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

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.


INTRODUCTION 58
Cellular invasion through basement membranes (BMs) is a critical step in 59 metazoan development and is important for human health and fitness. Early in hominid 60 development, trophoblasts must invade into the maternal endometrium for proper 61 blastocyst implantation (1). In the context of immunity, leukocytes become invasive upon 62 injury or infection to travel between the bloodstream and interstitial tissues (2,3). Atypical 63 activation of invasive behavior is associated with a variety of diseases, including 64 rheumatoid arthritis wherein fibroblast-like synoviocytes adopt invasive cellular behavior, 65 leading to the expansion of arthritic damage to previously unaffected joints (4,5). Aberrant 66 activation of cell invasion is also one of the hallmarks of cancer metastasis (6). 67 A variety of in vitro and in vivo models have been developed to study the process 68 of cellular invasion at the genetic and cellular levels. In vitro invasion assays typically 69 involve 3D hydrogel lattices, such as Matrigel, through which cultured metastatic cancer 70 cells will invade in response to chemo-attractants (7). Recently, microfluidic systems have 71 been integrated with collagen matrices to improve these in vitro investigations of cellular 72 invasion (8). While in vitro invasion models provide an efficient means to study the 73 mechanical aspects of cellular invasion, they are currently unable to replicate the complex 74 microenvironment in which cells must invade during animal development and disease. A 75 variety of in vivo invasion models have been studied, including cancer xenograft models 76 in mouse (9-11) and zebrafish (12, 13), each having their respective benefits and 77 drawbacks. Over the past ~15 years, Caenorhabditis elegans anchor cell (AC) invasion 78 has emerged as a powerful alternative model due to its visually tractable single-cell nature 79 (Fig 1 A) (14).  (22,24), which consists of cell cycle-independent (fos-1) and dependent (egl-43, hlh-2, and nhr-67) subcircuits, which together with hda-1 promote pro-invasive gene expression and maintain cell cycle arrest in the AC. In this and all subsequent figures, scale bar, 5 μm.
in cancer (31-34) and C. elegans mesoblast development (35). Examination using a CDK 136 activity sensor (36) revealed assembly-specific contributions to AC invasion: whereas 137 BAF promotes AC invasion in a cell cycle-dependent manner, PBAF contributes to 138 invasion in a cell cycle-independent manner. Finally, we utilized the auxin-inducible 139 degron (AID) system combined with PBAF RNAi to achieve strong PBAF subunit 140 depletion in the AC, which resulted in loss of both AC invasion and adhesion to the BM. 141 Together, these findings provide insight into how the SWI/SNF complex assemblies may 142 contribute to distinct aspects of proliferation and metastasis in human malignancies. 143

An RNAi screen of 269 CRFs identified SWI/SNF as a key regulator of AC invasion 146
To identify the suite of CRFs that, along with hda-1, contribute to AC invasion, we 147 generated an RNAi sub-library of 269 RNAi clones from the complete Vidal RNAi library 148 and a subset of the Ahringer RNAi library (37,38) targeting genes implicated in chromatin 149 state, chromatin remodeling, or histone modification (Table S1) (Fig 1 B). Because CRFs 150 act globally to control gene expression, we screened each RNAi clone by high-resolution 151 differential interference contrast (DIC) and epifluorescence microscopy in a uterine-specific RNAi hypersensitive background containing labeled BM (laminin::GFP) and an 153 AC reporter (cdh-3p::PH::mCherry) (Fig 1 A; Table S1) (14,22,24,39). This genetic 154 background allowed us to limit the effect of transcriptional knockdown of CRFs to only 155 affect the AC and the neighboring uterine tissue, and only for a time period following the 156 specification of the AC (39). As the neighboring uterine cells do not contribute to the 157 invasion program (14), AC invasion defects following RNAi treatments are indicative of 158 cell autonomous pro-invasive gene function (24,39). In wild-type animals, by the time the 159 1˚ fated P6.p vulval precursor cell has divided twice (P6.p 4-cell stage), 100% of ACs 160 have successfully breached the underlying BMs and made contact with the P6.p grand-161 daughters (14). While the AC also always invades in the uterine-specific RNAi 162 hypersensitive strain we utilized for our CRF screen, there is a low penetrance delay, 163 such that at the P6.p 4-cell stage, when we scored invasion, 2% (2/100 animals) still had 164 an intact BM. Thus, we used this baseline defect as a statistical reference point for this 165 genetic background and defined the cut-off threshold for significant defects in invasion 166 from RNAi depletion of CRFs in our screen as those RNAi clones that resulted in a ~13% 167 or greater defect in invasion (4/30 animals, Fisher's exact test = 0.0252). By this 168 threshold, we recovered 82 CRFs (30.5% of total CRFs screened) that significantly 169 regulate AC invasion, suggesting a general role for regulation of chromatin states in the 170 acquisition of invasive behavior (Table S2). Interestingly, five subunits of the broadly 171 conserved SWI/SNF chromatin remodeling complex were recovered as significant 172 regulators of AC invasion: swsn-1(SMARCC1/SMARCC2; 23% AC invasion defect), 173 swsn-5/snfc-5 (SMARCB1; 20% AC invasion defect), swsn-7 (ARID2; 23% AC invasion 174 defect), swsn-8/let-526 (ARID1A/ARID1B; 23% AC invasion defect), and pbrm-1 Improved RNAi vectors revealed distinct contributions of SWI/SNF subunits to AC 197 invasion Though many SWI/SNF assemblies have been described in mammalian and other 199 systems, including BAF, PBAF, esBAF, GBAF, nBAF, and npBAF) (53), to date, BAF and 200 PBAF are the only SWI/SNF assemblies that have been described in C. elegans. Both 201 assemblies consist of core subunits (SWSN-1, SWSN-4, SWSN-5) and accessory 202 subunits (DPFF-1, SWSN-2.1/HAM-3, SWSN-2.2, SWSN-3, SWSN-6, and PHF-10), 203 collectively referred to as common factors (46,54). These common factors are bound by 204 assembly-specific subunits in a mutually exclusive manner, which confers the distinct 205 character of each of the two assemblies (Fig 2 A) To investigate the contribution of individual SWI/SNF subunits to AC invasion and 217 to distinguish potentially distinct roles of the BAF and PBAF assemblies, we generated 218 improved RNAi constructs utilizing the T444T vector (62) to target representative subunits 219 of the core and both SWI/SNF assemblies ( Table S3). Knockdown of SWI/SNF subunits 220 in whole-body RNAi sensitive animals following treatment with T444T RNAi vectors 221 resulted in penetrant loss of invasion. The majority of ACs failed to invade following 222 treatment with RNAi targeting the core SWI/SNF ATPase subunit swsn-4 or core subunit 223 swsn-1 (90% and 94%, respectively; n=50 animals ; Fig 2 B, E). RNAi-mediated 224 knockdown of the BAF assembly subunit swsn-8 also resulted in significant loss of AC 225 invasion (32%; n=50 animals ; Fig 2 C, E). Knockdown of the PBAF assembly subunits 226 following RNAi depletion of SWI/SNF core (swsn-1 and swsn-4) (B), BAF (swsn-8) (C), and PBAF (pbrm-1, swsn-7, and swsn-9) (D) subunits. In cases where multiple cells expressed the AC reporter (2+ACs) in the same animal, each is indicated with a white arrowhead. In cases where multiple cells expressed the AC reporter (2+ACs), a representative image from the same treatment of a single AC that fails to breach the BM is displayed as an inset (  longer statistically separable from expression in the AC, whereas expression of SWSN-263 8::GFP in the VPCs remains significantly lower than in the AC (Fig S3 A'-C'). 264 We treated SWI/SNF endogenously labeled GFP-tagged strains with our RNAi 265 vectors to precisely quantify the efficiency of RNAi-mediated knockdown of target 266 SWI/SNF complex subunits and to correlate this loss with the resulting AC phenotypes. 267 Treatment with either swsn-4(RNAi) or swsn-8(RNAi) vectors resulted in robust depletion 268 of fluorescence expression of GFP::SWSN-4 (94% depletion) and SWSN-8::GFP (81% 269 depletion) in the AC (Fig S4 A-B, D) and penetrant loss of invasion (90% and 30%, 270 respectively; n=30 animals for each condition; Fig S4 E). We also noted instances where 271 multiple cells expressed the AC reporter (23% and 10%, respectively; n=30 animals for each condition; Fig S4 E). Treatment of the PBRM-1::eGFP strain with pbrm-1(RNAi) 273 revealed weaker but significant knockdown of PBRM-1 protein (49% depletion), and a 274 lower penetrance of invasion defects (17%; n=30 animals; Fig S4 C-E). It is unclear why may be the consequence of differential protein perdurance. Altogether, these results 279 confirm the dynamic expression of the SW/SNF core, BAF, and PBAF subunits in the AC 280 before, during, and after invasion and demonstrate the effectiveness of our improved 281 SWI/SNF-targeting RNAi vectors. 282 283

C. elegans SWI/SNF subunits exhibit low levels of intracomplex cross-regulation 284
Work in mammalian cell culture has revealed that the mSWI/SNF complex is 285 assembled in a step-wise fashion, with stability of the complex as a whole and association 286 of individual subunits depending on the prior expression and association of other subunits 287 (64). To date it is unknown whether in C. elegans individual SWI/SNF subunits activate 288 other SWI/SNF subunits. It is also unclear whether subunits of the two assemblies in C. 289 elegans -BAF and PBAF -stabilize the core protein subunits or vice-versa. Therefore, 290 we used our endogenously labeled GFP-SWI/SNF strains to ask whether transcriptional 291 knockdown of individual subunits of the core, BAF, or PBAF induce changes in protein 292 expression of other subunits at the time of AC invasion (Fig S5). First, to determine 293 whether representative subunits of the SWI/SNF assemblies promote or stabilize the 294 ATPase of the complex, we treated GFP::swsn-4 animals with either swsn-8(RNAi) or knockdown of the PBAF subunit pbrm-1 also resulted in a significant but weaker loss of 299 ATPase expression in the AC (11% GFP::SWSN-4 depletion; Fig S5 A, D). These results 300 suggest that individual subunits of either SWI/SNF assembly may contribute to the protein 301 stability and/or expression of the SWI/SNF ATPase in the C. elegans AC, with the BAF 302 complex playing a potentially dominant activating role with respect to the ATPase. 303 Next, we treated animals containing either the swsn-8 or pbrm-1 endogenous 304 GFP-reporters with enhanced RNAi to knockdown the expression of the SWI/SNF 305 ATPase or the representative subunit of the alternative SWI/SNF assembly. Interestingly, 306 while unaffected by knockdown of the PBAF assembly subunit pbrm-1, RNAi knockdown 307 of the ATPase swsn-4 resulted in a 42% increase in the expression of SWSN-8::GFP in 308 the AC (Fig S5 B, D). Finally, relative to the expression of the endogenous PBAF subunit 309 in the ACs of animals treated with the empty vector control RNAi, AC nuclei of PBRM-310 1::eGFP animals treated with swsn-4(RNAi) had significantly lower levels of protein 311 expression (38% PBRM-1::eGFP depletion), whereas ACs in swsn-8(RNAi) treated 312 animals expressed 13% more PBRM-1::eGFP (Fig S5 C-D). 313 Since knockdown of either swsn-4 or swsn-8 subunits resulted in two distinct 314 phenotypes -individual animals with single non-invasive ACs and animals with multiple 315 non-invasive cells expressing the AC-reporter -we next sought to determine whether 316 these two phenotypes were distinct with respect to SWI/SNF subunit expression. To do 317 this, we binned data from the intracomplex RNAi experimental series (above) into the two non-invasive phenotypes and compared the fluorescence expression levels of the 319 endogenous proteins within SWI/SNF RNAi conditions. Given the infrequency of the 320 multi-AC phenotype, statistical comparisons were necessarily limited to treatments in 321 which the population of animals contained at least 10 multi non-invasive ACs. Treatment 322 of SWSN-8::GFP with swsn-4(RNAi) resulted in a total of 24 multi non-invasive ACs (53 323 ACs total; n=41 animals) and no significant difference was detected in SWSN-8::GFP 324 expression between the nuclei of the single non-invasive AC phenotype and the multi 325 non-invasive AC phenotype groups (Fig S5 E). The second statistical comparison was 326 made between the two phenotypes in PBRM-1::eGFP animals treated with swsn-8(RNAi) 327 of ACs of the multi non-invasive phenotype group compared to the single non-invasive 331 phenotype (Fig S5 E). 332 Based on these results, a tentative model for epistatic interactions between the 333 SWI/SNF ATPase, BAF, and PBAF assembly subunits can be composed for the AC (Fig  334   S5 F). Our data indicate that some degree of SWI/SNF intra-and inter-complex regulation 335 occurs in the AC. We find that the most significant aspect of SWI/SNF intra-complex 336 regulation is exercised by the ATPase on the assembly specific subunits, where swsn-4 337 knockdown results in a significant increase in BAF/SWSN-8 and a significant decrease 338 PBAF/PBRM-1. SWI/SNF intercomplex regulation appears to be weaker in the AC as 339 knockdown of pbrm-1 does not affect SWSN-8::GFP expression, and knockdown of 340 swsn-8 results in a slight increase in PBRM-1::GFP expression.

E-F). 467
In animals treated with pbrm-1(RNAi), the CDK sensor also localized principally in the 468 nucleus of ACs that failed to invade (mean C/N ratio: 0.157+/-0.063, n=41 animals) and 469 only a single non-invasive AC was observed per animal (Fig 4 D, F). In contrast, following n=40 animals) in animals with single or multiple non-invasive ACs (Fig 4 B, F). 476 Interestingly, the swsn-4(RNAi) treatment resulted in a higher proportion of non-invasive 477 G0 phase (C/N ratio < 0.3) ACs (14%, n=48 cells) than were present in the swsn-8(RNAi) 478 treated population (8%, n=25) (Fig 4 B, E). This finding reemphasizes the dependence 479 of both SWI/SNF BAF and PBAF assemblies on the core ATPase of the complex, as the 480 distribution of cell cycle states in ACs following swsn-4(RNAi) treatment represents both the cell cycle-dependent and cell cycle-independent phenotypes seen in ACs deficient in 482 the swsn-8 and pbrm-1 subunits, respectively 483

Forced G0 arrest through ectopic CKI-1 rescues invasive potential in BAF-deficient 485 but not PBAF-deficient ACs 486
We have previously proposed and characterized a dichotomy that exists between 487 invasion and proliferation in the AC (22,24). As evidence of this, loss of two of the three 488 TFs that function in a cell cycle-dependent manner to maintain the AC in a cell cycle-489 arrested state (nhr-67/Tlx and hlh-2/Daughterless) can be rescued through induced 490 expression of a cyclin dependent kinase inhibitor, cki-1 (p21/p27) (22). These results 491 suggest that, at least in some cases, TF activity can be bypassed completely to promote 492 AC invasion by maintaining G0 arrest through direct cell cycle manipulation. To determine 493 the extent to which the BAF assembly contributes to AC invasion through regulation of 494 cell cycle arrest, we used a heat-shock inducible transgene to ectopically express CKI-495 1::mTagBFP2 in SWI/SNF-deficient ACs (Fig 5). Since the heat shock inducible 496 transgene is ubiquitous and expresses variably between different animals and different 497 tissues within an individual animal, we limited our analysis to animals with ACs with 498 obvious mTagBFP2 fluorescence expression. While forced arrest in G0 was insufficient to 499 significantly rescue AC invasion in animals treated with swsn-4(RNAi) (Fig 5 B, B', E) or 500 pbrm-1(RNAi) (Fig 5 D, D', E), ectopic cki-1 (CKI-1::mTagBFP2) expression in the AC 501 significantly rescued cellular invasion in animals treated with swsn-8(RNAi) (Fig 5 C, C', 502 E). Strikingly, in 85.7% (6/7) of cases where ACs had proliferated prior to ectopic CKI-1 503 expression, forced G0 arrest led to multiple ACs breaching the BM (Fig 5 F), a phenotype 504 we have reported previously using CKI-1 overexpression paired with loss of NHR-67. This 505 demonstrated that mitotic ACs maintain the capacity to invade if they are re-arrested into a G0 state (24). To corroborate our CKI-1 heat shock data, we used an AC-specific CKI-507 1 transgene (cdh-3p::CKI-1::GFP) to induce G0 cell cycle arrest in swsn-4-and swsn-8-508 depleted ACs (Fig S6). Similar to the heat shock results, lineage-restricted expression of 509 CKI-1::GFP failed to rescue invasion in animals deficient in swsn-4 (Fig S7 A-B). 510 However, transgenic cdh-3p::CKI-1::GFP animals treated with swsn-8(RNAi), had 511 lacked the G0 rescue transgene (Fig S7 B). Altogether, these data corroborate our DHB-513 based CDK sensor data (Fig 4), suggesting that the SWI/SNF assemblies differentially 514 contribute to AC invasion with BAF specifically required for G0 cell cycle arrest. 515

SWI/SNF chromatin remodeling promotes the invasive GRN in the AC 517
Previous work has demonstrated that the gene regulatory network (GRN) that 518 promotes AC invasion consists of both cell cycle-dependent and cell cycle-independent 519 TF subcircuits (22,68) (Fig 1 B). In the cell cycle-dependent subcircuit of the TF-GRN, contributes to the regulation of either or both AC GRN subcircuits (Fig 6). In the cell cycle- subunits pbrm-1, swsn-7, or swsn-9 resulted exclusively in single non-invasive cells 552 expressing the AC reporter. However, given that the enhanced pbrm-1(RNAi) resulted in 553 much weaker endogenous protein knockdown than the enhanced RNAis targeting either 554 the SWI/SNF ATPase (swsn-4) or BAF assembly subunit (swsn-8) in the AC (Fig S4), 555 and the dose-dependent phenotype following loss of the core ATPase (Fig 3), it is 556 possible that we failed to observe the mitotic non-invasive AC phenotype due to 557 insufficient PBAF subunit knockdown. To address this, we next asked whether strong 558 loss of PBAF subunit expression contributes to the mitotic non-invasive AC phenotype. 559 To accomplish this, we used an auxin inducible degron (AID)-RNAi combination 560 knockdown strategy (75,76). We generated a strain with pbrm-1 endogenously labeled 561 with mNeonGreen and an auxin inducible degron (AID) (pbrm-1::mNG::AID) in a genetic 562 background containing AC (cdh-3p::mCherry::moeABD) and BM (laminin::GFP) 563 reporters. We then quantified fluorescence expression in the AC in this strain. When 564 grown under standard conditions, 6.45% of the ACs had not invaded the BM by the P6.p 565 4 cell stage, suggesting a partial loss of function of pbrm-1 (n=30 ; Fig 7 A, E-F). This 566 partial loss of function phenotype is likely due to the insertion of the mNG::AID tag into 567 the genomic locus, causing a putative hypomorphic allele. Next, we introduced a 568 ubiquitous, mRuby-labeled TIR1 transgene (eft-3p::TIR1::mRuby) into the animals and assessed AC invasion under standard conditions (aux(-)) or in the presence of the auxin 570 hormone (aux(+) ; Fig 7 B). We observed no statistically significant difference in the 571 fluorescence expression of PBRM-1::mNG::AID protein in the AC, nor did we observe 572 any differences in AC invasion defects between the strains with and without the TIR1 573 transgene when grown on aux(-) media (TIR1+: 3% depletion, 16.67% invasion defect; 574 n=30; Fig 7 E-F). However, in both conditions, some ACs that invaded seemed to do so 575 only partially, as most of the ventral epidermal BM remained intact beneath ACs (Fig 7  576 B, black arrowhead). In the aux(+) condition, there was a significant reduction in PBRM- Next, we treated pbrm-1::mNG::AID animals containing ubiquitous TIR1 with 585 pbrm-1(RNAi) in both aux(-) and aux(+) conditions. As expected, treatment of the pbrm-586 1::mNG::AID strain with pbrm-1(RNAi) resulted in very low expression of the subunit in 587 the AC even in the absence of auxin and there was no significant difference in expression 588 between the Aux(-) and Aux(+) conditions (Fig 7 C,F). Interestingly, there was also no 589 significant difference in the penetrance of AC invasion defects between the pbrm-590 1::mNG::AID strain treated with control compared to the strain treated with pbrm-1(RNAi) 591 in the presence of auxin (Fig 7 G). Since the combination treatment of a hypomorphic Since the PBAF assembly in C. elegans consists of several subunits, pbrm-1 599 (PBRM1), swsn-7 (ARID2), and swsn-9 (BRD7/BRD9), we next investigated whether 600 combinatorial knockdown of PBAF subunits would enhance the penetrance of AC 601 invasion defects or result in the mitotic non-invasive AC phenotype. In the absence of 602 auxin, there was no significant difference in PBRM-1::mNG::AID expression in the AC of 603 animals treated with swsn-7(RNAi) compared to animals treated with empty vector control 604 (n=30 ; Fig 7 E), however there was a significant increase in loss of AC invasion (50% 605 invasion defect; n=30 ; Fig 7 F). Strikingly, in one case, the AC was completely detached 606 from the BM, as we detected no AC membrane protrusions (cdh-3p::mCherry::moeABD) 607 in contact with the ventral surface of the gonad (Fig 7 F) ; Fig 7 F-G). In contrast to treatment with 613 swsn-7(RNAi), in the swsn-9(RNAi) aux(-) condition, PBRM-1::mNG::AID expression in 614 ACs was significantly lower than that in the ACs of animals treated with empty vector 615 control aux(-) (39% depletion; n=30; Fig 7 E). It is unclear why transcriptional knockdown condition (19% depletion; n=30; Fig 7 E), however, we saw no statistically significant 621 difference in penetrance of AC invasion defects between the two conditions (30% vs. 622 43%; n=30; Fig 7 F). We also noted one animal with a detached AC in the swsn-9(RNAi) 623 aux (-) condition and zero in the aux(+) condition (Fig 7 F). Importantly, we only observed 624 one AC per animal across all combinatorial treatments, supporting the hypothesis that the 625 PBAF assembly does not contribute to G0 cell cycle arrest in the AC. fluorescence expression in ACs that displayed invasion defects (Fig S8 A-B). Animals 637 treated with pbrm-1(RNAi) had a modest but statistically significant loss of FOS-1::GFP 638 protein levels in non-invasive ACs (34% depletion; n=20 ; Fig S8 B), suggesting that the that the PBRM-1::mNG::AID allele was slightly hypomorphic, with ~17% invasion defects 644 in backgrounds with TIR1, we used the strain containing TIR1 as a sensitized 645 background. We found that even without the addition of auxin, co-depletion with fos- promotion of cellular invasion (24,79-83). In this study, we used the Caenorhabditis contribute to the process of cellular invasion. We performed a tissue-specific RNAi 664 feeding screen to assess 269 genes implicated in chromatin binding, chromatin 665 remodeling complexes, or histone modification. We do not claim that genes that we failed 666 to identify as regulators of cellular invasion in the screen are unimportant for the process; 667 however, RNAi-mediated loss of the majority of CRF genes in the screen did result in 668 some penetrance of AC invasion defects (Table S1). This finding was expected, as many 669 of the genes we screened are global regulators of the genome and broadly contribute to 670 various cell biological processes. We extracted a list of the most penetrant regulators of 671 cell invasion from the broader list (Table S2) (88)(89)(90). Additionally, we recovered nematode-specific genes including 677 nra-3, and cec-2, and genes whose human homologs have not been previously studied 678 in the context of cellular invasion to our knowledge, such as cec-3 (homologous human 679 protein is uncharacterized) and gna-2/GNPNAT. Since the majority of the CRF genes we Here we used the C. elegans AC invasion system as a model to investigate 716 whether the dose-dependent relationship between the ATPase and differentiated 717 phenotype extends to cellular invasion. The first indication that the functional dose of 718 SWI/SNF may have an instructive role in AC invasion was the relative enhancement of 719 all endogenously tagged subunits of the complex in the AC relative to neighboring VU 720 and VPC tissues (Fig S4). While it is tempting to interpret the enhancement of SWI/SNF 721 subunit expression in the AC as evidence for the dependence of cellular invasion on 722 SWI/SNF activity, it is also possible that this difference in expression is a consequence 723 of terminal differentiation, since at the time of invasion, the AC is terminally differentiated 724 unlike the VU and VPC). Further investigation is required to determine whether 725 endogenously tagged SWI/SNF subunits are enhanced in the AC relative other terminally 726 differentiated cells. 727 Additionally, we find that some degree of SWI/SNF intra-and inter-complex 728 regulation exists in the AC at the time of invasion with both SWI/SNF assemblies 729 cooperating to activate expression of the ATPase (Fig S5 B). It is possible that this added 730 level of complex autoregulation contributes to an "optimal" dose of the ATPase in a cell- Although we cannot be sure that combining swsn-4(RNAi) with an antiGFP-targeting 748 nanobody to deplete the SWI/SNF ATPase results in complete loss of protein expression, 749 we show that treatment with the improved swsn-4(RNAi) vector alone is sufficient to 750 phenocopy the null phenotype previously reported in late mesoblast (SM) development 751 (Fig S6). Additionally, enhancement of the AC mitotic phenotype statistically tracked with 752 a progressive step down in mean expression of the ATPase in the AC across our 753 experiments. Using endogenous fluorescent reporters for conserved pro-invasive genes, we also provide evidence that the SWI/SNF complex is involved broadly with the 755 regulation of the cell cycle-dependent and -independent subcircuits of the AC GRN (Fig  756   6). Altogether, this data supports the hypothesis that SWI/SNF cell-autonomously 757 contributes to cell cycle control in a dose-dependent manner and provides the first line of 758 evidence to link SWI/SNF ATPase dosage to the dichotomy between invasion and 759 proliferation (Fig 8). While previous work in our lab, based on localization of a DNA licensing factor, 764 CDT-1, has demonstrated indirectly that ACs must arrest in a G0/G1 cell cycle state 765 (22,24), we lacked a sensitive enough tool to distinguish between these two interphase 766 states. From our recent work utilizing a CDK sensor to examine the proliferation-767 quiescence decision in C. elegans, we can distinguish between pre-terminal cells in the 768 somatic gonad in G1 (mean C/N ratio: 0.67+/-0.10) as compared to terminally 769 differentiated G0 uterine cells (mean C/N ratio: 0.30+/-0.11) (36). Here, we compare CDK 770 activity measurements in the ACs of control animals with that of the terminal Pi lineage to 771 provide the first quantitative demonstration that ACs arrest in a CDK low G0 state to invade 772 (Fig 4 A, E, F). Furthermore, by combining the CDK sensor with loss of SWI/SNF 773 subunits, our data indicate that the SWI/SNF BAF assembly is specifically responsible for 774 regulation of G0 cell cycle arrest in the AC. 775  (S-4, swsn-4), PBAF (orange -S-7, swsn-7; S-9, swsn-9; P, pbrm-1), and BAF (blue -S-8, swsn-8) assemblies contribute to AC invasion.
factors, which are thought to collectively provide a platform of common factors that is 780 bound by assembly-specific subunits in a mutually exclusive manner. In C. elegans, the 781 association of SWSN-8 (BAF250a/ BAF250b/ ARID1A/ ARID1B) with the common factors 782 forms the SWI/SNF BAF assembly. Since we identified representative subunits from the 783 core of the complex and both SWI/SNF BAF and PBAF assemblies in our CRF RNAi 784 screen, we took a cell biological and genetic approach to investigate the role of the each 785 SWI/SNF assembly in promoting cellular invasion. Here, using a DHB-based CDK sensor 786 (36,70), we show that loss of either core or BAF assembly subunits specifically results in 787 mitotic ACs that failed to invade the BM. Our cell cycle sensor data first establishes that 788 wild-type AC invades in a G0 CDK low state, and second, that a major contribution of the 789 BAF assembly to AC invasion is through maintenance of this G0 arrest, as many ACs that 790 failed to invade the BM had increasing CDK activity, indicative of cells cycling in G1, S or 791 G2. Alternatively, 14% of ACs that failed to invade the BM following loss of swsn-792 4/ATPase of the complex had CDK activity ratios indicative of G0 cell cycle arrest, 793 suggesting a cell cycle-independent defect. In support of this, forced arrest of BAF-794 deficient ACs in G0 was sufficient to significantly rescue invasion, whereas CKI-1 795 induction failed to rescue invasion in ACs with RNAi-mediated loss of swsn-4/ATPase. 796 Altogether, our results indicate that the SWI/SNF complex contributes to AC invasion 797 through regulation of G0 cell cycle arrest via the BAF assembly. Further investigation will 798 require biochemical techniques to identify cell cycle regulators and TF targets of the BAF 799 assembly to provide a mechanistic explanation for how exactly BAF regulates the 800 chromatin landscape to promote invasion (Fig 8, blue arrow). Targeted DNA adenine methyltransferase identification (TaDa) is an attractive biochemical approach that may be 802 adaptable to the AC invasion system, as this approach has been characterized as an 803 effective, tissue-specific method to identify TF-target sequence interactions in the C. 804 elegans epidermis (106).  Table S5 for additional details). Hermaphrodite 899 adults were co-injected with guide plasmid (50 ng/μL), repair plasmid (50 ng/μL), and an 900 extrachromosomal array marker (pCFJ90, 2.5 ng/μL), and incubated at 25 ºC for several 901 days before screening and floxing protocols associated with the SEC system (112). 902

RNA interference (RNAi) 903
All 269 RNAi clones assessed in the chromatin remodeler screen were derived from the 904 commercially available Vidal or Ahringer RNAi libraries. Presence of inserts into the 905 L4440 RNAi vector was confirmed via colony PCR amplification of all L4440 vectors used 906 in the chromatin remodeler screen. Vectors which resulted in penetrant loss of invasion 907 (see Table S2) were also sequenced to confirm the identity of the insert targeting 908 chromatin remodeler genes in the L4440 vector using Sanger sequencing at the 909 Genomics Core Facility at Stony Brook University. An RNAi sub-library of SWI/SNF 910 subunits was constructed by cloning 950-1000 bp of synthetic DNA based on cDNA 911 sequences available on WormBase (www.wormbase.org) into the highly efficient T444T 912 RNAi vector (113,114). Synthetic DNAs were generated by Twist Biosciences as DNA 913 fragments and cloned into restriction digested T444T using NEB Gibson Assembly (see 914   Tables S6 for additional details). For all experiments, synchronized L1 stage animals 915 were directly exposed to RNAi through feeding with bacteria expressing dsRNA (115). its effects were analyzed as previously described(116). Briefly, L1 animals were first 919 synchronized by sodium hypochlorite treatment and transferred to NGM plates seeded 920 with the RNAi vector of interest. At the P6.p 1-cell stage, a time in development where 921 the AC has already undergone specification, animals were transferred to RNAi-seeded 922 plates treated with K-NAA. Animals were staged by DIC.  Figure S1. AC invasion is disrupted in temperature sensitive SWI/SNF hypomorphs. Single planes of confocal z-stacks representing AC invasion in swsn-1(os22) and swsn-4(os13) temperature sensitive mutants with fluorescently labeled AC (magenta, cdh-3>mCherry::moeABD) and BM (green, laminin::GFP) scored at the permissive temperature (A) and restrictive temperature (B). Significant loss of invasion was seen in both swsn-1(os22) (20% loss of invasion) and swsn-4(os13) (24% loss of invasion) hypomorphic ts strains when grown at the restrictive temperature 25°C and assessed at the P6.p 4-cell 1° VPC stage (B).  Quantification of endogenous GFP expression of SWI/SNF subunit in the AC, VU, and VPC over time. Statistical comparisons were made between the expression of each SWI/SNF subunit in the AC over time (magenta bracket and asterisks) or between the expression of each subunit in the AC relative to the expression of the same subunit in the neighboring VPCs or VUs at the same time (black asterisks) using Student's t-test (n≥30 for each stage and subunit; p values are displayed above compared groups). n.s. not significant.   -4, A), BAF assembly (swsn-8, B), and PBAF assembly (pbrm-1, C) in the AC (cdh-3p::mCherry::moeABD) following treatment with targeting either SWI/SNF assembly (A), or the core ATPase and alternative SWI/SNF assembly (B-C). White arrowheads indicate AC(s), yellow arrowheads indicate boundaries of breach in BM, and white brackets indicate 1 VPCs. (D) Quantification of fluorescence expression (mean gray value) of endogenous subunits in each condition. Statistical comparisons were made between the expression of each SWI/SNF subunit in the AC in control and RNAi-treated animals using Student's t-test (n≥30 for each stage and subunit; p values are displayed above compared data). n.s. not significant. (E) Quantification of fluorescence expression of endogenous GFP-tagged subunits of non-invasive ACs following loss of expression of alternative SWI/SNF subunits, binned per RNAi treatment by phenotype into single non-invasive AC (1AC) and mitotic non-invasive AC (2+ AC). Statistical comparisons (Student's t-test; p values are displayed above compared data) were limited to conditions with n>10 ACs in each phenotype. n.s. not significant. (F) Schematic summary of SWI/SNF core and assembly auto and cross regulation.