Background: The dynamic three-dimensional chromatin architecture of genomes and its co-evolutionary connection to its function—the storage, expression, and replication of genetic information—is still one of the central issues in biology. Here, we describe the much debated 3D architecture of the human and mouse genomes from the nucleosomal to the megabase pair level by a novel approach combining selective high-throughput high-resolution chromosomal interaction capture (T2C), polymer simulations, and scaling analysis of the 3D architecture and the DNA sequence. Results: The genome is compacted into a chromatin quasi-fibre with ~5 ± 1 nucleosomes/11 nm, folded into stable ~30–100 kbp loops forming stable loop aggregates/rosettes connected by similar sized linkers. Minor but significant variations in the architecture are seen between cell types and functional states. The architecture and the DNA sequence show very similar fine-structured multi-scaling behaviour confirming their co-evolution and the above. Conclusions: This architecture, its dynamics, and accessibility, balance stability and flexibility ensuring genome integrity and variation enabling gene expression/regulation by self-organization of (in)active units already in proximity. Our results agree with the heuristics of the field and allow “architectural sequencing” at a genome mechanics level to understand the inseparable systems genomic properties.
Background: Genome organization into subchromosomal topologically associating domains (TADs) is linked to cell-type-specific gene expression programs. However, dynamic properties of such domains remain elusive, and it is unclear how domain plasticity modulates genomic accessibility for soluble factors. Results: Here, we combine and compare a high-resolution topology analysis of interacting chromatin loci with fluorescence correlation spectroscopy measurements of domain dynamics in single living cells. We identify topologically and dynamically independent chromatin domains of ~1 Mb in size that are best described by a loop-cluster polymer model. Hydrodynamic relaxation times and gyration radii of domains are larger for open (161 ± 15 ms, 297 ± 9 nm) than for dense chromatin (88 ± 7 ms, 243 ± 6 nm) and increase globally upon chromatin hyperacetylation or ATP depletion. Conclusions: Based on the domain structure and dynamics measurements, we propose a loop-cluster model for chromatin domains. It suggests that the regulation of chromatin accessibility for soluble factors displays a significantly stronger dependence on factor concentration than search processes within a static network.
Unlike many other species, where the body plan is already pre-patterned in the oocyte or upon fertilization, in the early mouse embryo there is no asymmetry up to 8-cell stage when all cells in the embryo have the same morphology and developmental potential. As development proceeds initially identical cells of the embryo segregate into two distinct cell lineages: trophectoderm (TE) and the inner cell mass (ICM) (Wennekamp et al., 2013; Rossant and Tam, 2009; Yamanaka et al., 2006). While both apical-basal cell polarity (Hirate et al., 2013; Alarcon, 2010) and cell-cell adhesion (Stephenson, Yamanaka and Rossant, 2010) are required for this differentiation, the decisive cue that breaks symmetry between the cells and is sufficient for specifying the first cell fate remains to be identified (Wennekamp et al., 2013). To understand the mechanism underlying the symmetry breaking in the mouse embryo, in this study I have established a new experimental system in which a blastomere isolated at the 8-cell stage (1/8th blastomere) recapitulates the first lineage segregation between TE and ICM during its development into 4/32th mini-blastocyst. Using live-imaging and quantitative image analysis, I identified that inheritance of the apical domain during 1/8th-to-2/16th-cell stage division allows for predicting the process leading to TE fate specification. The majority of 8-cell blastomeres undergo asymmetric division defined by the differential segregation of the apical domain among daughter cells. In the 8-cell stage embryo, the apical domain, emerging at the center of the contact-free surface of the blastomere, recruits microtubule organizing centers to the sub-apical region, thereby forming one of the acentrosomal spindle poles and inducing the asymmetric division. After asymmetric 8-to-16-cell stage division, all cells that inherit the apical domain express a TE marker, Cdx2. In contrast, apolar cells can either acquire ICM fate, as previously described, or, if positioned on the embryo surface, form a new apical domain and turn on Cdx2. Thus, contrary to the previous model (Johnson and Ziomek, 1981b), cell fate is determined by its position within the embryo, but not by the division pattern. Finally, using 1/8th blastomere, I showed that cell contact, not mediated by Cdh1, facilitates cellular symmetry breaking and directs the apical domain formation in the center of the contact-free surface, and that the inheritance of this apical domain predicts the acquisition of TE fate.
To truly understand the self-replicating eukaryotic cell we need to make significant progress unraveling the interactome, the sum of all the interactions between the proteins and the metabolites of the cell. Here, I present two projects to that end: The first study maps protein complexes in a unicellular thermophilic eukaryote, Chaetomium thermophilum, using an innovative approach integrating several biological techniques. Thermophilic proteins are, by their nature, more stable than their mesophilic counterparts and C. thermophilum has been described as a potential model organism for structural studies. We use a size exclusion chromatography (SEC) to separate high-molecular weight protein complexes from cell lysate. Coeleuting proteins are identified by mass spectrometry and inferred to be in a protein complex together. Chemical crosslinking combined with mass spectrometry (XL-MS) is applied to the SEC fractions to provide direct biochemical confirmation of the predicted protein-protein interactions. Together these methods have allowed us to identify protein complexes with novel subunits and also functionally related coeluting proteins not hitherto known to form protein complexes. Additionally, negative-stain electron microscope (EM) images of protein mixtures from the SEC fractions are correlated with the elution patterns of the identified complexes to distinguish the structural signatures (Shapes) of specific complexes. This enabled manual picking of particles from cryo-EM micrographs to solve the molecular structure of C. thermophilum fatty acid synthase (FAS) to 4.7Å resolution directly from the SEC fractions without further purification. A novel binder of FAS was identified by EM and confirmed with XL-MS as a branching biotin dependent carboxylase, which together constitutes a potential metabolon for the production of branch chain fatty acids. This project facilitates the use of C. thermophilum as a model organism for structural biology and the methods may open the way for high-throughput structural biology. The second project used a high-content screen to discern the roles of lipid classes on the localization of proteins, protein complexes and biological processes in the cell. This approach attempts to shed light on this protein-metabolite network by genetically depleting selected lipid classes by perturbing their biosynthesis and using in vivo imaging to map localization changes of proteins in the cells and therefore identification of lipid dependent localizations. The database created in this project will facilitate the development of follow-up studies that will further discern the structural roles of lipids in the organization of the proteome.
Although several lipids have been shown to participate in intracellular signal transduction events and to influence central cellular processes, the bioactive actions of most lipids remain unexplored. This lack of knowledge is mainly due to a shortage of tools to manipulate lipid levels within living cells in a non-invasive way and to identify new protein interactors of single lipid species. This work presents the development of two methods to overcome these drawbacks applied to sphingosine (Sph). The origin of calcium signaling properties of Sph and its involvement in the pathophysiological development of the lysosomal storage disease Niemann-Pick type C (NPC) are reported. First, ‘caged’ variants of sphingosine were synthesized which enable the precise elevation of Sph levels in single living cells within seconds using light. This acute increase in Sph concentration led to an immediate release of lysosomal calcium through the actions of the two-pore channel 1 (TPC1). In cells derived from NPC patients, an accumulation of Sph in the endolysosomal compartments was visualized for the first time. Additionally, NPC cells exhibited reduced calcium signals upon Sph uncaging, indicating that Sph accumulation is upstream of a calcium defect in this disease. Sph-induced calcium release also initiated the nuclear translocation of transcription factor EB, which positively regulates the expression of autophagic and lysosomal biogenesis genes, further underlining the importance of lysosomal calcium release in direct lysosome-to-nucleus signaling pathways. In order to capture Sph-interacting proteins, a trifunctional Sph (TFS) was developed. TFS facilitates the release and immediate crosslinking of Sph to its interacting partners within the living cell. Mass-spectrometric analyses identified known Sph-binding proteins such as the ceramide synthase, as well as novel putative Sph-interactors. The general applicability of this method was proven by using trifunctional diacylglycerol as well as a trifunctional fatty acid. TFS was further employed in investigations of the subcellular localization and transport of Sph through the cell. NPC patient fibroblasts showed a striking accumulation of Sph in late endosomes and lysosomes. Sph transport out of these vesicles was severely hindered in the NPC condition. The kinetics of Sph efflux correlated with the severity of symptoms in different NPC patients, so this assay could potentially be used for monitoring and prognosis of NPC disease severity.