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Released from embargo on 4/22/25 per Professor Teresita Padilla-Benavides request. See LibAnswers QID 15889704.


Drupal Node ID: 48734
author email: eejohnston@wesleyan.edu
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The mammalian SWItch/Sucrose Non-Fermentable (mSWI/SNF) complex is a family of ATP-dependent chromatin remodelers with a primary role in transcriptional regulation. They control gene expression by restructuring chromatin through the displacement or exchange of histones, which alters the level of chromatin compaction and transcriptional activity. There are three main subfamilies of the mSWI/SNF complex, each with mutually exclusive subunits that distinguish them from one another. The canonical BRG1 or BRM-Associated Factor (BAF) is characterized by the Baf250a and Dpf2 subunits, the polybromo-domain BAF (PBAF) includes the Baf180, Brd7 and Baf200 subunits, and the non-canonical BAF (ncBAF) contains the Brd9 and Gltscr1l subunits. Each of these subfamilies have been shown to have differential roles within mammalian cells. Previous work in our lab investigated the roles of the mSWI/SNF subfamilies in the context of the skeletal muscle lineage via knock-down (KD) of these specific subunits. We showed that Baf250a and Brd9 have a significant role in myoblast proliferation and differentiation through regulation of the expression of genes involved in myogenesis and RNA metabolism, respectively. Conversely, Baf180 was shown to be dispensable for myoblast development and differentiation. However, transcriptomic analyses suggested a role for Baf180 in maintenance of cellular homeostasis in response to stress. Cellular stress involves the activation of multiple response pathways that may be triggered by environmental changes or internal stimuli. In this work, we specifically investigated metal stress. Transition metals, specifically copper (Cu) and zinc (Zn), are essential micronutrients regulated by a complex network of chaperones, transporters, enzymes, and transcription factors. Among these, Metal-regulatory transcription factor 1 (Mtf1) is one of the primary transcriptional regulators of metal levels, and it responds to stress by activating metalloprotective genes. Mtf1 also has an important role in myogenesis and muscle development, particularly in the presence of Cu. Considering this information, we hypothesized that the SWI/SNF complex, specifically Baf180 from the PBAF subfamily, works together with Mtf1 to maintain metal homeostasis in proliferating myoblasts. We utilized our well-established model of C2C12 myoblasts to investigate the interaction of Mtf1 with the different SWI/SNF subfamilies under various metal treatments using confocal microscopy and immunoprecipitation assays. In addition, we analyzed changes in labile Cu pools upon KD of the SWI/SNF subunits using fluorescent probes. Finally, we identified major candidate genes that are modulated by Mtf1 binding and the chromatin remodelers. Confocal microscopy analyses showed that the complex between Mtf1 and Baf180 has the highest levels of colocalization, which were enhanced in the Baf250a and Brd9 KD cell lines. Preliminary immunoprecipitation experiments validated the interaction between Baf180 and Mtf1 visualized via microscopy. Furthermore, the Cu probes showed an increase in Cu+2 levels in Baf180 KD myoblasts, indicating an alteration in metal homeostasis occurring in these cells. Additionally, Baf250a KD cells presented reduced levels of Cu+, which may partially explain the proliferation defects observed in these cells. This data supports our model in which Mtf1 and the PBAF complex work together to maintain metal homeostasis in the cell.

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