Project description:Malignant rhabdoid tumors (MRTs) are poorly differentiated pediatric cancers that arise in various anatomical locations and have a very poor outcome. The large majority of these malignancies are caused by loss of function of the SNF5/INI1 component of the SWI/SNF chromatin remodeling complex. However, the mechanism of tumor development associated with SNF5 loss remains unclear. Multiple studies have demonstrated a role for SNF5 in the regulation of cyclin D1, p16(INK4A), and pRb(f) activities suggesting it functions through the SWI/SNF complex to affect transcription of genes involved in cell cycle control. Previous studies in genetically engineered mouse models (GEMM) have shown that loss of SNF5 on a p53-null background significantly accelerates tumor development. Here, we use established GEMM to further define the relationship between the SNF5 and p53 tumor suppressor pathways. Combined haploinsufficiency of p53 and Snf5 leads to decreased latency for MRTs arising in alternate anatomical locations but not for the standard facial MRTs. We also observed acceleration in the appearance of T-cell lymphomas in the p53(+/-);Snf5(+/-) mice. Our studies suggest that loss of SNF5 activity does not bestow a selective advantage on the p53 spectrum of tumors in the p53(+/-);Snf5(+/-) mice. However, reduced p53 expression specifically accelerated the growth of a subset of MRTs in these mice.
Project description:Emerging evidence demonstrates that subunits of the SWI/SNF chromatin remodeling complex are specifically mutated at high frequency in a variety of human cancer types. SNF5 (SMARCB1/INI1/BAF47), a core subunit of the SWI/SNF complex, is inactivated in the vast majority of rhabdoid tumors (RT), an aggressive type of pediatric cancer. SNF5-deficient cancers are diploid and genomically stable, suggesting that epigenetically based changes in transcription are key drivers of tumor formation caused by SNF5 loss. However, there is limited understanding of the target genes that drive cancer formation following SNF5 loss. Here we performed comparative expression analyses upon three independent SNF5-deficient cancer data sets from both human and mouse and identify downregulation of the BIN1 tumor suppressor as a conserved event in primary SNF5-deficient cancers. We show that SNF5 recruits the SWI/SNF complex to the BIN1 promoter, and that the marked reduction of BIN1 expression in RT correlates with decreased SWI/SNF occupancy. Functionally, we demonstrate that re-expression of BIN1 specifically compromises the proliferation of SNF5-deficient RT cell lines. Identification of BIN1 as a SNF5 target gene reveals a novel tumor suppressive regulatory mechanism whose disruption can drive cancer formation.
Project description:Snf5 (Ini1/Baf47/Smarcb1), a core member of the Swi/Snf chromatin remodeling complex, is a potent tumor suppressor whose mechanism of action is largely unknown. Biallelic loss of Snf5 leads to the onset of aggressive cancers in both humans and mice. We have developed an innovative and widely applicable analytical technique for cross-species validation of cancer models and show that the gene expression profiles of our Snf5 murine models closely resemble those of human Snf5-deficient rhabdoid tumors. We exploit this system to produce what we believe to be the first report documenting the effects on gene expression of inactivating a Swi/Snf subunit in normal mammalian cells and to identify the transcriptional pathways regulated by Snf5. We demonstrate that the tumor suppressor activity of Snf5 depends on its regulation of cell cycle progression; Snf5 inactivation leads to aberrant up-regulation of E2F targets and increased levels of p53 that are accompanied by apoptosis, polyploidy, and growth arrest. Further, conditional mouse models demonstrate that inactivation of p16Ink4a or Rb (retinoblastoma) does not accelerate tumor formation in Snf5 conditional mice, whereas mutation of p53 leads to a dramatic acceleration of tumor formation.
Project description:Regulation of gene expression underlies cell differentiation and organogenesis. Both transcription factors and chromatin modifiers are crucial for this process. To study the role of the ATP-dependent SWI/SNF chromatin-remodeling complex in cell differentiation, we inactivated the gene encoding the core complex subunit SNF5/INI1 in the developing liver. Hepatic SNF5 deletion caused neonatal death due to severe hypoglycemia; mutant animals fail to store glycogen and have impaired energetic metabolism. The formation of a hepatic epithelium is also affected in SNF5-deficient livers. Transcriptome analyses showed that SNF5 inactivation is accompanied by defective transcriptional activation of 70% of the genes that are normally upregulated during liver development. These include genes involved in glycogen synthesis, gluconeogenesis and cell-cell adhesion. A fraction of hepatic developmentally activated genes were normally expressed, suggesting that cell differentiation was not completely blocked. Moreover, SNF5-deleted cells showed increased proliferation and we identified several misexpressed genes that may contribute to cell cycle deregulation in these cells. Our results emphasize the role of chromatin remodeling in the activation of cell-type-specific genetic programs and driving cell differentiation.
Project description:The SWI/SNF chromatin remodeling complex is highly conserved from yeast to human, and aberrant SWI/SNF complexes contribute to human disease. The Snf5/SMARCB1/INI1 subunit of SWI/SNF is a tumor suppressor frequently lost in pediatric rhabdoid cancers. We examined the effects of Snf5 loss on the composition, nucleosome binding, recruitment, and remodeling activities of yeast SWI/SNF. The Snf5 subunit is shown by crosslinking-mass spectrometry (CX-MS) and subunit deletion analysis to interact with the ATPase domain of Snf2 and to form a submodule consisting of Snf5, Swp82, and Taf14. Snf5 promotes binding of the Snf2 ATPase domain to nucleosomal DNA and enhances the catalytic and nucleosome remodeling activities of SWI/SNF. Snf5 is also required for SWI/SNF recruitment by acidic transcription factors. RNA-seq analysis suggests that both the recruitment and remodeling functions of Snf5 are required in vivo for SWI/SNF regulation of gene expression. Thus, loss of SNF5 alters the structure and function of SWI/SNF.
Project description:The SNF2, SNF5, and SNF6 genes of Saccharomyces cerevisiae are required for expression of a variety of differently regulated genes. Previous evidence implicated the SNF5 protein in transcriptional activation, and a DNA-bound LexA-SNF5 fusion protein was shown to activate expression of a nearby promoter. Here, we examine the functional relationship of the SNF2, SNF5, and SNF6 proteins. Activation by DNA-bound LexA-SNF5 fusion protein was greatly reduced in snf2 and snf6 mutants, indicating that activation by LexA-SNF5 requires SNF2 and SNF6 function. An spt6 mutation, which suppresses transcriptional defects caused by snf2, restored activation by LexA-SNF5 in a snf2 mutant. The SNF2 gene was sequenced and encodes a 194-kDa protein that is targeted to the nucleus. DNA-bound LexA-SNF2 fusion protein also activated transcription, dependent on SNF5 and SNF6. These findings suggest that SNF2, SNF5, and SNF6 function interdependently in transcriptional activation, possibly forming a heteromeric complex.
Project description:Malignant rhabdoid tumors (MRT), a pediatric cancer that most frequently appears in the kidney and brain, generally lack SNF5 (SMARCB1/INI1), a subunit of the SWI/SNF chromatin-remodeling complex. Recent studies have established that multiple SWI/SNF complexes exist due to the presence or absence of different complex members. Therefore, the effect of SNF5 loss upon SWI/SNF complex formation was investigated in human MRT cells. MRT cells and primary human tumors exhibited reduced levels of many complex proteins. Furthermore, reexpression of SNF5 increased SWI/SNF complex protein levels without concomitant increases in mRNA. Proteomic analysis, using mass spectrometry, of MRT cells before and after SNF5 reexpression indicated the recruitment of different components into the complex along with the expulsion of others. IP-Western blotting confirmed these results and demonstrated similar changes in other MRT cell lines. Finally, reduced expression of SNF5 in normal human fibroblasts led to altered levels of these same complex members. These data establish that SNF5 loss during MRT development alters the repertoire of available SWI/SNF complexes, generally disrupting those associated with cellular differentiation. These findings support a model where SNF5 inactivation blocks the conversion of growth-promoting SWI/SNF complexes to differentiation-inducing ones. Therefore, restoration of these complexes in tumors cells provides an attractive approach for the treatment of MRTs.SNF5 loss dramatically alters SWI/SNF complex composition and prevents formation of complexes required for cellular differentiation.
Project description:Nucleosome occupancy controls the accessibility of the transcription machinery to DNA regulatory regions and serves an instructive role for gene expression. Chromatin remodelers, such as the BAF complexes, are responsible for establishing nucleosome occupancy patterns, which are key to epigenetic regulation along with DNA methylation and histone modifications. Some reports have assessed the roles of the BAF complex subunits and stemness in murine embryonic stem cells. However, the details of the relationships between remodelers and transcription factors in altering chromatin configuration, which ultimately affects gene expression during cell differentiation, remain unclear. Here for the first time we demonstrate that SNF5, a core subunit of the BAF complex, negatively regulates OCT4 levels in pluripotent cells and is essential for cell survival during differentiation. SNF5 is responsible for generating nucleosome-depleted regions (NDRs) at the regulatory sites of OCT4 repressed target genes such as PAX6 and NEUROG1, which are crucial for cell fate determination. Concurrently, SNF5 closes the NDRs at the regulatory regions of OCT4-activated target genes such as OCT4 itself and NANOG. Furthermore, using loss- and gain-of-function experiments followed by extensive genome-wide analyses including gene expression microarrays and ChIP-sequencing, we highlight that SNF5 plays dual roles during differentiation by antagonizing the expression of genes that were either activated or repressed by OCT4, respectively. Together, we demonstrate that SNF5 executes the switch between pluripotency and differentiation.
Project description:Aberrant activation of the Hedgehog (Hh) pathway can drive tumorigenesis. To investigate the mechanism by which glioma-associated oncogene family zinc finger-1 (GLI1), a crucial effector of Hh signaling, regulates Hh pathway activation, we searched for GLI1-interacting proteins. We report that the chromatin remodeling protein SNF5 (encoded by SMARCB1, hereafter called SNF5), which is inactivated in human malignant rhabdoid tumors (MRTs), interacts with GLI1. We show that Snf5 localizes to Gli1-regulated promoters and that loss of Snf5 leads to activation of the Hh-Gli pathway. Conversely, re-expression of SNF5 in MRT cells represses GLI1. Consistent with this, we show the presence of a Hh-Gli-activated gene expression profile in primary MRTs and show that GLI1 drives the growth of SNF5-deficient MRT cells in vitro and in vivo. Therefore, our studies reveal that SNF5 is a key mediator of Hh signaling and that aberrant activation of GLI1 is a previously undescribed targetable mechanism contributing to the growth of MRT cells.