Co-condensation between transcription factor and cBAF selectively modulates chromatin remodeling and gene expression

Domain: physics
Published: 2025-12-11
Editorial outcome: major_revision

Abstract
Activation of gene transcription is a tightly coordinated process that requires the engagement of transcription factors (TFs) and chromatin remodelers, yet how these components integrate at specific genomic loci remains unclear. Here, we report that ARID1A, a key subunit of the chromatin remodeler cBAF complex, forms condensates through uniformly distributed tyrosine residues within its core intrinsically disordered region (IDR). A series of TFs which feature in the presence of tyrosine within their transcription activation domain (TAD), selectively interact with ARID1A through core IDR-TAD interaction, thereby enabling co-condensation at specific loci. Furthermore, we demonstrate that co-condensation between ARID1A and TFs, such as GATA2, is crucial for maintaining chromatin accessibility and activating genes essential for lung cancer cell proliferation. Collectively, our study establishes the essential role of ARID1A in spatial organization of TFs and cBAF through phase separation, and demonstrates that tyrosine-mediated selective TFs-cBAF co-condensation represents a pivotal mechanism for gene activation.

Introduction
Gene transcription is a fundamental process that plays critical roles in regulating cellular functionality and organismal development1. The activation of gene transcription is a highly coordinated event that necessitates the orchestrated engagement of various transcription factors (TFs) and the transcription apparatus to access DNA at specific gene promoters and enhancers2,3. Dysregulation of gene transcription is often associated with a variety of human diseases, including genetic disorders, developmental defects, and cancer4,5.

The DNA accessibility, the most important prerequisite for the initiation of gene transcription, is produced by chromatin remodelers, notably the mammalian SWI/SNF (BAF) complexes, which utilize ATP to reposition or evict nucleosomes from the DNA6. The BAF complexes are composed of 10–15 subunits assembled combinatorically from 29 genes and exist in three main forms: canonical BAF (cBAF), polybromo-associated BAF (PBAF), and non-canonical BAF (ncBAF)7,8. Approximately 20% of human malignancies harbor mutations in BAF subunits, rendering these complexes among the most frequently dysregulated targets in human cancer9. Although it is well established that BAF complexes maintain an accessible chromatin environment, the mechanism how they are directed to specific genomic regions are not fully understood.

The cBAF complexes interact with numerous TFs and epigenetic factors to modulate gene expression10–12. ARID1A, the largest subunit of cBAF, serves as a core scaffold that facilitates the complex assembly13,14. Recent studies have shown that ARID1A functions as a transcriptional activator by opening gene loci occupied by TF in a context-dependent manner15,16. For instance, in multiple myeloma, ARID1A associates with IRF4 on chromatin, facilitating IRF4 target gene expression, plasma cell differentiation, and myeloma survival17. In breast cancer, ARID1A occupies estrogen receptor (ER) α- and FOXA1-bound loci to regulate ER-dependent transcription, thereby maintaining luminal cell fate18. Additionally, ARID1A enables the cooperative and sequential binding of PU.1 and NF-kB at key cytokine genes to orchestrate B cell fate during the germinal center response19. These findings demonstrate the important role of ARID1A in modulating TF interactions to regulate gene expression across diverse biological contexts. However, the mechanism by which ARID1A engages with distinct classes of TFs to orchestrate context-specific chromatin remodeling and gene transcription remains unclear.

Emerging studies have illuminated that many nuclear processes occur within biomolecular condensates formed by liquid-liquid phase separation (LLPS)20,21. A prominent feature of these condensates is the enrichment of proteins containing intrinsically disordered regions (IDRs)22. The structural plasticity of IDRs enables the orchestration of multivalent interactions, and such IDR-driven multivalence serves as a fundamental physicochemical principle governing condensate nucleation and maturation. Accumulated evidence has shown that dynamic assemblies of transcriptional proteins enable robust transcriptional regulation23,24. The transcription coactivator TAZ and YAP, the transcription elongation factor P-TEFb as well as the super enhancers MED1 and BRD4 all form condensates to activate gene expression25–30. The transcriptional activation domains (TADs) of most TFs are typically disordered, which allows them to form phase-separated condensates with the mediator complex to activate gene expression31. While extensive studies have focused on individual nuclear condensates in gene transcription, the mechanism governing the selective partitioning of transcription machineries into distinct condensates remains yet to be elucidated.

In this study, we find that ARID1A promotes cBAF self-condensation and co-condensation with various TFs through its core IDR (cIDR), which enables the targeting of cBAF complex to specific gene loci. We identify a heterotypic interaction mediated by tyrosine within ARID1A cIDR and TADs of diverse TFs. Through a broad analysis, we demonstrate that TADs enriched in aromatic residues are indispensable for TFs-ARID1A co-condensation. By employing GATA2 and FOXA1 as model TFs, we uncover the critical roles of cBAF-TF co-condensation in regulating the chromatin accessibility and the expression of key genes required for lung cancer cell proliferation and invasion. Taken together, we propose that the IDR-mediated selective co-condensation between TFs and cBAF represents a principle for chromatin regulation and efficient gene activation.

Results
ARID1A forms liquid-like condensates in cells
We sought to investigate the role of phase separation in chromatin remodeling and TF activation during the initiation of gene transcription. Examining the structure-function of ARID1A reveals that, besides its folded glucocorticoid receptor (GR) domain, which mediates cBAF complex assembly, the remaining two-thirds of ARID1A is largely intrinsically disordered as predicted by IUPred analysis (Fig. 1a)32,33. To investigate whether ARID1A undergoes phase separation in cells, we first ectopically expressed GFP-ARID1A in HEK-293A, HeLa, or H1299 cells at a level comparable to that of endogenous ARID1A, and found that GFP-ARID1A formed discrete condensates in the nucleus (Supplementary Fig. 1a, b). We further restored GFP-ARID1A expression at the near-endogenous level in ARID1A knockout H1299 (H1299ARID1A-KO) and HEK-293T (HEK-293TARID1A-KO) cells, and observed that GFP-ARID1A also formed nuclear condensates (Supplementary Fig. 1c, d). Switching GFP to Flag tag did not affect the condensate formation by ARID1A (Supplementary Fig. 1e, f). Importantly, endogenous ARID1A also exhibited formation of nuclear puncta in cells (Supplementary Fig. 1g).

Next, we examined the biophysical property of ARID1A condensates. Live-cell imaging displayed that the ARID1A nuclear condensates readily fused into larger structures over time (Supplementary Fig. 1h). Fluorescence recovery after photobleaching (FRAP) assay revealed the dynamic nature of the observed condensates (Supplementary Fig. 1i). Together, these data demonstrate that ARID1A forms liquid-like condensates in cells.

cIDR is critical for ARID1A condensates formation
ARID1A contains multiple IDRs along with structured AT-rich interaction domain (ARID) and GR domain, which are important for interactions with DNA and other proteins (Fig. 1a). To determine the minimal region of ARID1A responsible for condensates formation, we generated several truncation variants by deleting specific regions of ARID1A. Given the large size of the N-terminal IDR (residues 1-1102), a complete deletion of this region would remove nearly half of the protein, which could disrupt other functional domains and complicate the interpretation of effects specifically related to IDR-mediated condensation. Therefore, the N-terminal IDR was subdivided into three segments based on the scoring plot that evaluated disorder propensity (Fig. 1a). Regions flanked by the lowest scoring thresholds were selected, as these are likely to represent distinct, functionally relevant modular units. We next examined the cellular distribution of these deletion mutants, ensuring that all constructs were expressed at comparable levels (Supplementary Fig. 2a). Deletion of the GR domain did not significantly impair the condensates formation (Fig. 1b, c). Similarly, removal of ARID domain resulted in a decreased number of puncta and an increase in their size (Fig. 1b, c), but it did not significantly impair droplet formation when assessed by the total area of droplet (Supplementary Fig. 2b). Deletion of IDR1, IDR2, or IDR3 considerably reduced but did not eliminate droplets formation (Fig. 1b, c). Notably, deletion of the middle IDR completely abolished ARID1A condensation (Fig. 1b, c), indicating that this IDR is the most critical and minimal region for ARID1A condensates formation. We therefore named it as the core IDR (cIDR).

To corroborate these findings, we performed an in vitro droplet assay using purified recombinant proteins (Supplementary Fig. 2c). Under similar conditions, purified GFP-cIDR appeared spontaneous formation of micron-scale droplets (Fig. 1d, e). GFP-IDR2 also formed droplets, but with a lesser propensity (Fig. 1d, e). As the concentration of protein or salt increased, and as the temperature rose, the GFP-cIDR droplets that formed in solution became larger and more numerous, suggesting that hydrophobic interactions, rather than electrostatic interactions, are the major driving force involved in this process (Fig. 1f, g and Supplementary Fig. 2d, e). Consistently, the addition of 5% 1,6-hexanediol, a compound that interferes with weak hydrophobic interactions, substantially diminished the formation of droplets (Supplementary Fig. 2f). Time-lapse microscopy experiment revealed that GFP-cIDR droplets exhibited fusion and coalescence over time (Fig. 1h). After selective bleaching of the center part of the droplets, fluorescence recovery was observed at a rapid timescale (Fig. 1i), indicating efficient diffusion of the cIDR liquid droplets.

Given the critical role of the ARID1A cIDR in driving condensate formation, we investigated whether this function could be performed by other LLPS-promoting IDRs. To explore this, we generated constructs in which the cIDR of ARID1A was substituted with well-characterized LLPS-promoting IDRs from TDP-43 (amino acids 274–414) and hnRNPA1 (amino acids 186–320), which contain 140 and 134 amino acids, respectively34,35 (Supplementary Fig. 2g). Confocal microscopy images revealed that these chimeric ARID1A proteins formed condensates in cells, suggesting that these alternative IDRs are able to induce ARID1A condensation.

Tyrosine of the cIDR is required for ARID1A condensates formation
To identify the essential amino acids within the cIDR that mediate ARID1A condensates formation, we mutated all cIDR-enriched residues, with the exception of proline, to alanine, respectively, and expressed these mutants at equivalent levels in cells (Supplementary Fig. 2h, i). We found that the ARID1A Q to A (QA) and M to A (MA) mutations did not affect ARID1A condensate formation, whereas Y to A (YA) mutation compromised ARID1A condensate formation (Fig. 1j). Consistently, purified cIDR QA or MA mutant proteins formed comparable droplets in vitro to wildtype cIDR, while YA mutation significantly abolished the ability to form droplets (Fig. 1k and Supplementary Fig. 2j). We further performed droplet sedimentation assay by mixing purified cIDR or its mutants with nuclear extracts. After centrifugation, cIDR, as well as QA and MA mutant, were distributed in the pellets, whereas YA mutant was largely found in the supernatant (Supplementary Fig. 2k). To further determine if tyrosine is necessary for cIDR condensate assembly in cells, we employed the optoDroplet system by fusing cIDR or its mutants with mCherry-labeled Arabidopsis photoreceptor cryptochrome 2 (CRY2). Puncta formation of cIDR, but not the control, was observed after blue light induction (Fig. 1l). Again, YA mutation, but not QA or MA mutation, completely inhibited light-induced droplets formation (Fig. 1l). Based on these findings, we conclude that tyrosine is the essential residue within cIDR for ARID1A condensate assembly.

ARID1A dictates cBAF condensation through both cIDR and structured domains
Since colocalization studies indicated that ARID1A condensates did not contain markers of promyelocytic leukemia (PML) bodies, Cajal bodies, nuclear speckle, or nucleoli (Supplementary Fig. 3a), we asked whether the ARID1A nuclear puncta contained cBAF subunits and other transcriptional coactivators. Immunostaining of endogenous cBAF subunits revealed that ARID1A condensates enriched cBAF subunits, such as SS18, SMARCA2, and DPF2, but not the PBAF subunit PHF10 or the ncBAF subunit BRD9, indicating that ARID1A specifically mediates cBAF condensation (Fig. 2a). To determine whether ARID1A phase separation was required for cBAF condensation, we used doxycycline (Dox) to induce the expression of wild type ARID1A or ARID1A-ΔcIDR and ARID1A YA mutants deficient in phase separation at similar levels in H1299 ARID1A-KO cells (Supplementary Fig. 3b). As expected, SS18 and SMARCA2 subunits were recruited to ARID1A condensate (Fig. 2b). However, they were evenly distributed in the nucleus of cells expressing ARID1A-ΔcIDR or ARID1AYA (Fig. 2b), although co-immunoprecipitation (co-IP) experiments showed that these two mutants remained bound to cBAF complex (Supplementary Fig. 3c, d). These data suggest that cIDR-mediated ARID1A phase separation dictates cBAF condensation in cells.

The GR domain of ARID1A is known to be sufficient for mediating the assembly of cBAF complex13,14. We confirmed that ARID1A, when deleted of the modular GR domain (ARID1A-ΔGR), failed to interact with other cBAF subunits, including SMARCA2, SMARCC1, SMARCE1, SMARCB1, SS18, and DPF2 (Supplementary Fig. 3c). When expressed in H1299 ARID1A-KO cells, ARID1A-ΔGR resulted in the exclusion of most cBAF subunits from the condensates (Supplementary Fig. 3e), and the similar results were observed in HEK-293T ARID1A-KO cells (Supplementary Fig. 3f). Interestingly, both SS18 and SMARCA2 were still incorporated into these condensates (Fig. 2c and Supplementary Fig. 3g). These data indicate that, in addition to GR domain-mediated high affinity interaction, a weak interaction between ARID1A IDR and SS18 or SMARCA2 also contributes to the cBAF complex assembly. Similar results were also observed with SMARCA4, indicating SMARCA4 behaves similarly to SMARCA2 in this sense (Supplementary Fig. 3h–j). Taken together, these results suggest that ARID1A forms nuclear condensates via cIDR-induced phase separation and recruits cBAF subunits through both GR domain and IDR.

The ARID1A nuclear condensates were also enriched for transcriptional coactivators P300, BRD4 and MED1, as well as the active RNA polymerase II phosphorylated at Ser2 or Ser5 of its C-terminal domain, suggesting that the ARID1A puncta were sites of active transcription (Fig. 2d, e). Consistent with this, we detected H3K27ac or H3K4me3, the actively transcribed chromatin mark, but not the transcriptionally repressive mark H3K9me3 in the ARID1A condensates (Fig. 2f). These data suggest that ARID1A forms condensates localized at active chromatin and enriched for key transcription cofactors.

ARID1A condensation regulates chromatin targeting, accessibility, and gene transcription
Since ARID1A condensates enriched the subunits of cBAF complex and key transcription cofactors, we assessed the role of ARID1A condensation in chromatin regulation and gene transcription. To this end, wild-type ARID1A and ARID1A-ΔcIDR were induced in H1299 ARID1A-KO cells using Dox. This induction achieved expression levels comparable to that of endogenous ARID1A in H1299 cells (Supplementary Fig. 3b). Through cleavage under targets and tagmentation (CUT&Tag) analysis, we found that the signals of ARID1A-binding regions were markedly decreased in ARID1A-ΔcIDR cells (Fig. 3a and Supplementary Fig. 4a). Accordingly, the assay for transposase-accessible chromatin with sequencing (ATAC-seq) demonstrated that deletion of cIDR reduced chromatin accessibility across the ARID1A-binding regions (Fig. 3b and Supplementary Fig. 4b). The peaks significantly impaired by cIDR deletion were predominantly located in enhancer (as defined by H3K27ac peaks) and promoter (Fig. 3c and Supplementary Fig. 4c), and were enriched in motifs corresponding to TFs such as FOXA1, P65, GATA2, and SOX2 (Fig. 3d). To clarify the enhancer subtype associated with ARID1A condensation, we delineated typical enhancers (TEs) and super-enhancers (SEs) based on H3K27ac abundance using the rank order super-enhancer (ROSE) algorithm (Supplementary Fig. 4d). cIDR deletion reduced ARID1A occupancy at H3K27ac-defined TEs and SEs by 18.1% and 29.6%, respectively, suggesting ARID1A condensates are associated with both TEs and SEs (Supplementary Fig. 4e). To assess whether the process of condensation, rather than mere presence of the cIDR, is essential for ARID1A chromatin binding, we analyzed the ARID1A-ΔIDR1-3 mutant, which retains cIDR but is deficient in condensation36. CUT&Tag similarly showed substantially decreased chromatin binding by this mutant compared with wild-type (Supplementary Fig. 4f, g). Peak annotation identified 12,849 ARID1A-binding genes, among which 2,459 showed significantly decreased binding upon deletion of cIDR (Fig. 3a and Supplementary Fig. 4h). These genes were enriched for multiple oncogenic and developmental signaling pathways, such as TNF, mTOR, Hippo, and Wnt (Fig. 3e). Among the 2459 cIDR-regulated genes, 2019 (82%) exhibited decreased ARID1A-binding when IDR1-3 was deleted, supporting the notion that ARID1A condensation is essential to their regulation (Supplementary Fig. 4i). Taken together, these data suggest that ARID1A condensation facilitates its chromatin targeting to specific gene loci and maintains the accessibility there.

We further explored the role of ARID1A condensation on gene transcription. Transcriptional profiling obtained through RNA sequencing (RNA-seq) showed that 1685 genes were significantly downregulated in ARID1A-ΔcIDR-expressing cells compared to ARID1A-expressing cells (Fig. 3f). These downregulated genes were associated with multiple signaling cascades, such as MAPK, Hippo, and Wnt, most of which were also enriched in CUT&Tag (Supplementary Fig. 4j). We further integrated RNA-seq and CUT&Tag data for analysis. After deletion of cIDR, up to 382 genes exhibited concomitant reductions in ARID1A binding and mRNA abundance (Supplementary Fig. 4k). Genes with decreased ARID1A binding that specifically resulted from cIDR deletion exhibited lower expression levels than those with ARID1A binding not affected by cIDR deletion (Supplementary Fig. 4l). These findings were exemplified by intragenic enhancers at the IL4R, TBC1D2, and NTNG2 loci, as visualized by Genomics Viewer (Fig. 3g). Quantitative PCR with reverse transcription (RT-qPCR) analyses confirmed that the expression levels of these genes were significantly downregulated in ARID1A-ΔcIDR-expressing cells relative to ARID1A-expressing cells (Fig. 3h). Collectively, these data demonstrate the critical role of ARID1A condensation in mediating chromatin targeting, remodeling, and the regulation of target gene transcription.

Co-condensation between ARID1A and multiple TFs through cIDR
Next, we sought to elucidate the mechanism governing the recruitment of ARID1A condensates to specific regions of chromatin. Previous studies have indicated that ARID1A collaborates with various TFs to modulate gene expression16–19. Certain TFs, such as IRF4, OCT4, and PU.1, recruit the ARID1A-containing BAF complexes to target genes, where they are required to establish and maintain nearby nucleosome accessibility12,17,19. These findings prompted us to investigate whether interactions with gene-specific TFs could direct the chromatin localization of ARID1A condensates. We focused on the interaction between ARID1A and several TFs, including GATA2, FOXA1, SOX2, and P65, that were enriched in both our CUT&Tag and ATAC-seq analyses. GFP-cIDR and mCherry-tagged TF proteins were purified from bacteria. When added individually to the droplet formation buffer, all purified mCherry-TFs formed droplets in a concentration-dependent manner in the presence of 10% PEG, as previously reported31 (Fig. 4a). We also examined TFs-cIDR co-condensation in the absence of crowding agent or with alternative crowding agents, including 10% dextran, BSA, and glycerol. The results showed that the ARID1A cIDR is sufficient to mediate co-condensation with some tested TFs either under no crowding or under specific crowding conditions, particularly with PEG or dextran, and to a lesser extent with BSA (Supplementary Fig. 5a–d).

We next determined the threshold concentrations for each protein (Supplementary Fig. 6a). Notably, GATA2 and cIDR proteins formed co-phase-separated droplets with PEG, even when the concentration of GATA2 was below the threshold (Supplementary Fig. 6b). In contrast, cIDR with YA mutation (cIDRYA) failed to induce droplet formation under similar conditions. Similar results were also observed with FOXA1, SOX2, and P65 (Supplementary Fig. 6c–e).

Given that replacing cIDR with alternative IDRs from TDP-43 or hnRNPA1 restored ARID1A condensate formation, we also examined the co-condensation between ARID1A chimeric proteins and TFs. Interestingly, co-localization analysis showed reduced partitioning of GATA2 or FOXA1 into the condensates formed by the chimeric proteins compared to wild-type ARID1A (Supplementary Fig. 7a, b). In vitro droplet formation assays also demonstrated that purified IDRs from TDP-43 or hnRNPA1 did not co-phase-separate with GATA2 or FOXA1 (Supplementary Fig. 7c, d). Taken together, these data suggest that cIDR can co-phase separate with multiple TFs and promote their droplet formation.

To clarify how cIDR interacts with GATA2, we first performed in vitro bead binding assay to detect their binding. In these experiments, GFP, GFP-cIDR or GFP-cIDRYA containing a TwinStrep-tagII were immobilized on the surface of Strep-Tactin beads, and then mCherry-GATA2 was added to the solution (Fig. 4b). If heterotypic interactions were present, GATA2 would be recruited to the bead surfaces coated with cIDR. Indeed, we observed that mCherry-GATA2 was recruited to the cIDR-coated beads but not those coated with cIDRYA, suggesting that cIDR can directly bind to GATA2 in vitro (Fig. 4b). To determine whether cIDR interacts with GATA2 in cells, we utilized a synthetic Lac operator (LacO) array integrated into the genome of U2OS cells expressing GFP-tagged Lac repressor (LacI)-fused proteins37. This strategy has been widely employed to interrogate IDR-mediated interactions in cells37. As expected, LacI-fused GFP-cIDR, GFP-cIDRYA, or GFP bound to the LacO array via targeted DNA binding, forming a concentrated local interaction hub in the nucleus (Fig. 4c, d). Colocalization analyses showed that GFP-cIDR, but not cIDRYA, recruited GATA2 to the foci (Fig. 4d). Importantly, either deletion of cIDR or YA mutation markedly prevented ARID1A co-condensation with GATA2 (Fig. 4e), or with other TFs, including FOXA1, SOX2, and P65 (Supplementary Fig. 8a–c). Taken together, these data suggest that ARID1A cIDR can interact with multiple TFs both in vitro and in cells.

Aromatic residues within TADs mediate the co-condensation between TFs and ARID1A
Next, we aimed to dissect how TFs bind to ARID1A. GATA2 harbors two TADs, TAD1 and TAD2, flanking its regulatory and zinc-finger domains38. When LacI-fused GFP-GATA2-TAD1, GFP-GATA2-TAD2, or GFP was expressed in cells, only GATA2-TAD1 enriched ARID1A to the LacO foci (Fig. 5a and Supplementary Fig. 9a). Similar results were obtained with TADs from other TFs, including FOXA1, SOX2, and P65, with the exception of P53 (Supplementary Fig. 9b). This recruitment was mediated by the tyrosine of cIDR, as mScarlet-cIDR, but not mScarlet-cIDRYA, was concentrated by LacI-fused GFP-GATA2-TAD1 or other GFP-TFs-TAD at the foci (Fig. 5b and Supplementary Fig. 9c–e). Moreover, the OptoDroplet assay indicated that TAD1-GFP-CRY2 co-condensed with cIDR-mCherry-CRY2, but not cIDRYA-mCherry-CRY2, upon light stimulation (Fig. 5c). Taken together, these data suggest TF TADs directly recruit ARID1A to specific genome loci.

To illustrate the molecular feature of GATA2 TAD1 relevant to cIDR or ARID1A interaction, we compared the amino acid sequence of GATA2 TAD1 and TAD2, and noted that TAD1, but not TAD2, contains tyrosine residues (Supplementary Fig. 9a). Interestingly, similar to GATA2 TAD2, P53 TAD is also depleted of tyrosine39 (Supplementary Fig. 9a). Considering the interaction between tyrosine and aromatic residue has been well-documented to mediate phase separation through π-π interaction23,24, we speculated that the aromatic residues of TAD1 may contribute to cIDR interaction. Thus, we mutated all aromatic residues to alanine within TAD1 and found that this mutation abolished ARID1A or cIDR recruitment to the loci (Fig. 5d, e). Similar results were also obtained with other TADs (Supplementary Fig. 9f, g). To ensure that full-length GATA2 and ARID1A behaved similarly to their IDRs, we performed immunofluorescence and found that ARID1A condensates enriched GATA2, but not GATA2-TAD1-AroMut in both H1299 and A549 cells (Supplementary Fig. 9h, i). Taken together, these data demonstrate that the aromatic residues of TF TADs are responsible for cIDR interaction.

To test whether the specific sequence features enriched by GATA2 and P53 TAD were sufficient for selective interaction, we generated GATA2-P53 chimeric proteins in which TAD1 of GATA2 was swapped with TAD of P53, and vice versa. If the sequence of TAD was the driver of specificity, then we should be able to redirect the protein containing the TAD to which cIDR is targeted. LacO array experiments showed that ARID1A cIDR recruited GATA2, but not P53, to the loci (Fig. 5f). Substituting GATA2 TAD1 with P53 TAD abolished the recruitment of GATA2 P53-TAD to the cIDR foci (Fig. 5f). In contrast, P53 GATA2-TAD1, in which the P53 TAD was substituted with GATA2 TAD1, gained the ability to partition to cIDR focus (Fig. 5f). These data suggest that the sequence features enriched in the different TADs are sufficient to drive specificity.

Enrichment of aromatic residues in TADs represents a prominent feature for ARID1A cIDR interaction
To assess the generalizability of TF selectivity for ARID1A co-condensation and further analyze the sequence features within diverse TF TADs, we performed a broad analysis of TF TADs capable of interacting with ARID1A. We restricted this analysis to TADs containing at least 30 consecutive amino acids predicted to be intrinsically disordered and validated by transcriptional reporter assays in the previous literature40. Thus, 307 TADs were obtained. Then we applied the computational tool FINCHES41, which predicts IDR-IDR interaction strengths based on the chemical physics of the Mpipi force field. Epsilon (ε) is a mean-field parameter that describes the average interaction between two IDRs, where negative values are attractive and positive values are repulsive. We calculated this predicted interaction parameter for each TAD by using cIDR as a bait and ranked the TADs from most attractive to most repulsive (Supplementary Fig. 10a and Supplementary Data 1). To explore if the amino acid composition or sequence patterning of TADs correlates with interaction strength, we conducted NARDINI+ analysis42 that considers a set of 90 IDR sequence features, including the enrichment of individual amino acids and the patterning of distinct pairs of residue types with respect to one another (Supplementary Fig. 10a). z-score (z)>0 (green) indicates nonrandom clustering of residues or enrichment of indicated features, and z-score<0 (purple) denotes nonrandom mixing of residues or depletion of indicated features. The results revealed that the enrichment scores for aromatic residues (F, W, Y) showed a significant positive correlation with the interaction strengths (Fig. 6a). Among these aromatic residues, the enrichment score for Y was the most correlated (Fig. 6b and Supplementary Fig. 10b, c). Conversely, the enrichment scores for aliphatic residues (A, I, L, M, V) and negative charged (D, E) residues correlated negatively (Fig. 6c and Supplementary Fig. 10d). No significant correlation was observed for positive charged residues (R, K) (Supplementary Fig. 10e). These findings suggest that the enrichment of aromatic residues, particularly Y, in TADs is a key feature associated with interaction with ARID1A cIDR.

To experimentally validate these predictions, we performed LacO-array recruitment and IF assays on representative TADs from the attractive and repulsive groups. TADs from ZNF326, TFAP2A, HOXB1, and EGR3 (attractive group) recruited ARID1A cIDR to the foci, whereas TADs from MBD2, ZXDC, GMEB2, and HNF1B (repulsive group) did not (Fig. 6d). Mutating aromatic residues in attractive TADs or substituting tyrosines within ARID1A cIDR abolished these interactions (Fig. 6e, f). Consistently, mutating aromatic residues in the TADs of these TFs significantly reduced interaction with full-length ARID1A (Fig. 6g and Supplementary Fig. 11a). Furthermore, co-condensation experiments showed that ARID1A formed condensates with ZNF326, TFAP2A, HOXB1, and EGR3, whereas mutations of FWY residues in these TADs prevented co-condensation in cells (Fig. 6h and Supplementary Fig. 11b). Taken together, the bioinformatic analyses and experimental results indicate that aromatic residues within TF TADs are responsible for a broad spectrum of TFs to co-condense with ARID1A through cIDR interaction.

ARID1A condensation promotes GATA2-mediated gene transcription and cell proliferation
Considering the GATA2 transcriptional network is critical for the viability of lung cancer cell43, we next asked whether ARID1A condensation is required for GATA2-mediated gene transcription and cell proliferation. Knockout of ARID1A significantly suppressed the proliferation and colony formation of H1299 cells, and these inhibitory effects could be reversed by restoration of wild type ARID1A, but not ARID1A-ΔcIDR or ARID1AYA (Supplementary Fig. 12a–c). These data suggest that the cIDR and the condensate formation property confer ARID1A the ability to promote the proliferation of lung cancer cells.

In H1299 cells, MTT and colony formation assays revealed that ectopic expression of GATA2 promoted cell proliferation, while knockout of GATA2 suppressed cell growth (Fig. 7a–c and Supplementary Fig. 12d–f). Overexpression of GATA2 increased the expression levels of target genes, including IL4R, TBC1D2, NTNG2, KRT13, and WNT7B, while knockout of GATA2 inhibited their expression (Fig. 7d and Supplementary Fig. 12g), suggesting that these genes are co-regulated by GATA2 and ARID1A. Importantly, knockout of ARID1A significantly inhibited GATA2-induced expression of these genes and the ability of cancer cells to grow (Fig. 7a–d). Both of these inhibitory effects were reversed by reintroduction of wild type ARID1A, but not ARID1A-ΔcIDR or ARID1AYA (Fig. 7e–h). Similar results were also observed in A549 lung cancer cells (Supplementary Fig. 13a–d). Furthermore, deletion of cIDR did not significantly affect GATA2 chromatin binding (Supplementary Fig. 13e), indicating that co-condensation with ARID1A is dispensable for GATA2 chromatin localization but required for efficient target gene activation. Taken together, these data demonstrate that ARID1A-GATA2 co-condensation is critical for GATA2 transcriptional activity and oncogenic function across different types of lung cancer cells.

ARID1A condensation promotes FOXA1-mediated gene transcription and tumor cell migration and invasion
In addition to GATA2, we investigated whether co-condensation between ARID1A and FOXA1 is required for FOXA1-mediated oncogenic function. Unlike GATA2, ectopic expression of FOXA1 did not significantly affect cell proliferation and colony formation in H1299 cells (Supplementary Fig. 14a–c). However, transwell experiments showed that FOXA1 markedly promoted tumor cell migration and invasion (Supplementary Fig. 14d–f). These FOXA1-induced phenotypes were substantially compromised by ARID1A-ΔcIDR and ARID1AYA mutants (Supplementary Fig. 14d–f). These data demonstrate that co-condensation between ARID1A and FOXA1 is critical for FOXA1 oncogenic activity, particularly migration and invasion.

We next asked whether co-condensation with ARID1A influences FOXA1 transcriptional activity. RT-qPCR analyses displayed that ARID1A-ΔcIDR and ARID1AYA suppressed FOXA1-induced target gene expression, including CRACDL and COL9A3 (Supplementary Fig. 14g). Thus, these results suggest that co-condensation with ARID1A is required for FOXA1-mediated gene transcription and oncogenic activity.

Pharmacological perturbation of ARID1A and TFs co-condensation inhibits gene transcription and tumor cell growth
Given the role of ARID1A-TF condensation in promoting tumor cell growth, we reasoned that a compound disrupting the phase separation of ARID1A could inhibit this process. Previous high-throughput phenotypic screen has identified the small molecule BD-K98645985 (BD-98), which specifically targets the cBAF complex and mimics the transcriptional profile observed following cBAF depletion44. Further experiments revealed that BD-98 selectively bound to ARID1A and prevented its function at the 5’ long terminal repeat (LTR) of HIV-1 for latency reversal45,46. However, the exact mechanism by which BD-98 inhibits ARID1A function remains unclear. To investigate the effect of BD-98, we selected a working concentration based on prior reports, where BD-98 was used at 5–30 μM to achieve biological effects without inducing significant toxicity or non-specific effects45,47,48. Specifically, we treated cells with 5 μM BD-98 and found that at this concentration, BD-98 did not affect the formation of GFP-ARID1A or endogenous ARID1A condensates in cells (Supplementary Fig. 15a, b), and GFP-cIDR droplets in vitro (Supplementary Fig. 15c). FRAP experiments showed that BD-98 had no influence on the dynamicity of GFP-ARID1A puncta in cells or GFP-cIDR droplets in vitro (Supplementary Fig. 15d, e). Moreover, BD-98 treatment did not significantly affect the expression levels of the indicated cBAF subunits, their interaction with ARID1A, or their partitioning into ARID1A condensates (Supplementary Fig. 15f, g). These data suggest that BD-98 does not affect ARID1A condensation.

Next, we asked whether BD-98 could affect ARID1A co-condensation with TFs. We first confirmed that BD-98 did not affect the condensation of GATA2 or other tested TFs on their own (Supplementary Fig. 15h–k). Importantly, BD-98 significantly inhibited the co-condensation of ARID1A and GATA2 in H1299 cells (Fig. 8a). Meanwhile, both the recruitment of GATA2 to GFP-ARID1A-cIDR loci and the recruitment of ARID1A to GFP-GATA2-TAD1 loci were remarkably abolished in cells upon BD-98 treatment (Fig. 8b, c). Consistently, BD-98 inhibited cIDR co-condensation with GATA2 in vitro (Fig. 8d). Furthermore, BD-98 inhibited GATA2-mediated gene transcription and cell proliferation (Fig. 8e–g and Supplementary Fig. 15l). Similar inhibitory effects on co-condensate formation, cell proliferation and GATA2 target gene expression upon BD-98 treatment were also observed in A549 cells (Supplementary Fig. 16a–e).

To further investigate the mechanism of BD-98, we performed the surface plasmon resonance (SPR) assay and found that BD-98 bound specifically to cIDR, but not to IDR1-3 or the GR domain, indicating that BD-98 acts on ARID1A through specific binding to cIDR (Fig. 8h and Supplementary Fig. 17a). Consistent with this, LacO array experiment displayed that BD-98 inhibited the recruitment of ARID1A to other TF TADs-bound loci, such as FOXA1, P65, and SOX2 (Supplementary Fig. 17b–d). Moreover, BD-98 also prevented the co-condensation between ARID1A and the tested TFs, including P65, SOX2, and FOXA1 (Supplementary Fig. 17e), and suppressed FOXA1 target gene expression (Supplementary Fig. 17f, g). Together, these data suggest that BD-98 targets ARID1A-TF co-condensation through binding to ARID1A cIDR, thereby impairing target gene activation and tumor cell proliferation.

Methods
Plasmids, antibodies, and reagents
The Flag-ARID1A and GFP-ARID1A constructs were generated by PCR and sub-cloned into the Dox-inducible PiggyBac transposon vector, pEGFP-C1 vector or pGFP-2×Strep vector. Mutant GFP-ARID1A or Flag-ARID1A containing various truncations and mutations in the ARID1A molecule were generated by PCR based on GFP-ARID1A or Flag-ARID1A. The truncated regions are as follows: IDR1 (amino acids 1–342), IDR2 (amino acids 343–824), IDR3 (amino acids 825–1012), IDR1-3 (amino acids 1–1012), ARID domain (amino acids 1013–1110), cIDR (amino acids 1111–1634), GR domain (amino acids 1635–2285). The chimeric GFP-ARID1A with cIDR replaced by hnRNPA1 IDR (amino acids 186–320), or TDP-43 IDR (amino acids 274–414) were generated by PCR based on GFP-ARID1A. The mCherry-GATA2, mCherry-P65, mCherry-SOX2, and mCherry-FOXA1 constructs were generated by PCR and subcloned into the pHis-mCherry vector. The HA-GATA2, HA-P53, HA-P65, HA-FOXA1, HA-SOX2, HA-TFAP2A, HA-ZNF326, HA-EGR3, HA-HOXB1, HA-MBD2, HA-HNF1B, HA-ZXDC, and HA-GMEB2 and the various variants were generated by PCR and subcloned into the pRK5 vector. All constructs were sequence verified using Sanger sequencing.

The following antibodies and reagents were obtained from commercial sources: anti-ARID1A (Cell Signaling Technology, D2A8U, 12354s, 1:1000); anti-ARID1A (GeneTex, GTX129433, 1:1000); anti-SMARCC1 (Santa Cruz, DXD7, sc-32763, 1:500); anti-SMARCC2 (Santa Cruz, E-6, sc-17838, 1:500); anti-SMARCB1 (Santa Cruz, A-5, sc-166165, 1:500); anti-SMARCD1 (Santa Cruz, 23, sc-135843, 1:500); anti-SMARCE1 (Cell Signaling Technology, E6H5J, 33360s, 1:1000); anti-SS18 (Cell Signaling Technology, D6I4Z, 21792s, 1:1000); anti-DPF2 (Santa Cruz, C-9, sc-514297, 1:500); anti-SMARCA2 (Cell Signaling Technology, D9E8B, 11966s, 1:1000); anti-SMARCA4 (Proteintech, 21634-1-AP, 1:1,000); anti-BRD9 (Cell Signaling Technology, E9R2I, 58906s, 1:1000); anti-PHF10 (GeneTex, GTX116314, 1:1000); anti-BCL7C (GeneTex, GTX115214, 1:1000); anti-BRD4 (Santa Cruz, A-7, sc-518021, 1:500); anti-P300 (Santa Cruz, F-4, sc-48343, 1:500); anti-HP1 (Santa Cruz, E-6, sc-515341, 1:500); anti-MED1 (Santa Cruz, H-7, sc-74475, 1:500); anti-SC35 (Abcam, SC-35, AB11826, 1:1000); anti-H3K27ac (Abcam, AB4729, 1:1000); anti-H3K4me3 (Abcam, EPR20551-225, AB213224, 1:1000); anti-H3K9me3 (Abcam, AB8898, 1:1000); anti-RNA Pol II S5 (Abcam, 3E8, AB252852, 1:1000); anti-RNA Pol II S2 (Abcam, 3E10, AB252855, 1:1000); anti-Flag (Proteintech, 20543-1-AP, 1:1000); anti-HA (Proteintech, 51064-2-AP, 1:1000); anti-Flag (Proteintech, 66008-4-Ig, 1:1000); anti-Actin (Proteintech, 66009-1-Ig, 1:10000); anti-GFP (Beyotime, AG281, 1:1000); anti-PML (Santa Cruz, PG-M3, sc-966, 1:1000); anti-Coilin (Santa Cruz, F-7, sc-55594, 1:1000); anti-NCL (Proteintech, 10556-1-AP, 1:1000); anti-GATA2 (Abcam, EPR2822, AB109241, 1:1000); Goat Anti-Rabbit IgG H&L Alexa Fluor 555 (Invitrogen, A21428, 1:500); Goat Anti-Mouse IgG H&L Alexa Fluor 555 (Invitrogen, A21422, 1:500); Goat Anti-Rabbit IgG H&L Alexa Fluor 488 (Invitrogen, A11008, 1:500); Goat Anti-Mouse IgG H&L Alexa Fluor 488 (Invitrogen, A11001, 1:500); Goat Anti-Rabbit IgG H&L Alexa Fluor 633 (Invitrogen, A21071, 1:500); PEG-8000 (Sigma, 89510); Dextran-70 (Sangon Biotech, A600375); BSA (Sangon Biotech, A500023); Doxycycline hyclate (Beyotime, ST039B); and BRD-K98645985 (MCE, HY-114268).

Cell culture, transfection, and infection
The HEK-293T (ATCC, CRL-3216), A549 (ATCC, CRM-CCL-185), and HeLa (ATCC, CRM-CCL-2) cells were cultured in high-glucose Dulbecco’s modified Eagle’s medium (DMEM) (Procell) supplemented with 10% fetal bovine serum (FBS) (VivaCell) and 50 µg ml−1 penicillin-streptomycin (Pen-Strep) (Sangon Biotech). The HEK-293A and U2OS LacO cell lines were kindly provided by Prof. Chun-Yan Lim (Guangzhou Medical University) and Prof. Huasong Lu (Zhejiang University), respectively. Both cell lines were cultured in DMEM supplemented with 10% FBS. H1299 cells (ATCC, CRL-5803) were cultured in Roswell Park Memorial Institute (RPMI) (Procell) with 10% FBS and 50 µg ml−1 Pen-Strep. All cell lines were cultured at 37°C with 5% CO2 in a humidified atmosphere and were free of mycoplasma contamination.

Transfection of cells was carried out using Lipofectamine 3000 (Thermo Fisher Scientific) according to the manufacturer’s instructions. To generate Dox-inducible cell lines expressing ARID1A or its mutants, PiggyBac plasmids expressing ARID1A or its mutants were co-transfected with a mammalian expression plasmid carrying a transposase gene cassette into ARID1A knockout cells using Lipofectamine 3000 and selected with hygromycin at 24h post-transfection for 5–7 days. Expression of the transgene was induced by addition of 50 ng ml−1 Dox for 48h. ARID1A knockout cell lines were generated using the CRISPR/Cas9 system. Briefly, a pair of targeting sequences (Supplementary Data 2) was synthesized and introduced into plentiCRISPRv2 (Addgene plasmid 52916). Then the plasmid encoding single guide RNA (sgRNA) was co-transfected with packaging plasmids pMD2.G and psPAX2 into HEK-293T cells using Polyethylenimine (PEI) (Polysciences). The viral medium was harvested, filtered through a 0.45 µm syringe filter, mixed with 4 µg ml−1 polybrene (Beyotime), and added to target cells. At 48h post-infection, cells were selected with puromycin for 48h and reseeded to 96-well plates to allow single-colony formation. Genomic DNA was extracted from individual colonies. The candidate knockout clones were verified by sequencing of the PCR fragments amplified by the primers and western blotting.

Protein expression and purification
For proteins purified from bacteria, plasmids containing Strep-GFP- or His-mCherry-tagged genes were transformed into E. coli BL21 competent cells. The bacterial cultures were grown at 37°C to an optical density of 0.6, then 0.2 mM isopropyl-β-D-thiogalactoside (IPTG) was added and expression was induced at 18°C for 16h. Bacterial pellets were collected, resuspended with lysis buffer (50 mM Tris-HCl, pH 7.5, 500 mM NaCl, 1 mM dithiothreitol (DTT), 1 mM phenylmethanesulfonyl fluoride (PMSF), and 1% Triton X-100), followed by sonication. For proteins purified from mammalian cells, HEK-293 GnTI cells transfected with plasmids containing Strep-GFP- or Strep-tagged genes were harvested into the lysis buffer (50 mM HEPES, pH 7.5, 1% Triton X-100, 150 mM NaCl, 1 mM MgCl2, 10% glycerol, and 1 mM DTT supplemented with protease inhibitors. The supernatant after centrifugation was collected for protein purification. The Strep-GFP- or Strep-tagged proteins were purified using the Strep-Tactin Superflow beads (IBA). The His-mCherry-tagged proteins were purified using a Ni-NTA column (GE Healthcare). The eluted proteins were applied onto a Superdex 200 column. After gel filtration, fractions containing pure proteins were pooled, concentrated with Amicon Ultra Centrifugal filters (Millipore), and the concentrations were determined using a Bradford Protein Quantification kit (Vazyme).

Immunoblotting and co-immunoprecipitation assay
For immunoblotting, whole cell lysates were prepared in radio-immunoprecipitation assay (RIPA) buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 1% Triton X-100, 5 mM EDTA) containing 50 mM NaF, 1 mM sodium vanadate, 2 mM DTT, and a cocktail of protease inhibitors (Roche). Clarified lysates were subjected to western analysis with the indicated antibodies and horseradish peroxidase-conjugated secondary antibodies. Proteins were visualized by enhanced chemiluminescence according to the manufacturer’s instructions (Tanon).

For co-IP assay, nuclei were extracted. Cell pellets were incubated in hypotonic buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 1 mM MgCl2, 1 mM DTT, and 1 mM PMSF) on ice for 10 min. Glass homogenizers were used to crush cell membranes. Nuclei were pelleted at 1600 × g for 10 min at 4°C, and resuspended in nuclear extraction buffer (50 mM Tris-HCl, pH 7.5, 300 mM NaCl, 1% NP-40, 1 mM EDTA, 1 mM MgCl2, 25% glycerol, 1 mM DTT, and a cocktail of protease inhibitors). The suspension was incubated at 4°C for 30 min, and then spun at 13,000 rpm at 4°C for 15 min. The supernatant was incubated with anti-FLAG M2 affinity gel (Sigma–Aldrich) on a rotator at 4°C overnight. The immunoprecipitates were washed and eluted with Flag peptide. The Flag eluates were subjected to western blot.

Immunofluorescence staining, live-cell imaging, and Opto-Droplet experiment
Cells grown on glass coverslips were fixed with 4% paraformaldehyde for 15 min and blocked with buffer containing 5% FBS and 0.3% Triton X-100 in PBS for 1h. The coverslips were then incubated with primary antibodies diluted in PBS containing 5% FBS overnight at 4°C, followed by Alexa Fluor 488- or 555-conjugated secondary antibodies (Thermo Fisher Scientific) diluted in PBS containing 4′,6-diamidino-2-phenylindole (DAPI) for 1h at room temperature. The coverslips were mounted on glass slides in mounting medium (Yeasen) and sealed. Immunofluorescence was analyzed using Zeiss LSM 900 confocal microscope. Colocalization analysis of green and red channels was conducted using ImageJ (NIH).

For live-cell imaging, HEK-293A cells were seeded on confocal dishes (Biosharp), transfected with GFP-ARID1A, and then examined under a Zeiss LSM 900 confocal microscope. During image acquisition, cells were maintained in an equilibrated observation chamber set at 37°C with 5% CO2. Images were captured at 2-s intervals and analyzed using ImageJ to identify fusion events.

For OptoDroplet assays, HEK-293T cells cultured in 35-mm glass-bottom dishes (Biosharp) were transfected with indicated plasmids. After 24h, cells were imaged using High Intelligent and Sensitive SIM (HIS-SIM) microscope in a 37°C humidified chamber with 5% CO2. Cells with similar fluorescence intensity were exposed to 488 nm light for both CRY2 activation and imaging of GFP signal, then to 555 nm light for imaging mCherry signal. Images were recorded for 2 min at 2-s intervals, and analyzed by Wiener (HIS-SIM) and FIJI.

Partition coefficient analysis
Analysis of factors enrichment at foci was performed using established protocols. All immunofluorescence images were recorded by ZEISS LSM 900. For quantifying partition of two factors of interest, the thresholds of intensity detected by FIJI were uniformed. The pipeline of CellProfiler V.4.5.2 showed that images were separated and converted into gray module. The Align module determined the position of each pair of images.

The Measure Correlation module measured the joint signals of immunofluorescence across the entire image, and compared them with the signals of factors of interest by Manders coefficient. Final results of colocalization were shown that the proportion of joint signals account for whole signals of factors (n ≥ 3).

FRAP
FRAP experiments were carried out on a ZEISS LSM 900 confocal microscope with a ×63 oil immersion objective. For FRAP in living cells, HEK-293A cells grown on 35 mm glass-bottom dishes were transfected with GFP-ARID1A plasmids. After 24h, GFP-ARID1A condensates were photobleached using a 100% laser power at 488 nm and recovery was recorded for the indicated duration. For in vitro FRAP, the GFP-cIDR droplets were photobleached using 488 nm laser on the glass slides.

The fluorescence intensity was measured using ImageJ. The pre-bleached fluorescence intensity was normalized to 1 and the signal after bleaching was normalized to the pre-bleached level.

In vitro condensation assay
Purified recombinant GFP-tagged or mCherry-tagged fusion proteins were diluted to varying concentrations in droplet formation buffer (20 mM Tris-HCl pH 7.5, 1 mM DTT) with the indicated salt concentrations. For co-condensation analysis, GFP-tagged and mCherry-tagged proteins were mixed and added to solutions at varying concentrations with indicated final salt and 10% PEG-8000 as crowding agent in droplet formation buffer. An 8 µl aliquot of the protein solution was loaded onto a glass slide, covered with a coverslip, and visualized using an inverted fluorescence microscope at room temperature. The sizes and numbers of the droplets were quantified using ImageJ.

Co-sedimentation assay
Purified recombinant GFP-fusion proteins and H1299 nuclear extracts were pre-cleared at 12,000 × g for 15 min to remove insoluble material. Purified GFP-cIDR or mutants and nuclear extract were mixed in tubes and incubated at room temperature for 15 min. Then the mixture was centrifuged at 12,000 × g for 10 min. The supernatant and pellet were collected and analyzed by western blot.

MTT and colony formation assays
For the MTT (3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide) assay, H1299 cells stably expressing Dox-inducible Flag-ARID1A or its variants were seeded at a density of 2500 cells per well in 96-well culture plates and treated with Dox (50 ng ml−1) for the indicated days. Cells were incubated with MTT (5 mg ml−1, 20 µl) for 2h. Dimethyl sulfoxide (100 µl) was then added into each well and resuspended until all crystals had been dissolved. The absorbance of the samples was measured with a spectrophotometer at 490 nm.

For colony formation assay, H1299 cells (3000 per well) stably expressing Dox-inducible Flag-ARID1A or its variants were seeded in 6-well plates. The cell culture media containing Dox (50 ng ml−1) was replaced every three days. After 14 days, the cells were stained with 0.5% crystal violet in 20% ethanol for 15 min. The number of colonies was photographed and counted using ImageJ software (NIH).

Transwell assay
Cells in serum-free media were seeded into Transwell with an 8 µm pore polycarbonate membrane.

For the invasion assay, the membrane was pre-coated with Matrigel (Corning, 356231) at a final concentration of 0.2 mg ml−1. The lower chamber was filled with 10% FBS and RPMI medium. After a 24h incubation, cells were fixed with 4% PFA for 10 min and stained with 0.1% crystal violet. The cells migrating to the lower chamber were imaged and statistically analyzed using GraphPad Prism 9.

Surface plasmon resonance assay
The SPR experiment was performed using the Biacore 1K system (Cytiva, Sweden). The sample compartment and flow cell temperature of the Biacore 1K system was set at 25°C. Recombinant protein was immobilized on a CM5 sensor chip using N-ethyl-N’-[3-dimethylaminopropyl] carbodiimide and N-hydroxysuccinimide following the standard amine-coupling method. Protein coupling conditions were protein in pH 4.0 sodium acetate solution with a chip activation time of 420 s, and blocking time of 420 s. The typical protein coupling efficiency was about 10,000 RU. HBS was used as the running buffer, containing 10 mM HEPES, 150 mM NaCl, and 5% DMSO. BD-98 was diluted from 10 to 50 µM with an association and dissociation time of 90 s with a flow rate of 30 µl min−1. DMSO calibration was carried out concurrently with the serial dilutions of BD-98 into the running buffer within various concentration ranges. Binding kinetics were calculated using Biacore Insight Evaluation Software (version 3.0.12.15655). The original data were evaluated in a 1:1 binding model with a fitted local affinity model.

RNA extraction, reverse transcription and qPCR
Total RNA was extracted using TRIzol (Vazyme) according to the manufacturer’s protocol. One microgram of total RNA was reverse-transcribed using HiScript II Q RT SuperMix for qPCR (Vazyme). RT-qPCR was performed using the Taq Pro Universal SYBR qPCR Master Mix (Vazyme) and CFX96 Real-Time PCR detection system (Roche). The PCR conditions were as follows: initial denaturation at 95°C for 30 s, then 45 cycles of denaturation at 95°C for 10 s and extension at 60°C for 30 s. Relative expression levels of mRNA were calculated using the 2−ΔΔCt method. The results were normalized to the expression of GAPDH. All primer sequences are shown in Supplementary Data 2.

RNA-seq
Total cellular RNAs were extracted using TRIzol, and cDNA libraries were prepared using high-quality RNA (RNA integrity number > 7). RNA-seq was performed on an Illumina Novaseq PE150 platform by OE Biotech, Inc., Shanghai. For RNA sequencing data, the quality of raw reads was assessed using FastQC (v0.11.9). The clean reads were mapped to the human reference genome (GRCh38.p13) using HISAT2 (2.1.0). Gene expression levels were quantified by StringTie (v.2.2.1) as fragments per kilobase per million (FPKM) mapped fragments in exon models. DESeq2 (v.4.2.0) was utilized for differential gene expression analysis. Genes with a p value < 0.05 and absolute fold change > 1.25 were considered to be significantly differentially expressed.

ATAC-seq
ATAC-seq was performed using Hyperactive ATAC-Seq Library Prep Kit (Vazyme, TD711-01) according to the manufacturer’s instructions. Briefly, 1×104 H1299 cells per sample were trypsinized and washed with PBS. Cells pelleted at 500 × g for 5 min were lysed in 50 µl pre-cooled lysis buffer (Resuspension Buffer, 0.1% NP40, 0.1% Tween 20, 0.01% Digitonin). Nuclei were collected at 500g for 10 min, resuspended in Tn5 transposase reaction solution (4 µl TTE Mix V50, 16.5 µl Tagment DNA & Wash Buffer, 0.1% Tween 20, 0.01% Digitonin, 10 µl 5× TruePrep Tagment Buffer L), and incubated at 37°C for 15 min. The transposition reaction was stopped by Stop Buffer at room temperature for 5 min. After purified by DNA Extract Beads, the tagmented DNAs were amplified using TruePrep Index Kit V2 for Illumina (Vazyme, TD202) and PCR. The ATAC-seq libraries were purified by DNA Clean Beads, and then were assessed by Agilent 5400 and sequenced on NovaSeq X Plus Series PE150.

The sequencing data were trimmed to remove the adapter and low-quality reads by cutadapt (v1.9.1)59. The clean reads were used as input to align to the hg38 genome by Bowtie 2 (v2.2.6)60. Unmapped and duplicated reads were removed using samtools (v1.7)61 and Picard (v1.126, http://broadinstitute.github.io/picard/). For generation of ATAC-seq peaks and tracks, the files were converted into narrowPeak and bigWig files by MACS2 (v2.1.0) and bedtools (v2.26)63. Peaks were displayed as a heatmap using deeptools (v3.5.1)64. Differential peaks were detected by Diffbind (v3.2)65 and edgeR.

CUT&Tag
1×105 cells were washed twice with PBS and permeabilized with 100 µl nuclear extraction (NE) buffer (20 mM HEPES-KOH pH 7.9, 10 mM KCl, 0.1% Triton X-100, 20% glycerol, 0.5 mM spermidine, 1× protease Inhibitor) for 10 min on ice. Nuclei were collected by centrifugation at 600 × g for 3 min, resuspended in 100 µl NE buffer, and then incubated with 10 µl activated concanavalin A beads (Vazyme) at room temperature for 10 min. The beads were washed twice with Digitonin 150 buffer (20 mM HEPES pH 7.5, 150 mM NaCl, 0.5 mM Spermidine, protease inhibitors, and 0.01% digitonin), and incubated with primary antibody in 100 µl Digitonin 150 buffer supplemented with 2 mM EDTA overnight at 4°C. The beads were washed with Digitonin 150 buffer, and then incubated with secondary antibody in 50 µl Digitonin 150 buffer at room temperature for 1h. Samples were incubated with pAG-Tn5 (Vazyme, S614) in 100 µl of Digitonin 300 buffer (20 mM HEPES pH 7.5, 300 mM NaCl, 0.5 mM Spermidine, protease inhibitors and 0.01% digitonin) at room temperature for 1h. Beads were washed twice with Digitonin 300 buffer, and then incubated with 100 µl Tagmentation buffer (Digitonin 300 buffer supplemented with 10 mM MgCl2) at 37°C for 1h. Ten microliters 10% SDS and 1 pg spike-in DNA (Vazyme, NS101) were added and incubated with the mixture at 55°C for 10 min. The tagmented DNA were extracted using VAHTS DNA Clean Beads (Vazyme, N411), and were amplified using TruePrep Index Kit V2 (Vazyme, TD202) and PCR. The libraries were purified by VAHTS DNA Clean Beads, and then were assessed by Agilent 5400 and sequenced on NovaSeq X Plus Series PE150.

The sequencing data were trimmed to remove the adapter and low-quality reads by cutadapt (v1.9.1). Paired-end reads were aligned to hg38 using Bowtie 2 (v2.2.6). Tracks were made as bedgraph files of normalized counts, which are the fraction of total counts at each base pair scaled by the size of the hg38 genome. E. coli chromatin (Vazyme) was used as a spike-in control for quantifying signals. For generation of CUT&TAG peaks and tracks, the files were converted into narrowPeak and bigWig files by MACS2 (v2.1.0) and bedtools (v2.26)63. Peaks were displayed as a heatmap using deeptools (v3.5.1)64. ARID1A-binding signals were clustered using k-means clustering and the differential peaks were detected by Diffbind (v3.2)65 and edgeR. Gene annotation of peaks was generated by ChIPseeker (v1.28.3)66. TF motif enrichment was analyzed by HOMER (v4.10)67.

Identification of enhancer subtype
Super-enhancers were defined by stitching peaks using ROSE with default settings, relying on H3K27ac intensities68,69. Enhancer peaks within a 12.5 kb range were merged and ranked by increasing H3K27ac signal. To prevent the inclusion of enhancers near transcription start sites (TSSs), a promoter exclusion zone of 5000 bp was applied, ensuring that enhancers located within 2500 bp of a TSS were not stitched together. Each potential SE showed the highest level of H3K27ac by plotting the inflection point and selecting the value for which the slope of the fitted curve exceeded 1. The elements below the inflection point on this curve where the slope equaled 1 were considered TEs.

FINCHES analysis of cIDR and TADs interaction strengths
Experimentally verified TADs of TFs were obtained from the published literature40. We selected TAD sequences which contain at least 30 consecutive amino acids predicted to be intrinsically disordered. Attractive (blue) and repulsive (red) interaction strengths (Epsilon, ε) between cIDR and TADs were calculated based on the chemical physics of the Mpipi force field using FINCHES with default settings41. Epsilon (ε) is a mean-field parameter that describes the average interaction between two IDRs, where negative values are attractive and positive values are repulsive.

Analysis of sequence features using NARDINI+
Sequence features of TADs were analyzed using NARDINI+42. NARDINI+ incorporates the NARDINI (Non-random Arrangement of Residues in Disordered Regions Inferred using Numerical Intermixing) and compositional grammar analyses, which results in a z-score vector of 90 sequence features. For compositional features, z-scores are computed by comparing the data against the distribution from the species-specific IDRome (Homo sapiens, ID: 9606). Compositional features assessed include the fraction of each amino acid type; fractions of aromatic, aliphatic, positive, negative, polar, charged residues (FCR), chain-expanding residues (FCE), and disorder-promoting residues; ratios of K–R and E–D; net charge per residue, isoelectric point, hydrophobicity, and polyproline-II propensity. Patch features indicate the fraction of the IDR composed of specific amino acid patches. A patch is defined as at least four occurrences of a specific residue or two occurrences of RG, with no more than two interruptions. W patches are excluded, as they do not exist in the human IDRome. Each compositional feature z-score is determined using the mean and standard deviation from the entire species-specific IDRome. z-score > 0 (green) indicates enrichment of indicated features, and z-score < 0 (purple) indicates depletion of indicated features.

For patterning analysis, a sequence is compared to 1000 random scrambles matching its composition to assess whether a patterning parameter is either more evenly mixed or more clustered than by random chance. Residues are categorized into eight groups: polar (S, T, N, Q, C, H), hydrophobic (I, L, M, V), positive (K, R), negative (E, D), aromatic (F, W, Y), alanine (A), proline (P), and glycine (G). A U-X z-score is extracted for each pair of residue types U and X. z-score > 0 (green) indicates a more fragmented pattern than expected, and z-score < 0 (purple) indicates the sequence is more uniform than expected.

Illustrations
Illustrations in Figs. 1l, 4b, c, 8i, and Supplementary Fig. 2k were created in BioRender and edited in Adobe Illustrator.

Statistics and reproducibility
All data were derived from at least three independent experiments and statistical details of the experiments are presented in the figures and captions. All comparisons were performed in GraphPad Prism 9.0. All attempts at replication were successful with similar results.

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "The manuscript titled “Co-condensation between transcription factor and cBAF selectively modulates chromatin remodeling and gene expression” provides a compelling and well-executed study that uncovers a selective co-condensation mechanism between transcription factors (TFs) and the cBAF complex through the ARID1A subunit. The authors identify an intrinsically disordered region (cIDR) in ARID1A that facilitates selective phase separation with aromatic-rich transcription factors, notably GATA2. This co-condensation enables cBAF recruitment to lineage-specific regulatory elements, influencing chromatin accessibility and gene expression. The study employs a wide range of state-of-the-art techniques including in vitro droplet assays, live-cell imaging, FRAP, CUT&Tag, ATAC-seq, RNA-seq, and CRISPR-based perturbations. This is a conceptually innovative and technically sound manuscript that offers an important contribution to the understanding of chromatin remodeling and transcriptional regulation via biomolecular condensation. However, there are many concerns need to be addressed before the manuscript is suitable for publication in Nature Communications.\n\nMajor concerns:\n• Limited exploration of other TF function other than GATA2: The paper has only focus on GATA2. Although FOXA1 motifs are enriched at ARID1A-bound regulatory regions and FOXA1 is known to play a central role in lineage-specific transcription, its role in ARID1A-mediated co-condensation is not deeply investigated. Additional experiments or expanded discussion around FOXA1’s functional relevance would help validate the generality of the proposed model and strengthen the claim of selective TF interaction. The whole manuscript mainly focused on GATA2 without a good justification about the reason for this choice.\n• Generalizability of TF selectivity: While several TFs are tested (GATA2, SOX2, p65, FOXA1), a broader assessment or motif-driven analysis of what defines “co-condensation-competent” TFs would further substantiate the selectivity mechanism and its predictive utility.\n• Lack of connection and in-depth interpretation between the current findings and previously published ARID1A phase separation data. The 2023 Cell paper (Cell 186, 4936–4955, October 26, 2023) focused on the N-terminal IDR domain (IDR1) and highlighted its key functional role, which appears to differ significantly from the findings reported in this manuscript. The authors should more thoroughly discuss the discrepancies and provide a clearer interpretation of how their results compare and contrast with the published data.\n\nMinor concerns:\n• Clarify whether ARID1A co-condensates are associated with specific nuclear compartments or enhancer subtypes (e.g., super-enhancers).\n• Provide more quantitative comparisons and replicate details in figure legends to enhance transparency and reproducibility.\n• Consider tightening the discussion to better highlight how this mechanism might integrate with known chromatin remodeling paradigms.\n• The authors should do a deletion for all IDR1-3 to check the effects.\n• The deletion of cIDR domain from ARID1A in Figure 3 and check its binding is sufficient to support cIDR domain is required for ARID1A chromatin binding. But it does not mean the binding required condensation behavior. The authors need to use another mutation that can keep the cIDR domain but deficient for condensation to do the ChIPseq binding again.\n• The authors should use a cell line that knockout the endogenous ARID1A and re-express with ARID1A-delta cIDR to check if the binding of GATA2 on chromatin is still normal or not.", "reviewer_2": "The authors Ye et al. examine the contribution of ARID1A’s cIDR to the formation of condensates of the cBAF complex in cells, its targeting on chromatin and co-condensation with specific transcription factors. They identify that especially Tyrosine residues are required for condensate formation and cBAF complex recruitment to the condensates, but are not required for overall complex formation. Lack of cIDR further negatively impacted ARID1A-binding to chromatin and chromatin accessibility across ARID1A-binding regions, as well as transcription. Co-condensation of transcription factors and cIDR was suggested as the mechanism of ARID1A recruitment to specific chromatin regions and shown by cIDR tethering and co-localization with TFs in cells, which was based on their aromatic residue content. Sequence features in TADs are the sole driver of TF binding to cIDR, as TFs gained the ability to partition with cIDR when obtaining a TAD from another TF that partitions with cIDR.\n\nThey show the significance of ARID1A-TF recruitment and co-condensation by the example of GATA2 in a cancer cell line, and show that this interaction is a driver of tumor cell growth and proliferation. They further identified the mechanism of action of a small molecule BD-98 which disrupts co-condensate formation of ARID1A and GATA2.\n\nThe study is, broadly, performed quite thoroughly. For example, several tags have been used in identical experiments, and similar results have been obtained, suggesting that the tags do not influence the results. Similarly, many in cellulo experiments have been performed in more than one cell line. However, several figures and legends need revision (s. minor comments).\n\nDespite the convincing scope of the presented work, some aspects have previously been described by Patil et al. (2023) and Xue et al. (2024) which impacts novelty of this report. ARID1A’s contribution to cBAF complex formation of condensates has been described, although a different IDR region has been pinned down in this manuscript vs. the previous ones. Similarly, the contribution of Y residues to this behavior is not surprising and has been previously described. Additionally, some IDRs of cBAF have been described to facilitate transcription factor-dependent targeting with similar experiments, highlighting the importance of this process in the development of some cancers. However, the authors add additional depth into specific regions of the TFs which are responsible for recruitment, and test different chimeras to show that it is the TF IDRs and aromatic regions which are responsible for the co-condensation and targeting of cBAF. They further elucidate the mechanism of action of a small molecule cBAF inhibitor that interferes selectively with cIDR-TF co-condensates. Both these aspects are unique to this publication and advance our current understanding.\n\nMajor comments\n- Line 140/ Extended Figure 2f: cIDR can be readily replaced by other LLPS-promoting IDRs in the context of phase separation and cBAF condensate formation. Since the whole sequence can be readily replaced, it makes it more difficult to appreciate the following steps of identifying the essential amino acids within cIDR. In this context, it could have been helpful/ more convincing to compare the cIDR composition with the chimera IDR composition and find overlaps.\nBesides their composition, there is no mentioning of the length of the replacing IDRs, are they comparable in size by residue numbers?\nNow, several LLPS-promoting IDR can replace cIDR condensate formation properties. Besides condensate formation properties, I would further suggest to test whether there is any specificity for cIDR and TF recruitment in comparison to these ARID1A chimera proteins. It would be interesting to see if co-condensation of cIDR and its interaction partners (like GATA2) would take place if cIDR is replaced by any of these LLPS-promoting IDR. This could help to explain how much specificity is needed for such process.\n- Similarly, based on its mode of action, I wonder how specific the drug BD-98 is. It would be interesting to test specificity in comparison to other co-condensates (either other LLPS-promoting IDR chimera or other TF), to underpin the statement that targeting co-condensation can be used as a sufficiently selective therapeutic strategy to target cancer.\n- Line 122/Figure 1c: the authors concluded that removal of ARID did not significantly impair condensate formation, but the number of puncta for ARID is significantly reduced. This statement might be true if total area of droplets is considered, as they observe a smaller number but a bigger size. But this is not what was analyzed in 1c.\n\nMinor comments\nSeveral figures and figure legends need to be revised:\n- Figure 1A is first introduced in line 100, but there is no description of what kind of data this is. Neither there is a description in the Figure legend. The description is finally found in line 120.\n- Figure 1B: Besides “merge”, there is no description of the use of a second color, I assume it is DAPI staining.\n- It is likely a mistake, but in Extended Figure 1d: “KO” has a band for ARID1A; whereas 1f has no band for “KO”.\n- In Extended Figure 2a, I am surprised that all deletion constructs of ΔIDR1, ΔIDR2, ΔcIDR and ΔGR are the same size. IDR1 should be about 34 kDa smaller than GR, for example. I understand that due to the big size of this protein, and due to charge difference for example, this difference might not be visible. However, if I am not mistaken, the size difference between WT and ΔIDR1 is 38 kDa and this difference is very clearly visible.\n- Extended Figure 2g: The Composition Profiler, it is not clear to me what was used as the reference?\n- Figure 2a, first&4th inlet is not zoomed in", "reviewer_3": "Overall Comments\nIn their submission, ‘Co-condensation between transcription factor and cBAF selectively modulates chromatin remodeling and gene expression,’ authors Ye, Xu, Sun, et al study how ARID1A – a defining structural component of the canonical BAF (cBAF) complex, interacts with specific transcription factors (TFs). The authors discover that ARID1A co-condenses with a wide variety of TFs, including FOXA1, GATA2, SOX2, and p65, thus facilitating the targeting of BAF to specific TF motifs and transcriptional activation. The authors find that co-condensation of cBAF with TFs (and condensation of cBAF alone) is dependent on tyrosine residues in the core intrinsically disordered region (cIDR) of ARID1A. The authors choose to focus specifically on the interactions between cBAF and GATA2 in this study, finding that co-condensation is mediated by interactions between tyrosine residues in the ARID1A cIDR and aromatic residues in the TAD1 domain of GATA2. Finally, the authors explore the physiological consequence of these interactions in cells, finding that co-condensation of GATA2 and ARID1A is necessary for the proliferation of H1299 lung cancer cells, and that co-condensation can be modulated by treatment with the small molecule BD-K98645985.\n\nBroadly speaking, these claims appear well supported by the provided evidence, and a sufficiently revised version of this manuscript should be of interest to the Nature Communications readership. The methods employed in evaluating co-condensation of ARID1A and TFs both in cellulo and in vitro, are generally rigorous and convincing. The use of cell and in vitro assays to show both necessity and sufficiency of specific domains for condensation is well done, though it would be nice to see droplet formation assays repeated either without the crowding reagent PEG, or with a different crowding agent (e.g. BSA or glycerol). Additionally, the division of the ARID1A N-terminal disordered region into 3 different IDRs seems arbitrary, and may confound the relative importance of this region and the ARID1A cIDR (see comments below). Finally, the results of Figures 6 - 7 (effects of co-condensation in cancer and potential merit as therapeutic target) are intriguing, but but would benefit from additional experiments/controls (see major comments below). Though this was not a driving factor for our assessment of this paper’s suitability for Nature Communications, we feel it important to highlight that a major finding of this paper (that ARID1A co-condenses with TFs to alter genome organization / transcriptional regulation) was covered by a previous study (Patil et al, Cell [2023], importantly already cited by this manuscript). A major difference between these studies appears to be the relative importance of the N-terminal IDR (IDR1-3) and the central IDR (cIDR) regions; thus, the authors should explore and comment on these differences in a revised manuscript (see major comments).\n\nMajor Comments:\nIt is unclear why the N-terminal disordered domain of ARID1A was split up into 3 pieces (IDR1, IDR2, and IDR3), rather than being characterized and ultimately deleted as a whole. Assuming this was not arbitrary, the authors should provide clear and explicit logic for why this was done.\n\nA previous study (Patil et al, reference 54 in this manuscript) found that deletion of the entire N-terminal disordered region (IDR1-3) had a major effect on ARID1A condensation and TF co-condensation. The same magnitude of effect was notably not seen in this study, which instead found the primary driver to be the cIDR region of ARID1A. Breaking up the N-terminal disordered region in three domains and testing them individually may have diluted the effect of the N-terminal disordered region, confounding the results of this study and preventing their direct comparison to the previous results. We would suggest the authors delete the entire N-terminal disordered region of ARID1A and/or purify the entire region and compare it to the cIDR domain. This will significantly strengthen the conclusions and potential novelty of the manuscript.\n\nThe authors note a potential interaction between SS18 and SMARCA2 based on how they are sorted between droplets where cIDR or GR ARID1A domains are disrupted. This is interesting, but would benefit from further characterization. Do SS18 / SMARCA2 co-IP with mutant complexes from cells or co-localize with WT / mutant ARID1A? Does SMARCA4 behave differently than SMARCA2 in this sense?\n\nThe effect of BD-98 on H1299 cell proliferation seems to be independent of GATA2 expression, making it unclear what the specific effect of BD-98 on GATA2-mediated proliferation is.\n\nFurther experiments/controls would be useful in showing that the effect of BD-98 is specifically interrupting interactions between GATA2 and cIDR by binding to ARID1A, rather than doing so non-specifically or through another mechanism. For example, does 5µM BD-98 alter condensation of GATA2 or other TFs alone? Does 5µM BD-98 alter the presence of other components in ARID1A condensates in cells? Is BD-98 thought to specifically bind to the cIDR region of ARID1A? Does BD-98 interrupt chromatin accessibility or transcription of ARID1A target genes that are not GATA2-dependent?\n\nWhy was 5µM BD-98 used here? A justification for choosing this concentration should be given based on prior work or shown experimentally.\n\nIt would be beneficial to show that GATA2-ARID1A co-condensation and BD-98 inhibition show similar trends in another lung cancer cell line, if available.\n\nMinor Comments:\nIt would be beneficial to the reader for the authors to highlight their extensive characterization of the GATA2-ARID1A interaction in the abstract, as their rigorous dissection of this interaction is a major source of novelty for this manuscript compared to previously published studies.\n\nIt would be useful to more thoroughly compare this study’s findings with those of previous studies (references 54-56) than the manuscript currently does in the discussion.\n\nLine 72-73: “Emerging studies have illuminated that most nuclear processes occur within biomolecular condensates formed by liquid-liquid phase separation (LLPS) 28, 29.”\nMost implies that it is quantitatively measured that over 50% of nuclear processes consistently occur in LLPS condensates (as opposed to other types of condensates or conditions) - is there explicit evidence to support this? It seems like a very strong statement.\n\nLine 404 – While the contribution of aromatic residues was well supported, the explicit contributions of π-π interactions was not explored in this paper."}, "author_response": "Point-by-point responses to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThe manuscript titled “Co-condensation between transcription factor and cBAF selectively\nmodulates chromatin remodeling and gene expression” provides a compelling and well-executed\nstudy that uncovers a selective co-condensation mechanism between transcription factors (TFs)\nand the cBAF complex through the ARID1A subunit. The authors identify an intrinsically\ndisordered region (cIDR) in ARID1A that facilitates selective phase separation with aromatic-rich\ntranscription factors, notably GATA2. This co-condensation enables cBAF recruitment to lineage-\nspecific regulatory elements, influencing chromatin accessibility and gene expression. The study\nemploys a wide range of state-of-the-art techniques including in vitro droplet assays, live-cell\nimaging, FRAP, CUT&Tag, ATAC-seq, RNA-seq, and CRISPR-based perturbations.\nThis is a conceptually innovative and technically sound manuscript that offers an important\ncontribution to the understanding of chromatin remodeling and transcriptional regulation via\nbiomolecular condensation. However, there are many concerns need to be addressed before the\nmanuscript is suitable for publication in Nature Communications.\nWe thank the reviewer for acknowledging the conceptual innovation and technical robustness of\nour study. We appreciate the constructive comments from the reviewer and the opportunity to\naddress the concerns raised. We have performed additional experiments to strength the key points,\nand have carefully revised the manuscript to improve the clarity. We believe these revisions have\nsignificantly enhanced the impact of our revised manuscript.\nMajor concerns:\n• Limited exploration of other TF function other than GATA2: The paper has only focus on\nGATA2. Although FOXA1 motifs are enriched at ARID1A-bound regulatory regions and FOXA1\nis known to play a central role in lineage-specific transcription, its role in ARID1A-mediated co-\ncondensation is not deeply investigated. Additional experiments or expanded discussion around\nFOXA1’s functional relevance would help validate the generality of the proposed model and\nstrengthen the claim of selective TF interaction. The whole manuscript mainly focused on GATA2\nwithout a good justification about the reason for this choice.\nWe thank the reviewer for raising this important point. We noted that a set of aromatic-rich TFs\ncould co-condensate with ARID1A, considering that the GATA2 transcripitional network has been\nwell documented to be crucial for cell proliferation including the lung cancer cell line that we used\nfor function analysis of ARID1A1, so we picked GATA2 as a representative TF for the initial\nfunctional relevance investigation. We agree that it is important to investigate other TF function\nto support a more general model. Thus, we performed additional experiments regarding the\nfunctional relavance of FOXA1 in ARID1A-mediated co-condensation as suggested by the\nreviewer.\nWe first provided more evidences demonstrating the selective interaction between FOXA1 and\nARID1A. In the previous version of the manuscript, we have shown that FOXA1 TAD selectively\ninteracted with ARID1A cIDR through aromatic residues using the LacO array assay\n(Supplementary Fig. 9b-g ). Here we further confirmed that ARID1A, but not ARID1A-DcIDR or\nARID1AYA, formed condensates with FOXA1 in H1299 cells (Fig. R1a). Using droplet formation\nassay, we also showed that cIDR, but not cIDRYA co-phase-separated with FOXA1 in vitro (Fig.\n,\nR1b). These data further strengthen the selective interaction between ARID1A and FOXA1 both\nin vitro and in cells. We added these data as Supplementary Fig. 8c and 6e in our revised\nmanuscript.\nFig. R1. ARID1A condensation promotes FOXA1-mediated gene transcription and tumor cell\nmigration and invasion. a, H1299ARID1A-KO cells stably expressing Flag-ARID1A or its mutants were\ntransfected with HA-FOXA1, and then subjected to immunostaining with anti-HA and anti-Flag\nantibodies. Line plots showing green and red fluorescence intensities along the dotted line in the\nmagnified image (right). Nuclei were stained with DAPI (blue). Scale bar, 2 µm. b, In vitro droplet co-\ncondensation using mCherry-tagged FOXA1 and GFP-cIDR or GFP-cIDRYA at indicated protein\nconcentration with 150 NaCl in the presence of 10% PEG-8000. Scale bar, 5 µm. c,d H1299ARID1A-KO\ncells stably expressing Flag-ARID1A or its mutants were transfected with Flag-FOXA1 or not, and\nthen subjected to western blot (c), and RT-qPCR analysis (d). e-g, H1299 cells transfected with Flag-\nFOXA1 or not were subjected to western blot (e), MTT (f) and colony formation assays (g). h-k,\nH1299ARID1A-KO cells stably expressing Flag-ARID1A or its mutants were transfected with Flag-\nFOXA1 or not, and then subjected to transwell assays to examine cell migration (h) or invasion (j).\nScale bar, 200 µm. Quantification of the number of migrated (i) or invasive (k) cells per field (right).\nFor experiments in d,g,i,k, data are mean ± s.e.m. of n = 3 biologically independent experiments. For\nexperiments in f, data are mean ± s.e.m. of n = 5 biologically independent experiments. Statistical\nsignificances were determined using unpaired two-tailed Student’s t-tests. ns, not significant. *P < 0.05,\n**P < 0.01, ***P < 0.001.\nNext, we determined if the co-condensation between FOXA1 and ARID1A is critical for FOXA1-\nmediated gene expression. H1299ARID1A-KO cells stably expressing ARID1A, ARID1A-DcIDR, or\nARID1AYA were transfected with FOXA1 or not, and then subjected to qRT-PCR analysis. We\nfound that ARID1A-DcIDR and ARID1AYA significantly inhibited the expression of FOXA1\ntarget genes, including CARCDL and COL93A (Fig. R1c,d). These results indicate that co-\ncondensation with ARID1A is required for FOXA1-stimulated gene transcription. We added these\ndata as Supplementary Fig. 14d,g in our revised manuscript.\nFinally, we explored the role of co-condensation on FOXA1-mediated oncogenic function. MTT\nand colony formation assays showed that unlike GATA2, ectopic expression of FOXA1 had no\nsignificant effect on the proliferation and colony formation in H1299 cells (Fig. R1e-g). However,\ntranswell experiments showed that FOXA1 overexpression substantially promoted tumor cell\nmigration and invasion (Fig. R1c,h-k). These FOXA1-induced phenotypes were significantly\ncompromised by ARID1A-DcIDR and ARID1AYA ( Fig. R1c,h-k). These data demonstrate that\nco-condensation between ARID1A and FOXA1 is critical for the oncogenic function of FOXA1,\nparticularly in tumor cell migration and invasion. We added these data as Supplementary Fig. 14a-\nf in our revised manuscript.\nTogether with these extended investigations on FOXA1, we believe that we have strengthened the\nproposal that co-codensation driven by the selective interaction between cIDR and TAD\nrepresents an important mechansim for specific gene activation. We have included these new data in our\nrevised manuscript.\n• Generalizability of TF selectivity: While several TFs are tested (GATA2, SOX2, p65, FOXA1),\na broader assessment or motif-driven analysis of what defines “co-condensation-competent” TFs\nwould further substantiate the selectivity mechanism and its predictive utility.\nWe thank the reviewer for the constructive suggestion. We think whether there are concesus motifs\nthat mediate the interactions between structured domains and IDRs or IDR-IDR interactions is an\nimportant question for the field. For our study, to perform a borader analysis of TFs that could co-\ncondensate with ARID1A, we first obtained the amino acid sequences of TF TADs that contain at\nleast 30 consecutive amino acids, which are predicted to be disordered as an IDR. In addition, the\nTADs should be experimentally verified through transcriptional reporter assays shown in the\nliterature2. Thus, 307 TADs were obtained. Then we employed the recently developed\ncomputational tool, FINCHES3, which enables direct predictions of IDR-IDR interaction strengths\nbased on the chemical physics of the Mpipi force field. Epsilon (e) is a mean-field parameter that\ndescribes the average interaction between two IDRs, where negative values are attractive and\npositive values are repulsive. We calculated this predicted interaction parameter for each TAD\nusing cIDR as a bait, and ranked the TADs from most attractive to most repulsive (Fig. R2a and\nSupplementary Table 1).\nTo identify if the amino acid composition or nonrandom sequence patterning features of TADs is\nassociated with the interaction strengths, we conducted the NARDINI+ analysis4, that considers a\ncomprehensive set of 90 different IDR sequence features. These include the enrichment of\nindividual amino acids and the patterning of distinct pairs of residue types with respect to one\nanother. Z score (z) > 0 indicates nonrandom clustering of residues or enrichment of indicated\nfeatures, and z < 0 indicates nonrandom mixing of residues or depletion of indicated features. The\nresults revealed that the enrichment scores for aromatic residues (F, W, Y) showed a significant\npositive correlation with the interaction strengths (Fig. R2b). Among these aromatic residues, the\nenrichment score for Y was the most correlated (Fig. R2c-e). Conversely, aliphatic (A, I, L, M, V)\nor negative charge (D, E) amino acids were negatively correlated (Fig. R2f,g). No significant\ncorrelation was observed for positve charged residues (R, K) (Fig. R2h). These analyses suggest\nthat the enrichment of aromatic residues in TAD represents a prominent feature that may dictate\nits interaction with ARID1A cIDR. We added these data as Fig. 6a-c and Supplementary Fig. 10a-\nc in our revised manuscript.\nTo experimentally validate this notion, we performed LacO-array and IF assays using 4 TADs\nfrom the attractive interaction group and and 4 TADs from the repulsive interaction group,\nrespectively. The results showed that the TADs of ZNF326, TFAP2A, HOXB1, or EGR3 in\nattractive group, but not those of MBD2, ZXDC, GMEB2, or HNF1B in repulsive group, enriched\nARID1A cIDR to the foci (Fig. R3a). Either mutation of aromatic residues within TADs of the\nTFs in attractive group or mutation of tyrosines within cIDR remarkably abolished the interactions\nbetween TADs and cIDR (Fig. R3a-c). Consistently, mutation of aromatic residues within TADs\nof ZNF326, TFAP2A, HOXB1, or EGR3 in attractive group inhibited the interaction with full-\nlength ARID1A (Fig. R4a,b). Finally, we checked the co-condensation between full-length TFs\nand ARID1A. Accordingly, ARID1A co-condensated with TFAP2A, ZNF326, EGR3, or HOXB1,\nwhile muation of FWY in the TAD of each transcription factor prevented the co-condensation with\nARID1A in cells (Fig. R4c,d). We added these data as Fig. 6d-h and Supplementary Fig. 11a,b in\nour revised manuscript.\nCollectively, our broader bioinformatic analysis and experimental verification suggest that the\nenrichment of FWY residues in TAD, but maybe not a specific motif, is important for the\nselectivity for ARID1A cIDR interaction. These data have been added in our revised manuscript.\nFig. R2 Enrichment of aromatics in TAD is predicted to be correlated to the cIDR interaction\nstrengths. a, NARDINI+ analysis of sequence features across the TADs. IDR grammar key is shown\nright. Attractive (blue) and repulsive (red) interaction strengths (e) between cIDR and each TAD were\ncalculated using FINCHES. Negative values (blue) are attractive and positive values (red) are repulsive,\nand TAD are ranked by decreasing interaction. Z-scores of enriched (green) and depleted (purple)\nsequence feature of each TAD were determined using NARDINI+. Z score > 0 (green) indicates\nnonrandom clustering of residues or enrichment of indicated features, and z score < 0 (purple) indicates\nnonrandom mixing of residues or depletion of indicated features. b-h, Pearson correlation between\ninteraction strengths (e) and the indicated sequence features (z-score). Pearson correlation coefficients\nand associated P values are shown.\nFig. R3 Enrichment of aromatic residues in TADs is a general feature for selective interaction\nbetween TF and ARID1A. a,b, Representative images of U2OS-LacO cells transfected with the\nindicated GFP-LacI-TFs-TAD or GFP-LacI-TFs-TAD-Aro Mut construct and mScarlet-cIDR (a) or\nmScarlet-cIDRYA (b). Scale bar, 2 µm. c, Boxplots of the partition coefficients for each group in a,b. n\n= 20 cells. Data are mean ± s.e.m. All data in each group were pooled from 3 biologically independent\nreplicates. Statistical significances were determined using one-way ANOVA with Dunnett’s multiple\ncomparisons test. ****P < 0.0001.\nFig. R4 Aromatic interaction promotes the co-condensation between ARID1A and multiple TFs.\na, Representative images of U2OS-LacO cells transfected with Flag-ARID1A and GFP-LacI-TFs-TAD\nor GFP-LacI-TFs-TAD-Aro Mut. Flag-ARID1A was stained using anti-Flag antibody (red). Scale bar,\n2 µm. b, Boxplots of the partition coefficients in each group (a). c, HEK-293TARID1A-KO cells stably\nexpressing Flag-ARID1A were transfected with the indicated HA-TF or HA-TF-TAD-Aro Mut, and\nthen subjected to immunostaining with anti-Flag (green) and anti-HA (red) antibodies. Nuclei were\nstained with DAPI (blue). Scale bar, 2 µm. Line plots showing green and red fluorescence intensities\nalong the dotted line in the magnified image. d, Boxplots of the Manders coefficients in each group (c).\nFor experiments in b,d, n = 20 cells in each group pooled from 3 biologically independent replicates.\nData are mean ± s.e.m. Statistical significances were determined using unpaired two-tailed Student’s t-\ntests. ****P < 0.0001.\n• Lack of connection and in-depth interpretation between the current findings and previously\npublished ARID1A phase separation data. The 2023 Cell paper (Cell 186, 4936-4955, October 26,\n2023) focused on the N-terminal IDR domain (IDR1) and highlighted its key functional role, which\nappears to differ significantly from the findings reported in this manuscript. The authors should\nmore thoroughly discuss the discrepancies and provide a clearer interpretation of how their results\ncompare and contrast with the published data.\nWe appreciate the reviewer’s suggestion to deepen this discussion. Both the current study and the\nCell 2023 paper highlight the crticial role of ARID1A phase separation in chromatin remodeling\nand transpriction activation. However, our findings provide new insights into the mechanisms\nundering ARID1A condensation and its co-condensation with multiple TFs.\nRegarding the mechasim of ARID1A condensate formation, while the Cell study demonstrated\nthat the N-terminal IDR domain is imporant for ARID1A phase seperation and its functional\nimplications, our study identified the same residues within the cIDR (which was not investigated\nin the Cell paper) as also being essential for the ARID1A condensation. These discrepancies\nbetween our results may arise from the differences in how the IDRs are predicted and characterized,\nas well as variations in domain function assessments. However, our results do not contradict the\nrole of N-IDR, our additional data (see response to minior comments) indicate that both cIDR and\nN-IDRare essential for ARID1A condensation and function in cells, suggesting that mutiple IDRs\ncontribute to ARID1A regulation.\nIn terms of TF recruitment, we discovered that the cIDR mediates TF co-condensation through the\naromatic interaction driven by cIDR-TAD, which differs from the Cell study’s focus on N-IDR\nthat relies on an AGQ patch to interact with TFs. Our data highlight that the tyrosine residues in\ncIDR in not only involved in homotypic interaction of cIDR and condensation, but also interact\nwith TFs, thereby driving the selective co-condensation between ARID1A and TFs. Notably,we\ndemonstrate that cIDR, but not IDR1-3 (N-IDR), could be recruited by GATA2 or FOXA1 TAD\nto the foci in LacO array cells. Deletion of cIDR, but not IDR1-3, abolished the enrichment of\nARID1A to GATA2 TAD- or FOXA1 TAD-bound loci (see response to minor comments). These\nfindings reveal that the cIDR has a specific role in co-condensation with TFs such as GATA2 and\nFOXA1. Coupled with our data showing that cIDR can co-condensate with a broad set of TFs that\ncontain TADs enriched with aromatic residues, our findings suggest a generalizable mechanism\nfor TF recruitment mediated by cIDR, which is likely crucial for the regulation of specific gene\ntranscription. Furthermore,we show that the small molecule BD-98 could specifically target cIDR,\nbut not IDR1-3, which inhibits co-condensation and impairs tumor cell proliferation.\nWe believe that our elucidation of 1) the contributions of multiple IDRs in ARID1A condensation\nand function; 2) the co-codensation between cIDR and a general set of TFs; 3) the molecular\nmechanisms underlying the cIDR-mediated interaction significantly extends current understanding.\nTo clarify these points, we have revised the discussion section to inlucde a more comprehensive\ncomparision with the published literature.\nMinor concerns:\n• Clarify whether ARID1A co-condensates are associated with specific nuclear compartments or\nenhancer subtypes (e.g., super-enhancers).\nIn our previous experiments, we found that ARID1A condensates were not localized to some well-\nknown nuclear structures, including promyelocytic leukemia (PML) bodies, Cajal bodies, nuclear\nspeckle, heterochromatin, or nucleoli (Supplementary Fig. 3a). Actually, ARID1A condensates\nwere co-localized with the enhancer marker H3K27ac, but not repressive region marked by\nH3K9me3 (Figure 2f). This suggest that ARID1A co-condensates may also be localized to the\nactive enhancers. To confirm this, we further performed IF experiment to examine the localization\nof ARID1A co-condensates in the nucleus. Using super-resolution structured illumination\nmicroscopy, we observed that the ARID1A and GATA2 co-condensates were indeed localized to\nenhancers marked by H3K27ac, but not repressive region marked by H3K9me3 (Fig. R5a).\nTo clarify the enhancer subtype associated with ARID1A condensation, we delineated typical\nenhancers (TEs) and super-enhancers (SEs) in H1299 cells based on H3K27ac abundance using\nthe ROSE algorithm (Fig. R5b). cIDR deletion reduced ARID1A binding at H3K27ac-defined TEs\nand SEs by 18.1% and 29.6%, respectively (Fig. R5c). Thus, these data suggest that ARID1A\ncondensates are localized to both TEs and SEs. We added this data as Supplementary Fig. 4d,e in\nour revised manuscript.\nFigure R5. ARID1A co-condensates are localized to both typical enhancers and super-\nenhancers. H1299ARID1A-KO cells stably expressing Flag-ARID1A were transfected with HA-\nGATA2 and subjected to immunostaining with the indicated antibodies. Nuclei were stained with\nDAPI (blue). Scale bar, 2 µm. b, Distribution of H3K27ac CUT&Tag density at enhancer regions,\nranked by increasing signal. Enhancers appearing above the inflection point of the curve are\nconsidered super-enhancers (SEs). c, Distribution of ARID1A and ARIDA-ΔcIDR binding\noccurred at TEs and SEs.\n• Provide more quantitative comparisons and replicate details in figure legends to enhance\ntransparency and reproducibility.\nWe have thoroughly revised the figure legends to provide more quantitative comparisons and\nreplicate details in all the figure legends. We believe these revisions could enhance transparency\nand reproducibility.\n• Consider tightening the discussion to better highlight how this mechanism might integrate with\nknown chromatin remodeling paradigms.\nAs suggested by the reviewer, we have revised the discussion section to better illustrate the\nrelevance of our findings within the exsiting framework of chromatin regulation. Specifically, we\nhave emphasized how our findings elucidate the mechanisms by which specific transcription\nfactors recruit the borad remodeling complex cBAF to gene loci to promote chromatin accessibility\nand subsequent gene transcription, which extends the current models of chromatin regulation.\n• The authors should do a deletion for all IDR1-3 to check the effects.\nThanks to the reviewer’s suggestion. Considering the extensive length of IDR1-3 (residues 1-1102),\nwhich constitutes nearly half of the protein, we initially focused on making deletions of IDR1, 2,\n3 and cIDR seperately, as we wanted to identify the minimal regions essential for ARID1A\ncondensation and to generate muntants that primarily disrupt condensation for functional analysis.\nAs suggested by the reviewer, we deleted all IDR1-3 regions and observed that this deletion\ncompletely abolished ARID1A condensate formation, similar to what was seen with cIDR deletion\n(Fig. R6a-c). This data suggest a synergistic role of multiple IDRs in mediating ARID1A phase\nseperation. We added these data as Supplementary Fig. 18a-c in our revised manuscript.\nDue to the disrupted ARID1A condensation, the deletion of all IDR1-3 also significantly impaired\nARID1A chromatin binding (Fig. R6d,e). These effects were comparable to those observed with\ncIDR deletion (Fig. R6f). These data further confirm the importance of condensate formation in\nARID1A’s function. We added these data as Supplementary Fig. 4f,g,i in our revised manuscript.\nWe next assessed their abilities to selectively interact with GATA2 and FOXA1 using LacO array\nassay. Interestingly, we found that cIDR, but not IDR1-3, could be recruited by GATA2 or FOXA1\nTAD to the foci in LacO array cells (Fig. R6g). Consistently, deletion of cIDR, but not IDR1-3,\nabolished the enrichment of ARID1A to GATA2 TAD- or FOXA1 TAD-bound loci (Fig. R6h).\nWe added these data as Supplementary Fig. 18i,j in our revised manuscript.\nThese data indicate that while both IDR1-3 and cIDR can mediate condensate formation, the cIDR\nexhibits a specific role in co-condensation with TFs such as GATA2 and FOXA1. Combined with\nour data showing that cIDR can co-codensate with a broad set of TFs that contain TADs enriched\nwith aromatic residues (Fig. R2, R3, and R4), these results suggest a generalizable mechanism for\nTF recruitment mediated by cIDR, which is likely crucial for the regulation of specific gene\ntranscription.\nFig. R6 A comprehensive comparison of IDR1-3 and cIDR functions. a,b, HEK-293T cells\ntransfected with GFP-ARID1A or the indicated mutants were subjected to western blot with anti-GFP\nantibody (a) and confocal imaging (b). Nuclei were stained with DAPI (blue). Scale bar, 2 µm. c,\nQuantification of the average size of puncta per nucleus as shown in b. n = 20 cells in each group\npooled from 3 biological independent replicates. d, H1299 and H1299ARID1A-KO cells stably expressing\nFlag-ARID1A or its mutants induced by Dox were subjected to western blot with anti-ARID1A\nantibody. e, Profiles (left) and heatmaps (right) of CUT&Tag signal intensities showing the genomic\noccupancy of ARID1A in H1299ARID1A-KO cells stably expressing Flag-ARID1A or Flag-ARIDA-\nΔIDR1-3, respectively. Peaks are centered on transcription start site (TSS) of ARID1A-binding regions,\nranked by increasing occupancy. f, Venn diagram showing the overlap of genes with decreased\nARID1A-binding caused by deletion of cIDR and IDR1-3. g, Representative images of U2OS-LacO\ncells transfected with the indicated GFP-LacI-TFs-TAD constructs and mScarlet-cIDR or mScarlet-\nIDR1-3. Scale bar, 2 µm. Boxplots of the partition coefficients in each group is shown (right). n = 10\ncells in each group pooled from 3 biological independent replicates. h, Representative images of U2OS-\nLacO cells transfected with the indicated GFP-LacI-TFs-TAD constructs and Flag-ARID1A or its\nmutants. Scale bar, 2 µm. Boxplots of the partition coefficients in each group is shown (right). n = 14\ncells in each group pooled from 3 biological independent replicates. For experiments in c,g,h, data are\nshown as mean ± s.e.m. Statistical significances were determined using unpaired two-tailed Student’s\nt-tests (c,g) or one-way ANOVA with Dunnett’s multiple comparisons test (h). ns, not significant.\n****P < 0.0001.\n• The deletion of cIDR domain from ARID1A in Figure 3 and check its binding is sufficient to\nsupport cIDR domain is required for ARID1A chromatin binding. But it does not mean the binding\nrequired condensation behavior. The authors need to use another mutation that can keep the cIDR\ndomain but deficient for condensation to do the ChIPseq binding again.\nTo address this concern, we performed Cut&Tag assays using the ARID1A-ΔIDR1-3 mutant,\nwhich retains the cIDR domain but is deficient in condensation. We induced near-endogenous\nexpression of Flag-ARID1A or Flag-ARID1A-ΔIDR1-3 in H1299ARID1A-KO cells with doxycycline\n(Fig. R6d). Subsequently, we employed Cut&Tag analysis to assess the chromatin binding. The\nresults showed a significant decrease in ARID1A-binding signals in ARID1A-ΔIDR1-3-exprssing\ncells compared to that in ARID1A-expressing cells (Fig. R6e). Among 2748 cIDR-dependent\nARID1A-binding genes, 2019 of them were also downregulated by IDR1-3 deletion (Fig. R6f).\nTogether, these data suggest that ARID1A’s chromatin binding largely depends on its\ncondensation ability. We have included this data as Supplementary Fig. 4f,g,i in the revised\nmanuscript.\n• The authors should use a cell line that knockout the endogenous ARID1A and re-express with\nARID1A-delta cIDR to check if the binding of GATA2 on chromatin is still normal or not.\nAs suggested by the reviewer, we employed H1299ARID1A-KO cells with inducible expressed Flag-\nARID1A or Flag-ARID1A-ΔcIDR, and used doxcycline to restore ARID1A or ARID1A-ΔcIDR\nexpression at the near-endogenous level. Cut&Tag assay was performed to check the binding of\nGATA2 on chromatin. The data displayed that deletion of cIDR did not affect the binding of\nGATA2 on chromatin (Fig. R7), suggesting that the co-condensation with ARID1A is dispensable\nfor the chromatin localization of GATA2, but is required for its-mediated efficient taget gene\nexpression. We have included this data as Supplementary Fig. 13e in the revised manuscript.\nFig. R7 cIDR deletion does not affect the binding of GATA2 on chromatin. Profiles (left) and\nheatmaps (right) of CUT&Tag signal intensities showing the genomic occupancy of GATA2 in\nH1299ARID1A-KO cells stably expressing Flag-ARID1A or Flag-ARIDA-ΔcIDR, respectively. Peaks are\ncentered on transcription start site (TSS) of GATA2-binding regions, ranked by increasing occupancy.\nReviewer #2 (Remarks to the Author):\nThe authors Ye et al. examine the contribution of ARID1A’s cIDR to the formation of condensates\nof the cBAF complex in cells, its targeting on chromatin and co-condensation with specific\ntranscription factors. They identify that especially Tyrosine residues are required for condensate\nformation and cBAF complex recruitment to the condensates, but are not required for overall\ncomplex formation. Lack of cIDR further negatively impacted ARID1A-binding to chromatin and\nchromatin accessibility across ARID1A-binding regions, as well as transcription. Co-condensation\nof transcription factors and cIDR was suggested as the mechanism of ARID1A recruitment to\nspecific chromatin regions and shown by cIDR tethering and co-localization with TFs in cells,\nwhich was based on their aromatic residue content. Sequence features in TADs are the sole\ndriver of TF binding to cIDR, as TFs gained the ability to partition with cIDR when obtaining a TAD\nfrom another TF that partitions with cIDR.\nThey show the significance of ARID1A-TF recruitment and co-condensation by the example of\nGATA2 in a cancer cell line, and show that this interaction is a driver of tumor cell growth and\nproliferation. They further identified the mechanism of action of a small molecule BD-98 which\ndisrupts co-condensate formation of ARID1A and GATA2.\nThe study is, broadly, performed quite thoroughly. For example, several tags have been used in\nidentical experiments, and similar results have been obtained, suggesting that the tags do not\ninfluence the results. Similarly, many in cellulo experiments have been performed in more than\none cell line. However, several figures and legends need revision (s. minor comments).\nDespite the convincing scope of the presented work, some aspects have previously been described\nby Patil et al. (2023) and Xue et al. (2024) which impacts novelty of this report. ARID1A’s\ncontribution to cBAF complex formation of condensates has been described, although a different\nIDR region has been pinned down in this manuscript vs. the previous ones. Similarly, the\ncontribution of Y residues to this behavior is not surprising and has been previously described.\nAdditionally, some IDRs of cBAF have been described to facilitate transcription factor-dependent\ntargeting with similar experiments, highlighting the importance of this process in the development\nof some cancers. However, the authors add additional depth into specific regions of the TFs which\nare responsible for recruitment, and test different chimeras to show that it is the TF IDRs and\naromatic regions which are responsible for the co-condensation and targeting of cBAF. They\nfurther elucidate the mechanism of action of a small molecule cBAF inhibitor that interferes\nselectively with cIDR-TF co-condensates. Both these aspects are unique to this publication and\nadvance our current understanding.\nWe thank the reviewer for the thorough evaluation. We appreciate the recognition of the\nsignificance of our findings and the thoroughness of our experimental approach.\nRegarding the novelty of our study, we acknowledge that some concepts, such as the role of\nARID1A phase seperation in cBAF complex formation and function and the contribution of\ntyrosine residues within IDR1-3 (residues 1-1102) in mediating ARID1A condensation, have been\ndescribed previously. However, our work provides new insights into the mechanisms underlying\nARID1A condensation and its co-condensation with multiple TFs. Specifically, our study\nidentifies the critical role of cIDR, alongside the N-terminal IDR, in mediating ARID1A\ncondensation and function, suggesting a synergistic contribution of multiple IDRs. We also\nidentify that the cIDR, but not N-IDR, specifically mediates the co-condensation with TFs such as\nGATA2 and FOXA1. More importantly, we demonstrate that TADs enriched with aromatic\nresidues are crucial for a broad set of TF’s co-condensation with the cIDR, which reveals the\ngeneralizability of TF-cIDR recruitment. Further, we show that the small molecule BD-98 could\nspecifically target cIDR-TF condensates, thereby impairing tumor cell proliferation (additional\ndata support these notions have been included in the revised manuscript). Overall, we believe that\nour findings extend the current understanding established by the previous studies, providing deeper\nmechanistic insights and potential theraputic implications.\nMajor comments\n- Line 140/ Extended Figure 2f: cIDR can be readily replaced by other LLPS-promoting IDRs in\nthe context of phase separation and cBAF condensate formation. Since the whole sequence can be\nreadily replaced, it makes it more difficult to appreciate the following steps of identifying the\nessential amino acids within cIDR. In this context, it could have been helpful/ more convincing to\ncompare the cIDR composition with the chimera IDR composition and find overlaps.\nThank you for this thoughtful suggestion. To determine whether the phase separation ability of\ncIDR is important for ARID1A condensation, we generated the chimeric proteins in which the\ncIDR was replaced with other well characterized LLPS-promoting IDRs, including those from\nTDP-43 (amino acids 274-414) and hnRNPA1 (amino acids 186-320). Swapping IDRs to create\nchimeric proteins is a widely used approach in the field, commonly used to explore the functional\nimportance of specific IDRs in condensate formation. Our results showed that both chimeras\nreadily form condensates. However, different IDRs may drive phase separation through distinct\nmolecular mechanisms and may rely on different amino acid residues. In our study, we identified\ntyrosine residues within the cIDR are critical for ARID1A condensates formation, it is not clear\nwhether the IDRs substituted in our chimeras utilize similar or different redisues to mediate phase\nseparation.\nIn response to the reviewer’s suggestion, we conducted an amino acid composition analysis using\nNARDINI+4 to compare the cIDR and the chimeric IDRs (Fig. R8a). The results showed that\nresidues such as F, G, N, Y, S are enriched in hnRNPA1 IDR; G, N, M, F, W, Q, S, A are enriched\nin TDP-43 IDR, and M, Y, Q, P, N, F, G are enriched in cIDR. Thus, G, N, and aromatic residues\nare overlapped among these IDRs (Fig. R8b). Previous studies have reported the important role of\naromatic residues within hnRNPA1 and TDP43 IDRs for condensate formation5,6, suggesting that\nsimilar residues may also contribute to condensation in the context of these chimeric proteins. To\nmake our study more focused, we did not include this data in our revised manuscript.\nFigure R8. Compositional features of cIDR, TDP43 IDR, and hnRNPA1 IDR. a, IDR grammar\nkey. b, NARDINI+ analysis of compositional features across the TDP43 IDR, hnRNPA1 IDR, and\ncIDR. Z score (z) > 0 indicates enrichment of indicated features, and z < 0 denotes\ndepletion of indicated features.\nBesides their composition, there is no mentioning of the length of the replacing IDRs, are they\ncomparable in size by residue numbers?\nThe length of TDP-43 IDR (amino acids 274-414) and hnRNPA1 IDR (amino acids 186-320) is\n140 and 134 amino acids, respectively. Although these are shorter than cIDR (524 aa), they are\nstill capable of mediating condensate formation. We have added this information in the revised\nmanuscript (Line 158).\nNow, several LLPS-promoting IDR can replace cIDR condensate formation properties. Besides\ncondensate formation properties, I would further suggest to test whether there is any specificity\nfor cIDR and TF recruitment in comparison to these ARID1A chimera proteins. It would be\ninteresting to see if co-condensation of cIDR and its interaction partners (like GATA2) would take\nplace if cIDR is replaced by any of these LLPS-promoting IDR. This could help to explain how\nmuch specificity is needed for such process.\nWe further examined the co-condensation between ARID1A chimera proteins and the TF GATA2.\nInterestingly, our co-localization analysis revealed reduced partitioning of GATA2 into the\ncondensates formed by the chimeric proteins compared to wild-type ARID1A (Fig. R9a). In vitro\ndroplet formation assays also demonstrated that purified TDP-43 IDR or hnRNPA1 IDR did not\nco-phase-separate with GATA2 (Fig. R9c). Additionally, similar results were also observed with\nanother binding partner FOXA1 (Fig. R9b,d). These data suggest a unique role for cIDR in\nmediating transcription factor (TF) co-condensation. We have included this data as Supplementary\nFig. 7a-d in the revised manuscript.\nWhile replacing cIDR with alternative IDRs can restore condensate formation, our data indicate\nthat the sequence specifity of cIDR is critical for partitioning of partner proteins into condensates.\nThe tyrosine residuces within cIDR are necessary, but may not be sufficient, for TFs co-\ncondensation; residues interspersed between these hydrophobic tyrosines also appear important.\nThese residues may help maintain hydrophobic residues in an exposed conformation, promoting\nspecific interactions with TFs. Taken together, these results indicate that the sequence specificity\nof cIDR, beyond mere phase separation capacity, is critical for the selective co-condensation of\nATID1A with TFs.\nFig. R9 ARID1A chimera proteins are deficient in co-condensation with GATA2 or FOXA1.\na,b,\nH1299ARID1A-KO cell transfected with HA-GATA2 and Flag-ARID1A or its chimera were subjected to\nimmunostaining with anti-Flag (green) and anti-HA (red) antibodies. Nuclei were stained with DAPI\n(blue). Scale bar, 2 µm. Line plots showing green and red fluorescence intensities along the dotted line\nin the magnified image (bottom). Boxplots of the Manders coefficients in each group (right). n = 10\ncells in each group pooled from 3 biologically independent replicates. c,d, In vitro droplet formation\nassay of purified mCherry-GATA2 (0.5 μM), mCherry-FOXA1 (1 μM), and GFP-cIDR or its chimera\n(5 μM) with 150 mM NaCl and 10% PEG-8000. Scale bar, 5 µm. For experiments in a,b, all data in\neach group were pooled from 3 biologically independent replicates. Data are mean ± s.e.m. Statistical\nsignificances were determined using one-way ANOVA with Dunnett’s multiple comparisons test.\n****P < 0.0001.\n- Similarly, based on its mode of action, I wonder how specific the drug BD-98 is. It would be\ninteresting to test specificity in comparison to other co-condensates (either other LLPS-promoting\nIDR chimera or other TF), to underpin the statement that targeting co-condensation can be used as\na sufficiently selective therapeutic strategy to target cancer.\nThanks to the reviewer’s question. BD-98 is a potent inhibitor of ARID1A-contaning BAF\ncomplex and is widely used to inhibit ARID1A function; however, the precise mechanism by\nwhich it exerts its inhibitory effects remains unclear. In response to the reviewer’s concern about\nthe specificity of BD-98 on targeting co-condensation, we performed a series of experiments\ndemonstrating that BD-98 specifically binds to ARID1A cIDR and prevents the incorporation of\nTFs such as GATA2.\nWe first examined the effect of BD-98 on the condensation of GATA2 and other TFs including\nFOXA1, SOX2, and P65. The in vitro droplet formation assay demonstrated that addition of BD-\n98 did not significantly affect the condensation of these TFs alone, indicating that its effect is not\nnon-specific to TF condensation (Fig. R10a-d). We have included this data as Supplementary Fig.\n15h-k in the revised manuscript.\nConsidering ARID1A chimeric proteins with replaced IDRs failed to co-condensate with GATA2,\nwe did not test the effect of BD-98 in that context. We then tested whether BD-98 could inhibit\nARID1A co-condensation with other TFs, including FOXA1, P65, and SOX2. We found that BD-\n98 inhibited the interaction and co-condensation between ARID1A and all these TFs, supporting\nits role in targeting cIDR-mediated co-condensation (Fig. R10e-h). We have included this data as\nSupplementary Fig. 17b-e in the revised manuscript.\nWe further demonstrated that BD-98 specifically binds to the cIDR of ARID1A, but not to IDR1-\n3 or the GR domain using surface plasmon resonance assay, indicating that the action of BD-98\non ARID1A is specifically through direct binding to cIDR (Fig. R10i,j). Taken together, these\nfindings support that BD-98 selectively targets ARID1A cIDR to prevent the incorporation of TFs\ninto condensates, highlighting its potential as a therapeutic agent. We have included this data as\nFig. 8h and Supplementary Fig. 17a in the revised manuscript.\nFig. R10 BD-98 inhibits ARID1A co-condensation with multiple TFs by specifically targeting cIDR.\na-d, Purified mCherry-TFs incubated with 5 μM BD-98 or not was subjected to droplet formation assay\nwith 150 mM NaCl at the indicated protein concentration. Scale bar, 5 μm. e-g, Confocal microscopy\nimages of U2OS-LacO cells transfected with Flag-ARID1A and the indicated GFP-LacI-TFs-TAD\ntogether with or without 5 μM BD-98 treatment. Scale bar, 2 μm. Boxplots of the partition coefficients in\neach group (right). n = 20 cells in each group pooled from 3 biological independent replicates. h,\nConfocal microscopy images of H1299 cells transfected with Flag-ARID1A and the indicated HA-TFs\ntogether with or without 5 μM BD-98 treatment. Line plots showing green and red fluorescence intensities\nalong the dotted line in the magnified image (bottom). Nuclei were stained with DAPI (blue). Scale bar,\n2 µm. Boxplots of the Manders coefficient in each group (right). n = 9 cells in each group pooled from 3\nbiological independent replicates. i, Coomassie blue staining of purified Strep-tagged recombinant\nproteins as indicated. j, Purified proteins were incubated with the indicated concentrations of BD-98 and\nthen subjected to SPR analysis. Fitting curves from the SPR assay demonstrate the dose-dependent\nbinding of BD-98 to cIDR, but not IDR1-3 and GR domain.\n- Line 122/Figure 1c: the authors concluded that removal of ARID did not significantly impair\ncondensate formation, but the number of puncta for ARID is significantly reduced. This statement\nmight be true if total area of droplets is considered, as they observe a smaller number but a bigger\nsize. But this is not what was analyzed in 1c.\nOur initial conclusion was primarily based on the overall ability of ARID to form condensates, as\nsupported by the analysis in Figure 1c, which showed a reduced number of puncta but increased\nsize by the removal of ARID. As the reviewer suggested, we have analyzed the total area of\ncondensates in cells expressing GFP-ARID1A or GFP-ARID1A-DARID. The results indicate that\nthe total area of droplets is not significantly affected by the removal of ARID compared to wildtype\nARID1A (Supplementary Fig. 2b), further supporting the statement. We have included the\nstatistical analysis of the total droplet area and corresponding description in the revised manuscript\nto strengthen our conclusion.\nMinor comments\nSeveral figures and figure legends need to be revised:\n- Figure 1A is first introduced in line 100, but there is no description of what kind of data this is.\nNeither there is a description in the Figure legend. The description is finally found in line 120.\nThank you for pointing this out. Figure 1a is the schematic illustration demonstrating the\nintrinsically disordered regions and domains of ARID1A, with IDRs analyzed by IUPred. We have\nrevised the text to clearly describe Figure 1a (current Line 106) and added a detailed description\nin the corresponding figure legend (Line 1041).\n- Figure 1B: Besides “merge”, there is no description of the use of a second color, I assume it is\nDAPI staining.\nYes, the second color in “merge” panel is DAPI staining. We have added this description to the\nrevised figure legend. Meanwhile, we have checked all figures in our manuscript to ensure this\ninformation is consistently provided.\n- It is likely a mistake, but in Extended Figure 1d: “KO” has a band for ARID1A; whereas 1f has\nno band for “KO”.\nIt should be “WT” in Extended Figure 1d (current Supplementary Fig. 1d). We have corrected it.\n- In Extended Figure 2a, I am surprised that all deletion constructs of ΔIDR1, ΔIDR2, ΔcIDR and\nΔGR are the same size. IDR1 should be about 34 kDa smaller than GR, for example. I understand\nthat due to the big size of this protein, and due to charge difference for example, this difference\nmight not be visible. However, if I am not mistaken, the size difference between WT and ΔIDR1\nis 38 kDa and this difference is very clearly visible.\nThank you for your careful review and pointing this out. In our original experiments, we verified\nthat the protein sizes observed on the blots were consistent with the theoretical molecular weights.\nHowever, during the manuscript preparion process, an incorrect blot image was inadvertently\nincluded, which led to the apparent discrepancy in protein sizes. We apologize for the confusion.\nBased on the calculations using the Compute pI/Mw tool (https://web.expasy.org/compute_pi/)\nand the corresponding amino acid sequences, the theoretical molecular weights are approximately\n269 kDa for WT, 237 kDa for ΔIDR1, 218 kDa for ΔIDR2, 213 kDa for ΔcIDR, and 197 kDa for\nΔGR. In the revised figure (Supplementary Fig. 2a), the band size of ΔIDR1 is clearly smaller than\nWT but larger than ΔGR, and the other constructs display band sizes consistent with these\npredictions, with minor variations likely attributable to the large molecular weight and charge-\nrelated effects on electrophoretic mobility as pointed out by the reviewer.\n- Extended Figure 2g: The Composition Profiler, it is not clear to me what was used as the reference?\nIn the previous Extended Figure 2g (current Supplementary Fig. 2h), the SwissProt 51 dataset was\nused as the reference. We added this information in the revised figure legend for clarity.\n- Figure 2a, first & 4th inset is not zoomed in.\nCorrected. Thanks again.\nReviewer #3 (Remarks to the Author):\nIn their submission, “Co-condensation between transcription factor and cBAF selectively\nmodulates chromatin remodeling and gene expression,” authors Ye, Xu, Sun, et al study how\nARID1A - a defining structural component of the canonical BAF (cBAF) complex, interacts with\nspecific transcription factors (TFs). The authors discover that ARID1A co-condenses with a wide\nvariety of TFs, including FOXA1, GATA2, SOX2, and p65, thus facilitating the targeting of BAF\nto specific TF motifs and transcriptional activation. The authors find that co-condensation of cBAF\nwith TFs (and condensation of cBAF alone) is dependent on tyrosine residues in the core\nintrinsically disordered region (cIDR) of ARID1A. The authors choose to focus specifically on the\ninteractions between cBAF and GATA2 in this study, finding that co-condensation is mediated by\ninteractions between tyrosine residues in the ARID1A cIDR and aromatic residues in the TAD1\ndomain of GATA2. Finally, the authors explore the physiological consequence of these\ninteractions in cells, finding that co-condensation of GATA2 and ARID1A is necessary for the\nproliferation of H1299 lung cancer cells, and that co-condensation can be modulated by treatment\nwith the small molecule BD-K98645985.\nBroadly speaking, these claims appear well supported by the provided evidence, and a sufficiently\nrevised version of this manuscript should be of interest to the Nature Communications readership.\nThe methods employed in evaluating co-condensation of ARID1A and TFs both in cellulo and in\nvitro, are generally rigorous and convincing. The use of cell and in vitro assays to show both\nnecessity and sufficiency of specific domains for condensation is well done, though it would be\nnice to see droplet formation assays repeated either without the crowding reagent PEG, or with a\ndifferent crowding agent (e.g. BSA or glycerol). Additionally, the division of the ARID1A N-\nterminal disordered region into 3 different IDRs seems arbitrary, and may confound the relative\nimportance of this region and the ARID1A cIDR (see comments below). Finally, the results of\nFigures 6 - 7 (effects of co-condensation in cancer and potential merit as therapeutic target) are\nintriguing, but but would benefit from additional experiments/controls (see major comments\nbelow). Though this was not a driving factor for our assessment of this paper’s suitability for\nNature Communications, we feel it important to highlight that a major finding of this paper (that\nARID1A co-condenses with TFs to alter genome organization/transcriptional regulation) was\ncovered by a previous study (Patil et al, Cell [2023], importantly already cited by this manuscript).\nA major difference between these studies appears to be the relative importance of the N-terminal\nIDR (IDR1-3) and the central IDR (cIDR) regions; thus, the authors should explore and comment\non these differences in a revised manuscript (see major comments).\nWe thank the reviewer for the thoughtful and constructive feedback on our manuscript. We have\ncarefully addressed the questions raised by the reviewer, and have updated our manuscript\naccordingly.\nFor the droplet-formation assays without PEG or with a different crowding agent,actually, no\ncrowding agent was used to identify the specific domain responsible for AIDR1A condensate\nformation, but the TFs-cIDR co-condensation assays were initially performed with 10% PEG. As\nthe suggested by the reviewer, we repeated the assays to examine TFs-cIDR co-condensation with\nno crowding agent or alternative crowding agents including 10% dextran, 10% BSA and 10%\nglycerol (Fig. R11). We found that GATA2 and FOXA1, but not SOX2 or P65, co-condensed with\nthe cIDR without any crowding agent, indicating a stronger ability of GATA2 and FOXA1 for\ncIDR co-condensation than SOX2 and P65. In the presence of 10% dextran, all four TFs co-\ncondensed with cIDR, similar to the effect of PEG. Using 10% BSA, we observed that GATA2,\nFOXA1, and SOX2 co-condensed with cIDR, whereas P65 did not. Interestingly, none of the TFs\nco-condensed with cIDR in the presence of 10% glycerol. Together, these results indicate that the\nARID1A cIDR is sufficient to mediate co-condensation with the tested TFs under specific\ncrowding conditions, notably PEG or dextran, and to a lesser extent with BSA. The co-\ncondensation behavior, however, is sensitive to the type of the crowding agent used. We have\nincluded this data as Supplementary Fig. 5a-d in the revised manuscript.\nFig. R11 Co-condensation between ARID1A cIDR and multiple TFs in vitro. a-d, In vitro droplet\nformation assay of purified GFP-cIDR (5 μM) and the indicated transcription factors (5 μM) with 150\nmM NaCl in the absence or presence of various crowding reagents. The crowding reagents include 10%\nPEG-8000, Dextran, BSA, and glycerol. Scale bar, 5 µm.\nRegarding the division of the ARID1A N-terminal disordered region, we have provided a more\nthorough rationale for our division into three distinct IDRs, and also conducted additional\nexperiments to better elucidate the relative contributions of these regions to the functionality of\nARID1A (see response to major comments).\nWe also performed additional experiments to further confirm the effects of co-condensation\nhighlighted in Figure 6 and 7 (also see response to major comments).\nFor comparison with the previous study (Patil et al, Cell [2023]), while both our studies underscore\nthe critical role of ARID1A phase separation in chromatin remodeling and transcription activation,\nour work provides new insights into the mechanisms underling ARID1A condensation and its co-\ncondensation with multiple TFs. Specifically, our study identifies the critical role of cIDR,\nalongside the N-IDR, in mediating ARID1A condensation and function, suggesting a synergistic\ncontribution of multiple IDRs. More importantly, we demonstrate that TADs enriched with\naromatic residues are crucial for a broad set of TF’s co-condensation with the cIDR, which reveals\nthe generalizability of TF-cIDR recruitment. Further, we show that the small molecule BD-98\ncould specifically target cIDR-TF condensates, thereby impairing cell proliferation.We have\nrevised the discussion section to better highlight these differences and to emphasize how our\ndemonstration of co-codensation with multiple TFs, along with elucidating the molecular\nmechanisms of cIDR-mediated recruitment, significantly extend with the current understanding\nestablished by the previous studies.\nWe believe that these revisions significantly enhance the clarity and impact of our manuscript.\nThank you again for your valuable comments to help us improve our work.\nMajor Comments:\nIt is unclear why the N-terminal disordered domain of ARID1A was split up into 3 pieces (IDR1,\nIDR2, and IDR3), rather than being characterized and ultimately deleted as a whole. Assuming\nthis was not arbitrary, the authors should provide clear and explicit logic for why this was done.\nThank you for raising this important point. When we initiated the project, we first identified that\nARID1A formed discrete nuclear condensates in cells. Our subsequent goal was to investigate the\nfunction of ARID1A condensates in chromatin remodeling and gene transcription, as well as to\nunderstand how ARID1A undergoes phase separation. To do this, we aimed to identify the\nminimal region that essential for ARID1A phase separation and generate mutants deficient in this\nability for functional studies.\nConsidering the extensive length of the N-terminal IDR (residues 1-1102), a complete deletion of\nthis region would remove nearly half of the protein, potentially impairing other function domains\nand complicating the interpretation of effects specifically related to IDR-mediated condensation.\nTherefore, we subdivided the N-terminal IDR into three segments based on the scoring plot that\nevaluated disorder interaction strengths. We selected regions flanked by the lowest scores around\nthe cutoff line (Fig 1a), which are likely to represent discrete, functionally relevant modular units.\nBoth IDR1 and IDR2 are of similar size, and are comparable to the cIDR, while IDR3 is smaller.\nOur results show that deletion of cIDR completely abolishes condensate formation, whereas\ndeletion of IDR1, IDR2, or IDR3 does partially, indicating that the cIDR is most important and\nminimal region for ARID1A condensates formation.\nNonetheless, as the reviewer pointed out next, dividing the N-terminal disordered region into three\ndomains and testing them individually could potentially dilute the overall effect of the entire N-\nIDR. Thus, we performed additional experiments to carefully compare the capacity of the full N-\nIDR versus cIDR in driving ARID1A condensation, co-condensation with TFs, and their functions\nin chromatin remodeling and gene transcription (see response to next comment). These additional\ndata help to clarify the contribution of each region and strengthen our conclusions regarding the\nspecific importance of the cIDR in co-condensation with a broad set of TFs containing TADs\nenriched with aromatic residues.\nA previous study (Patil et al, reference 54 in this manuscript) found that deletion of the entire N-\nterminal disordered region (IDR1-3) had a major effect on ARID1A condensation and TF co-\ncondensation. The same magnitude of effect was notably not seen in this study, which instead\nfound the primary driver to be the cIDR region of ARID1A. Breaking up the N-terminal disordered\nregion in three domains and testing them individually may have diluted the effect of the N-terminal\ndisordered region, confounding the results of this study and preventing their direct comparison to\nthe previous results. We would suggest the authors delete the entire N-terminal disordered region\nof ARID1A and/or purify the entire region and compare it to the cIDR domain. This will\nsignificantly strengthen the conclusions and potential novelty of the manuscript.\nThank you for your insightful suggestion.We have performed additional experiments to better\nunderstand the contributions of the entire N-IDR (IDR1-3) compared to the cIDR.\nWe first examined the role of IDR1-3 and cIDR in mediating ARID1A condensation and\nfunction.Our results showed that deletion of IDR1-3 or cIDR abolished ARID1A condensate\nformation in cells (Fig.R12a-c). The OptoDroplet assay showed that IDR1-3-mCherry-CRY2 and\ncIDR-mCherry-CRY2 comparably formed droplets under blue light stimulation (Fig. R12d,e). In\nvitro droplet formation assay using purified proteins also showed that these two IDRs formed\ndroplets with a similar size and number (Fig. R12f-h). These data indicate that the entire N-\nterminal IDR and cIDR have similar abilities in condensate formation. We have included this data\nSupplementary Fig. 18a-h in the revised manuscript.\nFurthermore, due to disrupted ARID1A condensation, the deletion of all IDR1-3 also significantly\nimpaired ARID1A chromatin binding (Fig. R12i,j), which was comparable to those observed with\ncIDR deletion (Fig. R12k). These data reinforce the critical role of condensate formation in\nARID1A’s functional activity. We have included this data as Supplementary Fig. 4f,g,i in the\nrevised manuscript.\nWe next assessed their ability to interact with GATA2 and FOXA1, the two TFs we primarily\nexplored in our study. Interestingly, we found that cIDR, but not IDR1-3, could be recruited by\nGATA2 or FOXA1 TAD to the foci in LacO array cells (Fig. R12l). Furthermore, deletion of cIDR,\nbut not IDR1-3, abolished the enrichment of ARID1A to GATA2 TAD- or FOXA1 TAD-bound\nloci (Fig. R12m). These data indicate that while both N-terminal IDR and cIDR can mediate\ncondensate formation, the cIDR exhibits a specific role in co-condensation with TFs such as\nGATA2 and FOXA1. Combined with our data showing that cIDR can co-condensate with a broad\nset of TFs that contain TADs enriched with aromatic residues (Fig. R2, R3 and R4), these results\nsuggest a generalizable mechanism for TF interaction mediated by cIDR, which is likely crucial\nfor the regulation of specific gene transcription. We have included this data as Supplementary Fig.\n18i,j in the revised manuscript.\nFig. R12 A comprehensive comparison of IDR1-3 and cIDR functions. a,b, HEK-293T cells\ntransfected with GFP-ARID1A or the indicated mutants were subjected to western blot with anti-GFP\nantibody (a) and confocal imaging (b). Nuclei were stained with DAPI (blue). Scale bar, 2 μm. c,\nQuantification of the average size of puncta per nucleus as shown in b. n = 20 cells in each group\npooled from 3 biological independent replicates. d, Live-cell imaging of HEK-293T cells transfected\nwith plasmids expressing cIDR-mCherry-CRY2 or IDR1-3-mCherry-CRY2 before and after light\nactivation. Scale bar, 2 μm. e, Quantification of the number and average size of droplets per nucleus a\nshown in d. n = 10 cells in each group pooled from 3 biological independent replicates. f, Coomassie\nblue staining of purified GFP-tagged recombinant proteins as indicated. g, In vitro droplet formation\nassay of purified GFP-cIDR and GFP-IDR1-3 proteins at various protein concentrations with 150 mM\nNaCl. Scale bar,10 μm. h, Quantification of the size per droplet. n = 100 droplets in each group as\nshown in g, pooled from 3 biological independent replicates. i, H1299 and H1299ARID1A-KO cells stably\nexpressing Flag-ARID1A or its mutants induced by Dox were subjected to western blot with anti-\nARID1A antibody. j, Profiles (left) and heatmaps (right) of CUT&Tag signal intensities showing the\ngenomic occupancy of ARID1A in H1299ARID1A-KO cells stably expressing Flag-ARID1A or Flag-\nARIDA-ΔIDR1-3, respectively. Peaks are centered on transcription start site (TSS) of ARID1A-\nbinding regions, ranked by increasing occupancy. k, Venn diagram showing the overlap of genes with\ndecreased ARID1A-binding caused by deletion of cIDR and IDR1-3. l, Representative images of\nU2OS-LacO cells transfected with the indicated GFP-LacI-TFs-TAD constructs and mScarlet-cIDR or\nmScarlet-IDR1-3. Scale bar, 2 μm. Boxplots of the partition coefficients in each group is shown (right).\nn = 10 cells in each group pooled from 3 biological independent replicates. m, Representative images\nof U2OS-LacO cells transfected with the indicated GFP-LacI-TFs-TAD constructs and Flag-ARID1A\nor its mutants. Scale bar, 2 μm. Boxplots of the partition coefficients in each group is shown (right). n\n= 14 cells in each group pooled from 3 biological independent replicates.For experiments in c,e,h,l,m,\ndata are shown as mean ± s.e.m. Statistical significances were determined using unpaired two-tailed\nStudent’s t-tests (c,e,h,l) or one-way ANOVA with Dunnett’s multiple comparisons test (m). ns, not\nsignificant. ****P < 0.0001.\nThe authors note a potential interaction between SS18 and SMARCA2 based on how they are\nsorted between droplets where cIDR or GR ARID1A domains are disrupted. This is interesting,\nbut would benefit from further characterization. Do SS18/SMARCA2 co-IP with mutant\ncomplexes from cells or co-localize with WT/mutant ARID1A? Does SMARCA4 behave\ndifferently than SMARCA2 in this sense?\nWe appreciate the reviewer’s insightful comments. It has been well-established that the structural\nGR domain is essential for the assembly of cBAF complex. Our co-IP analysis confirmed that\ndeletion of GR domain disrupted the interaction between ARID1A and other cBAF subunits.\nConsistently, while deletion of GR domain did not imapir ARID1A condensate formation, it\ninhibited the recruitment of most cBAF subunits into condensates, except SS18 and SMARCA2.\nThis suggest a potential weak multivalent interaction between SS18/SMARCA2 and other regions\nof ARID1A, which was disrupted during the process of co-IP assay but can be retained by\nimmunuofluorescence.\nTo further explore this potential interaction, as the reviewer suggested, we first performed co-IP\nto examine the interaction between SS18/SMARCA2 and ARID1A or mutant ARID1A complex.\nDeletion of GR domain, but not cIDR domain, indeed abolished the interaction between ARID1A\nand SS18/SMARCA2 (Fig. R13a). However, co-localization analyses showed that both SS18 and\nSMARCA2 could still be recruited to ARID1A-ΔGR condensates (Fig. R13b), suggesting that\nmultivatlent interactions between ARID1A IDRs and SS18/SMARCA4 also contribute to the\ncBAF complex formation. Additionally, similar results were also observed with SMARCA4 (Fig.\nR13c,d), indicating SMARCA4 behaves similarly to SMARCA2 in this sense. These data have\nbeen updated in the revised manuscript. We have included this data as supplementary Fig. 3c,g,h,j\nin the revised manuscript.\nFig. R13 The interaction between ARID1A-ΔGR and SS18/SMARCA2 or SS18/SMARCA4. a,\nCo-IP analysis of interaction between the indicated endogenous cBAF complex subunits and Flag-\nARID1A or Flag-ARID1A-ΔGR in 293TARID1A-KO cells by western blotting. b, Confocal images of\ncolocalization of ARID1A, SMARCA2 and mScarlet-SS18 in H1299ARID1A-KO cells expressing Flag-\nARID1A or Flag-ARID1A-ΔGR induced by Dox. SMARCA2 was stained with anti-SMARCA2\nantibody (Magenta). ARID1A or ARID1A-ΔGR was stained with anti-Flag antibody (green). Line\nplots showing green, red, and magenta fluorescence intensities along the dotted line in the magnified\nimage (bottom). Nuclei were stained with DAPI (blue). Scale bar, 2 µm. c, Co-IP analysis of interaction\nbetween the indicated endogenous cBAF complex subunits and Flag-ARID1A or Flag-ARID1A-ΔGR\nin 293TARID1A-KO cells by western blotting. d, Confocal images of colocalization of ARID1A,\nSMARCA4 and mScarlet-SS18 in HEK-293TARID1A-KO cells expressing Flag-ARID1A or Flag-\nARID1A-ΔGR induced by Dox. SMARCA4 was stained with anti-SMARCA4 antibody (Magenta).\nARID1A or ARID1A-ΔGR was stained with anti-Flag antibody (green). Line plots showing green, red,\nand magenta fluorescence intensities along the dotted line in the magnified image (bottom). Nuclei\nwere stained with DAPI (blue). Scale bar, 2 µm.\nThe effect of BD-98 on H1299 cell proliferation seems to be independent of GATA2 expression,\nmaking it unclear what the specific effect of BD-98 on GATA2-mediated proliferation is.\nThank you for your comment. We acknowledge that the effects of BD-98 on H1299 cell\nproliferation may not be solely dependent of GATA2 expression. As we explained in our\nsubsequent response, we found that BD-98 also downregulated the expression of FOXA1 target\ngenes. This suggests that BD-98 could influence the recruitment of other TFs that important for\nproliferation beyond GATA2. Additionally, the surface plasmon resonance (SPR) assays indicated\nthat BD-98 specifically interacts with ARID1A cIDR (see response to the next comment). Thus,\nwe think that the specificity of BD-98 likely arises from its direct binding to ARID1A cIDR, which\ndisrupts the heterotypic interaction with multiple TFs, incuding GATA2, thereby contributing to\nthe inhibition of cell proliferation.\nFurther experiments/controls would be useful in showing that the effect of BD-98 is specifically\ninterrupting interactions between GATA2 and cIDR by binding to ARID1A, rather than doing so\nnon-specifically or through another mechanism. For example, does 5 µM BD-98 alter\ncondensation of GATA2 or other TFs alone? Does 5 µM BD-98 alter the presence of other\ncomponents in ARID1A condensates in cells? Is BD-98 thought to specifically bind to the cIDR\nregion of ARID1A? Does BD-98 interrupt chromatin accessibility or transcription of ARID1A\ntarget genes that are not GATA2-dependent?\nThank you for your valuable suggestions. To further confirm the specificity of BD-98, we\nconducted a series of experiments as the reviewer suggested.\nWe first examined the effects of BD-98 on the condensation of GATA2 and other TFs including\nFOXA1, SOX2, and P65. The in vitro droplet formation assay demonstrated that the addition of\nBD-98 did not significantly affect the condensation of these TFs alone (Fig. R14a-d), indicating\nthat its effect is not non-specific to TF condensation. We have included this data as Supplementary\nFig. 15h-k in the revised manuscript.\nTo determine whether BD-98 alters the presence of other components in ARID1A condensates in\ncells, we analyzed the enrichment of cBAF components to ARID1A condensates in cells after BD-\n98 treatment. The results showed that BD-98 had no significant effect on the partitioning of the\ncBAF components (Fig. R14e), suggesting BD-98 does not impair the integrity of cBAF complex.\nWe have included this data as Supplementary Fig. 15g in the revised manuscript.\nWe then investigated whether BD-98 specifically binds to the cIDR region of ARID1A by using\nSPR assay. The results showed that BD-98 specifically binds to cIDR, but not to IDR1-3 or the\nGR domain (Fig. R14f,g), indicating that the action of BD-98 on ARID1A is specifically through\nits binding to cIDR. We have included this data as Fig.8h and Supplementary Fig. 17a in the\nrevised manuscript.\nFinally, we examined the effect of BD-98 on the transcription of ARID1A and FOXA1 target gene\nCARCDL and COL93A. qRT-PCR analysis displayed that BD-98 significantly decreased the\nexpression levels of these genes (Fig. R14h,i). This data suggests that BD-98 interrupts the\ntranscription of ARID1A target genes beyond GATA2 regulation. We have included this data as\nSupplementary Fig. 17f in the revised manuscript.\nTogether, these findings suggest that BD-98 specifically interupts interactions between ARID1A\nand TFs by binding to the cIDR, rather than through a nonspecific or alternative mechanism.\nFig. R14 BD-98 specifically targets cIDR to inhibit ARID1A-TFs co-condesation. a-d, Purified\nmCherry-TFs incubated with 5 μM BD-98 or not was subjected to droplet formation assay\nwith 150 mM NaCl at the indicated protein concentration. Scale bar, 5 μm. e, H1299ARID1A-\nKO cells stably expressed with Flag-ARID1A induced by Dox were treated with 5 μM BD-98 or not ,\nand then subjected to immunostaining with antibodies against each cBAF subunits. Boxplots of\nManders coefficients in each group are shown, n = 15 cells. f, Coomassie blue staining of purified\nStrep-tagged recombinant proteins as indicated. g, Purified proteins were incubated with\nthe indicated concentrations of BD-98 and then subjected to SPR analysis. Fitting curves\nfrom the SPR assay demonstrate the dose-dependent binding of BD-98 to cIDR, but not\nIDR1-3 and GR domain. h,i, H1299 cells transfected with Flag-FOXA1 or not were treated with 5\nμM BD-98, and then subjected to western blot (h) and RT-qPCR (i) analysis. n = 3 biological\nindependent replicates. For experiments in e,i, data are mean ± s.e.m. Statistical significances were\ndetermined using unpaired two-tailed Student’s t-tests. ns, not significant. *P < 0.05, **P < 0.01, ***P\n< 0.001.\nWhy was 5µM BD-98 used here? A justification for choosing this concentration should be given\nbased on prior work or shown experimentally.\nIn our study, we sought to use a concentration of BD-98 that would effectively disrupt ARID1A-\nGATA2 co-condensation and inhibit cell proliferation without causing non-specific effects.\nThis concentration choice is in line with previous studies, where BD-98 was used in cellular assyas at\n10 µM in B16F10 melonoma cell7, 5 or 20 uM in neuronal cell8, and 30 µM in mESCs9.\nWe initially tested BD-98 at concentrations of 5 and 10 µM, and found that 5 µM was sufficient\nto markedly inhibit cell prolieferation and disrupt ARID1A-GATA2 co-condensation in H1299\ncells. Therefore, we used 5 µM BD-98 throughout the study. This rationale has been added to the\nrevised text.\nIt would be beneficial to show that GATA2-ARID1A co-condensation and BD-98 inhibition show\nsimilar trends in another lung cancer cell line, if available.\nFollowing the reviewer’s suggestion, we investigated GATA2-ARID1A co-condensation and the\neffects of BD-98 in another lung cancer cell line A549, since GATA2 is also crucial for the\nproliferation of this cell type1. Our data showed that in A549ARID1A-KO cells, the co-condensation\nbetween GATA2 and ARID1A was disrupted by reintroduction of ARID1A mutants including\nARID1A-DcIDR or ARID1AYA (Fig. R15a). Consistent with our previous findings, ARID1A-\nDcIDR and ARID1AYA significantly inhibited GATA2-mediated gene expression, cell\nprolieferation, and colony formation (Fig. R15b-e). Additionally, pharmacological perturbation of\nGATA2-ARID1A co-condensation with BD-98 showed similar trend patterns in A549 cells (Fig.\nR15f-j). These results confirm that the trends observed in H1299 cell are also present in another\nlung cancer cell, suggesting that the effect of BD-98 is conserved across different lung cancer cell\ntypes. We have included this data as Supplementary Fig. 9i, 13a-d, 16a-e in the revised manuscript.\nFig. R15 ARID1A and GATA2 co-condensation promotes target gene transcription and the\nproliferation in A549 cells. a, A549ARID1A-KO cells stably expressing Flag-ARID1A were transfected with\nHA-GATA2 or HA-GATA2-TAD1-Aro Mut, and subject to immunostaining with anti-Flag (green) and\nanti-HA (red) antibodies. Nuclei were stained with DAPI (blue). Scale bar, 2 µm. Boxplots of the Manders\ncoefficients in each group (right). b-e, A549ARID1A-KO cells stably expressing Flag-ARID1A or its mutants\nwere infected with lentivirus expressing Flag-GATA2 or not, and then subjected to western blot (b),\nMTT (c), colony formation (d), and RT-qPCR analysis (e). f, Confocal microscopy images of A549 cells\ntransfected with Flag-ARID1A and HA-GATA2 together with or without 5 μM BD-98 treatment. Line\nplots showing green and red fluorescence intensities along the dotted line in the magnified image (middle).\nNuclei were stained with DAPI (blue). Scale bar, 2 µm. Boxplots of Manders coefficient in each group\n(right). g, A549 cells infected with lentivirus expressing Flag-GATA2 or not were treated with 5 μM BD-\n98, and then subjected to western blot with anti-ARID1A and anti-Flag antibodies. h-j, MTT (h), colony\nformation assay (i) and RT-qPCR analysis of indicated mRNA level (j) in H1299 cells treated with 5 μM\nBD-98 or not. For experiments in a,f, data are mean ± s.e.m. n =15 cells in each group pooled from 3\nbiologically independent replicates. For experiments in c,h, data are mean ± s.e.m. of n = 5 biologically\nindependent experiments. For experiments in d,e,i,j, data are mean ± s.e.m. of n = 3 biologically\nindependent experiments. Statistical significances were determined using unpaired two-tailed Student’s\nt-tests. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001.\nMinor Comments:\nIt would be beneficial to the reader for the authors to highlight their extensive characterization of\nthe GATA2-ARID1A interaction in the abstract, as their rigorous dissection of this interaction is\na major source of novelty for this manuscript compared to previously published studies.\nThanks for the advice. We have revised the abstract to highlight the extensive characterization of\nthe GATA2-ARID1A interaction.\nIt would be useful to more thoroughly compare this study’s findings with those of previous studies\n(references 54-56) than the manuscript currently does in the discussion.\nThank you for your suggestion. In the revised manuscript, we have expanded our discussion to\nmore thoroughly compare our findings with the previous studies. Specifically, we emphasize the\nroles of multiple IDRs in mediating ARID1A condensation and funciton, and we clarify how our\ndemonstration of co-condensation with multiple TFs and the molecular mechanism of cIDR-\nmediated recruitment extend the current understanding established by these studies.\nLine 72-73: “Emerging studies have illuminated that most nuclear processes occur within\nbiomolecular condensates formed by liquid-liquid phase separation (LLPS) 28, 29”. Most implies\nthat it is quantitatively measured that over 50% of nuclear processes consistently occur in LLPS\ncondensates (as opposed to other types of condensates or conditions) - is there explicit evidence to\nsupport this? It seems like a very strong statement.\nThank you for pointing this out. Our intention was to highlight the emerging importance and\nwidespread recognition of biomolecular condensates formed by LLPS in nuclear processes. We\nagree that the original statement using “most” was overly strong. We have changed “most” into\n“many”, which emphasizes the growing evidence without implying a definitive quantitative\npredominance (Line 73).\nLine 404-While the contribution of aromatic residues was well supported, the explicit\ncontributions of π-π interactions was not explored in this paper.\nThank you again. We removed the description of “π-π interactions” in the revised sentence (current\nLine 494).\nReferences\n1. Kumar MS, et al. The GATA2 transcriptional network is requisite for RAS oncogene-driven non-small cell\nlung cancer. Cell 149, 642–655 (2012).\n2. Soto LF, et al. Compendium of human transcription factor effector domains. Mol Cell 82, 514–526 (2022).\n3. Ginell GM, et al. Sequence-based prediction of intermolecular interactions driven by disordered regions.\nScience 388, eadq8381 (2025).\n4. Cohan MC, Shinn MK, Lalmansingh JM, Pappu RV. Uncovering Non-random Binary Patterns Within\nSequences of Intrinsically Disordered Proteins. J Mol Biol 434, 167373 (2022).\n5. Martin EW, et al. Valence and patterning of aromatic residues determine the phase behavior of prion-like\ndomains. Science 367, 694–699 (2020).\n6. Schmidt HB, Barreau A, Rohatgi R. Phase separation-deficient TDP43 remains functional in splicing. Nat\nCommun 10, 4890 (2019).\n7. Maxwell MB, et al. ARID1A suppresses R-loop-mediated STING-type I interferon pathway activation of\nanti-tumor immunity. Cell 187, 3390–3408.e3319 (2024).\n8. Cornejo KG, et al. Activity-assembled nBAF complex mediates rapid immediate early gene transcription by\nregulating RNA polymerase II productive elongation. Cell Rep 43, 114877 (2024).\n9. Marian CA, et al. Small Molecule Targeting of Specific BAF (mSWI/SNF) Complexes for HIV Latency\nReversal. Cell Chem Biol 25, 1443–1455.e1414 (2018)."}, {"round": 2, "reviewer_comments": {"reviewer_1": "The authors have addressed all the concerns that I have raised during my first review and this manuscript has been greatly approved.", "reviewer_2": "The revisions have further enhanced the scope and significance of the manuscript by Ye et al. The authors successfully clarified how their findings differ from previous publications. Their conclusions are now well supported by the additional evidence provided.\n\nIn this revised version, specificity of cIDR co-localization with transcription factors (TFs) as well as the consequences on cell proliferation have been studied in greater depth, with additional representative examples. The authors also provide new insights into the mode of action of the small molecule BD-98 by demonstrating a direct binding interaction between BD-98 and cIDR through SPR analysis. Moreover, the additional data convincingly establish that BD-98 inhibits co-condensation of multiple TFs, extending beyond their previous example of Gata2.\n\nAll my concerns were addressed by the authors. I therefore recommend the manuscript for publication.", "reviewer_3": "We are satisfied with the authors' revisions and applaud their willingness to engage with all of our comments."}, "author_response": "Point-by-point responses to the reviewers’ comments\nReviewer #1 (Remarks to the Author):\nThe authors have addressed all the concerns that I have raised during my first review and this manuscript\nhas been greatly approved.\nWe sincerely thank the reviewer for their positive assessment and constructive suggestions throughout the\nreview process, which has significantly strengthened our manuscript.\nReviewer #2 (Remarks to the Author):\nThe revisions have further enhanced the scope and significance of the manuscript by Ye et al. The authors\nsuccessfully clarified how their findings differ from previous publications. Their conclusions are now well\nsupported by the additional evidence provided.\nIn this revised version, specificity of cIDR co-localization with transcription factors (TFs) as well as the\nconsequences on cell proliferation have been studied in greater depth, with additional representative\nexamples. The authors also provide new insights into the mode of action of the small molecule BD-98 by\ndemonstrating a direct binding interaction between BD-98 and cIDR through SPR analysis. Moreover, the\nadditional data convincingly establish that BD-98 inhibits co-condensation of multiple TFs, extending\nbeyond their previous example of Gata2.\nAll my concerns were addressed by the authors. I therefore recommend the manuscript for publication.\nWe are delighted that the reviewer finds our revised manuscript significantly enhanced. We sincerely thank\nthe reviewer for the valuable comments and insightful suggestions to greatly improve our manuscript.\nReviewer #3 (Remarks to the Author):\nWe are satisfied with the authors' revisions and applaud their willingness to engage with all of our comments.\nWe sincerely thank the reviewer for their valuable suggestions and encouraging remarks. We are pleased\nthat they are satisfied with our comprehensive revisions."}]

Decision letter