The SWI/SNF chromatin remodeling assemblies BAF and PBAF differentially regulate cell cycle exit and cellular invasion in vivo

Abstract

Chromatin remodelers such as the SWI/SNF complex coordinate metazoan development through broad regulation of chromatin accessibility and transcription, ensuring normal cell cycle control and cellular differentiation in a lineage-specific and temporally restricted manner. Mutations in genes encoding the structural subunits of chromatin, such as histone subunits, and chromatin regulating factors are associated with a variety of disease mechanisms including cancer metastasis, in which cancer co-opts cellular invasion programs functioning in healthy cells during development. Here we utilize Caenorhabditis elegans anchor cell (AC) invasion as an in vivo model to identify the suite of chromatin agents and chromatin regulating factors that promote cellular invasiveness. We demonstrate that the SWI/SNF ATP-dependent chromatin remodeling complex is a critical regulator of AC invasion, with pleiotropic effects on both G0 cell cycle arrest and activation of invasive machinery. Using targeted protein degradation and enhanced RNA interference (RNAi) vectors, we show that SWI/SNF contributes to AC invasion in a dose-dependent fashion, with lower levels of activity in the AC corresponding to aberrant cell cycle entry and increased loss of invasion. Our data specifically implicate the SWI/SNF BAF assembly in the regulation of the G0 cell cycle arrest in the AC, whereas the SWI/SNF PBAF assembly promotes AC invasion via cell cycle-independent mechanisms, including attachment to the basement membrane (BM) and activation of the pro-invasive fos-1/FOS gene. Together these findings demonstrate that the SWI/SNF complex is necessary for two essential components of AC invasion: arresting cell cycle progression and remodeling the BM. The work here provides valuable single-cell mechanistic insight into how the SWI/SNF assemblies differentially contribute to cellular invasion and how SWI/SNF subunit-specific disruptions may contribute to tumorigeneses and cancer metastasis.

Loss of the E3 ubiquitin ligases UBR-5 or HECD-1 restoresCaenorhabditis elegansdevelopment in the absence of SWI/SNF function

“…To test whether the suppressor mutations we identified specifically suppress the developmental arrest in swsn-1 mutants or if they might generally suppress the loss of SWI/SNF function, we assayed AC invasion in wild-type, swsn-1 mutants, and various combinations of double and triple mutant animals. We found that swsn-1 single mutants exhibited defective AC invasion phenotype in 20% (10/50 animals) of animals , consistent with previously published findings (Smith et al, 2022). This phenotype was partially rescued in swsn-1; snfc-5 double (5.2% invasion defects, 4/77 animals) and swsn-1; snfc-5; ubr-5 triple mutants (6.78% invasion defects, 5/74 animals), but not in swsn-1; ubr-5 double mutants (23% invasion defects, 7/30 animals) .…”

“…Mutations in SWI/SNF subunits cause numerous defects in animal development and physiology. For example, the swsn-1 mutation also causes defective anchor cell (AC) invasion, a process required for establishing the uterine-vulval connection during larval development critical for adult egg-laying (Smith et al, 2022) (see schematic of AC invasion in Figure 1F). To test whether the suppressor mutations we identified specifically suppress the developmental arrest in swsn-1 mutants or if they might generally suppress the loss of SWI/SNF function, we assayed AC invasion in wild-type, swsn-1 mutants, and various combinations of double and triple mutant animals.…”

Dynamic compartmentalization of the pro-invasive transcription factor NHR-67 reveals a role for Groucho in regulating a proliferative-invasive cellular switch inC. elegans

“…To achieve temporal control over POP-1 expression to tease apart its two opposing roles, we inserted an AID tag into the N-terminus of the pop-1 locus; however, this resulted in gonadal defects even in the absence of both TIR1 and auxin. Instead, using tools at hand, we paired eGFP-tagged POP-1 with a uterine-specific anti-GFP nanobody (Smith et al, 2022; Wang et al, 2017). The anti-GFP nanobody is fused to ZIF-1 and serves as an adapter, recognizing GFP-tagged proteins and promoting their ubiquitination by the Cullin2-based E3 ubiquitin ligase, which ultimately targets them for degradation via the proteasome (Figure 6–figure supplement 1A) (Wang et al, 2017).…”

“…GFP-tagged ama-1 (Hills-Muckey et al, 2021) as well as mKate2-tagged hpl-1 and hpl-2 (Patel and Hobert, 2017) were also disseminated in prior publications. The single-copy transgenes expressing the CDK sensor and TIR1 variants under ubiquitously expressed ribosomal promoters ( rps-27 and rpl-28 , respectively) as well as the tissue-specific GFP-targeting nanobody are described in previous work (Adikes et al, 2020; Hills-Muckey et al, 2021; Smith et al, 2022; Wang et al, 2017) and are located at neutral genomic sites, ttTi4348 or ttTi5605 (Frøkjær-Jensen et al, 2013). The same is true for the heat shock inducible constructs for HLH-2 and NHR-67 (Medwig-Kinney et al, 2020).…”

A light sheet fluorescence microscopy protocol for Caenorhabditis elegans larvae and adults

“…We also demonstrate how this protocol is applicable to imaging a variety of proteins and structures, including extracellular matrix proteins (type IV collagen and laminin), the nuclear envelope, and the distal tip cell (DTC). We expect the adoption of these methods will enable better live-imaging studies of important dynamic cell and developmental processes, such as germ stem cell biology, cell migration, cell division, and cell invasion (Sherwood and Plastino, 2018;Gordon et al, 2020;Smith et al, 2022). Furthermore, this protocol is generalizable and applicable to other organisms with little or no modifications.…”