BAF60A KO ATAC seq (primary cells)-Highly discriminative globin gene activation by the non-canonical BAF chromatin remodeling complex
The regulation of beta-like globin genes, including the switch from fetal (HBG) to adult (HBB and HBD) genes has served as a paradigm of developmental transcriptional control. While HBG repressors such as BCL11A and LRF have been well characterized, recent discoveries of HIF1a and BGLT3 as HBG-specific activators have raised interest in finding new activators as potential therapeutic targets. The ATP-dependent chromosome remodeler BRG1 has previously been demonstrated as necessary for hemoglobin gene transcription. In loss of function studies in adult-like HUDEP2 cells, we found that BRG1 depletion preferentially decreased fetal HBG and the minor adult HBD. BRG1 forms multisubunit complexes with combinations of 27 subunits in 3 families - canonical BAF, polybromo BAF, and non-canonical BAF (ncBAF) - to regulate chromatin accessibility and gene transcription in a cell and condition-specific manner. Using CRISPR loss of function studies in HUDEP2 cells and primary adult erythroid cells, we identified the non-canonical BAF complex as a positive regulator of HBG and HBD. Loss of the non-canonical BAF complex subunits BRD9 and BAF60A/SMARCD1 preferentially decreased HBG and HBD in HUDEP2 cells and primary adult erythroid cultures, while accelerating erythroid differentiation and hemoglobinization. Pharmacological depletion of BRD9 led to acute decreases in chromatin accessibility at HBD and HBG promoters and reduced their primary transcripts, while longer treatment recapitulated  genetic ablation effects. This surprising demonstration of BAF complex selectivity within a multi-gene cluster extends our understanding of globin gene transcriptional regulation and identifies a potential target for therapeutic manipulation of beta-like globins. Overall design: We performed ATAC seq analysis in 4 HUDEP-2 clonal lines generated using single cell cloning of a pool of HUDEP-2 cells. ATAC-seq was conducted following the Omni-ATAC protocol (https://www.nature.com/articles/nmeth.4396). In brief, 100,000 cells underwent lysis using a buffer containing 0.1% NP-40, 0.1% Tween-20, and 0.01% digitonin, followed by incubation with Tagment DNA Enzyme (Illumina cat # 20034197) at 37°C for 30 minutes. DNA was subsequently purified using the QIAGEN MinElute Reaction Cleanup Kit. For library preparation, fragmented DNA was amplified with Phusion High-Fidelity PCR Master Mix supplemented with HF Buffer (Thermo Fisher Scientific, catalog no. F531S) and indexed primers featuring unique dual barcodes. Libraries were purified with AMPure XP beads (Beckman Coulter, catalog no. A63881) at a 1:1 ratio, adhering to the manufacturer's guidelines.