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 >250 different constructs containing eCRs based on individual and combinatorial reader domains from numerous proteins covering over thirty different chromatin reader domain families. More than 220 mouse stem cell lines were generated and validated to stably express eCRs, either as GFP fusion for live imaging and ChIP-seq, or with TurboID fusions for biotin-based proximity proteomics via ChromID (Villasenor, Nat Biotech 2020 - Figure 2). 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 100 genome-wide maps for different eCRs, reflecting the genomic localisation of the isolated chromatin reader domains in mouse stem cells to identify their genomic binding locations. Furthermore, we have generated more than forty different 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).
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).
In more recent work, we used ChromID to profile the proteome associated with elongating RNA Pol2 in presence and absence of the H3K36me3 writer SETD2 (Ambrosi et al, biorxiv 2024) and we have developed a DNA damage reader eCR that can be used for live tracking of DNA damage and for the identification of proteins associated with DNA damage sites (Cardoso da Silva et alum biorxiv 2024).
