The overall goals of this proposal are to understand how the ATP-dependent chromatin remodeler CSB is distinct from other chromatin remodelers and how its unique features enable this enzyme to protect cells from genotoxic stress. ATP-dependent chromatin remodelers use energy from ATP hydrolysis to alter DNA- histone contacts, leading to changes in nucleosome position, composition or conformation. Thus, they have critical functions in regulating fundamental processes, such as transcription and DNA repair. The importance of these enzymes to health is exemplified by the variety of diseases that result from defects in their activities, which include developmental syndromes and cancer. Mutations in the CSB remodeler lead to Cockayne syndrome: a devastating premature aging disorder associated with numerous developmental and neurological defects. There are more than 30 known and predicted ATP-dependent chromatin remodelers in humans; CSB, however, is the only remodeler that is essential for transcription-coupled DNA repair (TCR), a process that rapidly removes transcription-stalling DNA lesions. Moreover, CSB is critical for relieving oxidative stress. We have made significant advances in understanding how this chromatin remodeler is uniquely equipped to carry out its functions. (1) The association of CSB with chromatin is tightly regulated. In general, only 10% of CSB is chromatin bound, but upon UV irradiation or oxidative stress, more than 90% of CSB becomes chromatin associated; this is in stark contrast to other, well-studied remodelers such as SWI/SNF and ISWI that are constitutively bound to chromatin. (2) Using separation-of-function derivatives, we have demonstrated that the chromatin remodeling activity of CSB is essential for its function in transcription-coupled DNA repair (TCR). (3) The chromatin remodeling activity of CSB is tunable: CSB, on its own, displays weak remodeling activity (~20-fold less than SWI/SNF or ACF). Strikingly, together with NAP1-like histone chaperones, CSB remodels chromatin robustly, to a level similar to SWI/SNF or ACF. (4) CSB interacts with the CTCF protein and through this interaction becomes significantly enriched at CTCF binding sites upon oxidative stress. Given that CTCF is a key player in regulating higher- order chromatin-structure, CSB may collaborate with CTCF to reorganize chromatin structure in response to oxidative stress to facilitate coordinated gene expression or efficient DNA repair. The experiments outlined in this proposal will exploit these unique properties of CSB to understand how this ATP-dependent chromatin remodeler is uniquely equipped to protect cells from genotoxic stress. Moreover, this work may shed new light on the causes and mechanism of Cockayne syndrome.
The numerous diseases associated with defective chromatin structure regulation, including cancer and developmental disorders, underscore the importance of chromatin remodelers to public health. Results from this study will not only offer crucial insights into the underlying mechanism of Cockayne syndrome but will shed light on the fundamental principles that regulate chromatin structure and the maintenance of genome integrity. Moreover, the results of this study will have broad implications to open up new avenues for the development of a wide spectrum of epigenetic-based therapies.
