SMARCB1 missense mutants disrupt SWI/SNF complex stability and remodeling activity

Chromatin remodeling complexes, such as the SWItch/Sucrose Non-Fermentable (SWI/SNF) complex, play key roles in regulating gene expression by modulating nucleosome positioning. The core subunit SMARCB1 is essential for these functions, as it anchors the complex to the nucleosome acidic patch, enabling effective chromatin remodeling. While biallelic inactivation of SMARCB1 is a hallmark of several aggressive pediatric malignancies, the functional implication of missense mutations is not fully understood. Current diagnostic approaches focus on detecting the presence or absence of SMARCB1 by immunohistochemistry (IHC) often without consideration of mutation status as such data is lacking. Here, we present the first comprehensive deep mutational scanning (DMS) of SMARCB1, encompassing 8,418 amino acid substitutions, to systematically assess their functional impact. We show that missense mutations in the RPT2 domain of SMARCB1 disrupt SMARCB1 tumor suppressor function by destabilizing the SWI/SNF complex. Notably, we identify mutations in RPT2 that impair chromatin remodeling and transcriptional regulation to an extent comparable to nonsense mutations, despite maintaining detectable protein expression, thus challenging the conventional diagnostic reliance on IHC. Importantly, these mutations seem to act by disrupting winged-helix domain flexibility. These findings provide a deeper understanding of the role of SMARCB1 in chromatin remodeling and cancer biology, highlighting the limitations of current mutation classification approaches. By establishing a high-throughput functional framework, this study offers a critical resource for elucidating SMARCB1’s mutational landscape and its implications for cancer diagnostics.

染色质重塑复合物（chromatin remodeling complexes），如SWI/SNF复合物（SWItch/Sucrose Non-Fermentable, SWI/SNF），可通过调控核小体定位在基因表达调控中发挥关键作用。其核心亚基SMARCB1对上述功能至关重要：该亚基可将复合物锚定至核小体酸性补丁区域，从而实现高效的染色质重塑。尽管SMARCB1的双等位基因失活是多种侵袭性儿童恶性肿瘤的标志性特征，但错义突变的功能意义尚未完全阐明。当前的诊断方法多通过免疫组化（immunohistochemistry, IHC）检测SMARCB1的存在与否，却未考虑突变状态——这是因为相关数据的匮乏。本研究首次针对SMARCB1开展了全面的深度突变扫描（deep mutational scanning, DMS），涵盖8418种氨基酸替换，以系统评估各类突变的功能影响。研究显示，SMARCB1的RPT2结构域内的错义突变可通过破坏SWI/SNF复合物的稳定性，削弱其肿瘤抑制功能。值得注意的是，本研究鉴定出RPT2结构域内的部分突变，其对染色质重塑与转录调控的损害程度可与无义突变相媲美，尽管这些突变仍可检测到蛋白表达——这一发现对当前依赖免疫组化的常规诊断思路提出了挑战。进一步机制分析显示，此类突变似乎通过破坏翼螺旋结构域的柔性发挥作用。本研究成果加深了人们对SMARCB1在染色质重塑与癌症生物学中作用的理解，同时揭示了当前突变分类方法的局限性。本研究建立的高通量功能研究框架，可为解析SMARCB1的突变谱及其在癌症诊断中的应用价值提供关键的研究资源。