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ChromArch Report Summary

Project ID: 637987
Funded under: H2020-EU.1.1.

Periodic Reporting for period 1 - ChromArch (Single Molecule Mechanisms of Spatio-Temporal Chromatin Architecture)

Reporting period: 2015-05-01 to 2016-10-31

Summary of the context and overall objectives of the project

Chromatin packaging into the nucleus of eukaryotic cells is highly sophisticated. It not only serves to condense the genomic content into restricted space, but mainly to encode epigenetic traits ensuring temporally controlled and balanced transcription of genes and coordinated DNA replication and repair. Owing to fluorescence in situ hybridization (FISH) assays and novel chromatin conformation capture (3C) techniques, it became evident that regulatory traits are not only encrypted along the one-dimensional sequence of DNA, but within the three-dimensional arrangement of chromatin. This non-random chromatin organization ranging from the state of compaction by nucleosomes over topologically associated domains to the relative location of whole chromosomes is of utmost importance for the correct read-out and control of genetic information. Transcription of a gene for example can be significantly increased by long-range chromatin interactions between enhancers and promoters, and misarrangements of chromatin structures are associated with severe diseases.

A rich variety of biomolecules involved in organizing the genome has been identified, including nuclear lamina, non-coding RNA molecules and architectural proteins. However, despite increasing interest in spatio-temporal chromatin organization, mechanistic details of their contributions to establishing and maintaining chromatin topology are not well understood. Many open question beyond the identification of participating factors are unanswered. How long does it take for the distal ends of a chromatin sequence to form a loop, how long do functional connections, for example enhancer-promoter interactions, persist and what are the molecular mechanisms of tethering two genetic regions together?

We aim at unveiling molecular mechanisms of chromatin organization by quantitative in vivo and in vitro single molecule experiments. 3C techniques can detect changes in chromatin organization upon cell differentiation or during the cell cycle, but important information on the frequency of interactions within a cell population and the temporal stability of distant chromatin associations remain elusive due to averaging over many cells and the destructive nature of these methods. We thus use single cell and single molecule fluorescence microscopy to measure the dynamics of organizational structures within chromatin and to study the molecular mechanisms of biomolecules mediating chromatin topology in the nucleus. In complementary single molecule force spectroscopy experiments we study mechanisms of chromatin structure formation in vitro. Our goal is to enhance the mechanistic understanding of three-dimensional chromatin architecture and its regulatory effects on nuclear functions and to inspire experiments on the potential therapeutic utility of controlled modification of regulatory traits mediating chromatin topology.

Work performed from the beginning of the project to the end of the period covered by the report and main results achieved so far

During the first reporting period, biochemical groundwork has been performed to prepare and enable single molecule fluorescence microscopy of biomolecules mediating chromatin topology. Initial measurements have been conducted and point towards previously unknown mechanisms of action of these biomolecules. Moreover, preparatory work for fluorescence measurements of specific chromatin structures has been started. In addition, design and setup of an in vitro force spectroscopy instrument is under way, and initial test experiments have been conducted. First results obtained with the new setup inspired novel design considerations for an improved instrument.

Progress beyond the state of the art and expected potential impact (including the socio-economic impact and the wider societal implications of the project so far)

The experiments on biomolecules involved in structuring the genome start to reveal novel insights into their mechanisms of action. At the end of the project it is expected that we will be able to understand in detail how these molecules engage in organizing chromatin structures. Our findings will inspire future experiments on the regulatory effect of three-dimensional chromatin architecture and potential therapeutic approaches upon its controlled modification.
The work on the force spectroscopy instrument revealed potential improvements of current state of the art designs. Once implemented, previously impractical experimental assays will become possible and enable novel biological and biophysical insights.
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