A disordered region controls cBAF activity via condensation and partner recruitment

Abstract

Intrinsically disordered regions (IDRs) represent a large percentage of overall nuclear protein content. The prevailing dogma is that IDRs engage in non-specific interactions because they are poorly constrained by evolutionary selection. Here, we demonstrate that condensate formation and heterotypic interactions are distinct and separable features of an IDR within the ARID1A/B subunits of the mSWI/SNF chromatin remodeler, cBAF, and establish distinct "sequence grammars" underlying each contribution. Condensation is driven by uniformly distributed tyrosine residues, and partner interactions are mediated by non-random blocks rich in alanine, glycine, and glutamine residues. These features concentrate a specific cBAF protein-protein interaction network and are essential for chromatin localization and activity. Importantly, human disease-associated perturbations in ARID1B IDR sequence grammars disrupt cBAF function in cells. Together, these data identify IDR contributions to chromatin remodeling and explain how phase separation provides a mechanism through which both genomic localization and functional partner recruitment are achieved.

Keywords: ARID1A; ARID1B; ATP-dependent chromatin remodeling; IDRs; cBAF complexes; condensates; intrinsically disordered regions; mammalian SWI/SNF complexes; phase separation; transcription factors.

Figure 1.. The IDRs of ARID1A/B are dispensable for cBAF assembly and in vitro nucleosome remodeling.

A. Human cBAF complex (PDBDEV_00000056) with putative ARID1A N-terminal region of unassigned cryo-EM density and C-terminal CBR highlighted. B. Disease-associated mutations mapped onto ARID1A/B and disorder as PONDR score. C. Distribution of disease-associated missense and indel mutations in ARID1A/B’s N-terminus. D. Schematic of HA-tagged ARID1A expression constructs. E. Immunoblots of nuclear protein input and anti-HA IPs in AN3CA (ARID1A/B-deficient) cells expressing HA-tagged ARID1A WT or mutant variants. F. TMT mass spectrometric signal for cBAF components from anti-HA ARID1A WT or mutant immunoprecipitation. G. Top, restriction enzyme accessibility assay (REAA) time course using 2.5 nM purified cBAF carrying ARID1A WT or mutant variants; Bottom, REAA using 0–5 nM cBAF (t=30min) (n=2 experimental replicates each). H. ATPase (ADP-Glo) measurements for indicated conditions and timepoints. ns, not significant by one-way ANOVA test.

Figure 2.. ARID1A IDRs dictate cBAF complex condensation in vitro and in cells, which is enhanced by DNA binding.

A. Left, in vitro condensation experiments of indicated 0.66 μM eGFP-tagged cBAF complexes; Right, condensate area per field of view. B. Percent condensate-covered area with 100 nM DNA, nucleosomes, or RNA. C. Confocal imaging of eGFP-tagged cBAF complexes containing ARID1A WT or mutant variants in live AN3CA cells. D. Saturation concentration, condensate count and area ARID1A puncta in AN3CA cells. PS: Phase Separation. E. Immunoblot for ARID1A and other cBAF subunits in AN3CA cells −/+ doxycycline alongside human and murine cell types. F. Immunofluorescence of AN3CA cells without or with doxycycline induction of exogenous eGFP-tagged ARID1A. PCC between eGFP-ARID1A and anti-ARID1A immunostaining. Bottom: Immunostain for endogenous ARID1A in KLE (human endometrial), C2C12 myoblast (mouse), MCF10A (human breast cancer), and primary rat neurons. G. Top, schematic of Corelet system used to evaluate self-interaction propensity of IDRs; Bottom, schematic of IDR-containing constructs evaluated. H. Representative images of U2OS cell nuclei without (-light) and with (+light) light-induced oligomerization. I. Top, phase diagram schematic; Bottom, phase diagrams of ARID1A constructs; shaded area indicates two-phase region. In A and B, P-values calculated by one-way ANOVA test. In D, by unpaired student’s t-test.

Figure 3.. ARID1A IDRs and DNA-binding functions govern cBAF occupancy, DNA accessibility and gene expression in cells.

A. Chromatin occupancy of cBAF complexes marked by HA (ARID1A), SMARCA4, and SMARCC1, H3K27ac enhancer mark occupancy and DNA accessibility (ATAC) at cBAF-occupied sites in AN3CA cells, divided into 4 clusters using k-means clustering. B. Distance-to-TSS distribution of merged CUT&Tag and ATAC-Seq peaks for all conditions, across Clusters 1–4 from (A). C. Principal Component Analysis (PCA) of cBAF-occupied enhancer sites across conditions as assayed by SMARCA4 and SMARCC1 signals. D. Representative CUT&Tag and ATAC-Seq tracks at the MAP2, NCAPH, and intergenic enhancer loci in AN3CA cells across Empty and ARID1A WT or mutant conditions. E. Overlap of accessible sites by ATAC-Seq in empty vector control (Empty) versus ARID1A WT or mutant conditions in AN3CA cells. Gained sites relative to empty condition are highlighted in bold. F. Transcription factor motif enrichment analysis (HOMER) at Clusters 2, 3, and 4 from (A). G. Box and whisker plot for all conditions comparing expression levels of top differentially expressed genes (DEGs) upon ARID1A WT introduction versus empty control.

Figure 4.. ARID1A IDRs mediate local proximity of cBAF complex with cellular transcriptional machinery, enabling ARID domain-dependent TF binding.

A. Immunoblot for input and anti-HA IP from AN3CA cells expressing HA-ARID1A fused to biotin ligase TurboID (TbID). B. Distribution of biotinylated proteins fold changes. C. Volcano plots comparing biotinylated protein levels. D. Immunofluorescence analysis of ARID1A and p300 in AN3CA cells. E. Volcano plots comparing detected protein levels following IP-Mass Spec. F. Overlap of ARID1A WT-carrying cBAF interactomes measured using proximity labeling or IP-Mass Spec. G. Protein class enrichment of detected cBAF interacting proteins via IP-MS (DNA interactors in red). H. Input and selected transcription factor (cJUN, NFIA, TEAD1) reciprocal IPs using AN3CA cells expressing empty vector or WT- and mutant-ARID1A.

Figure 5.. Sequence-specific heterotypic interactions of ARID1A IDR1 are required for cBAF-mediated chromatin and gene regulation.

A. Schematic of ARID1A FUS^IDR and DDX4^IDR fusion mutant variants. B. Representative images of eGFP-tagged constructs in live AN3CA cells. C. Count and average area of condensates. Statistical test: one way ANOVA. D. FRAP curves, Immobile fraction, and half time of recovery (T_1/2) quantification for indicated constructs. Error bars: standard deviation. n = 3 biological trials, 15 cells each. Statistical test, one way ANOVA. E. Chromatin occupancy of cBAF complexes marked by HA (ARID1A), SMARCA4 and SMARCC1, H3K27ac enhancer mark occupancy and DNA accessibility (ATAC-Seq) at Cluster 2 and 3 sites from Fig. 3A. F. Fold change of differentially expressed genes (DEGs) relative to empty vector. G. Volcano plots comparing detected protein levels by IP-MS. Hits meeting the cut off of log2 fold change <−1 and >1 and p-value <0.25 are blue and red, respectively. H. Immunofluorescence analysis of ARID1A and p300. I. Top, metaplots of SMARCA4 occupancy over cBAF sites (shared SMARCA4/SMARCC1 sites) ΔIDR1 (left) or CBR-only (right) cBAF complex target sites; Bottom, metaplots of ATAC-Seq accessibility. J. Example tracks of SMARCA4 occupancy and DNA accessibility in the ARID1A CBR-only, FUS^IDR, and DDX4^IDR mutant conditions at the BRD2 and CD320 genomic loci.

Figure 6.. Sequence patterning analysis enabled separation of condensation and heterotypic interaction functions in ARID1A IDR1.

A. Clustering analysis of non-random amino acid sequence features performed across all IDRs within mSWI/SNF proteins. Z-scores for enriched/’blocky’ or depleted/’well-mixed’ sequence features are shown as a green-to-purple color scale. Red arrow: ARID1A/B IDRs. IDR sequence feature key in panel B. B. Left, enrichment of amino acid sequence features across Clusters 1–4 of mSWI/SNF IDR patterns; Right, IDR sequence feature key. C. Schematic for 42YS and AQG scramble ARID1A IDR1 rationally designed mutant variants. D. NARDINI plots of ARID1A IDR1 WT, AQG scramble and 42YS mutant IDRs. Amino acid key on left. E. Immunoblot for input and anti-HA IP from AN3CA cells. F. Live cell imaging of eGFP-tagged cBAF complexes containing WT ARID1A and the 42YS or AQG scramble IDR1 variants. G. Condensation metrics for ARID1A WT and mutants (3 biological trials of n=25 cells each); error bars represent SEM. **p=0.002 by unpaired t-test. H. Clustered heatmap of chromatin occupancy of cBAF complexes marked by HA (ARID1A), SMARCA4 and H3K27ac enhancer mark occupancy and DNA accessibility (ATAC-Seq) across empty, WT ARID1A and the 42YS or AQG scramble IDR1 ARID1A mutants. I. Overlap between Cluster B lost sites from H and Clusters 2,3 lost sites from Figure 3A. J. Top DEGs in WT and 42YS and AQGscram conditions relative to empty control. K. TbID proximity labeling results for the AQG scramble and 42YS mutants compared to ARID1A WT. Hits meeting the cut off log2 fold change < −1 and >1 and p-value <0.2 are labeled in blue. L. Immunofluorescence of p300 and eGFP-tagged cBAF complexes containing WT ARID1A or AQG scramble. M. Nuclear protein input and anti-TF IP-immunoblot studies.

Figure 7.. Mutations in ARID1B IDR1 sequence pattern disrupt condensation and genomic targeting of cBAF.

A. Mutational frequencies in ARID1A/B IDRs associated with neurodevelopmental disorders (NDD) from DECIPHER. B. NDD-associated mutations (DECIPHER) plotted across the 26 sequence blocks within IDR1 of ARID1B. C. Schematic of ARID1B WT, block deletion, and NDD mutants. D. Immunoblot for nuclear input and anti-HA IP experiments in AN3CA cells expressing HA-tagged ARID1B WT or mutants. E. Representative images of eGFP-tagged ARID1B in AN3CA cells. F. Condensation metrics of ARID1B in AN3CA cells. G. SMARCA4 genomic localization over severely lost sites in Block 9 deletion (left) and Block 13 deletion (right). H. PCA of ATAC-Seq peaks across ARID1B WT and mutant conditions. I. Example tracks of cBAF localization and ATAC accessibility over NCAPH and IL1B loci in ARID1B WT and mutant conditions. J. Transcription factor motif enrichment analysis (HOMER) of Cluster Y sites (Figure S7). K. Change in NFI TF family TMT-MS signal in the S320_G327del mutant condition relative to WT ARID1B. L. Differential gene expression changes for top upregulated genes in WT versus Block 9 and 13 deletions and mutant conditions. M. Relative gene expression changes of top differential genes across WT and mutant conditions. N. Model highlighting the role of the ARID1A N-terminus.