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Chromatin readout: Dissecting the protein-chromatin interaction code in living cells

Periodic Reporting for period 1 - ChromatinLEGO (Chromatin readout: Dissecting the protein-chromatin interaction code in living cells)

Reporting period: 2020-09-01 to 2021-10-31

Eukaryotic genomes are packaged into highly ordered structures, consisting of nucleosomes as the basic unit of chromatin. Both, DNA and histones are decorated by various chemical modifications that positively or negatively influence biological processes such as transcription, genome organisation, DNA repair or DNA replication. These modifications are deposited and removed by “writer” and “eraser” proteins in a highly regulated fashion. Furthermore, regulatory proteins can read single or multiple modification, enabling the precise execution of various biological processes at defined genomic regions based on spatiotemporal readout of chromatin marks (Figure 1). How these "readers" mediate specific recruitment of regulatory factors to the genome based on chromatin modifications is not fully understood, remaining an important and central challenge for the field of chromatin biology and gene regulation.

Detailed knowledge about the underlying interaction logic between regulatory proteins and chromatin is essential to understand how chromatin modifications influence important regulatory events that occur in the nucleus, such as gene transcription, DNA replication, DNA repair etc. Furthermore, alterations in protein-chromatin interactions, either caused by aberrant genomic distribution of chromatin marks or mutations in chromatin reader proteins are frequently observed in numerous human diseases. Therefore, a detailed understanding about the mechanisms at work would enable to uncover novel disease-related mechanisms and reveal potential therapeutic targets.

We propose a systematic approach where protein-chromatin interactions are dissected into the individual building blocks that mediate specificity: the chromatin reader domains of proteins and their corresponding chromatin modifications. This allows us to reduce the interaction complexity obtained from studying entire proteins and complexes, which so far has convoluted the underlying principles of protein-chromatin interactions. We want to measure specificities of these individual building blocks in a quantitative and comprehensive manner and reveal their individual contribution to more complex interactions observed in nature. In particular we will 1) systematically explore how chromatin reader domains and their synthetic combinations guide protein localisation along the genome by using engineered Chromatin Readers (eCRs), and 2) make use of their selectivity to generate synthetic proteins that enable us to study the local protein composition at chromatin states in living cells based on proximity-biotin ligation using ChromID.
We have established workflows for streamlined generation and validation of plasmids and their targeted integration to a defined position in the mouse stem cell genome. These processes allowed us to generate so far over 130 different constructs containing eCRs based on individual and combinatorial reader domains from numerous proteins. More than 80 mouse stem cell lines were generated and validated to stably express eCRs, either as GFP fusion for live imaging or with TurboID fusions for biotin-based proximity proteomics (e.g. ChromID, Villasenor Nat Biotech 2020 - Figure 2).

With these cell lines we first apply live imaging to identify their nuclear localisation in non-fixed cells and time-lapse microscopy to measure their localisation dynamics during cell cycle. In combination with iterative indirect immunofluorescence imaging (4i, Gut et al Science 2018), we obtain multiplexed protein localisations to uncover the association of eCRs to nuclear markers. This setup allows to characterise the different chromatin readers based on their dynamic localisation in the nucleus.

So far, we have generated over 40 genome-wide maps for different eCRs, reflecting the genomic localisation of the isolated chromatin reader domains in mouse stem cells. With these datasets, we have identified genomic binding locations that are specific for certain readers, or shared between reader families. We contrasted these binding maps to available genome-wide maps for numerous chromatin and DNA modifications that we have already generated in the same ES cell lines to identify their potential chromatin preferences. In addition, we are currently expanding this dataset to represent over hundred readers and identify their localisation preferences.

Finally, we have generated more than ten ChromID datasets based on domains specific for histone-methylation, -acetylation and DNA-methylation, indicating the suitability of this proximity biotinylation technique to detect proteins associated with these chemical modifications (Figure 2).
We expect to provide a comprehensive and quantitative dataset on chromatin-reader domain binding preferences obtained from using a systematic setup (over 300 readers). This unique framework will not only reveal the affinities and site-site-specific localisation of these domains in vivo but will also provide a framework for computational studies aimed to compare specificities between and within domain families. These results will enable us to understand how each of these reader domains function on their own, and then, based on this information, to understand how they cooperate to specify genome-wide targeting of multi-domain proteins.

Furthermore, the specific binding properties of these chromatin readers will enable us to address a current challenge in chromatin biology: How do chromatin marks influence the local proteome at defined chromatin states along the genome. By using specific readers we will guide promiscuous biotin ligases to genomic regions and nuclear compartments defined by specific chromatin states. This method, which we termed ChromID (Figure 2), will help us to obtain a detailed and complete view on the proteins associated with defined chromatin states in stem cells. We expect to identify numerous interesting protein candidates, which we will further characterise using functional assays.
Chromatin modifications can influence dynamic localisation of regulatory proteins to the genome.
ChromID enables recording of protein-chromatin interactions using biotin ligases and mass spec