specific regulatory elements to their target genes. Although the linear composition of many genomes is largely known, their three dimensional (3D) organization and dynamics are largely unknown. Hence, in order to unravel gene regulation, it is necessary to understand the chromatin structure and organization. Furthermore, developmental procedures are controlled by complex combinatorial transcription factor (TF) networks. Hence, unveiling those networks will provide a better insight towards understanding those developmental procedures. The work described in this thesis aims to study the genome conformation/interactome and their effect on gene regulation and to unveil the role of transcription factor proteins (TFs) in complex developmental processes

Cardiovascular Disease and Chronic Inflammation in End Stage Kidney Disease

Background: Chronic Kidney Disease (CKD) is one of the most severe diseases worldwide. In patients affected by CKD, a progressive destruction of the nephrons is observed not only in structuralbut also in functional level. Atherosclerosis is a progressive disease of large and medium-sized arteries. It is characterized by the deposition of lipids and fibrous elements and is a common complication of the uremic syndrome because of the coexistence of a wide range of risk factors. High blood pressure, anaemia, insulin resistance, inflammation, high oxidative stress are some of the most common factors that cause cardiovascular disease and atherogenesis in patients suffering from End Stage Kidney Disease (ESRD). At the same time, the inflammatory process constitutes a common element in the apparition and development of CKD. A wide range of possible causes can justify the development of inflammation under uremic conditions. Such causes are oxidative stress, oxidation, coexistentpathological conditions as well as factors that are due to renal clearance techniques. Patients in ESRD and coronary disease usually show increased acute phase products. Pre-inflammatory cytokines, such as IL-6 and TNF-a, and acute phase reactants, such as CRP and fibrinogen, are closely related. The treatment of chronic inflammation in CKD is of high importance for the development ofthe disease as well as for the treatment of cardiovascular morbidity.Conclusions: The treatment factors focus on the use of renin-angiotensic system inhibitors, acetylsalicylic acid, statins and anti-oxidant treatment in order to prevent the action of inflammatorycytokines that have the ability to activate the mechanisms of inflammation

Enhancers and silencers: an integrated and simple model for their function

Regulatory DNA elements such as enhancers, silencers and insulators are embedded in metazoan genomes, and they control gene expression during development. Although they fulfil different roles, they share specific properties. Herein we discuss some examples and a parsimonious model for their function is proposed. All are transcription units that tether their target promoters close to, or distant from, transcriptional hot spots (or 'factories')

Targeted Chromatin Capture (T2C): A novel high resolution high throughput method to detect genomic interactions and regulatory elements.

Background: Significant efforts have recently been put into the investigation of the spatial organization and the chromatin-interaction networks of genomes. Chromosome conformation capture (3C) technology and its derivatives are important tools used in this effort. However, many of these have limitations, such as being limited to one viewpoint, expensive with moderate to low resolution, and/or requiring a large sequencing effort. Techniques like Hi-C provide a genome-wide analysis. However, it requires massive sequencing effort with considerable costs. Here we describe a new technique termed Targeted Chromatin Capture (T2C), to interrogate large selected regions of the genome. T2C provides an unbiased view of the spatial organization of selected loci at superior resolution (single restriction fragment resolution, from 2 to 6 kbp) at much lower costs than Hi-C due to the lower sequencing effort. Results: We applied T2C on well-known model regions, the mouse β-globin locus and the human H19/IGF2 locus. In both cases we identified all known chromatin interactions. Furthermore, we compared the human H19/IGF2 locus data obtained from different chromatin conformation capturing methods with T2C data. We observed the same compartmentalization of the locus, but at a much higher resolution (single restriction fragments vs. the common 40 kbp bins) and higher coverage. Moreover, we compared the β-globin locus in two different biological samples (mouse primary erythroid cells and mouse fetal brain), where it is either actively transcribed or not, to identify possible transcriptional dependent interactions. We identified the known interactions in the β-globin locus and the same topological domains in both mouse primary erythroid cells and in mouse fetal brain with the latter having fewer interactions probably due to the inactivity of the locus. Furthermore, we show that interactions due to the important chromatin proteins, Ldb1 and Ctcf, in both tissues can be analyzed easily to reveal their role on transcriptional interactions and genome folding. Conclusions: T2C is an efficient, easy, and affordable with high (restriction fragment) resolution tool to address both genome compartmentalization and chromatin-interaction networks for specific genomic regions at high resolution for both clinical and non-clinical research

Exploiting native forces to capture chromosome conformation in mammalian cell nuclei

Mammalian interphase chromosomes fold into a multitude of loops to fit the confines of cell nuclei, and looping is tightly linked to regulated function. Chromosome conformation capture (3C) technology has significantly advanced our understanding of this structure-to-function relationship. However, all 3C-based methods rely on chemical cross-linking to stabilize spatial interactions. This step remains a “black box” as regards the biases it may introduce, and some discrepancies between microscopy and 3C studies have now been reported. To address these concerns, we developed “i3C”

Pre-B Cell Receptor Signaling Induces Immunoglobulin κ Locus Accessibility by Functional Redistribution of Enhancer-Mediated Chromatin Interactions

During B cell development, the precursor B cell receptor (pre-BCR) checkpoint is thought to increase immunoglobulin κ light chain (Igκ) locus accessibility to the V(D)J recombinase. Accordingly, pre-B cells lacking the pre-BCR signaling molecules Btk or Slp65 showed reduced germline Vκ transcription. To investigate whether pre-BCR signaling modulates Vκ accessibility through enhancer-mediated Igκ locus topology, we performed chromosome conformation capture and sequencing analyses. These revealed that already in pro-B cells the κ enhancers robustly interact with the ∼3.2 Mb Vκ region and its flanking sequences. Analyses in wild-type, Btk, and Slp65 single- and double-deficient pre-B cells demonstrated that pre-BCR signaling reduces interactions of both enhancers with Igκ locus flanking sequences and increases interactions of the 3′κ enhancer with Vκ genes. Remarkably, pre-BCR signaling does not significantly affect interactions between the intronic enhancer and Vκ genes, which are already robust in pro-B cells. Both enhancers interact most frequently with highly used Vκ genes, which are often marked by transcription factor E2a. We conclude that the κ enhancers interact with the Vκ region already in pro-B cells and that pre-BCR signaling induces accessibility through a functional redistribution of long-range chromatin interactions within the Vκ region, whereby the two enhancers play distinct roles

Targeted chromatin conformation analysis identifies novel distal neural enhancers of ZEB2 in pluripotent stem cell differentiation

The transcription factor zinc finger E-box binding protein 2 (ZEB2) controls embryonic and adult cell fate decisions and cellular maturation in many stem/progenitor cell types. Defects in these processes in specific cell types underlie several aspects of Mowat-Wilson syndrome (MOWS), which is caused by ZEB2 haplo-insufficiency. Human ZEB2, like mouse Zeb2, is located on chromosome 2 downstream of a ±3.5 Mb-long gene-desert, lacking any protein-coding gene. Using temporal targeted chromatin capture (T2C), we show major chromatin structural changes based on mapping in-cis proximities between the ZEB2 promoter and this gene desert during neural differentiation of human-induced pluripotent stem cells, including at early neuroprogenitor cell (NPC)/rosette state, where ZEB2 mRNA levels increase significantly. Combining T2C with histone-3 acetylation mapping, we identified three novel candidate enhancers about 500 kb upstream of the ZEB2 transcription start site. Functional luciferase-based assays in heterologous cells and NPCs reveal co-operation between these three enhancers. This study is the first to document in-cis Regulatory Elements located in ZEB2's gene desert. The results further show the usability of T2C for future studies of ZEB2 REs in differentiation and maturation of multiple cell types and the molecular characterization of newly identified MOWS patients that lack mutations in ZEB2 protein-coding exons

TRIM33 switches off Ifnb1 gene transcription during the late phase of macrophage activation

Despite its importance during viral or bacterial infections, transcriptional regulation of the interferon-β gene (Ifnb1) in activated macrophages is only partially understood. Here we report that TRIM33 deficiency results in high, sustained expression of Ifnb1 at late stages of toll-like receptor-mediated activation in macrophages but not in fibroblasts. In macrophages, TRIM33 is recruited by PU.1 to a conserved region, the Ifnb1 Control Element (ICE), located 15 kb upstream of the Ifnb1 transcription start site. ICE constitutively interacts with Ifnb1 through a TRIM33-independent chromatin loop. At late phases of lipopolysaccharide activation of macrophages, TRIM33 is bound to ICE, regulates Ifnb1 enhanceosome loading, controls Ifnb1 chromatin structure and represses Ifnb1 gene transcription by preventing recruitment of CBP/p300. These results characterize a previously unknown mechanism of macrophage-specific regulation of Ifnb1 transcription whereby TRIM33 is critical for Ifnb1 gene transcription shutdown

Developing a competence framework for nurses in pharmaceutical care: A Delphi study

Background: Nurses play an important role in pharmaceutical care. They are involved in: detecting clinical change; communicating/discussing pharmacotherapy with patients, their advocates, and other healthcare professionals; proposing and implementing medication-related interventions; and ensuring follow-up of patients and medication regimens. To date, a framework of nurses' competences on knowledge, skills, and attitudes as to interprofessional pharmaceutical care tasks is missing. Objectives: To reach agreement with experts about nurses' competences for tasks in interprofessional pharmaceutical care. Methods: A two-phase study starting with a scoping review followed by five Delphi rounds was performed. Competences extracted from the literature were assessed by an expert panel on relevance by using the RAND/UCLA method. The experts (n = 22) involved were healthcare professionals, nurse researchers, and educators from 14 European countries with a specific interest in nurses' roles in interprofessional pharmaceutical care. Descriptive statistics supported the data analysis. Results: The expert panel reached consensus on the relevance of 60 competences for 22 nursing tasks. 41 competences were related to 15 generic nursing tasks and 33 competences were related to seven specific nursing tasks. Conclusions: This study resulted in a competence framework for competency-based nurse education. Future research should focus on imbedding these competences in nurse education. A structured instrument should be developed to assess students' readiness to achieve competence in interprofessional pharmaceutical care in clinical practice.The research was supported by the Erasmus+ Programme of the European Union (grant number 2018–1-BE02-KA203–046861) and MDMJ accountants, an accountancy service in Belgium that financially supported the Belgian authors, without any conflicts of interest

Nurse students’ competences in interprofessional pharmaceutical care in Europe: cross-sectional evaluation

Background: Safe pharmaceutical care requires competent nurses with specific knowledge, skills and attitudes. It is unclear whether nursing students are adequately prepared to perform pharmaceutical care in practice. Mapping their pharmaceutical care competences can lead to a better understanding of the extent to which curricula fit expectations of the labour market. Objectives: To assess pharmaceutical care competences of final-year nursing students of different educational levels. Design: A cross-sectional survey design. Settings: In 14 European countries, nursing schools who offer curricula for level 4 to 7 students, were approached. Participants: Through convenience sampling 1741 final-year student nurses of level 4 to 7 were included. Sampling strategies were country-specific. Methods: A web-platform was developed with an assessment of the level in which students mastered pharmaceutical care competences. Knowledge questions, case studies (basic/advanced level), self-reported practical skills and attitudes were evaluated. Results: Mean scores for knowledge questions differed significantly (p<0.001) between level 5 (56/100), level 6 (68/100) and level 7 students (72/100). For basic cases level 5 students reached lower scores (64/100) compared with level 6 (71/100) and level 7 (72/100) students (p=0.002 and p=0.005). For more advanced cases no difference between levels was observed (overall mean 61/100). Most students (63-90%) considered themselves skilled to perform pharmaceutical care and had positive attitudes towards their participation in pharmaceutical care (65-97%). Conclusions: Relatively low knowledge scores were calculated for final-year student nurses. In some domains, lower levels of students might be insufficiently prepared to take up responsibilities in pharmaceutical care. Our assessment can be used as a tool for educators to evaluate how prepared nursing students are for pharmaceutical care. Its further implementation for students of different educational levels will allow benchmarking between the levels, both within and between countries.This work was supported by the Erasmus+ Programme of the European Union [grant number 2018-1-BE02-KA203-046861] and Consensus accountants, an accountancy service in Belgium that financially supported the Belgian authors, without any conflicts of interest