Structural and Dynamic Changes of Chromatin Remodeling at a Developmental Switch

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DESCRIPTION (provided by applicant): Recent studies have shown that development of the mammalian nervous system requires regulated changes of subunit composition in the family of ATP-dependent chromatin remodeling BAF complexes. Neural progenitor cells have a distinct 'npBAF' complex defined by its subunit composition, which is essential for self- renewal. When neural progenitors give rise to neurons, npBAF complexes containing Baf45a and Baf53a are replaced by neuron-specific ¿nBAF¿ complexes with homologous subunits (Baf45b and Baf53b). This regulated switch is required for mitotic exit, activity-dependent dendrite outgrowth, and other post-mitotic, neuron-specific functions. Although several lines of evidence indicate the complex undergoes a dramatic change in genomic distribution at the developmental switch, the long-standing question of exactly how subunit composition drives the function of the BAF complex remains unanswered. Our studies will be directed at understanding the biophysical and mechanistic consequences of subunit switching, to reveal the mechanisms used by the complex to support these two essential epigenetic states. We have developed a comprehensive approach to examine the mechanistic role of the developmental switch by creating a mouse expressing the photoswitchable fluorescent protein Dendra2 fused to the BAF complex's central ATPase Brg. To examine the role of the npBAF/nBAF developmental switch on complex stability and turnover, we will use this Brg-Dendra2 mouse strain to measure the complex's turnover in live cells at the neural progenitor stage before the developmental switch, and in differentiated neurons after the switch. Additionally, previous studies suggest that differentiation is accompanied by changes in the physical mobility of chromatin-related proteins. To identify the effect of the subunit switch the dynamics of the complex, we will use fluorescence decay after photoactivation (FDAP) in live cells to measure changes in BAF complex nuclear mobility before and after the developmental switch. Finally, we will use a super-resolution optical microscopy technique, 3D-PALM, to examine structural changes arising from the npBAF/nBAF developmental switch. High-resolution localization of individual complexes using 3D-PALM will allow us to compare sub-nuclear structure, clustering, and other parameters to describe the structural effects of the complex's developmental regulation. In each of these aims, we will identify the BAF subunits responsible for the complex's change. At the conclusion of our studies, we will have defined the biophysical interactions and mechanisms modulated by an essential epigenetic switch to regulate specific aspects of neural development. Revealing the biophysical basis for developmental regulation of the complex will yield valuable insight into the molecular mechanisms of pluripotency and differentiation.