Structural and functional properties of mSWI/SNF chromatin remodeling complexes revealed through single-cell perturbation and genomic profiling

Mammalian SWI/SNF (mSWI/SNF or BAF) chromatin remodeling complexes play critical roles in regulating DNA accessibility and gene expression and are comprised of three final-form assemblies, cBAF, PBAF, and ncBAF, which exhibit distinct modular organization, chromatin targeting, and roles in disease. However, the contributions of each subcomplex and their constituent subunits to gene expression remain incompletely defined. Here, we performed a large-scale CRISPR-Cas9 knockout screen targeting mSWI/SNF subunits individually and in selected combinations, followed by single-cell RNA-sequencing (Perturb-seq) and SHARE-Seq. We uncover complex- and module-specific contributions across cell regulatory pathways, define paralog subunit relationships, and identify shifted functions upon perturbations. Finally, we superimpose single-cell perturbation signatures over bulk primary human tumor gene expression profiles, defining unique subunit-specific signatures and identifying tumor features that mirror BAF loss-of-function. Taken together, these data highlight the utility of large-scale single-cell genomics to uncover gene expression and chromatin accessibility signatures in human disease states. Overall design: 1. Perturb-seq experiments: To map the roles of different subunits of the mSWI/SNF complex, we performed Perturb-seq on MOLM13 cells, following either individual perturbation of subunits (“single” experiment) or combinatorial perturbation of selected subunit combinations (“combo” experiment). Cells were profiled with the 10X Chromium Single Cell 3' RNA-seq v3 kit, with 6000 cells loaded per channel. The “single” and “combo” experiments were profiled with 15 and 3 channels, respectively, and were sequenced paired-end on an Illumina Hi-Seq instrument at a ratio of 3 channels of 10x to 1 lane of sequencing (5 for the “single” experiment, and 1 for the “combo” experiment), using paired end reads as follows. For the “single” experiment, read 1 is 28 bases long (16 for cell barcode, 12 for UMI), read 2 is 91 bases long and sample index read is 8 bases long. For the “combo” experiment, read 1 is 28 bases long (16 for cell barcode, 12 for UMI), read 2 is 91 bases long and sample index read is 8 bases long. Perturbations were assigned to cells using dial-out PCR. Dial-out PCR products were sequenced on an Illumina Mi-seq, paired-end (for all experiments, read 1 is 28 bases and read 2 is 60 bases long), with read 1 having the same structure as in the high-throughput sequencing of the libraries described above. Each experiment required only one Mi-Seq run to process the dial-out libraries. 2. SHARE-seq experiment: To map the changes in accessibility and gene regulation controlled by mSWI/SNF complexes, we performed SHARE-seq on MOLM13 cells following CRISPR-mediated knockout of specific mSWI/SNF subunits, obtaining both RNA-seq and ATAC-seq readouts in the same single cell. RNA and ATAC libraries were sequenced using a 150 cycle high output Illumina kit on a NextSeq500 (Illumina) instrument. We performed SHARE-seq for 5 conditions: control guide, ARID1A, SMARCD2, SMARCA4 and BRD9, each with 2 replicate wells in an arrayed format. All cells from the SHARE-seq experiment were sequenced together in the same library, and can be demultiplexed by their well index (see Methods).