Biochemistry, Structure, and Function of SMARCB1-mutant mSWI/SNF Chromatin Remodeling Complexes in Human Disease

Abstract

Mammalian SWI/SNF (mSWI/SNF or BAF) chromatin remodeling complexes are multi-protein molecular machines that alter chromatin architecture to regulate gene expression, however the subunit-specific mechanisms by which this activity is achieved remain incompletely defined. SMARCB1 (BAF47) is a highly conserved mSWI/SNF complex subunit, and its complete loss is implicated in ~100% of cases of the rare and lethal pediatric cancer, Malignant Rhabdoid Tumor (MRT). Furthermore, recent sequencing studies have revealed an accumulation of single amino acid mutations in the highly conserved, yet functionally uncharacterized C-terminal domain (CTD) of SMARCB1, which cause the neurodevelopmental disorder, Coffin-Siris syndrome (CSS). Given its conservation through yeast, we hypothesized that these disease-associated mutations disrupt the fundamental chromatin binding and nucleosome remodeling activity of mSWI/SNF complexes. In order to assess this, we used a variety of biochemical, structural biology, and functional genomics experiments to define the molecular and genome-wide regulatory consequences of complete loss of SMARCB1, implicated in MRT, and recurrent single amino acid SMARCB1-CTD mutations, implicated in CSS. By comparing SMARCB1-null cell lines with SMARCB1 rescued conditions, we found that SMARCB1 loss results in reduced mSWI/SNF complex genome-wide occupancy (ChIP-seq) and that rescue initiates transcriptional activation by counteracting polycomb-mediated repression (RNA-seq) (Nakayama, Pulice, Valencia et al., Nature Genetics, 2017). Through 3D-protein NMR structural efforts, we discovered that the SMARCB1-CTD forms an alpha helix with a positively charged face. Coupled with peptide pull-down assays we identified that that this positive cluster binds directly to the negatively charged nucleosome acidic patch, and that all CSS-associated mutations disrupt this interaction. Lastly, single amino acid SMARCB1-CTD mutations significantly abrogate mSWI/SNF complex-mediated chromatin remodeling activity in vitro (nucleosome remodeling assays) and DNA accessibility in human cells (ATAC-seq), without changes in genome-wide complex localization (ChIP-seq) (Valencia et al., Cell 2019). These studies underscore the power of using human genetics to reveal molecular mechanisms of disease that could inform novel therapeutic opportunities for the treatment of cancers or comorbid issues of neurodevelopmental disorders. Furthermore, these studies provide the first molecular mechanism underlying the intellectual disability disorder CSS, and unmask a major, evolutionarily conserved function of SMARCB1 that dates back over 900 million years.