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Genetic causes of atherosclerosis and their molecular and cellular implications at the susceptibility locus on chromosome 21
Get PDFAtherosclerosis is a chronic inflammatory disease of large and medium-sized vessels. It is induced and maintained by various cellular and molecular mechanisms. Besides environmental factors, the genetic background of an individual contributes to the predisposition for the development of atherosclerosis. With the help of genome-wide association studies (GWAS), many genetic variants associated with atherosclerosis have already been unraveled. However, for most of the variants, their causative role in atherogenesis is still widely unclear, not least because many GWAS hits lie in non-protein-coding parts of the genome which are, thus far, not so well understood.
It was the aim of this thesis to create a comprehensive overview of currently known atherosclerosis susceptibility loci from published GWAS and, subsequently, to focus on one non-protein-coding risk locus to explore its genetic, molecular and cellular implications for atherogenesis.
Therefore, in a first step, we compiled all GWAS (n = 124) associated with atherosclerosis and its risk factors published up to May 2016. The resulting polymorphisms (SNPs) (n = 216) were used to define haplotype blocks (n = 120), as a tool for examining candidate genetic effector mechanisms. 11 out of 120 atherosclerosis risk-associated haplotype blocks solely contained noncoding RNAs. Focusing on these, and after ranking by effect size, we identified a prominent (OR 1.14) and so far uncharacterized atherosclerosis risk locus on chromosome 21q22.11, and marked it for further examination. It contained two lead SNPs with robust atherosclerosis association, rs9982601 (p-value 1.33x10-13) and rs28451064 (p-value 1.33x10-15). Since both SNPs resided within a lncRNA (AP000318.2) but were in linkage disequilibrium with lower ranking atherosclerosis associations in a relatively large noncoding region (> 100 kilobases), we generated two knockouts of differing sizes in human induced pluripotent stem cells (iPSCs) by CRISPR/CAS9 technology, in order to address potential effector mechanisms. Knockout and control wild type hiPSCs were differentiated into cells relevant for atherosclerosis, namely vascular smooth muscle (SMCs) and endothelial cells (ECs). We detected enhanced expression of neighbouring genes when regions encompassing parts of the lncRNA-encoding core locus were deleted. This effect was most prominently seen in SMCs and pertained to genes 3’ and 5’ to the risk locus, including protein-coding genes, over a genomic distance of at least 700 kilobases. In terms of function, we observed altered apoptosis as well as decreased proliferation and migration, and increased adhesion for the knockout comprising the atherosclerosis risk interval. These are features observed in atherosclerotic lesions, hence underlining the atherogenic character of the knockout. Collectively, our data from knockout analyses narrowed down the effector element to reside between chr21:35589815-35653842 and gave rise to the hypothesis that the atherosclerosis-causing mutation on chr21q22.11 deactivates a noncoding, gene-regulatory element, thereby leading to proatherogenic gene expression nearby, and downstream altered proatherogenic mechanisms in cells that constitute the vascular wall
Auswirkungen von regelmäßiger Bewegung, akuter sportlicher Extrembelastung und Erholung anhand eines Marathonlaufes auf neurokognitive, psychopathologische und kardiovaskuläre Funktionen
Get PDFSelf-organization in heterogeneous biological systems
Get PDFSelf-organization is an ubiquitous and fundamental process that underlies all living systems. In cellular organisms, many vital processes, such as cell division and growth, are spatially and temporally regulated by proteins -- the building blocks of life. To achieve this, proteins self-organize and form spatiotemporal patterns. In general, protein patterns respond to a variety of internal and external stimuli, such as cell shape or inhomogeneities in protein activity. As a result, the dynamics of intracellular pattern formation generally span multiple spatial and temporal scales. This thesis addresses the underlying mechanisms that lead to the formation of heterogeneous patterns. The main themes of this work are organized into three parts, which are summarized below.
The first part deals with the general problem of mass-conserving reaction-diffusion dynamics in spatially non-uniform systems. In section 1 of chapter II, we study the dynamics of the E. coli Min protein system -- a paradigmatic model for pattern formation. More specifically, we consider a setup with a fixed spatial heterogeneity in a control parameter, and show that this leads to complex multiscale pattern formation. We develop a coarse-graining approach that enables us to explain and reduce the dynamics to the "hydrodynamic variables'' at large length and time scales. In another project, we consider a system where spatial heterogeneities are not imposed externally, but self-generated by the dynamics via a mechanochemical feedback loop between geometry and reaction-diffusion system (section 2 of chapter II). We show that the resulting dynamics can be explained from the phase-space geometry of the reaction-diffusion system.
The second part focuses on how patterns in realistic cell geometries are controlled by shape and biochemical cues. We examine axis selection of PAR polarity patterns in C. elegans, where we show that spatial variations in the bulk-surface ratio and a tendency of the system to minimize the pattern interface yield robust long-axis polarization of PAR protein patterns (section 1 of chapter III). In a second project, we develop a theoretical model that explains the localization of the B. subtilis Min protein system (section 2 of chapter 3). We show that a biochemical cue -- which acts as a template for pattern formation -- guides and stabilizes Min patterns.
In the third part, we study the coupling between lipid membranes and curvature-generating proteins. We demonstrate that myosin-VI motor proteins cooperatively bind to saddle-shaped regions of lipid membranes, and thereby induce large-scale membrane remodeling (section 1 of chapter IV). To understand the dynamics, we develop a coarse-grained geometric model and show that the emergence of regular spatial structures can be explained by a "push-pull'' mechanism: protein binding destabilizes the membrane shape at all length scales, and this is counteracted by line tension. Inspired by this system, we then investigate a general model for the dynamics of growing protein-lipid interfaces (section 2 of chapter IV). A key feature of the model is that the protein binding kinetics is explicitly coupled to the morphology of the interface.
We show that such a coupling leads to turbulent dynamics and a roughening transition of the interface that is characterized by universal scaling behaviour
Role of DDK kinase in DNA double-strand break repair and insights into the DDK-Cdc5/PLK1 kinase complex
Get PDFThe eukaryotic cell cycle consists of an ordered sequence of tightly regulated events to restrict specific activities within specific cycle phases. Key regulators are cell cycle kinases. Budding yeast harbor three essential cell cycle kinases conserved in humans: Dbf4-dependent kinase Cdc7 (DDK), Cyclin-dependent kinase (CDK) and the single yeast Polo-like kinase, Cdc5 (PLK1 in human).
DNA double-strand breaks (DSBs) are a severe form of DNA damage. Two main pathways evolved for the repair of such toxic lesions are homologous recombination (HR) and non-homologous end joining (NHEJ). HR often uses the homologous sequence of the sister chromatid as template for error-free DSB repair. Therefore, HR is upregulated in S, G2 and M phase when a sister chromatid is present, while NHEJ is the preferred repair pathway in G1. The crucial switch from repair via NHEJ to HR is considered to be the processing of the broken ends during DNA end resection, which primes for repair by HR and inhibits repair by NHEJ. Part of this regulation comes from CDK phosphorylation of Sae2-MRX (CtIP-MRN in human), which initiates DNA end resection. However, it is increasingly clear that additional cell cycle regulated mechanism might be involved in the regulation of DNA end resection initiation.
To identify novel functions of DDK, we performed phosphoproteomic experiments and discovered that DDK phosphorylates several proteins involved in DSB repair via HR, among which also factors involved in DNA end resection. We therefore followed a first line of research focused on the possible role of DDK in regulating DNA end resection. We showed that DDK is required for resection and HR, unveiling a previously unknown role of DDK in the cell cycle regulation of DSB repair. Mechanistically, we focused on phosphorylation of Sae2. We showed DDK-dependent phosphorylation of Sae2 in vivo and in vitro, and found that via phosphorylation of Sae2, DDK can stimulate the nucleolytic activity of the Sae2-MRX complex. Given the importance of DDK as regulator of resection, we tested if we could bypass the cell cycle regulation of DNA end resection and HR by forcing DDK expression in G1 cells. We observed that DDK expression in G1 cells lead to premature phosphorylation of Sae2, and by performing DNA end resection and HR assays we observed that synthetic activation of DDK in G1 leads to a moderate activation of HR, highlighting the central role of DDK in DSB repair pathway choice.
Cell cycle kinases also regulate the resolution of recombination structures by the Mus81- Mms4 nuclease during late steps of HR. DDK and Cdc5 can physically interact with each other and it was previously shown that this two-kinase complex is required for phosphorylation and activation of Mus81-Mms4. In a second project we therefore focused on the DDK-Cdc5 kinase complex and how it works as two-kinase complex to phosphorylate Mus81-Mms4 and other proteins. In a candidate approach, we identified a novel phosphorylation substrate of the kinase complex, the DNA replication factor Sld2, suggesting the DDK-Cdc5 complex might be a more general regulator of M phase. We developed protocols to purify to homogeneity from yeast cells the DDK-Cdc5 complex and the single kinases. Through a series of in vitro experiments, we showed that the DDK- Cdc5 complex was overall active as well as the single kinases. Different phosphorylation substrates were specifically phosphorylated by either one of the two kinases, either when on their own or as part of the complex, suggesting that within the complex each of the kinases could act as scaffold or adaptor for substrates. Lastly, we showed that also the human proteins DDK and PLK1 (human ortholog of Cdc5) physically interact, indicating that the DDK-Cdc5/DDK-PLK1 complex is an evolutionary conserved composite cell cycle regulator.
Taken together, the work presented in this thesis uncover a novel role of DDK in regulating DSB repair and offer insights into the evolutionary conserved DDK-Cdc5 kinase complex
