| dc.description.abstract | The Conaway lab previously identified and purified a human ATP-dependent chromatin remodeling complex with similarity to the Saccharomyces cerevisiae INO80 complex (65) and demonstrated that it is composed of (i) a Snf2 family ATPase (hIno80) related in sequence to the S. cerevisiae Ino80 ATPase, (ii) 7 additional evolutionarily conserved subunits orthologous to yeast INO80 complex subunits, and (iii) 6 apparently metazoan-specific subunits. In the first part of my thesis, we present evidence that the human INO80 complex is composed of three modules that assemble with three distinct domains of the hIno80 ATPase. These modules include (i) one that is composed of the N-terminus of the hIno80 protein and all of the metazoan-specific subunits and is not required for ATP-dependent nucleosome remodeling, (ii) a second that is composed of the hIno80 HSA/PTH domain, the actin-related proteins Arp4 and Arp8, and the GLI-Kruppel family transcription factor YY1, and (iii) a third that is composed of the hIno80 Snf2 ATPase domain, the Ies2 and Ies6 proteins, the AAA+ ATPases Tip49a and Tip49b, and the actin-related protein Arp5. Through purification and characterization of hINO80 complex subassemblies, we demonstrate that ATP-dependent nucleosome remodeling by the hINO80 complex is catalyzed by a core complex comprised of the hIno80 protein HSA/PTH and Snf2 ATPase domains acting in concert with YY1 and the complete set of its evolutionarily conserved subunits. In the follow-up chapter, we seek to define the requirement for assembling core subunits Ies2, Ies6, Arp5, Tip49a and Tip49b, and distinguish their functional contribution to INO80 chromatin remodeling process. We obtained evidence that the ATPase insertion regions of INO80 family ATPases are necessary and sufficient for assembling all of the five ATPase-associating subunits Ies2, Ies6, Arp5, Tip49a and Tip49b. The missing or inclusion of this insertion module correlates with loss or gain of nucleosome binding capacity of the INO80 subcomplexes, suggesting they contribute to nucleosome binding. Consistent with this hypothesis, the subcomplexes missing the insertion module were not able to bind to nucleosome, thus they were deficient in nucleosome-stimulated ATPase and ATP dependent nucleosome remodeling activities. Within the insertion module, Ies6 and Arp5 form a heterodimer, and are mutually dependent for assembly into INO80. The heterodimer is dispensable for INO80's ATPase activity, but is required for the optimal nucleosome remodeling, presumably via its contribution in nucleosome binding. On the contrary, Ies2 assembles independently of the Arp5-Ies6 dimer, and is absolutely required for the catalytic activities of the INO80 complex, while dispensable for the binding affinity to nucleosomes. Our studies described in this thesis shed light on the structure and function of the human INO80 chromatin remodeling complex. | |
