ABSTRACT Ubiquitin (Ub) is a highly conserved eukaryotic protein that is attached to other cellular proteins as a post- translational modification (PTM). Ubiquitination signals a large variety of downstream events, proteasome- dependent degradation being the most familiar. Histones are among the most abundant ubiquitinated sub- strates, but that typically does not lead to their degradation. Instead, the functional consequences are diverse and depend on the type of histone ubiquitinated as well as the specific lysine to which the Ub is attached. Like other epigenetic signals, ubiquitination is reversible, and both ubiquitination and deubiquitination of histones feature prominently in the regulation of transcriptional activation, gene silencing, and DNA damage responses. Defects in the readers, writers, and erasers of histone ubiquitination (e.g., RNF168, polycomb, BRCA1, USP22, and BAP1) have been strongly implicated in cancer development. A hallmark of PTMs used in signaling and other regulatory pathways is that they are highly dynamic, yet the understanding of PTM kinetics in the context of a living cell is, with few exceptions, very superficial. For crit- ical processes like the cell cycle and responses to DNA damage, Ub and other PTM dynamics have been largely unexplored due to the lack of appropriate reagents. Typically, probes for specific proteins use antibod- ies, antibody derivatives, or protein scaffolds developed to bind single protein epitopes, but deficiencies regard- ing availability, performance, reproducibility, and compatibility with intracellular expression make them general- ly unsuitable as live-cell PTM sensors. To help fill this gap, a new class of sensors will be developed to visualize nucleosome ubiquitination in live cells. The sensor designs will employ an avidity-based strategy in which independent nucleosome and Ub binding domains with modest affinities will be joined together to pro- mote high-affinity binding to nucleosomes ubiquitinated at specific positions. Additions of fluorescent or lumi- nescent reporters to these constructs will generate sensor proteins that can be expressed and monitored in live cells. Structure-based modeling and Molecular Dynamics calculations in concert with in vitro binding assays will be used to optimize sensor affinity and selectivity. Sensors will be expressed in human cells and evaluated with respect to ability to detect the cognate target while discriminating against other ubiquitinated nucleo- somes, and to confirm that competition with endogenous proteins does not adversely affect cell physiology. They then will be used to monitor the sequence and kinetics of the different types of histone ubiquitination that occur in response to DNA double-strand breaks. The strategy, methods, and principles uncovered in this work can be applied more widely to sensor design for, as examples, other ubiquitinated proteins or histone PTMs.