ABSTRACT Ubiquitin (Ub), a small protein that is conserved in all eukaryotes, is attached to cellular proteins as a post- translational modification (PTM) that signals a large variety of downstream events, proteasome-dependent degradation being the most familiar. Histones are among the most abundant ubiquitinated substrates, and their ubiquitination typically does not lead to degradation. Instead, the functional consequences are diverse and de- pend on the type of histone ubiquitinated as well as the specific lysine to which Ub is attached. Like other epi- genetic signals, ubiquitination of histones is reversible; histone deubiquitinating enzymes (DUBs) feature prom- inently in the regulation of transcriptional activation, silencing, and DNA damage response. We have previously shown that a highly conserved DUB, UCH37, is associated with two large multi-subunit complexes, the 26S proteasome and the INO80 chromatin remodeler, and that these complexes have oppos- ing effects on UCH37 deubiquitination activity. This proposal focuses on how the human INO80 complex re- stricts the specificity of UCH37 and how deubiquitination of histone proteins by INO80-UCH37 may promote transcription and DNA double-strand break (DSB) repair. We will use our knowledge of hINO80 biochemistry and UCH37 structure and enzymology to address the following questions: (i) What is the substrate specificity of INO80-UCH37 (Aim 1)? (ii) What is the mechanism of hINO80-UCH37 activation and remodeling-coupled deubiquitination (Aim 2)? (iii) What is the function of hINO80-UCH37 and H2A.Z ubiquitination in transcription and DNA DSB repair (Aim 3)? By employing a combination of in vitro biochemistry, single-molecule studies, and in vivo genomics approaches, we will investigate the interplay between histone variant H2A.Z, deubiquiti- nation by UCH37, and chromatin remodeling by INO80. Together, these factors contribute to a chromatin envi- ronment that regulates the transcription and DNA repair machinery.