A genome-wide CRISPR screen reveals a role for the non-canonical nucleosome remodeling BAF complex in Foxp3 expression and regulatory T cell function

Regulatory T cells (Treg) play a pivotal role in suppressing auto-reactive T cells and maintaining immune homeostasis. Treg development and function are dependent on the transcription factor Foxp3. Here we performed a genome-wide CRISPR loss-of-function screen to identify Foxp3 regulators in mouse primary Treg cells. Foxp3 regulators were enriched in genes encoding subunits of the SWI/SNF nucleosome remodeling and SAGA chromatin modifying complexes. Among the three SWI/SNF-related complexes, the Brd9-containing non-canonical (nc)BAF complex promoted Foxp3 expression, whereas the PBAF complex was repressive. Chemical-induced degradation of Brd9 led to reduced Foxp3 expression and reduced Treg function in vitro. Brd9 ablation compromised Treg function in inflammatory disease and tumor immunity in vivo. Furthermore, Brd9 promoted Foxp3 binding and expression of a subset of Foxp3 target genes. Our findings provide an unbiased analysis of the genetic networks regulating Foxp3 and reveal ncBAF as a target for therapeutic manipulation of Treg function. ChIP-seq for Foxp3, BRG1, BRD9 and PHF10 in natural T regulatory cells (nTregs) from Foxp3Thy1.1/Rosa-Cas9 mice to assess their genomic localization; replicates are indicated. ChIP-seq for Foxp3 and H3K27ac in nTregs either transduced with sgNT, sgFoxp3, sgBrd9, sgPbrm or treated with DMSO vehicle or dBRD9 to determine the role of Brd9 and Pbrm1 in regulating Foxp3 binding and H3K27ac levels. ChIP-seq for Foxp3 in nTregs exogenously expressing either MIGR empty vector or Foxp3 construct, with transduction of either sgNT or sgBrd9. ATAC-seq in nTregs transduced with sgNT (non-targeting) or sgBrd9 to assess changes in chromatin accessibility after loss of Brd9. Two replicates per sample. RNA-seq in nTregs treated with DMSO vehicle or dBRD9. RNA-seq in nTregs transduced with sgNT, sgFoxp3 or guides targeting different SWI/SNF subunits. Two to three replicates per sample.

调节性T细胞（Regulatory T cells, Treg）在抑制自身反应性T细胞、维持免疫稳态中发挥关键作用。Treg的发育与功能依赖于转录因子Foxp3。本研究通过全基因组CRISPR功能缺失筛选，在小鼠原代Treg细胞中鉴定Foxp3的调控因子。Foxp3调控因子富集于编码SWI/SNF核小体重塑复合物与SAGA染色质修饰复合物亚基的基因。在三类SWI/SNF相关复合物中，含Brd9的非经典（non-canonical, nc）BAF复合物可促进Foxp3表达，而PBAF复合物则发挥抑制作用。化学诱导的Brd9降解可在体外降低Foxp3表达并削弱Treg功能。体内Brd9敲除会损害炎症疾病与肿瘤免疫中的Treg功能。此外，Brd9可促进Foxp3结合并调控部分Foxp3靶基因的表达。
本研究结果为调控Foxp3的遗传网络提供了无偏倚分析，并揭示ncBAF可作为靶向调控Treg功能的治疗靶点。
对来自Foxp3Thy1.1/Rosa-Cas9小鼠的天然调节性T细胞（natural T regulatory cells, nTregs）开展染色质免疫沉淀测序（Chromatin Immunoprecipitation Sequencing, ChIP-seq），以评估这些蛋白的基因组定位；实验设置生物学重复并予以标注。
对分别转导非靶向向导RNA（sgNT）、sgFoxp3、sgBrd9、sgPbrm，或经二甲基亚砜（Dimethyl Sulfoxide, DMSO）溶剂对照、dBRD9处理的nTregs开展Foxp3与H3K27ac的ChIP-seq，以明确Brd9与Pbrm1在调控Foxp3结合及H3K27ac水平中的作用。
对分别转导sgNT或sgBrd9、且外源表达MIGR空载体或Foxp3重组质粒的nTregs开展Foxp3的ChIP-seq。
对转导sgNT（非靶向向导RNA）或sgBrd9的nTregs开展转座酶可及性测序（Assay for Transposase-Accessible Chromatin using sequencing, ATAC-seq），以评估Brd9缺失后染色质开放状态的变化。每个样本设置2个生物学重复。
对经DMSO溶剂对照或dBRD9处理的nTregs开展RNA测序（RNA Sequencing, RNA-seq）。对转导sgNT、sgFoxp3或靶向不同SWI/SNF复合物亚基的向导RNA的nTregs开展RNA-seq。每个样本设置2~3个生物学重复。