Revealing the mechanism of replication initialization with a sequential microscopy-based approach
Final Report Abstract
In the course of my DFG Research Fellowship, I worked on three projects that focused on different aspects of the functional consequence of chromatin organization. The first project centered on the spatial organization of enhancers and promoters (genetic elements that regulate gene transcription) within a topologically associating domain (TAD), and how this organization was linked to gene transcription in distinct cell types. Surprisingly, we found a similar network of interactions between enhancers and promoters in both cells that are either transcriptionally active or repressed. Altogether, our results suggest that the 3D chromatin architecture can play a double role, as contacts serve to reinforce both gene activation and repression. In addition, we found that the interaction between enhancers and promoters arise early during development of the fruit fly Drosophila melanogaster embryo, even before TADs – that are thought to insulate promoters from unrelated, neighbouring enhancers – emerge. Recently, it became clear that chromatin organization is highly variable and largely differs from cell to cell. Thus, the second project aimed at shedding light on the interplay between single-cell chromatin organization and transcriptional state. We found a large heterogeneity in chromatin architecture that was similar in both transcriptionally active and repressed cells. This means that it is very unlikely to find two cells that have the same chromatin organization, even if their transcriptional program is very similar. Also, we didn’t observe a strong correlation between the presence of a TAD boundary and the transcriptional state in single cells, indicating that other mechanisms than encapsulating enhancer-promoter interactions in TADs might be at work to isolate promoters from surrounding regulatory elements. The third project addressed the spatial organization of origins of replication. In order to produce new cells, all living organisms need to efficiently and faithfully replicate their DNA. In eukaryotic organisms, such as mice or humans, the prevalent model predicts that an organization of chromatin in space and time contributes to the activation of a distinct subset of origins of replication, the specific genomic sequences where DNA replication is initiated. To test this model directly, we aimed to map the position of origins in mouse embryonic stem cells (mESCs). For this, we successfully implemented a labeling and imaging strategy and proved the feasibility of sequential labeling of origins in mESCs. This paves the way for future studies that will dissect the mechanisms of origin activation.
Publications
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2021. “Cis-Regulatory Chromatin Loops Arise before TADs and Gene Activation, and Are Independent of Cell Fate during Early Drosophila Development.” Nature Genetics 53 (4): 477–86
Sergio Martin Espinola, Markus Götz, Maelle Bellec, Olivier Messina, Jean-Bernard Fiche, Christophe Houbron, Matthieu Dejean, Ingolf Reim, Andrés M. Cardozo Gizzi, Mounia Lagha, and Marcelo Nollmann
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2021. “The Impact of Space and Time on the Functional Output of the Genome.” Cold Spring Harbor Perspectives in Biology, a040378
Marcelo Nollmann, Isma Bennabi, Markus Götz, and Thomas Gregor
