Periodic Reporting for period 2 - ChromatinLEGO (Chromatin readout: Dissecting the protein-chromatin interaction code in living cells)
Période du rapport: 2021-11-01 au 2023-04-30
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.
With these cell lines we first applied live imaging to identify their nuclear localisation and time-lapse microscopy to measure their localisation dynamics during cell cycle and differentiation. This setup already allowed us to identify and characterise interesting chromatin readers that dynamically localise in the nucleus in a cell type specific manner or upon specific conditions (i.e. DNA damage, cell cycle phase, DNA methylation inhibition), generating interesting hypotheses that we are currently following up.
So far, we have generated over 60 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.
We have generated more than twenty ChromID datasets based on domains specific for histone-methylation, -phosphorylation, -acetylation, and DNA-methylation, indicating the suitability of this proximity biotinylation technique to detect proteins associated with these chemical modifications (Figure 2). Depending on the biological question and eCR used, ChromID measurements are performed in mouse stem cells, neuronal cells, in presence of DNA damaging agents, or upon interference with DNA methylation.
We have also successfully applied eCRs and ChromID to investigate binding of ZFP57, a known DNA methylation binder at imprinted genes, to identify its genomic localisation in wild type ES cells and ES cells lacking DNA methylation. Finally, we performed ChromID experiments to identify regulatory proteins associated with methylated regulatory sites of imprinted genes using a ZFP57-TurboID fusion. This revealed known and novel factors involved in regulation of genomic imprinting, which we further identified using CRISPR screens as an orthogonal approach, and characterised for their molecular function (Butz, Nat. Genetics 2022).
We furthermore collaborated with the laboratory of Sachdev Sidhu to enhance the affinities of Cbx chromodomains for methylated histone marks by mutating key residues and using phage display. We tested a novel "super-binder" that has a higher affinity for H3K27me3 and can be used in combination with CRISPRi to silence target genes (Veggiani, Nat Communications 2022).
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.