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Report

Teaser, summary, work performed and final results

Periodic Reporting for period 3 - ChromArch (Single Molecule Mechanisms of Spatio-Temporal Chromatin Architecture)

Teaser

Summary

Chromatin packaging into the nucleus of eukaryotic cells is highly complex. 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 associating 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

The architectural protein CTCF is involved in organizing topologically associating domains and chromatin loops. To gain a better understanding of its mechanism of action, we measured the time that CTCF is bound to chromatin. To this end we transducted fluorescently tagged CTCF molecules into cells and monitored their movement. We observed three subpopulations of CTCF exhibiting significantly different residence times. Our data suggest that CTCF scans DNA unspecifically in search for two different subsets of specific target sites and provide information on the timescales over which topologically associating domains might be restructured (~15 min). In cell cycle-resolved measurements we observed a drop of specific interactions with chromatin in S-phase, indicating that specific interactions need to be dissolved for replication to proceed. This work was published in Biophysical Journal in 2017.

Similar to CTCF, most transcription factors studied so far exhibit unspecific and specific binding to DNA. Using a simple state-based theoretical model that coarse-grains facilitated diffusion, we calculated that these interaction times allow for optimally fast target site search, but generally lead to low occupation frequencies of the specific target site. Due to its long residence time of several minutes, ~40% of CTCF molecules are specifically bound. This work was published on arXiv in 2017.

To obtain a better understanding of the importance of transcription factor DNA residence times on transcription regulation, we designed a series of transcription repressors differing in their DNA residence time by utilizing the modular DNA binding domain of transcription activator-like effectors (TALEs). The repressors competitively inhibited DNA binding of an endogenous transcription activator in a residence time-dependent manner. Thus transcription factor DNA residence time is a regulatory factor of transcription. This work was published in Nucleic Acids Research in 2017.

Zebrafish embryos do not transcribe their genome during the first period of rapid cell divisions until the ~1000 cell stage. During these cell divisions each cell and its nucleus decrease in volume. We investigated transcription factor binding during this developmental period and observed that the frequency of specific transcription factor - DNA interactions increased after each cell devision cycle, in accordance with the decreasing nuclear reaction volume. This suggests that a mechanism based on the law of mass action contributes to timing of zygotic transciption onset in the embryo. This work was published on arXiv in 2017.

Final results

Our single molecule experiments on CTCF and theoretical considerations of DNA residence times revealed important details on the mechanism of action of this architectural protein:
- how it searches and finds its specific sites amongst myriads of unspecific sequences
- how long specific sites are occupied and thus how long loop structures are stabilized
- how cells deal with CTCF-bound chromatin during replication.

The experiments varying the DNA residence time of a transcription factor directly demonstrate the importance of this time parameter for the regulation of transcription. Analogously, other processes relying on protein-DNA interaction, such as CTCF-mediated loop formation, will depend on the lifetime of the protein-DNA bond. In addition, these experiments established the DNA binding domain of TALEs as tool to study temporal aspects of gene regulation and chromatin architecture.

Our single molecule experiments on TBP-DNA interactions in live developing Zebrafish embryos brought forth several very important new experimental possibilities and scientific insights:
- by using reflected light sheet microscopy, single molecule tracking can now be performed in the natural envireonment of live developing embryos for several hours
- by using interlaced time-lapse microscopy, biomolecular interaction kinetics such as protein-DNA interactions can be quantified even during rapid cell division cycles
- the nucleus plays a role as decreasing reaction volume during early embryo development. Intranuclear biomolecules are thus concentrated during development, with tremendous impact on the fraction of biomolecules entering a complexed state.