Biological Applications

Identifying proteins bound to distinct chromatin regions decorated with specific modifications has been a long-standing challenge in the chromatin field. With ChromID, we demonstrate the potential of coupling synthetic chromatin readers with proximity biotinylation to overcome this challenge.

We used eCRs to recruit a bacterial “biotin labeling” enzyme to distinct regions in the genome based on chromatin readout to identify which proteins associate with certain chromatin marks. As a result, the proteins associated with or bound near the respective target chromatin modification were labeled with biotin. This strategy allowed us to isolate biotin-labeled proteins and detect them via mass spectrometry. By applying this method, which we termed ChromID, we could identify various proteins associated with several histone and DNA modifications in mouse stem cells. Our results revealed previously unknown crosstalk between specific chromatin environments, led to the discovery of novel proteins associated with individual chromatin modifications, and uncovered the proximal interactome at bivalent promoters marked by H3K4me3 and H3K27me3. The applicability of ChromID in living cells and eCRs as synthetic readers further opens the exciting possibility to perform similar experiments in other systems to chart the epi-proteome during dynamic cellular processes.

Lastly, live-cell imaging with eGFP-tagged eCRs enabled us to explore the localization of histone modifications in the cell nucleus. In the future, this strategy will allow us to study the dynamics of histone marks during cell division and explore their sub-nuclear localization at high resolution.