Dose-dependent functions of SWI/SNF BAF in permitting and inhibiting cell proliferationin vivo

Abstract

20SWI/SNF complexes regulate transcription through chromatin remodeling and opposing gene silencing by Polycomb-group (PcG) proteins. Genes that encode SWI/SNF subunits are 22 frequently mutated in human cancer. The selective advantage, subunit bias, and common heterozygosity of such mutations remains poorly understood. Here, we characterized how 24 functional loss of various SWI/SNF subunits and PcG EZH2 affect proliferation-differentiation decisions in vivo, making use of the reproducible development of the nematode C. elegans. We 26 applied a lineage-specific genetics strategy to create partial or complete SWI/SNF subunit loss, as well as double gene knockout with PcG EZH2. Our data show that a high SWI/SNF BAF 28 dosage is needed to oppose Polycomb-mediated transcriptional repression and to arrest cell division during differentiation. In contrast, even in the absence of the PcG EZH2-related 30 methyltransferase, a low level of the SWI/SNF BAF complex is necessary and sufficient to sustain cell proliferation and hyperplasia. Our data provide experimental support for the theory 32 that during carcinogenesis partial SWI/SNF BAF loss-of-function mutations are selected because they eliminate a tumor suppressor activity while maintaining an essential transcription regulatory 34 function. 36 suppressor (Rb) protein family of transcriptional co-repressors, CDK-inhibitory proteins (CKIs) 42 that bind and block cyclin-dependent kinases (CDKs), and the anaphase-promoting complex in association with the coactivator Cdh1/FZR1 (APC/C-FZR1), which promotes protein 44 degradation (Ruijtenberg and van den Heuvel 2016). In addition to these general regulators of the cell cycle, lineage-specific transcription factors and chromatin regulators coordinate the arrest of 46 cell division with terminal differentiation. In particular, SWI/SNF (switch/sucrose nonfermenting) chromatin remodeling complexes have been found to play an important role in this 48 process (Ruijtenberg and van den Heuvel 2015; Yu et al. 2013; Joliot et al. 2014; Albini et al. 2015). 50 SWI/SNF chromatin remodeling complexes are large, multi-subunit protein complexes, initially 52 identified as positive regulators of gene expression in yeast (for review, (Mathur and Roberts 2018)). The conserved components of SWI/SNF complexes were independently identified in 54 Drosophila melanogaster as antagonists of Polycomb-mediated transcriptional repression, and found in mammals through homology searches. Multiple distinct SWI/SNF subcomplexes are 56 modularly assembled from a variety of different subunits. These SWI/SNF complexes contain an ATPase core subunit and use the energy generated by ATP hydrolysis to alter nucleosome 58

PBRM-1/PBAF-regulated genes in a multipotent progenitor

“…Consistent with this, the addition of PBRM-1 degradation in the mesoderm produced a phenotype that resembled maternal and zygotic loss of pbrm-1 function in the somatic gonad. Our observations are consistent with a previous study that showed dose-dependent functions of the SWI/SNF complex in the regulation of cell division in the M mesoblast: incomplete loss of swsn-1 via Cre/lox recombination or GFPnanobody-directed protein degradation produced an over-proliferation phenotype, while the combination of both produced an under-proliferation phenotype (van der Vaart et al 2020). Together, these two studies argue strongly for the use of both genetic deletion and protein degradation to produce a strong loss of gene function in specific tissues.…”

Bubble guts–Controlling apical membrane morphology in the C. elegans intestine

“…The CRY2 variant used, CRY2(olig), contains an E490G mutation to increase clustering ability (Taslimi et al, 2014). The GFP nanobody (VHH(GFP)) sequence was PCR amplified from plasmid pVP130 (Vaart et al, 2020), which was codon-optimized from the original sequence (Wang et al, 2017). The Pwrt-2 promotor was amplified from plasmid pRS177, the Pelt-2 promoter was amplified from plasmid pSMR12, the Phsp-16.48 promoter from plasmid pJRK83, and the par-6 coding sequence from pJRK11.…”