The role of SWI/SNF chromatin remodeling complexes in germ line transcription and nuclear organization - SUMMARY Errors in homologous chromosome segregation during meiosis are the leading cause of birth defects, spontaneous pregnancy loss, and contribute to infertility. Proper chromosome segregation, which requires pairwise associations of the homologous chromosomes are governed by genes which expression pattern are exquisitely regulated in developing gametes. Chromatin remodeling complexes play a crucial role in facilitating gene transcription during meiotic cell division by regulating access to chromatin. However, the mechanisms through which these complexes control the recruitment and activity of the transcription machinery at gene promoters remain unclear. Our central hypothesis posits that PBAF/BAF/ncBAF (SWI/SNF) complexes at promoters are essential for recruiting and activating the transcription machinery. Additionally, we propose that SWI/SNF complexes regulate the expression and activity of other critical chromatin features, such as non-coding RNAs and transposons. To test our model, we have outlined the following specific aims: Firstly, we will explore the roles of specific subunits of PBAF (PBRM1 and ARID2), BAF (ARID1A), and ncBAF (BRD9), along with the common catalytic subunit BRG1, in meiotic DNA double-strand break repair, crossover formation, and the association and disjunction of homologous chromosomes. Using imaging techniques and genetically modified mice, we aim to differentiate between the unique and shared functions of PBAF, BAF, and ncBAF as regulators of meiosis. In Aim 2, we will investigate the relationship between PBAF/BAF/ncBAF complexes and promoters in developing gametes, as well as other genomic elements, by generating genome-wide binding profiles for these complexes and transcription machinery components in mouse spermatocytes. Subsequently, we will study the impact of deleting specific subunits on the distribution and composition of PBAF, BAF, and ncBAF at promoters, as well as on gene transcription (RNA-seq). In the third aim, we will utilize high-throughput fluorescence in situ hybridization (FISH) on whole-mounted seminiferous tubules to examine how 3D nuclear organization changes as meiosis progresses and the consistency of these changes between cells. To evaluate the role of SWI/SNF complexes in nuclear organization, we will employ a panel of FISH probes strategically placed across the genome, including at promoters, enhancers, and meiotic super-enhancers, in both wild-type and knockout cells lacking PBRM 1, ARID2, ARID1A, BRD9, or BRG1. Our findings will provide valuable insights into the role of SWI/SNF complexes controlling global nuclear changes versus specific alterations mediated by BAF/PBAF/ncBAF subtypes in the expression of meiotic genes, DNA repair, and homologous chromosome pairing.
