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

Chromatin modifications are key regulators of genome function. They can be directly recognised by specialised protein reader domains, leading to coordinated recruitment of regulatory proteins to the genome in a dynamic, spatiotemporal manner. Despite many efforts to characterise chromatin-mediated protein recruitment, the underlying principles that determine specificity and how chromatin marks influence the proteome composition at genomic sites in living cells, remain unclear. Here I propose to uncover the underlying logic that mediates specificity between regulatory proteins and chromatin states by using a reductionistic approach that enables us to study these interactions in a controlled and comprehensive manner in living cells. Towards this we combine high-throughput stem cell engineering with functional genomics and computational methods to achieve the following aims: First, we aim to identify and characterise the genome-wide binding preferences of a comprehensive panel of chromatin reader domains (CRD) by using a novel strategy for comparative profiling of multiple protein-genome interactions in parallel. Second, we will systematically dissect the context-dependent determinants that mediate individual and combinatorial CRD binding to the genome. Finally, we will utilise the selectivity of CRDs to uncover the local proteome at defined chromatin states in ES and neuronal cells, revealing novel components involved in the regulation and organisation of the epigenome. The overarching goal of ChromatinLEGO is to elucidate in a systematic, quantitative and unified manner, how protein-genome interactions are guided by specific chromatin modifications. Through identifying the chromatin-dependent recruitment principles of regulatory factors, and by dissecting the underlying mechanisms that specify these interactions, this study will provide novel paradigms and important advances to our current understanding of chromatin function in vivo.