A genome-wide CRISPR screen reveals a role for the BRD9 ... · 2 15 Summary 16 Regulatory T cells (Tregs) play a pivotal role in suppressing auto-reactive T cells 17 and maintaining

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A genome-wide CRISPR screen reveals a role for the BRD9-containing non-1

canonical BAF complex in regulatory T cells 2

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Chin-San Loo1,3,#, Jovylyn Gatchalian2,#, Yuqiong Liang1, Mathias Leblanc1, Mingjun 4

Xie1, Josephine Ho2, Bhargav Venkatraghavan1, Diana C. Hargreaves2*, and Ye 5

Zheng1* 6

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1. NOMIS Center for Immunobiology and Microbial Pathogenesis, Salk Institute for 8

Biological Studies 9

2. Molecular and Cellular Biology Laboratory, Salk Institute for Biological Studies 10

3. Division of Biological Sciences, University of California, San Diego 11

# Co-first authors 12

* Co-corresponding authors 13

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(which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission. The copyright holder for this preprintthis version posted February 27, 2020. . https://doi.org/10.1101/2020.02.26.964981doi: bioRxiv preprint

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Summary 15

Regulatory T cells (Tregs) play a pivotal role in suppressing auto-reactive T cells 16

and maintaining immune homeostasis. Treg development and function are 17

dependent on the transcription factor Foxp3. Here we performed a genome-wide 18

CRISPR/Cas9 knockout screen to identify the regulators of Foxp3 in mouse 19

primary Tregs. The results showed that Foxp3 regulators are highly enriched in 20

genes encoding SWI/SNF and SAGA complex subunits. Among the three 21

SWI/SNF-related complexes, the non-canonical or ncBAF (also called GBAF or 22

BRD9-containing BAF) complex promoted the expression of Foxp3, whereas the 23

PBAF complex repressed its expression. Gene ablation of BRD9 led to 24

compromised Treg function in inflammatory disease and tumor immunity. 25

Functional genomics revealed that BRD9 is required for Foxp3 binding and 26

expression of a subset of Foxp3 target genes. Thus, we provide an unbiased 27

analysis of genes and networks regulating Foxp3, and reveal ncBAF complex as a 28

novel target that could be exploited to manipulate Treg function. 29

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Introduction 31

Regulatory T cells (Tregs) play a crucial role in maintaining immune system 32

homeostasis by suppressing over-reactive immune responses(Josefowicz et al., 2012; 33

Sakaguchi et al., 2008). Defects in Tregs lead to autoimmune disorders and 34

immunopathology, while certain tumors are enriched with Tregs that suppress anti-35

tumor immune responses(Tanaka and Sakaguchi, 2017). Foxp3, a member of the 36

Forkhead transcription factor family, is a critical regulator that orchestrates the 37


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molecular processes involved in Treg differentiation and function(Zheng and Rudensky, 38

2007). Therefore, understanding the regulation of Foxp3 expression could reveal novel 39

therapeutic targets to potentially change Treg numbers or alter their function. It has 40

been established that the T cell receptor (TCR) and IL-2 signaling pathways play critical 41

roles in Foxp3 induction(Chinen et al., 2016; Lee et al., 2012). TGF-β signaling is also 42

essential for Foxp3 induction in periphery-derived Tregs and in vitro induced Tregs, 43

although its role in thymus-derived Treg development is still under debate (Chen et al., 44

2003; Liu et al., 2008; Ouyang et al., 2010). Accordingly, a number of downstream 45

transcription factors have been identified that regulate Foxp3 induction in vitro or in vivo, 46

including STAT5a/b, CBF-β/RUNX1/3, NFAT1, SMAD3/4, cRel, and CREB (Burchill et 47

al., 2007; Kim and Leonard, 2007; Kitoh et al., 2009; Long et al., 2009; Rudra et al., 48

2009; Tone et al., 2008; Yang et al., 2008). Compared to the large number of studies 49

focused on the mechanism of Foxp3 induction, relatively less is known about the factors 50

that maintain Foxp3 expression in mature Treg cells. An intronic enhancer in Foxp3 51

named CNS2 (conserved non-coding sequence 2), also known as TSDR (Treg-specific 52

demethylated region), is a key cis-regulatory element required for stable Foxp3 53

expression(Polansky et al., 2008; Zheng et al., 2010). CNS2 is heavily methylated in 54

naive and activated conventional T cells by DNA methyl-transferase 1 (DNMT1), and 55

deletion of Dnmt1 leads to aberrant expression of Foxp3 in conventional T 56

cells(Josefowicz et al., 2009). Once Foxp3 expression is induced during Treg 57

development, the CNS2 region is rapidly demethylated, opening it up for the binding of 58

transcription factors(Polansky et al., 2008). Foxp3 can bind to CNS2, as well as an 59


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additional upstream enhancer named CNS0(Kitagawa et al., 2017), and stabilize its own 60

expression in a positive feedback loop(Feng et al., 2014; Li et al., 2014b). 61

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Post-translational modifications (PTM) of the Foxp3 protein, including phosphorylation, 63

acetylation, and ubiquitination, are also a crucial part of the regulatory circuit that 64

controls Foxp3 function and stability (van Loosdregt and Coffer, 2014). Among the 65

regulators of Foxp3 PTMs, a pair of enzymes, ubiquitin ligase STUB1 and ubiquitin 66

hydrolase USP7, were reported to promote or inhibit degradation of Foxp3 via 67

ubiquitination, respectively (Chen et al., 2013; van Loosdregt et al., 2013). Finally, 68

intracellular metabolism, and specifically the metabolic regulator mTOR (mammalian 69

target of Rapamycin), has emerged as a key regulator of Foxp3 expression and Treg 70

function. Early studies showed that weakened mTOR signaling leads to increased 71

Foxp3 expression in iTregs in vitro (Delgoffe et al., 2009). However, recent studies 72

using genetic models showed that complete ablation of mTOR in Tregs leads to 73

compromised homeostasis and function of effector Tregs (Chapman et al., 2018; Sun et 74

al., 2018). Despite these and other significant advances in understanding the molecular 75

mechanisms regulating Foxp3, we lack a comprehensive picture of the regulatory 76

networks that control Foxp3 expression. 77

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In this study, we performed a genome-wide CRISPR/Cas9 knockout screen to identify 79

the regulators of Foxp3 in mouse primary natural Treg cells. The screen results not only 80

confirmed a number of known Foxp3 regulators but also revealed many novel factors 81

that control Foxp3 expression. Gene ontology analysis showed that Foxp3 regulators 82


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are highly enriched in genes encoding subunits of the SAGA and SWI/SNF complexes, 83

which we further validated by single gRNA knockout and flow cytometry analysis. The 84

mammalian SWI/SNF complex is a multi-subunit complex with a core ATPase protein, 85

either SMARCA4 (BRG1) or SMARCA2 (BRM), that uses energy derived from ATP 86

hydrolysis to remodel nucleosomes on chromatin. Mouse genetic studies have 87

demonstrated that conditional knockout of Smarca4 leads to impaired differentiation of T 88

lymphocytes (Gebuhr et al., 2003; Zhao et al., 1998). In addition, a previous report 89

demonstrated that genetic deletion of Smarca4 in Tregs using the Foxp3-Cre driver 90

results in the development of a fatal inflammatory disorder reminiscent of Foxp3 mutant 91

scurfy mice (Chaiyachati et al., 2013). The authors showed that while Treg development 92

and Foxp3 expression was normal in Smarca4 deficient Tregs, Treg function was 93

nevertheless compromised due to impaired activation of TCR target genes, for example 94

chemokine receptor genes in Tregs. This is consistent with the rapid association of 95

SMARCA4-containing SWI/SNF complexes with chromatin following TCR activation in T 96

cells (Zhao et al., 1998). 97

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Biochemical studies have demonstrated that SMARCA4 is associated with both the 99

canonical BAF complex (BAF) and Polybromo1-associated BAF complex (PBAF) (Xue 100

et al., 2000; Yan et al., 2005). In addition, recent studies in embryonic stem cells 101

(ESCs)(Gatchalian et al., 2018) and cancer cell lines (Alpsoy and Dykhuizen, 2018; 102

Michel et al., 2018; Wang et al., 2019) have identified a BRD9-containing non-canonical 103

complex or ncBAF complex (also referred to as GBAF or BRD9-containing BAF), which 104

contains several shared subunits including SMARCA4, but is distinct from the BAF and 105


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PBAF complexes. Apart from uniquely incorporating BRD9, the ncBAF complex also 106

contains GLTSCR1 or the paralog GLTSCR1L and lacks BAF- and PBAF-specific 107

subunits ARID1A, ARID1B, ARID2, SMARCE1, SMARCB1, SMARCD2, SMARCD3, 108

DPF1-3, PBRM1, BRD7, and PHF10. The distinct biochemical compositions of these 109

three SWI/SNF complex assemblies suggest functional diversity. However, it is not 110

known which SWI/SNF complex assemblies are expressed in Tregs and the potential 111

roles of specific SWI/SNF variants in regulating Foxp3 expression and Treg 112

development have not been studied in depth. 113

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Here, we find that the BRD9-containing ncBAF complex promotes the expression of 115

Foxp3, whereas the PBAF complex represses its expression. Furthermore, deletion of 116

Brd9 or PBAF component Pbrm1 in Tregs results in reduced or enhanced suppressor 117

activity, respectively, suggesting divergent regulatory roles of ncBAF and PBAF 118

complexes in controlling Foxp3 expression and Treg function. Consistent with this 119

model, we find that chemically-induced degradation of BRD9 by dBRD9 leads to 120

reduced Foxp3 expression and compromised Treg function. Genome-wide binding 121

studies revealed that BRD9 co-localizes with Foxp3, including at the CNS0 and CNS2 122

enhancers at the Foxp3 locus. Furthermore, targeting BRD9 by sgRNA or dBRD9 123

reduces Foxp3 binding at the Foxp3 locus and a subset of Foxp3 binding sites genome-124

wide, which results in differential expression of many Foxp3-dependent genes, 125

indicating that BRD9 participates in the regulation of the Foxp3-dependent 126

transcriptional program. Finally, we show that deletion of Brd9 in Tregs reduced 127

suppressor activity in an in vivo model of T cell transfer induced colitis, and improved 128


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anti-tumor immune responses in an MC38 colorectal cancer cell induced cancer model. 129

In summary, we perform an unbiased genome-wide screen to identify genes and 130

networks regulating Foxp3, and reveal ncBAF complex as a novel target that could be 131

exploited to manipulate Treg function in vitro and in vivo. 132

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Results 134

Genome-wide CRISPR screen in natural regulatory T cells identifies regulators of 135

Foxp3 136

To screen for genes that regulate Foxp3 expression, we developed a pooled retroviral 137

CRISPR sgRNA library by subcloning an optimized mouse genome-wide lentiviral 138

CRISPR sgRNA library (lentiCRISPRv2-Brie) (Doench et al., 2016) into a newly 139

engineered retroviral vector pSIRG-NGFR, which allowed us to efficiently transduce 140

mouse primary T cells and to perform intracellular staining for Foxp3 without losing the 141

transduction marker NGFR after cell permeabilization (Figure S1). Using this library, we 142

performed CRISPR knockout screens on Tregs to identify genes that regulate Foxp3 143

expression. We activated CD4+Foxp3+ Tregs isolated from Rosa-Cas9/Foxp3Thy1.1 144

knock-in mice (Liston et al., 2008; Platt et al., 2014) with CD3 and CD28 antibodies and 145

IL-2 (Figure 1A). Treg cells were transduced 24 hours post-activation with the pooled 146

retroviral sgRNA library at multiplicity of infection of less than 0.2 to ensure only one 147

sgRNA was transduced per cell. NGFR+ transduced Treg cells were collected on day 3 148

and day 6 to identify genes that are essential for cell proliferation and survival. In 149

addition, the bottom quintile (NGFR+Foxp3Low) and top quintile (NGFR+Foxp3High) 150

populations were collected on day 6 to identify genes that regulate Foxp3 expression. 151


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We validated the screen conditions by transducing Tregs with sgRNAs targeting Foxp3 152

itself, as well as previously reported positive (Cbfb) (Rudra et al., 2009) and negative 153

(Dnmt1) (Lal et al., 2009) regulators of Foxp3 (Figure 1B-D). Guide RNA sequences 154

integrated within the genomic DNA of sorted cells were recovered by PCR amplification, 155

constructed into amplicon libraries, and sequenced with a NextSeq sequencer. 156

157

The relative enrichment of sgRNAs between samples and hit identification were 158

computed by MAGeCK, which generates a normalized sgRNA read count table for each 159

sample, calculates the fold change of sgRNA read counts between two cell populations, 160

and further aggregates information of four sgRNAs targeting each gene to generate a 161

ranked gene list (Li et al., 2014a). Prior to hit calling, we evaluated the quality of screen 162

samples by measuring the percentage of mapped reads to the sgRNA library and total 163

read coverage, which showed a high mapping rate (79.8-83.4%) with an average of 164

236X coverage and a low number of missing sgRNAs (0.625-2.5%) (Figure S2). With 165

the cutoff criteria of log2 fold change (LFC) >±0.5 and p-value less than 0.01, we 166

identified 254 potential positive Foxp3 regulators enriched in the Foxp3Low population 167

and 490 potential negative Foxp3 regulators enriched in the Foxp3High population 168

(Figure 2A, 2B, and Table S1). In a parallel analysis, we also identified 22 and 1497 169

genes that affect cell expansion and contraction, respectively (p-value < 0.002, LFC>1, 170

Figure S3 and Table S2). As expected, we identified genes belonging to pathways 171

known to regulate Foxp3 expression both transcriptionally (Cbfb, Runx3) (Rudra et al., 172

2009) and post-transcriptionally through the regulation of Foxp3 protein stability (Usp7, 173

Stub1) (Chen et al., 2013; van Loosdregt et al., 2013) (Figure 2C). 174


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We next compared the potential positive and negative regulators with genes involved in 176

cell contraction and expansion to exclude hits that might affect Foxp3 expression 177

indirectly by affecting cellular fitness in general, leaving 197 positive Foxp3 regulators 178

and 327 negative Foxp3 regulators (Figure 2D and Table S3). Gene ontology analysis 179

of positive Foxp3 regulators revealed a number of notable functional clusters including 180

SAGA-type complex, negative regulation of T cell activation, RNA Polymerase II 181

holoenzyme, positive regulation of histone modification, and SWI/SNF complex (Figure 182

2E, Table S4). Among negative Foxp3 regulators, genes are highly enriched in clusters 183

related to negative regulation of TOR signaling, transcriptional repressor complex, 184

mRNA decay and metabolism, and hypusine synthesis from eIF5A-lysine (Figure 2F, 185

Table S4). Several of these pathways, including mTOR signaling, Foxp3 186

ubiquitination/deubiquitination, and transcriptional regulation, have been implicated in 187

Foxp3 regulation previously, suggesting that our screen is robust for the validation of 188

known pathways and the discovery of novel regulators of Foxp3. Among novel 189

regulators, we identified many genes encoding subunits of the SAGA (Ccdc101, Tada2b, 190

Tada3, Usp22, Tada1, Taf6l, Supt5, Supt20) and SWI/SNF (Arid1a, Brd9, Smarcd1) 191

complexes (Table S4), strongly suggesting that these complexes could have 192

indispensable roles for Foxp3 expression. We thus further validated and characterized 193

the SAGA and SWI/SNF related complexes to understand their roles in Foxp3 194

expression and Treg function. 195

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Validation of the SAGA complex as a novel regulator of Foxp3 expression and 197

Treg suppressor activity 198

The SAGA complex possesses histone acetyltransferase (HAT) and histone 199

deubiquitinase (DUB) activity, and functions as a transcriptional co-activator through 200

interactions with transcription factors and the general transcriptional 201

machinery(Helmlinger and Tora, 2017; Koutelou et al., 2010). We identified Ccdc101, 202

Tada2b, and Tada3 in the HAT module, Usp22 in the DUB module, and Tada1, Taf6l, 203

Supt5, and Supt20 from the core structural module among positive Foxp3 regulators 204

that do not affect cell expansion or contraction (Figure S4A). We sought to validate the 205

potential regulatory function of SAGA complex subunits by using sgRNAs to knock out 206

individual subunits in Tregs and measure Foxp3 expression (Figure S4B, S4C). We 207

found that deletion of every subunit tested resulted in a significant and 19-29% 208

reduction in Foxp3 mean fluorescence intensity (MFI). We then further tested the 209

function of SAGA subunit Usp22 in an in vitro suppression assay, which measures the 210

suppression of T cell proliferation when conventional T cells are co-cultured with Tregs 211

at increasing ratios. We found that Tregs transduced with sgRNAs targeting Usp22 had 212

compromised Treg suppressor activity compared with Tregs transduced with a non-213

targeting control sgRNA, with significantly more proliferation of T effector cells (Teff) at 214

every ratio of Treg to Teff ratio tested (Figure S4D). These results provide independent 215

validation of our genome-wide screen analyses for this class of chromatin regulators 216

and demonstrate that the SAGA complex is essential for normal Foxp3 expression and 217

that disrupting the SAGA complex by sgUsp22 reduces Treg suppressor function. 218

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Identification of the BRD9-containing ncBAF complex as a specific regulator of 220

Foxp3 expression 221

We next wanted to characterize the role of SWI/SNF complex variants (BAF, ncBAF, 222

and PBAF complexes) in Foxp3 expression. While these complexes share certain core 223

subunits, such as the ATPase SMARCA4, each complex also contains specific subunits, 224

for example the selective incorporation of the bromodomain containing protein BRD9 in 225

ncBAF complexes (Figure 3A). Since the tissue-specific distribution and functional 226

requirement for ncBAF complexes in primary T cells is not known, we performed co-227

immunoprecipitation assays to probe the composition of SWI/SNF-related complexes in 228

Tregs. As expected, immunoprecipitation of SMARCA4, a core component of all three 229

SWI/SNF complexes, revealed association of common subunits SMARCC1 and 230

SMARCB1, as well as specific subunits ARID1A, BRD9, and PBRM1. 231

Immunoprecipitations against ARID1A, BRD9, and PHF10 revealed the specific 232

association of these subunits with BAF, ncBAF, and PBAF complexes, respectively 233

(Figure 3A). These results established that all three SWI/SNF complexes are present 234

with the expected composition in Tregs. 235

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In our screen, we identified Brd9, Smarcd1, Arid1a and Actl6a among positive 237

regulators of Foxp3, whereas SWI/SNF shared subunits Smarca4, Smarcb1, Smarce1, 238

and Actl6a were identified in cell contraction (Table S3). This suggests a potential 239

regulatory role for ncBAF and/or BAF complexes. To explore the specific function of 240

BAF, ncBAF, and PBAF complexes in Foxp3 expression, we cloned independent 241

sgRNAs to knockout unique subunits for each complex, and measured Foxp3 MFI in 242


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sgRNA transduced Tregs. We observed an essential role for the ncBAF complex in 243

Foxp3 expression in Tregs. Specifically, knockdown of ncBAF specific subunits, 244

including Brd9 and Smarcd1, significantly diminished Foxp3 expression by nearly 40% 245

in Tregs (Figure 3B, 3C). Knockdown of ncBAF-specific paralogs Gltscr1 and Gltscr1l 246

individually resulted in a slight reduction in Foxp3 expression, which was further 247

reduced in the Gltscr1/Gltscr1l double knockout, suggesting that these two paralogs can 248

compensate in the regulation of Foxp3 expression (Figure 3C). In contrast, knockdown 249

of PBAF specific subunits, including Pbrm1, Arid2, Brd7, and Phf10, significantly 250

enhanced Foxp3 expression by as much as 17% (Figure 3C, green). Knockdown of 251

BAF specific subunits Arid1a, Arid1b, Dpf1, or Dpf2 did not significantly affect Foxp3 252

expression (Figure 3C, blue). To determine if ARID1A and ARID1B could be 253

compensating for one another, we performed Arid1a/Arid1b double deletion and found 254

that deletion of either or both ARID paralogs resulted in slight, but non-significant 255

reduction in Foxp3 MFI (Figure 3C, blue). These data suggest that ncBAF and PBAF 256

have opposing roles in the regulation of Foxp3 expression. To further explore the role of 257

different SWI/SNF complexes in Treg genome-wide transcription, we performed RNA 258

sequencing from Tregs with knockdown of variant-specific subunits with one or two 259

independent guide RNAs and conducted principal component analysis, which showed 260

that the ncBAF, PBAF, and BAF also have distinct effects at whole transcriptome level 261

in Tregs (Figure 3D). 262

263

We then made use of a recently developed chemical BRD9 protein degrader 264

(dBRD9)(Remillard et al., 2017) as an orthogonal method to probe BRD9 function. 265


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dBRD9 is a bifunctional molecule that links a small molecule that specifically binds to 266

the bromodomain of BRD9 and another ligand that recruits the cereblon E3 ubiquitin 267

ligase. We confirmed that treatment of Tregs with dBRD9 resulted in reduced BRD9 268

protein levels (Figure S5A). Similar to sgRNA depletion of Brd9, dBRD9 treatment 269

significantly decreased Foxp3 expression in Treg cells in a concentration-dependent 270

manner, without affecting cell viability or proliferation (Figure 3E, Figure S5B). These 271

data demonstrate the requirement for BRD9 in maintenance of Foxp3 expression using 272

both genetic and chemically-induced proteolysis methods. 273

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BRD9 regulates Foxp3 binding at the CNS0 and CNS2 enhancers and a subset of 275

Foxp3 target sites 276

To dissect the molecular mechanism of how ncBAF and PBAF complexes regulate 277

Foxp3 expression in Treg cells, we performed chromatin immunoprecipitation followed 278

by genome-wide sequencing (ChIP-seq) in Tregs using antibodies against the ncBAF-279

specific subunit BRD9, the PBAF-specific subunit PHF10 and the shared enzymatic 280

subunit SMARCA4. Data generated from these ChIP-seq experiments revealed that 281

BRD9, SMARCA4, and PHF10 co-localize at CNS2 in the Foxp3 gene locus and at 282

CNS0 found within the Ppp1r3f gene immediately upstream of Foxp3 (Figure 4A). Since 283

CNS2 was previously shown to regulate stable Foxp3 expression through a positive 284

feedback loop involving Foxp3 binding(Feng et al., 2014; Li et al., 2014b), and Foxp3 is 285

additionally bound at CNS0 in Tregs(Kitagawa et al., 2017), we rationalized that ncBAF 286

and/or PBAF complexes might affect Foxp3 expression by regulating Foxp3 binding at 287

CNS2/CNS0. We therefore performed Foxp3 ChIP-seq in Tregs transduced with sgNT, 288


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sgFoxp3, sgBrd9 or sgPbrm1. We observed a dramatic reduction in Foxp3 binding at 289

CNS2/CNS0 in sgFoxp3 transduced cells, as expected, and there was also marked 290

reduction of Foxp3 binding at CNS2/CNS0 in Brd9-depleted Tregs (Figure 4A). In 291

contrast, we observed a subtle increase in Foxp3 binding at CNS2/CNS0 in Pbrm1 292

sgRNA transduced Tregs, which could explain why Pbrm1 emerged as a negative 293

regulator of Foxp3 expression in our validation studies (Figure 4A). These data suggest 294

that BRD9 positively regulates Foxp3 expression by promoting Foxp3 binding to its own 295

enhancers. 296

297

We then extended this analysis to examine the cooperation between BRD9 and Foxp3 298

genome-wide. Notably, we find co-binding of BRD9, SMARCA4, and PHF10 with Foxp3 299

at a subset of Foxp3-bound sites (Figure 4B, 4C). All four factors localize to promoters, 300

intronic, and intergenic regions of the genome and their binding correlates well with 301

chromatin accessibility as measured by assay of transposase-accessible chromatin with 302

sequencing (ATAC-seq) (Figure 4B, S6A). Motif analysis of Foxp3-bound sites revealed 303

an enrichment for motifs recognized by ETS and RUNX transcription factors consistent 304

with what has been previously shown(Samstein et al., 2012). ETS and RUNX motifs 305

were also among the most significant motifs at both BRD9-bound sites, along with an 306

enrichment of the CTCF motif as we and others previously reported(Gatchalian et al., 307

2018; Michel et al., 2018) (Figure S6B). These results demonstrate that ncBAF and 308

PBAF complexes are co-localized with Foxp3 at Foxp3 binding sites genome-wide. 309

310


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To assess the requirement for BRD9 or PBRM1 in Foxp3 targeting genome-wide, we 311

analyzed Foxp3 binding in Tregs transduced with sgNT, sgFoxp3, sgBrd9, or sgPbrm1 312

at all Foxp3 binding sites (Figure 4D). As expected, we find that Foxp3 binding is lost at 313

over 85% of its binding sites in sgFoxp3-transduced Treg cells (Figure 4E). Foxp3 314

binding at a subset of these sites is also significantly reduced in sgBrd9-transduced 315

Tregs (FC 1.5, Poisson p < 0.0001), suggesting that BRD9 is required for Foxp3 binding 316

at a subset of its target sites (Figure 4E). This is a specific function of BRD9, as Foxp3 317

binding does not change in Pbrm1-depleted Tregs at these BRD9-dependent sites 318

(Figure 4F). ChIP-seq for the active histone mark H3 lysine27 acetylation (H3K27ac) 319

revealed that BRD9 and Foxp3 cooperate to maintain H3K27ac at over 1,800 shared 320

sites (Figure 4G). At BRD9-dependent Foxp3 sites, for example, we observed a 321

reduction in H3K27ac in sgFoxp3 and sgBrd9-transduced Tregs, but not in sgPbrm1-322

transduced Tregs (Figure 4H). Using dBRD9, we confirmed that BRD9 binding to 323

chromatin is reduced (Figure S6C). We further recapitulated our observation that BRD9 324

loss results in diminished Foxp3 binding to chromatin at a subset of Foxp3 target sites 325

(Figure 4I, 4J, S6D), including at CNS2 and CNS0 (Figure 4A). These data demonstrate 326

that BRD9 co-binds with Foxp3 at the Foxp3 locus to positively reinforce its expression. 327

BRD9 additionally promotes Foxp3 binding and H3K27ac levels at a subset of Foxp3 328

target sites both by potentiating Foxp3 expression and through direct epigenetic 329

regulation at BRD9/Foxp3 co-bound sites. 330

331

BRD9 co-regulates the expression of Foxp3 and a subset of Foxp3 target genes 332


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Based on co-binding of BRD9 and Foxp3 at Foxp3 target sites, we assessed the effects 333

of BRD9 ablation on the transcription of Foxp3 target genes. We performed RNA-seq in 334

Tregs transduced with sgFoxp3, sgBrd9, or sgNT. Consistent with Foxp3's role as both 335

transcriptional activator and repressor, we observed down-regulation and up-regulation 336

of 793 and 532 genes, respectively, in Foxp3 sgRNA transduced Tregs, which are 337

enriched in ‘cytokine production’, ‘regulation of defense response’, and ‘regulation of cell 338

adhesion’ (Figure 5A, 5B). Of these, 67% are directly bound by Foxp3 in our ChIP-seq 339

dataset and 60% are co-bound by Foxp3 and BRD9 (Figure 5C). Deletion of BRD9 340

resulted in transcriptional changes that strongly correlated with the transcriptional 341

changes in sgFoxp3 transduced Tregs (r2 = 0.534, Linear regression analysis; Figure 342

5D). Indeed, gene set enrichment analysis (GSEA) demonstrated that the sgBrd9 up-343

regulated genes are significantly enriched among genes that increase upon Foxp3 344

knockdown, while the sgBrd9 down-regulated genes are enriched among genes that 345

decrease in sgFoxp3 Tregs (Figure 5E). We also performed RNA-seq for Tregs treated 346

with either vehicle or the dBRD9 degrader and observed a similar significant enrichment 347

for dBRD9 affected genes among the Foxp3 up- and down-regulated genes (Figure 5F). 348

Notably, the BRD9-dependent target gene sets generated from our RNA-seq data were 349

among the most significantly enriched dataset of 9,229 immunological, gene ontology 350

and curated gene sets when analyzed against the sgFoxp3 transduced Treg expression 351

data (Figure 5G). In addition, both datasets were significantly enriched for genes that 352

are differentially expressed between Tregs and conventional T cells(Feuerer et al., 353

2010), and between Foxp3 mutant Tregs from scurfy mice and wild-type Tregs(Hill et al., 354


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2007). These data define a role for BRD9 in Tregs through specifically regulating the 355

expression of Foxp3 itself and a subset of Foxp3 target genes. 356

357

ncBAF complex is required for normal Treg suppressor activity in vitro and in 358

vivo. 359

The divergent roles of ncBAF and PBAF complexes in regulating Foxp3 expression 360

suggested that these complexes might also differentially affect Treg suppressor function. 361

We performed sgRNA knockdown of ncBAF-specific Brd9 and Smarcd1 or PBAF-362

specific Pbrm1 and Phf10 in Tregs and measured their function by conducting an in 363

vitro suppression assay. Tregs depleted of Brd9 or Smarcd1 exhibited significantly 364

reduced suppressor function, whereas depletion of Pbrm1 or Phf10 resulted in 365

significantly enhanced suppressor function (Figure 6A, S7A). These data demonstrate 366

that the opposing regulation of Foxp3 expression by ncBAF and PBAF complexes 367

results in decreased/increased Treg suppressor activity upon ncBAF or PBAF subunit 368

deletion, respectively. Similar to sgRNA depletion of Brd9, Tregs treated with dBRD9 369

also showed significantly and specifically compromised Treg suppressor function in vitro 370

(Figure S7B). These results underscore the requirement for BRD9 in Foxp3 expression 371

maintenance and Treg suppressor activity, and further demonstrate that dBRD9 372

reduces Treg suppressor activity without impairing T effector responses in vitro. 373

374

To test if BRD9 also affects Treg function in vivo, we utilized a T cell transfer-induced 375

colitis model. In this model, Rag1 knockout mice were either transferred with CD45.1+ 376

CD4+ CD25-CD45RBHigh effector T cell (Teff) only, or co-transferred with Teff along with 377


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CD45.2+ Tregs transduced with Brd9 sgRNA (sgBrd9) or control sgRNA (sgNT) (Figure 378

6B). Mice transferred with Teff cells alone lost body weight progressively due to 379

development of colitis. Co-transfer of Tregs transduced with sgNT protected recipient 380

mice from weight loss, whereas co-transfer of sgBrd9 transduced Tregs failed to protect 381

recipients from losing weight (Figure 6C). The mice transferred with Brd9-depleted 382

Tregs showed significant colitis pathology at seven weeks compared to mice that 383

received control Tregs (Figure 6D). Furthermore, Brd9 depletion also led to 384

compromised Treg stability after transfer, manifested by reduced Foxp3+ cell 385

frequencies within the CD45.2+CD4+ transferred Treg population (Figure 6E). These 386

results demonstrate that BRD9 is an essential regulator of normal Foxp3 expression 387

and Treg function in a model of inflammatory bowel disease in vivo. 388

389

In addition to their beneficial role in preventing autoimmune diseases, Tregs have also 390

been shown to be a barrier to anti-tumor immunity. We therefore wondered whether we 391

could exploit the compromised suppressor function shown in Brd9 deficient Tregs to 392

disrupt Treg-mediated immune suppression in tumors. We used the MC38 colorectal 393

tumor cell line to induce cancer due to the prominent role Tregs play in this cancer 394

model(Delgoffe et al., 2013). Rag1 knockout mice were used as recipients for adoptive 395

transfer of Treg depleted-CD4 and CD8 T cells (Teff) only, or co-transfer of Teff with 396

Tregs transduced with either sgBrd9 or control sgNT. MC38 tumor cells were implanted 397

subcutaneously on the following day (Figure 7A). Transfer of sgNT Tregs allowed for 398

significantly faster tumor growth compared to mice that received Teff cells only (“No 399

Treg”) due to suppression of the anti-tumor immune response by Tregs (Figure 7B, 7C). 400


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Furthermore, tumor growth in mice that received sgBrd9 transduced Tregs was 401

significantly slower than mice that received sgNT Tregs consistent with our findings that 402

Brd9 knockdown reduces Treg suppressor activity (Figure 7B, 7C). Both CD4 and CD8 403

T cell tumor infiltration significantly increased in mice that received sgBrd9 transduced 404

Tregs compared to sgNT Tregs (Figure 7D, 7E). Additionally, the percent of IFN-γ 405

producing intra-tumor CD4 and CD8 T cells in mice that received sgBrd9 transduced 406

Tregs was significantly greater than the sgNT Treg condition, and comparable to the 407

transfer of Teff alone (“No Treg”) (Figure 7F, 7G). Consistent with our findings that 408

BRD9 is required for Treg persistence in vivo (Figure 6E), the percentage of transferred 409

Treg cells was reduced in mice that received sgBrd9 transduced Tregs relative to sgNT 410

Tregs (Figure 7H). Overall, a 2-3 fold increase in the ratio of CD8 T cells to Tregs in 411

tumor and spleen was observed in the sgBrd9 versus the sgNT condition, consistent 412

with the enhanced anti-tumor immune response in mice that received sgBrd9 413

transduced Tregs (Figure 7I). This experiment demonstrates that BRD9 promotes stable 414

Treg function in MC38 tumors and knockdown of Brd9 in Tregs improves anti-tumor 415

immunity in this context. 416

417

Discussion 418

In this study, we performed a genome-wide CRISPR screen to identify positive and 419

negative regulators of Foxp3 expression in mouse natural Tregs. Among positive 420

regulators, we identified Cbfb and Runx3, consistent with previous reports showing a 421

requirement for CBF-β/Runx3 in Foxp3 expression and Foxp3-dependent target gene 422

expression(Kitoh et al., 2009; Rudra et al., 2009). Among the novel positive regulators, 423


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we discovered subunits from two chromatin remodeling complexes, the BRD9-424

containing ncBAF and SAGA complexes. Independent validation and functional assays 425

demonstrated an essential role for the ncBAF complex and SAGA complex in Foxp3 426

expression and Treg suppressor function. 427

428

Our screens also confirmed several known negative regulators of Foxp3, including DNA 429

methyl-transferase Dnmt1 and the ubiquitin ligase Stub1. Additionally, we identified 430

multiple negative regulators of the mTOR pathway as Foxp3 negative regulators (Tsc2, 431

Flcn, Ddit4, Sesn2, Nprl2), confirming an essential role for mTOR in homeostasis and 432

function of activated Tregs(Chapman et al., 2018; Sun et al., 2018). Among novel 433

negative Foxp3 regulators, we uncovered genes encoding regulators of RNA 434

metabolism, which have no previously reported function in Foxp3 expression. For 435

example, Mettl3 and Mettl14 form a methyltransferase complex that is essential for the 436

m6A methylation of RNA, which is recognized as an important regulatory mechanism for 437

a wide range of biological processes, including RNA stability, protein translation, stem 438

cell self-renewal, cell lineage determination, and oncogenesis(Yue et al., 2015). Our 439

screen suggests a potentially novel role for RNA m6A methylation in post-transcriptional 440

regulation of Foxp3. Together, our genome-wide screens provide the first 441

comprehensive picture of the complex regulatory network controlling Foxp3 expression 442

levels, and reveal previously unknown pathways and factors that warrant further 443

investigation. 444

445


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Following the identification of SWI/SNF subunit genes among Foxp3 regulators, we 446

endeavored to characterize the roles of the three SWI/SNF-related complexes by 447

deleting subunits unique to each of the ncBAF, BAF, and PBAF complexes. We 448

observed specific and divergent roles of ncBAF and PBAF complexes in regulating 449

Foxp3 expression in Tregs. In contrast, deletion of BAF-specific subunits had a slight, 450

but non-significant effect on Foxp3 expression. Nevertheless, several SWI/SNF core 451

subunits were recovered in our screen among genes that regulate Treg cell contraction, 452

suggesting that BAF complexes may regulate Treg activation or proliferation in 453

response to TCR stimulation used to activate and culture Tregs in our screen. This is 454

consistent with a role for Smarca4 in Treg activation and control of autoimmunity in vivo 455

independent of affecting Foxp3 expression, which is not changed in Foxp3-456

Cre:Smarca4f/f Tregs(Chaiyachati et al., 2013). Thus, deletion of Smarca4 or other BAF 457

complex subunits likely results in overall defects in Treg fitness, whereas deletion of 458

ncBAF subunits appears to have a selective effect on Foxp3 expression and its target 459

genes. Mechanistically, we find that the ncBAF complex co-binds and cooperates with 460

Foxp3 to potentiate its binding to the CNS2 and CNS0, enhancers of the Foxp3 locus. 461

In addition to the Foxp3 locus itself, our ChIP-seq analysis revealed that ncBAF also 462

binds to regulatory elements in a subset of Foxp3 target genes to regulate their gene 463

expression. One possibility is that reduced Foxp3 expression results in lowered Foxp3 464

binding at a select group of target genes, but what differentiates the dosage-dependent 465

binding of Foxp3 at these sites compared to other unaffected sites remains unclear. 466

467


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Finally, we tested the vivo relevance of our findings by disrupting the ncBAF subunit 468

Brd9 in Tregs in mouse models of inflammatory bowel disease and cancer. Knockdown 469

of Brd9 in Tregs weakened their suppressor function in a model of T cell induced colitis, 470

leading to exacerbated disease progression. In the context of cancer, we found that 471

transfer of Brd9 deficient Tregs failed to restrict anti-tumor immune responses in the 472

MC38 cell induced cancer model, leading to slower tumor growth. Currently, there is a 473

concerted effort to develop compounds targeting a number of SWI/SNF complex 474

subunits to modulate their function. Our data show that bromodomain-directed 475

degradation of BRD9 by dBRD9 recapitulates the effects of Brd9 genetic deletion, 476

suggesting that the ncBAF complex can be targeted with small molecules to control 477

Foxp3 expression and Treg function. Thus, through the unbiased screen of Foxp3 478

regulators, we have identified novel proteins that can potentially be targeted to 479

manipulate Treg homeostasis and function in autoimmune diseases and cancer. 480

481

482


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689


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Acknowledgments 690

We would like to thank C. Gordon for mouse colony management, B. Kuo and X. Hu for 691

assistance in plasmid extraction and mouse dissection, N. Hah and G. Chou for 692

assistance in RNA-seq, ChIP-seq, CRISPR screen experiments, E. Shifrut and A. 693

Marson (UCSF) for providing us the R script to generate the sgRNA distribution 694

histograms, A. Williams and M. Shokhirov for bioinformatic assistance, and M. Downes 695

and R. M. Evans for helpful discussion. C.S.L. was partly supported by the Albert G. 696

and Olive H. Schlink Foundation. J.G. was supported by the Salk Institute T32 Cancer 697

Training Grant T32CA009370 and the NIGMS NRSA F32 GM128377-01. D.C.H. was 698

supported by the National Institutes of Health (GM128943-01, CA184043-03), the V 699

Foundation for Cancer Research V2016-006, and the Leona M. and Harry B. Helmsley 700

Charitable Trust. Y.Z. was supported by the NOMIS Foundation, the Crohn’s and Colitis 701

Foundation, the Leona M. and Harry B. Helmsley Charitable Trust, and National 702

Institute of Health (AI107027 and OD023689). This work was also supported by 703

National Cancer Institute funded Salk Institute Cancer core facilities (CA014195). 704

705

Author Contributions 706

Conceptualization: C.S.L., J.G., D.C.H. and Y.Z. Methodology: C.S.L. Investigation: 707

C.S.L., J.G., Y.L., M.X., J.H., B.V. Resources: D.C.H. and Y.Z. Formal analysis: C.S.L., 708

J.G., M.L. Data Curation: C.S.L., J.G. Supervision: D.C.H. and Y.Z. Funding acquisition: 709

D.C.H. and Y.Z. Writing – original draft preparation: C.S.L., J.G., D.C.H. and Y.Z. 710

Writing – review and editing: C.S.L., J.G., D.C.H. and Y.Z. 711

712


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Figure Legends 713

Figure 1 | A genome-wide CRISPR screen in Treg cells 714

A, Workflow of the CRISPR screen in Tregs. B-D, Validation of the CRISPR screen 715

conditions. B, FACS plots showing Foxp3 expression in Tregs after knocking out Foxp3 716

(sgFoxp3), positive regulator Cbfb (sgCbfb), and negative regulator Dnmt1 (sgDnmt1). 717

Red and green gates were set based on Foxp3 low 20% and high 20% in sgNT Treg, 718

respectively. C, Mean fluorescence intensity (MFI) of Foxp3 and D, Relative Log2FC of 719

cell count comparing Foxp3Low to Foxp3High after deletion of the indicated target gene 720

(n=3 per group). See also Figure S1 and S2. 721

722

Figure 2 | Identification of novel Foxp3 regulators in Treg cells 723

A, B, A scatter plot of the Treg screen result showing positive regulators (A) and 724

negative regulators (B). Genes that have met cutoff criteria (P-value<0.01, and 725

Log2FC >± 0.5) are shown as red dots for positive regulators and green dots for 726

negative regulators. C, Distribution of sgRNA Log2FC comparing Foxp3Low to Foxp3High. 727

Red stripes represent sgRNAs from positive Foxp3 regulators, whereas green stripes 728

represent sgRNAs from negative Foxp3 regulators. D, Venn diagram showing the 729

overlap of Foxp3 regulators with genes involved in cell contraction or expansion. E, F, 730

Gene ontology analysis of positive Foxp3 regulators (E) and negative Foxp3 regulators 731

(F). See also Figures S3 and S4; Tables S1, S2, S3, and S4. 732

733

Figure 3 | The three SWI/SNF complex assemblies have distinct regulatory roles 734

for Foxp3 expression in Tregs 735


https://doi.org/10.1101/2020.02.26.964981

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A, A diagram showing three different variants of SWI/SNF complexes: BAF, ncBAF, and 736

PBAF. BAF-specific subunits (ARID1A, DPF1-3) are colored blue, ncBAF-specific 737

subunits (BRD9, SMARCD1, GLTSCR1L, GLTSCR1) colored orange, and PBAF-738

specific subunit (PBRM1, ARID2, BRD7, PHF10) colored green. Shared components 739

among complexes are colored gray. Immunoprecipitation assay of ARID1A, BRD9, and 740

PHF10, and BRG1 in Tregs. The co-precipitated proteins were probed for shared 741

subunits (SMARCA4, SMARCC1, SMARCB1), BAF-specific ARID1A, ncBAF-specific 742

BRD9, and PBAF-specific PBRM1. B, FACS histogram of Foxp3 expression in Tregs 743

after sgRNA knockout of the indicated SWI/SNF subunits. C, Mean fluorescence 744

intensity (MFI) of Foxp3 after sgRNA knockout of the indicated SWI/SNF subunits. Data 745

represents mean and standard deviation of biological replicates (n = 3-21). D, Principal 746

component analysis of RNA-seq data collected from Tregs transduced with guides 747

against the indicated SWI/SNF subunits. In cases where two independent guides were 748

used to knockdown a gene, the second guide for targeting gene indicated as “-2”. E, 749

MFI of Foxp3 expression in Tregs after treatment with either DMSO or 0.16-10 μM 750

dBRD9 for 4 days. Data represent mean ± s.d. Statistical analyses were performed 751

using unpaired two-tailed Student's t test (ns: p≥0.05, *p<0.05, **p<0.01, ***p<0.001, 752

****p<0.0001). 753

754

Figure 4 | BRD9 deletion reduces Foxp3 binding at CNS0, CNS2 enhancers and a 755

subset of Foxp3 target sites 756

A, Genome browser tracks of SMARCA4, BRD9, PHF10 ChIP-seq and ATAC-seq 757

signal, as well as Foxp3 ChIP-seq in sgNT, sgFoxp3, sgBrd9 and sgPbrm1 Tregs and 758


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Foxp3 in DMSO and dBRD9 treated Tregs (2.5 μM dBRD9 for 4 days). Foxp3 locus is 759

shown with CNS0 and CNS2 enhancers indicated in gray ovals. B, Heat map of Foxp3, 760

BRD9, SMARCA4, PHF10 ChIP-seq and ATAC-seq signal ± 3 kb centered on Foxp3-761

bound sites in Tregs, ranked according to Foxp3 read density. C, Venn diagram of the 762

overlap between ChIP-seq peaks in Tregs for BRD9, Foxp3, and PHF10 763

(hypergeometric p value of BRD9:Foxp3 overlap = e-30704, hypergeometric p value of 764

PHF10:Foxp3 overlap = e-13182, hypergeometric p value of BRD9:PHF10 overlap = e-765

12895). D, Heat map of Foxp3 ChIP-seq signal in sgNT, sgFoxp3, sgBrd9 and sgPbrm1 766

Tregs ± 3 kilobases (kb) centered on Foxp3-bound sites in sgNT, ranked according to 767

read density. E, Venn diagram of the overlap (hypergeometric p value = e-11,653) 768

between sites that significantly lose Foxp3 binding (FC 1.5, Poisson p value < 0.0001) in 769

sgFoxp3 and sgBrd9, overlaid on all Foxp3-bound sites in sgNT (in gray). F, Histogram 770

of Foxp3 ChIP read density ± 1 kb surrounding the peak center of sites that significantly 771

lose Foxp3 binding in both sgFoxp3 and sgBrd9 (n=1,699) in sgNT, sgFoxp3, sgBrd9 772

and sgPbrm1. G, As in E, but for sites that lose H3K27ac (FC 1.5, Poisson p value < 773

0.0001, hypergeometric p value of overlap = e-7,938). H, As in F, but for H3K27ac ChIP 774

read density. I, As in E, but for sites that significantly lose Foxp3 binding in dBRD9 775

treated Tregs versus DMSO (FC 1.5, Poisson p value < 0.0001). J, As in F, but for 776

DMSO and dBRD9 treated cells. 777

778

Figure 5 | BRD9 co-regulates the expression of Foxp3 and a subset of Foxp3 779

target genes 780


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33

A, Volcano plot of log2 fold change RNA expression in sgFoxp3/sgNT Tregs versus 781

adjusted p value (Benjamin-Hochberg). Number of down- and up-regulated genes are 782

indicated, which are colored blue and red, respectively. B, Significance of enrichment of 783

Foxp3-dependent genes in each gene ontology. C, Pie chart of Foxp3 and BRD9 784

binding by ChIP-seq for Foxp3-dependent genes. D, Scatterplot of the mRNA log2 fold 785

changes in sgFoxp3/sgNT and sgBrd9/sgNT for Foxp3-dependent genes. Linear 786

regression analysis was performed to calculate the r2. Best fit is represented as an 787

orange dashed line. E, Gene set enrichment analysis (GSEA) enrichment plot for up- 788

and down-regulated genes in sgBrd9/sgNT compared with RNA-seq data of genes that 789

significantly change in sgFoxp3/sgNT Tregs. ES: Enrichment Score, NES: Normalized 790

Enrichment Score, FWER: Familywise Error Rate. F, As in E, but for up- and down-791

regulated genes in dBRD9/DMSO Tregs. G, GSEA of the sgFoxp3/sgNT RNA-seq data; 792

plot shows the familywise error rate (FWER) p value versus the normalized enrichment 793

score (NES). See also Table S5. 794

795

Figure 6 | The ncBAF complex regulates Treg suppressor function in vitro and in 796

vivo. 797

A. In vitro suppression assay of Tregs with sgRNA knockout of Brd9, Smarcd1, Pbrm1, 798

and Phf10 (n=3 per group, data represent ± s.d.). sgNT was used as non-targeting 799

control. B-F. Experiment to measure Treg function of sgNT or sgBrd9 knockout Treg 800

cells relative to no Tregs in a T cell transfer induced colitis model. B, Experimental 801

procedure. C, Body weight loss. D, Colon histology (left) and colitis scores (right). E, 802

Percentage of Foxp3+ cells in transferred CD45.2+CD4+ Treg population at end point. 803


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(n=4-6 per group. Data represent mean ± s.e.m.) Statistical analyses were performed 804

using unpaired two-tailed Student's t test (ns: p≥0.05, *p<0.05, **p<0.01, ***p<0.001). 805

806

Figure 7 | Targeting BRD9 in Treg improves anti-tumor immunity. 807

A, Experiment procedure to measure function of sgNT or sgBrd9 knockout Treg cells 808

relative to no Tregs in MC38 tumor model. B, Tumor growth curve. C, Tumor weight at 809

end point. D,E, Bar graph of total CD4 T cells (D) and CD8 T cells (E) percentage in 810

CD45+ immune cell population. F,G, Bar graph of IFN-γ+ cell percentage in CD4 T cells 811

(F) and in CD8 T cells (G). H, Bar graph of CD4+eGFP+Foxp3+ donor cells in CD4+ T 812

cells. I, Ratio of CD8/Treg. (n=5-7 per group. Data represent mean ± s.e.m.) Statistical 813

analyses were performed using unpaired two-tailed Student's t test (ns: p≥0.05, *p<0.05, 814

**p<0.01, ***p<0.001). 815

816

817


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Methods 818

List of antibodies 819

Target protein Antibody source Application (dilution)

CD4-Alexa fluor 700 eBioscience 56-0042-82 Flow (1:400)

CD4-PerCP-Cy5.5 TONBO 65-0042-U100 Flow (1:400)

CD8-PE eBioscience 12-0081-85 Flow (1:400)

CD8-BV510 Biolegend 100752 Flow (1:400)

CD45.1-BV605 Biolegend 110735 Flow (1:400)

CD45.2-Alexa 700 Biolegend 109822 Flow (1:400)

Foxp3-eFluor 450 eBioscience 48-5773-82 Flow (1:400)

NGFR-PE Biolegend 345106 Flow (1:400)

NGFR-APC Biolegend 345108 Flow (1:400)

Thy1.1-PE eBioscience 12-0900-83 Flow (1:400)

CD44-BV650 Biolegend 103049 Flow (1:400)

CD62L-BV605 Biolegend 104438 Flow (1:400)

IFNg-APC eBioscience 17-7311-82 Flow (1:400)

Ghost Viability Dye TONBO 13-0865-T100 Flow (1:800)

Foxp3 In-house WB (1:2000); ChIP (1:100)

BRG1/SMARCA4 Abcam 110641 WB (1:2000); IP, ChIP (1:100)

BAF155/SMARCC1 Santa Cruz sc-10756 WB (1:1000)

BAF47/SMARCB1 Santa Cruz sc-166165 WB (1:1000)

BRD9 Active Motif 61537 WB (1:2000); IP, ChIP (1:100)

PBRM1 Bethyl A301-591A WB (1:2000)


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PHF10 Thermo Fisher PA5-30678 IP, ChIP (1:100)

ARID1A Santa Cruz sc-32761 WB (1:1000)

Histone H3K27ac Abcam ab4729 ChIP (1:100)

IgG Cell Signaling 2729S IP (1:100)

anti-mouse secondary Thermo Fisher A21058 WB (1:20,000)

anti-rabbit secondary Thermo Fisher SA535571 WB (1:20,000)

820

List of sgRNA sequence 821

Plasmid name Target gene sgRNA sequence

pSIRG-NGFR-sgFoxp3 Foxp3 TCTACCCACAGGGATCAATG

pSIRG-NGFR-sgCbfb Cbfb GCCTTGCAGATTAAGTACAC

pSIRG-NGFR-sgDnmt1 Dnmt1 TAATGTGAACCGGTTCACAG

pSIRG-NGFR-sgArid1a Arid1a GCAGCTGCGAAGATATCGGG

pSIRG-NGFR-sgArid1a-2 Arid1a TACCCAAATATGAATCAAGG

pSIRG-NGFR-sgArid1b Arid1b TGAGTGCAAAACTGAGCGCG

pSIRG-NGFR-sgArid1b-2 Arid1b CAGAACCCCAACATATAGCG

pSIRG-NGFR-sgDpf1 Dpf1 TCTTCTACCTCGAGATCATG

pSIRG-NGFR-sgDpf2 Dpf2 GAAGATACGCCAAAGCGTCG

pSIRG-NGFR-sgPbrm1 Pbrm1 AAAACACTTGCATAACGATG

pSIRG-NGFR-sgPbrm1-2 Pbrm1 CAATGCCAGGCACTACAATG

pSIRG-NGFR-sgArid2 Arid2 ACTTGCAGTAAATTAGCTCG

pSIRG-NGFR-sgBrd7 Brd7 CAGGAGGCAAGCTAACACGG

pSIRG-NGFR-sgPhf10 Phf10 GTTGCCGACAGACCGAACGA


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pSIRG-NGFR-sgBrd9 Brd9 ATTAACCGGTTTCTCCCGGG

pSIRG-NGFR-sgBrd9-2 Brd9 GGAACACTGCGACTCAGAGG

pSIRG-NGFR-sgGltscr1 Gltscr1 GTTCTGTGTAAAATCACACT

pSIRG-NGFR-sgGltscr1l Gltscr1l ATGGCTTTATGCAACACGTG

pSIRG-NGFR-sgSmarcd1 Smarcd1 CAATCCGGCTAAGTCGGACG

pSIRG-NGFR-sgEny2 Eny2 AGAGCTAAATTAATTGAGTG

pSIRG-NGFR-sgAtxn7l3 Atxn7l3 GCAGCCGAATCGCCAACCGT

pSIRG-NGFR-sgUsp22 Usp22 GCCATCGACCTGATGTACGG

pSIRG-NGFR-sgCcdc101 Ccdc101/Sgf29 CCAGGTTTCCCGATCCAGAG

pSIRG-NGFR-sgTada3 Tada3 GAAGGTCTGTCCCCGCTACA

pSIRG-NGFR-sgTada1 Tada1 TTTCCTTCTCGACACAACTG

pSIRG-NGFR-sgTaf6l Taf6l TCATGAAACACACCAAACGA

pSIRG-NGFR-sgSupt20 Supt20 TTAGTAGTCAATCTGTACCC

pSIRG-NGFR-sgSupt5 Supt5 GATGACCGATGTACTCAAGG

pSIRG-NGFR-sgNT Non-targeting AAAAAGTCCGCGATTACGTC

pSIRG-eGFP-sgBrd9 Brd9 ATTAACCGGTTTCTCCCGGG

pSIRG-eGFP-sgNT Non-targeting AAAAAGTCCGCGATTACGTC

822

Mice 823

C57BL/6 Rosa-Cas9/Foxp3Thy1.1 mice were generated by crossing Rosa26-LSL-Cas9 824

knockin mice(Platt et al., 2014) (The Jackson Laboratory #024857) with Foxp3Thy1.1 825

reporter mice(Liston et al., 2008). Male Cas9/Foxp3Thy1.1 mice at 8-12 weeks age were 826

used to isolate Tregs for the CRISPR screen, and no gender preference was given for 827


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38

other experiments. C57BL.6 Ly5.1+ congenic mice and Rag1-/- mice purchased from 828

the Jackson Laboratory were used for Treg suppression assay and adoptive T cell 829

transfer in colitis and tumor models. All mice were bred and housed in the specific 830

pathogen-free facilities at the Salk Institute for Biological Studies and were conducted 831

under the regulation of the Institutional Animal Care and Use Committee (IACUC) and 832

institutional guidelines. 833

834

Retroviral vectors and sgRNA library construction 835

Self-inactivating retroviral vector pSIRG-NGFR was generated by modifying pSIR-836

dsRed-Express2(Fujita and Fujii, 2014) (Addgene #51135), which enables us to clone 837

sgRNA as efficient as lentiCRISPRv2, to enrich transduced cells via magnetic beads 838

isolation, and to perform intracellular staining without losing transduced reporter marker. 839

We first mutated all BbsI sites in pSIR-dsRed-Express2, then inserted a sgRNA 840

expressing cassette containing the U6 promoter, guide RNA scaffold and a 500bp filler 841

embedded with BbsI cloning site. The dsRed cassette was replaced by cDNA sequence 842

of human NGFR with truncated intracellular domain. We also generated pSIRG vector 843

with eGFP (pSIRG-eGFP) for the purpose of T cells transfer in tumor study, minimizing 844

potential immune rejection. The pSIRG-eGFP was generated by cutting pSIRG-NGFR 845

with XcmI to remove NGFR cassette and replaced by eGFP cDNA by Gibson cloning. 846

For cloning single guide RNA into the pSIRG vector, an annealed sgRNA oligos can be 847

directly inserted into BbsI-digested pSIRG-NGFR by T4 ligation similar to the cloning 848

method utilized by lentiCRISPRv2(Sanjana et al., 2014). To create a pooled sgRNA 849

library in pSIRG-NGFR, we first amplified sgRNA sequences from an optimized mouse 850


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39

CRISPR knockout library lentiCRISPRv2-Brie (Addgene #73632). A total of eight 50 μL 851

PCR reactions were performed to maximize coverage of sgRNA complexity. Each 50 μL 852

PCR reaction contained Q5 High-Fidelity DNA polymerase and buffer (NEB #M0491), 853

15ng of lentiCRISPRv2-Brie, and targeted primers (Forward: 854

GGCTTTATATATCTTGTGGAAAGGACGAAACACCG, Reverse: 855

CTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC). PCR was performed at 98°C 856

denature, 67°C annealing, 72°C extension for 12 cycles. The sgRNA library amplicons 857

were then combined and separated in 2 % agarose gel, and purified by the QIAquick 858

Gel Extraction Kit (Qiagen #28704). The purified sgRNA amplicons was inserted into 859

the BbsI-digested pSIRG-NGFR by NEBuilder HIFI assembly (NEB #E2621S). The 860

sgRNA representative of the retroviral CRISPR library (pSIRG-NGFR-Brie) was 861

validated by deep sequencing and comparing to the original lentiCRISPRvs-Brie. The 862

coverage of the new pSIRG-NGFR sgRNA library was evaluated by the PinAPL-Py 863

program (Spahn et al., 2017) (see Extended Data Figure 1). 864

865

T cell isolation and culture 866

For large scale Treg culture, we first expanded Treg in Rosa-Cas9/Foxp3Thy1.1 mice by 867

injecting IL-2:IL-2 antibody immune complex according protocol described in Webster 868

KE et. al(Webster et al., 2009). Spleen and lymph node Tregs were labeled with PE-869

conjugated Thy1.1 antibody and isolated by magnetic selection using anti-PE 870

microbeads (Mitenyl #130-048-801). All isolated Tregs were activated by plate bound 871

anti-CD3 and anti-CD28 antibodies and cultured with X-VIVO 20 media (LONZA #04-872

448Q) supplemented by 1X Pen/Strep, 1X Sodium pyruvate, 1X HEPES, 1X GlutaMax, 873


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55 μM beta-mercaptoethanol in the presence of IL-2 at 500 U/mL. For experiments with 874

BRD9 degradation, Tregs were treated at day 0 with 2.5μM dBRD9 (Tocris #6606) and 875

cultured for four days for RNA- and ChIP-seq and 0.16-10μM treated at day 0 and 876

cultured dBRD9 for four days for Foxp3 MFI, cell viability and cell proliferation assays. 877

Live cells were enriched by Ficoll-Paque 1.084 (GE Health 17-5446-02) for RNA-seq 878

and ChIP-seq. 879

880

Retroviral production and T cell transduction 881

HEK293T cells were seeded in 6-wells plate at 0.5 million cells per 2mL DMEM media 882

supplemented by 10% FBS, 1% Pen/Strep, 1X GlutaMax, 1X Sodium Pyruvate, 1X 883

HEPES, and 55 μM beta-mercaptoethanol. One day later, cells from each well was 884

transfected with 1.2 μg of targeting vector pSIRG-NGFR and 0.8 μg of packaging vector 885

pCL-Eco (Addgene, #12371) by using 4 μL of FuGENE HD transfection reagent 886

(Promega #E2311) according manufactured protocol. Cell culture media was replaced 887

by 3 mL fresh DMEM complete media at 24 hours and 48 hours after transfection. The 888

retroviral supernatant was collected at 48 and 72 hours post transfection for T cell 889

infection. For experiments with CRISPR sgRNA targeted knockdown, Cas9+ Tregs 890

were first seeded in 24-wells plate coated with CD3 and CD28 antibodies. At 24 hour 891

post-activation, 70% of Treg media from each well was replaced by retroviral 892

supernatant, supplemented with 4 μg/mL Polybrene (Milipore # TR-1003-G), and spun 893

in a benchtop centrifuge at 1,258 x g for 90 minutes at 32°C. After centrifugation, Treg 894

media was replaced with fresh media supplemented with IL-2 and cultured for another 895

three days. Transduced cells were analyzed for Foxp3 and cytokine expression in 896


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41

eBioscience Fix/Perm buffer (eBioscience #00-5523-00) using flow cytometry. 897

Transduced NGFR+ cells were FACS-sorted for subsequent RNA- and ChIP-seq 898

experiments. 899

900

Genome-wide CRISPR screen in Treg 901

Approximately 360 million Treg cells isolated from Rosa-Cas9/Foxp3Thy1.1 mice were 902

used for the Treg screen. On day 0, Tregs were seeded at 1x106 cells/mL into 24-wells 903

plate coated with anti-CD3/28 and cultured with X-VIVO complete media with IL-2 (500 904

U/ml). On day 1, sgRNA retroviral library transduction was performed with a MOI<0.2. 905

On day 3, approximately 4 million (~50X coverage) NGFR+ transduced cells were 906

collected in three replicates as the starting state sgRNA input. Treg cells reached 907

confluence on day 4. NGFR+ transduced cells were isolated via magnetic selection by 908

anti-PE beads (Mitenyl #130-048-801), and then plated onto new 24-wells plates coated 909

with anti-CD3/CD28, and cultured in X-VIVO complete media with IL-2 (500 U/ml). On 910

day 6, approximately 4 million NGFR+ transduced cells were collected in three 911

replicates as the ending state sgRNA output. The remaining cells were fixed, 912

permeabilized, and stained for intracellular Foxp3. Approximately 2 million Foxp3High 913

(top 20%) and 2 million Foxp3Low (bottom 20%) cell populations were sorted in three 914

replicates by a FACS Aria cell sorter for genomic DNA extraction and library 915

construction. 916

917

Preparation of sgRNA amplicons for Next-Generation Sequencing 918


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42

To extract genomic DNA, we first lysed cells with homemade digestion buffer (100mM 919

NaCl, 10mM Tris, 25mM EDTA, 0.5% SDS, 0.1mg/mL Proteinase K) overnight in 50 °C. 920

On the following day, the lysed sample was mixed with phenol: chloroform: isoamyl 921

alcohol (25:24:1, v/v) in 1:1 ratio, and spun at 6000rpm for 15 min at room temperature. 922

The supernatant containing genomic DNA was transferred into a new tube and mixed 923

with twice volume of 100% ethanol, then spun at 12,500 rpm for 5 min in room 924

temperature to precipitate DNA. Supernatant was removed, and the precipitated DNA 925

was dissolved in ddH2O. DNA concentration was measured by Nanodrop. To generate 926

sgRNA amplicons from extracted genomic DNA, we used a two-step PCR protocol 927

which was adopted from the protocol published by Shalem et. al. (Shalem et al., 2014). 928

We performed eight 50 μL PCR reactions containing 2 μg genomic DNA, NEB Q5 929

polymerase, and buffer, and targeted primers (Forward: 930

GGCTTTATATATCTTGTGGAAAGGACGAAACACCG, Reverse: 931

CTAGCCTTATTTTAACTTGCTATTTCTAGCTCTAAAAC). PCR was performed at 98°C 932

denature, 70°C annealing, 15s extension for 20 cycles. The products from the first PCR 933

were pooled together, and purified by AMPure XP SPRI beads according to 934

manufacturer’s protocol, and quantified by Qubit dsDNA HS assay. For the second 935

round PCR, we performed eight 50 μL PCR reactions containing 2 ng purified 1st round 936

PCR product, barcoded primer (see primer set from (Shalem et al., 2014), Priming site 937

of reverse primer was changed to CTTCCCTCGACGAATTCCCAAC), NEB Q5 938

polymerase, and buffer. PCR was performed at 98°C denature, 70°C annealing, 15 s 939

extension for 12 cycles. The 2nd round PCR products were pooled, purified by AMPure 940


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43

XP SPRI beads, quantified by Qubit dsDNA HS assay, and sequenced by NEXTSeq 941

sequencer at single end 75 bp (SE75). 942

943

Data analysis of pooled CRISPR screen 944

The screening hit identification and quality control was performed by MAGeCK-VISPR 945

program(Li et al., 2015; Li et al., 2014a). The abundance of sgRNA from a sample fastq 946

file was first quantified by MAGeCK “Count” module to generate a read count table. For 947

hit calling, we used MAGeCK “test” module to generate a gene-ranking table that 948

reporting RRA gene ranking score, p-value, and log2 fold change. The size factor for 949

normalization was adjusted according to1000 non-targeting control assigned in the 950

screen library. All sgRNAs that are zero read were removed from RRA analysis. The 951

log2 fold change of a gene was calculated from a mean of 4 sgRNA targeting per gene. 952

The scatter plots showing the screen results were generated by using the R script 953

EnhancedVolcano (https://github.com/kevinblighe/EnhancedVolcano). The R script that 954

generated the sgRNA distribution histogram was provided by E. Shifrut and A. Marson 955

(UCSF)(Shifrut et al., 2018). A gene list from Foxp3 regulators (either positive or 956

negative) without affecting cell proliferation was subjected to Gene Ontology analysis 957

using Metascape(Zhou et al., 2019). Genes were analyzed for enrichment for Functional 958

Set, Pathway, and Structural Complex. 959

960

In vitro Treg suppression assay 961

Tregs were transduced by retrovirus expressing sgRNA targeting gene of interest and 962

cultured in X-VIVO complete media supplemented with IL-2 (500 U/ml). Four days after 963


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transduction, transduced cells were sorted and mixed with FACS sorted CD45.1+ naive 964

CD4 T cells (CD4+ CD25– CD44Low CD62LHigh) labeled with CellTrace Violet (Thermo 965

Fisher Scientific #C34571) in different ratio in the presence of irradiated T cell depleted 966

spleen cells as antigen-presenting cells (APC). Three days later, Treg suppression 967

function was measured by the percentage of non-dividing cells within the CD45.1+ 968

effector T cell population. For dBRD9 treatment experiment, dBRD9 was first dissolved 969

in DMSO (10 mM stock) and added into Treg:Teff:APC mixture at 2.5μM. For Foxp3 970

overexpression rescue experiment, Tregs were first transduced with sgNT or sgBrd9 at 971

24 hour post-activation, and then transduced with MIGR empty vector or MIGR-Foxp3 972

at 48 hour post-activation. Double transduced Tregs were FACS sorted on day 4 based 973

on NGFR+ and GFP+ markers and then mixed with CellTrace labeled effector T cells in 974

the presence of APC. Treg suppression readout was measured after three days of co-975

culture. 976

977

Adoptive T cells transfer-induced colitis model 978

Tregs were transduced by retrovirus expressing sgRNA targeting gene of interest, and 979

cultured in X-VIVO complete media and IL-2 (500 U/ml). Four days after transduction, 980

the NGFR+ transduced Treg cells were FACS sorted before transferred into recipient 981

mice. To induce colitis, 2 million effector T cells (CD45.1+ CD4+ CD25– CD45RBHigh) and 982

1 million sgRNA knockout Tregs (CD45.2+ CD4+ Thy1.1+ NGFR+) were mixed together 983

and transferred into Rag1 knockout recipient mice. The body weight of recipient mice 984

was monitored weekly for signs of wasting symptoms. Mice were harvested 7 weeks 985


https://doi.org/10.1101/2020.02.26.964981

45

after T cell transfer. Spleens were used for profiling immune cell populations by FACS. 986

Colons were collected for histopathological analysis. 987

988

Colon histopathological analysis 989

Histopathological analysis was performed in a blinded manner and scored using the 990

following criteria. Eight parameters were used that include (i) the degree of inflammatory 991

infiltrate in the LP (0-3); (ii) Goblet cell loss (0–2); (iii) reactive epithelial 992

hyperplasia/atypia with nuclear changes (0–3); (iv) the number of IELs in the epithelial 993

crypts (0–3); (v) abnormal crypt architecture (distortion, branching, atrophy, crypt loss) 994

(0–3); (vi) number of crypt abscesses (0–2); (vii) mucosal erosion to frank ulcerations 995

(0–2) and (viii) submucosal spread to transmural involvement (0-2). The severity of 996

lesion was scored independently in 3 regions (proximal, middle and distal colon) over a 997

maximal score of 20. The overall colitis score was based as the average of each 998

regional score (maximal score of 20). 999

1000

Adoptive T cells transfer and MC38 tumor model 1001

Similar to the “Adoptive T cells transfer-induced colitis model”, Tregs were activated in 1002

vitro and transduced with pSIRG-eGFP expressing sgNT or sgBrd9. Four days after 1003

transduction, the eGFP+ transduced Treg were FACS sorted. Concurrently, Treg 1004

depleted CD4 and CD8 T cells isolated from Rosa-Cas9/Foxp3Thy1.1 mice were used as 1005

effector T cells. A total of 1 million pSIRG-sgRNA transduced eGFP+ Tregs, 1 million 1006

effector CD8 T cells, and 2 million Treg-depleted CD4 T cells were mixed and 1007

transferred into Rag1 knockout recipient mice. on the following day, mice were 1008


https://doi.org/10.1101/2020.02.26.964981

46

implanted with 0.5 million MC38 cells (a gift from the laboratory of Dr. Susan Kaech) by 1009

subcutaneous injection on the flank of mouse. When palpable tumor appeared, tumor 1010

size was measured every two day by electronic calipers. At the end point, spleen and 1011

tumor were collected for immune profiling. For tumor processing, tumor tissues were 1012

minced into small pieces and digested with 0.5 mg/mL Collagenase IV (Sigma #C5138) 1013

and DNAase I (Roche #4716728001) for 20 minutes and passed through 0.75 μm cell 1014

strainer to collect single cell suspension. Isolated cells were stimulated with 1015

PMA/Ionomycin and Golgi plug for 5 hours, and then were subjected to Foxp3 and 1016

cytokines staining with eBioscience Fix/Perm buffer (eBioscience #00-5523-00). 1017

1018

Nuclear protein extraction 1019

Nuclear lysates were collected from Treg cells following a revised Dignam 1020

protocol(Andrews and Faller, 1991). After cellular swelling in Buffer A (10 mM Hepes pH 1021

7.9, 1.5 mM MgCl2, 10 mM KCl) supplemented with 1 mM DTT, 1 mM PMSF, 1 µM 1022

pepstatin, 10 µM leupeptin and 10 µM chymostatin, cells were lysed by homogenization 1023

using a 21-gauge needle with six to eight strokes. If lysis remained incomplete, cells 1024

were treated with 0.025 - 0.05% Igepal-630 for ten minutes on ice prior to nuclei 1025

collection. Nuclei were spun down at 700 x g for five minutes then resuspended in 1026

Buffer C (20 mM Hepes pH 7.9, 20% glycerol, 420 mM NaCl, 1.5 mM MgCl2, 0.2 mM 1027

EDTA) supplemented with 1 mM DTT, 1 mM PMSF, 1 µM pepstatin, 10 µM leupeptin 1028

and 10 µM chymostatin. After thirty minutes of end-to-end rotation at 4°C, the sample 1029

was clarified at 21,100 x g for ten minutes. Supernatant was collected, flash frozen in 1030

liquid nitrogen and stored in the -80°C freezer. 1031


https://doi.org/10.1101/2020.02.26.964981

47

1032

Co-Immunoprecipitation 1033

Nuclear lysates were thawed on ice then diluted with two-thirds of original volume of 50 1034

mM Tris-HCl pH 8, 0.3% NP-40, EDTA, MgCl2 to bring down the NaCl concentration. 1035

Proteins were quantified using Biorad DC Protein Assay (Cat #5000112) according to 1036

manufacturer’s instructions. For the co-IP reaction, 200-300 µg of proteins were 1037

incubated with antibody against normal IgG, SMARCA4, BRD9, ARID1A or PHF10 1038

overnight at 4°C, with end-to-end rotation. Precipitated proteins were bound to 50:50 1039

Protein A: Protein G Dynabeads (Invitrogen) for one to two hours and washed 1040

extensively with IP wash buffer (50 mM Tris pH 8, 150 mM NaCl, 1 mM EDTA, 10% 1041

glycerol, 0.5% Triton X100). Proteins were eluted in SDS-PAGE loading solution with 1042

boiling for five minutes and analyzed by western blotting. 1043

1044

Western blot 1045

Protein samples were run on 4-12% Bis-Tris gels (Life Technologies). After primary 1046

antibody incubation which is typically done overnight at 4°C, blots were probed with 1047

1:20,000 dilution of fluorescently-labeled secondary antibodies in 2% BSA in PBST (1X 1048

Phospho-buffered saline with 0.1% Tween-20) for an hour at room temperature (RT). 1049

Fluorescent images were developed using Odyssey and analyzed using Image Studio 2. 1050

Protein quantitation was performed by first normalizing the measured fluorescence 1051

values of the proteins of interest against the loading control (TBP) then normalizing 1052

against the control sample (vehicle treated). 1053

1054


https://doi.org/10.1101/2020.02.26.964981

48

RNA-seq sample preparation 1055

RNA from 1-3 x 106 cells was extracted and purified with TRIzol reagent (Thermo Fisher) 1056

according to manufacturer’s instructions. RNA-seq libraries were prepared using 1057

Illumina TruSeq Stranded mRNA kit following manufacturer’s instructions with 5 µg of 1058

input RNA. 1059

1060

RNA-seq analysis 1061

Single-end 50 bp reads were aligned to the mouse genome mm10 using STAR 1062

alignment tool (V2.5)(Dobin et al., 2013). RNA expression was quantified as raw integer 1063

counts using analyzeRepeats.pl in HOMER(Heinz et al., 2010) using the following 1064

parameters: -strand both -count exons -condenseGenes -noadj. To identify differentially 1065

expressed genes, we performed getDiffExpression.pl in HOMER, which uses the 1066

DESeq2 R package to calculate the biological variation within replicates. Cut-offs were 1067

set at log2 FC = 0.585 and FDR at 0.05 (Benjamin-Hochberg). Principal Component 1068

Analysis (PCA) was performed with the mean of transcript per million (TPM) values 1069

using Cluster 3.0 with the following filter parameters: at least one observation with 1070

absolute value equal or greater than two and gene vector of four. TPM values were log 1071

transformed then centered on the mean. 1072

1073

Gene Set Enrichment Analysis 1074

GSEA software(Mootha et al., 2003; Subramanian et al., 2005) was used to perform the 1075

analyses with the following parameters: number of permutations = 1000; enrichment 1076

statistic = weighted; and metric for ranking of genes = difference of classes (Input RNA-1077


https://doi.org/10.1101/2020.02.26.964981

49

seq data was log-transformed). For Figure 5G, input RNA-seq data contained the 1078

normalized log-transformed reads of the 1,325 differentially expressed genes (DEGs) in 1079

sgFoxp3/sgNT Tregs. The compiled gene list included GSEA Gene Ontology, 1080

Immunological Signature, Curated Gene, and the up- and down-regulated DEGs in 1081

sgBrd9/sgNT Tregs. The resulting normalized enrichment scores and FWER p values 1082

were combined to generate the graph. 1083

1084

ChIP-seq sample preparation 1085

Treg cells were collected and cross-linked first in 3 mM disuccinimidyl glutarate (DSG) 1086

in 1X PBS for thirty minutes then in 1% formaldehyde for another ten minutes, both at 1087

RT, for chromatin binding protein ChIP or in 1% formaldehyde only for histone 1088

modification ChIP. After quenching the excess cross-linker with a final concentration of 1089

125 mM glycine, the cells were washed in 1X PBS, pelleted, flash-frozen in liquid 1090

nitrogen, and stored at -80°C. Cell pellets were thawed on ice and incubated in lysis 1091

solution (50 mM HEPES-KOH pH 8, 140 mM NaCl, 1 mM EDTA, 10% glycerol, 0.5% 1092

NP40, 0.25% Triton X-100) for ten minutes. The isolated nuclei were washed with wash 1093

solution (10 mM Tris-HCl pH 8, 1 mM EDTA, 0.5 mM EGTA, 200 mM NaCl) and 1094

shearing buffer (0.1% SDS, 1 mM EDTA, 10 mM Tris-HCl pH 8) then sheared in a 1095

Covaris E229 sonicator for ten minutes to generate DNA fragments between ~ 200-1096

1000 base pairs (bp). After clarification of insoluble material by centrifugation, the 1097

chromatin was immunoprecipitated overnight at 4°C with antibodies against Foxp3, 1098

SMARCA4, BRD9, PHF10 or H3K27ac. The next day, the antibody bound DNA was 1099

incubated with Protein A+G Dynabeads (Invitrogen) in ChIP buffer (50 mM HEPES-1100


https://doi.org/10.1101/2020.02.26.964981

50

KOH pH 7.5, 300 mM NaCl, 1 mM EDTA, 1% Triton X-100, 0.1% DOC, 0.1% SDS), 1101

washed and treated with Proteinase K and RNase A. Cross-linking was reversed by 1102

incubation at 55°C for two and a half hours. Purified ChIP DNA was used for library 1103

generation (NuGen Ovation Ultralow Library System V2) according to manufacturer’s 1104

instructions for subsequent sequencing. 1105

1106

ChIP-seq analysis 1107

Single-end 50 bp or paired-end 42 bp reads were aligned to mouse genome mm10 1108

using STAR alignment tool (V2.5)(Dobin et al., 2013). ChIP-Seq peaks were called 1109

using findPeaks within HOMER using parameters for histone (-style histone) or 1110

transcription factor (-style factor) (Christopher Benner, HOMER, 1111

http://homer.ucsd.edu/homer/index.html, 2018). Peaks were called when enriched > two-1112

fold over input and > four-fold over local tag counts, with FDR 0.001 (Benjamin-1113

Hochberg). For histone ChIP, peaks within a 1000 bp range were stitched together to 1114

form regions. ChIP-Seq peaks or regions were annotated by mapping to the nearest 1115

TSS using the annotatePeaks.pl command. Differential ChIP peaks were found by 1116

merging peaks from control and experiment groups and called using 1117

getDiffExpression.pl with fold change ≥ 1.5 or ≤ -1.5, Poisson p value < 0.0001. 1118

1119

Motif analysis 1120

Sequences within 200 bp of peak centers were compared to motifs in the HOMER 1121

database using the findMotifsGenome.pl command using default fragment size and 1122

motif length parameters. Random GC content-matched genomic regions were used as 1123


https://doi.org/10.1101/2020.02.26.964981

51

background. Enriched motifs are statistically significant motifs in input over background 1124

by a p-value of less than 0.05. P-values were calculated using cumulative binomial 1125

distribution. 1126

1127

ATAC-seq sample preparation 1128

ATAC-seq was performed according to previously published protocol(Buenrostro et al., 1129

2013). Briefly, 50,000 Treg cells were collected in duplicates and washed first with cold 1130

1X PBS then with Resuspension buffer (RSB; 10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 1131

mM MgCl2). Cells were lysed in RSB supplemented with 0.1% Igepal-630 and nuclei 1132

were isolated by centrifugation at 500 x g for ten minutes. Nuclei were incubated with 1133

Tn5 transposase in Tagment Buffer (Illumina) for thirty minutes at 37°C. Purified DNA 1134

was ligated with adapters, amplified and size selected using AMPure XP beads 1135

(Beckman) for sequencing. Library DNA was sequenced using paired end 42 bp reads. 1136

1137

ATAC-seq analysis 1138

Paired end 42 bp reads were aligned to mouse genome mm10 using STAR alignment 1139

tool (V2.5). ATAC-seq peaks were called using findPeaks within HOMER using the style 1140

parameter dnase. Peaks were called when enriched > four-fold over genomic 1141

background and > four-fold over local tag counts, with FDR 0.001 (Benjamin-Hochberg). 1142

1143


https://doi.org/10.1101/2020.02.26.964981

Day 0

Cas9 TregsIsolation

Pooled sgRNAsretroviral infectionMOI < 0.2

1 2 3

CollectingTransduced T cells(sgRNAs Input)

Refreshingmedia &reactivation

CollectingTransduced T cells(sgRNAs Output)

Fix/Perm andFoxp3 staining

4 5 6

Foxp3

Low

High

IL2

A

sgNT sgFoxp3 sgCbfb sgDnmt1

Foxp3

B

sgNT

sgFoxp

3

sgCbfb

sgDnmt1

0

200

400

600

800

MFI

Fox

p3

C

sgNT

sgFoxp

3

sgCbfb

sgDnmt1

-4

-2

0

2

4

6

8

Log2

FC o

f cel

l cou

nt

Loo, et al. Figure 1

D


https://doi.org/10.1101/2020.02.26.964981


A B

C

2.40E-07

2.40E-07

0.00273

0.06094

Foxp3Low Vs Foxp3High

EnrichedDepleted

0.0

0.5

1.0

−10 −5 0 5 10Log2FC [sgRNA]

0.00798

0.00758

P-value

Foxp3

Cbfb

Runx3

Usp7

Dnmt1

Stub1

163

1278

56

3271971

21Expansion

Contraction

NegativePositive

D

0 4 8 12 16

protein polyubiquitinationchromosome, telomeric region

dendritic cell differentiationregulation of DNA-binding transcription factor activity

nuclear receptor transcription coactivator activitySWI/SNF complex

regulation of histone acetylationRNA polymerase II, holoenzyme

negative regulation of T cell activationSAGA-type complex

-Log10(P)

E

-10 -8 -6 -4 -2 0

mRNA metabolic processforebrain radial glial cell differentiationpositive regulation of transferase activitycellular response to hormone stimulusprotein export from nucleusregulation of small molecule metabolic processHypusine synthesis from eIF5A-lysineDeadenylation-dependent mRNA decaytranscriptional repressor complexnegative regulation of TOR signaling

Log10(P)

F Negative Foxp3 regulator

Positive Foxp3 regulator

Il2ra

Commd4Tbx21

Slc39a7

Tox4 Runx3

Taf5l

Zap70

Gtf2a2

Rc3h1 Taf11

Rrm1BatfJak1

Ccdc101

Brd9

Usp22

Cbfb

Ssu72SympkUbe2m

Tada1Smarcd1Wdr82Foxp3

Log2 Fold Change-4 -2 0 2 4 6

6

4

2

0

-Log

10P

Positive Foxp3 regulator (254 hits)

6

4

2

0

-Log

10P

Cnot11Bcor Ccdc84

Dhps Cnot10Mettl14

Aip Stk11Wdr25

Dbr1Edrf1Drap1

Prpf19DohhDdit4 Snrnp40 Usp5

Otud5 Eif5a Rps3Mettl3 Rpl19

Rpl32 Defa2

4 2 0 -2 -4 -6Log2 Fold Change

Negative Foxp3 regulator (490 hits)


https://doi.org/10.1101/2020.02.26.964981


A

CInputIgG SMARCA4

ARID1A

BRD9PHF10

IP for BAF subunits

SMARCA4

SMARCC1

SMARCB1

ARID1A

BRD9

PBRM1

sgArid1a sgArid1b

sgBrd9 sgSmarcd1

sgBrd7 sgArid2 sgPbrm1

Foxp3

BAF

ncBAF

PBAF

sgPhf10

sgGltscr1 sgGltscr1l sgGltscr1/1l

sgDpf1 sgDpf2sgArid1a/b

****

n.s.

n.s.

*******

****

**** ****

n.s.n.s. n.s.

Nor

mal

ized

Fox

p3 M

FI

****

ARID1AARID1B

A4A2

C1 C2ACTB

ACTL6A

B1

E1D1-D3SS18

DPF1-3

Bcl7a-c

Bcl11a,b

BAF

D1

SS18A4A2

C1 C1ACTB

ACTL6A

Bcl7b,c

BRD9

GLTSCR1LGLTSCR1

ncBAF

BRD7

ARID2

A4A2

C1 C2ACTB

ACTL6A

E1D1-D3

PHF10

PBAF

PBRM1B1

-60 -40 -20 0 20 40

-20

0

20

40

PC1 (20.3%)

PC2

(11.

5%)

NT

Arid1aArid1a-2

Arid1a/bArid1a-2/b-2

Dpf2Dpf1/2

Smarcd1Brd9

Brd9-2Gltscr1 Gltscr1/Gltscr1l

Pbrm1

Pbrm1-2

Brd7

Phf10Arid2

D

B

PCA of transcriptomic changes in Tregs

E

sgNT

sgArid

1a

sgArid

1b

sgArid

1a/1b

sgDpf1

sgDpf2

sgDpf1/

2

sgBrd

9

sgSmarc

d1

sgGlts

cr1

sgGlts

cr1l

sgGlts

cr1/1l

sgBrd

7

sgArid

2

sgPbrm

1

sgPhf10

0.0

0.5

1.0

1.5

2.0

*n.s.

****

0

2000

4000

6000

8000

Foxp

3 M

FI

DMSO

0.16u

M

0.31u

M

0.63u

M

1.25u

M2.5

uM5u

M10

uM

dBRD9


https://doi.org/10.1101/2020.02.26.964981


C

Rea

d de

nsity

cen

tere

d on

Fox

p3 p

eaks

(n =

11,

287)

sgNT sgFoxp3 sgBrd9 sgPbrm1

-3 kb 3 kb

0 1-10

4

8

12

distance from peakcenter (kb)

0 1-1distance from peak

center (kb)

048

1612

sgNTsgFoxp3sgBrd9

Sites with decreased Foxp3 binding

Foxp

3 C

hIP-

seq

dens

ity

H3K27ac at BRD9-dependentFoxp3 target sites

H3K

27ac

ChI

P-se

qde

nsity

sgPbrm1

BRD9-dependent Foxp3target sites

J

sgNTsgFoxp3sgBrd9sgPbrm1

Foxp3 binding

Down insgFoxp3n = 9,730

Down insgBrd9

n = 1,809sgNT, n = 11,287

1,699

B

sgFoxp3n = 10,609

Overlap of H3K27ac down sites

I

sgBrd9n = 2,880

sgNT, n = 25,678

1,847

966

8,548

Foxp3

Rea

d de

nsity

cen

tere

d on

Fo

xp3

peak

s (n

= 2

0,09

6)

BRD9SMARCA4

PHF10ATAC

-3 kb 3 kb

D

F

G H

E

Decreased Foxp3binding with dBRD9

Down indBRD9

n = 7,245

DMSO, n = 17,135

7,154

0

16

FOXP

3 C

hIP-

seq

dens

ity

BRD9-dependent Foxp3sites

0 1-1distance from peak

center (kb)

DMSOdBRD9

4

8

12

ConservationFo

xp3

sgNTsgFoxp3

sgBrd9sgPbrm1

ATACSMARCA4

BRD9PHF10

DMSOdBRD9

Ppp1r3fFlicr

Foxp3Ccdc22

1.6 _0.7 _1.5 _0.6 _1.8 _1.8 _1.8 _1.8 _2.1 _2.1 _

CNS0 CNS2

A

Overlap of binding sites, nTreg

BRD9n = 21,916

FOXP3n = 20,096

PHF10n = 6,791

3,362

7,00110,565 8,763

9861,475

968


https://doi.org/10.1101/2020.02.26.964981


A C

G

-10 -5 0 5 10

10-300

10-250

10-200

10-150

10-100

10-50

100

sgFoxp3 vs. sgNT Log2 Fold Change

adj p

val

ue

Foxp3-dependent genes

n = 793 n = 532

Il21

Gbp2

Il5

Il13

Il4

Igfbp4 Ccr8

B

0 5 10 15 20 25-log10 of P value

regulation of MAPK cascadeangiogenesistaxispositive regulation of cell deathnegative regulation of catalytic activityregulation of innate immune responsecytokine biosynthetic processpositive regulation of locomotioncytokine-cytokine receptor interactionleukocyte differentiationregulation of cell adhesionregulation of defense responsecytokine production

GO Enrichment of Foxp3 DEGs Foxp3 and BRD9 binding at Foxp3-dependent genes

-10 -5 0 5 10-4-20246

Log2FC sgFoxp3/sgNT

Log2

FC s

gBrd

9/sg

NT

r = 0.5342

Foxp3-dependent genesD E

0 250 500 750 1000 1250

-0.8

-0.6

-0.4

-0.20.0

Rank in ordered data set

Run

ning

ES

Up in sgBrd9 Genes

sgNT (positive correlation)

sgFoxp3 (negative correlation)

0.0

0.2

0.4

0.6

0.8

Run

ning

ES

Down in sgBrd9 Genes

5.02.50.0

-5.0-2.5

Ran

ked

list

met

ric

0 250 500 750 1000 1250Rank in ordered data set



5.02.50.0

-5.0-2.5

Ran

ked

list

met

ric

NES = -3.30FWER p = 0

NES = 3.13FWER p = 0

Down in sgFoxp3/sgNT

Up in sgFoxp3/sgNT

9,229 gene lists

0 0.6 1.2FWER p value

Norm

. Enrichment Score

-4

-2

0

2

4

Down,dBRD9

Up, dBRD9

Down,sgBrd9

Up,sgBrd9

Gene Set Enrichment

Up, FOXP3 mutant

Down, FOXP3 mutant

Down, Tregvs Tcell

Down, CD8vs Treg

JAK STATsignaling

F

0 250 500 750 1000 1250

-0.6

-0.4

-0.2

0.0Up in dBrd9 Genes Down in dBrd9 Genes

Run

ning

ES

Rank in ordered data set

Ran

ked

list

met

ric

-4.0-6.0

-2.0

4.02.0

0

NES = -2.52FWER p = 0



0.00.10.20.30.40.5

NES = 2.25FWER p = 0R

unni

ng E

S

Foxp3 and BRD9

BRD9 only

Foxp3 only

Neither

81169272

803

0 250 500 750 1000 1250Rank in ordered data set

Ran

ked

list

met

ric

-4.0-6.0

-2.0

4.02.0

0sgNT (positive correlation)



https://doi.org/10.1101/2020.02.26.964981

sgNT sgBrd9 Teff only

150 uM 150 uM 150 uM

A


B C

D

sgNT sgBrd90

20

40

60

80

CD

45.2

Fox

p3+

%

sgNT sgBrd9

Foxp3

CD

4

sgNT

sgBrd9

Teff o

nly0

5

10

15

20

Colit

is sc

ore

0 1 2 3 4 5 6 7-20

-15

-10

-5

0

5

Weeks

Bod

y w

eigh

t cha

nge

(%)

sgNT

sgBrd9

Teff only

E

1:1 1:2 1:4 1:80

20

40

60

80

100

Treg:Teff ratioN

on-d

ivid

ing

Cel

ls (%

) sgNT

sgBrd9**

******

***

1:1 1:2 1:4 1:80

20

40

60

80

100

Treg:Teff ratio

Non

-div

idin

g C

ells

(%) sgNT

sgSmarcd1******

******

1:1 1:2 1:4 1:80

20

40

60

80

100

Treg:Teff ratio

Non

-div

idin

g C

ells

(%) sgNT

sgPbrm1******

ns

ns

1:1 1:2 1:4 1:80

20

40

60

80

100

Treg:Teff ratio

Non

-div

idin

g C

ells

(%) sgNT

sgPhf10ns

*****

**


https://doi.org/10.1101/2020.02.26.964981


0 8 10 12 14 16 180

200

400

600

800

1000

Day

Tum

or s

ize

(mm

3 )sgNT

sgBrd9

No Treg

*

**

*

sgNT

sgBrd

9

No Treg

0.0

0.5

1.0

1.5

Tum

or w

eigh

t (g)

Tumor Spleen0

20

40

60

80

100

CD

4+ IF

Nγ+

%

Tumor Spleen0

20

40

60

80

100

CD

8+ IF

Nγ+

%

sgNT

sgBrd9

No Treg

Tumor Spleen0

5

10

15

20

25

CD

8/Tr

eg ra

tio

sgNT

sgBrd9

B C

D E

F G

H

Tumor Spleen0

10

20

30

40

CD

4 of

CD

45+

(%)

Tumor Spleen0

10

20

30

40

50

CD

8 0f

CD

45+

(%)

I

A

Tumor Spleen0

5

10

15

CD

4+ e

GFP

+ Fo

xp3+

(%)

sgNT

sgBrd9

No Treg


https://doi.org/10.1101/2020.02.26.964981

Documents

A genome-wide CRISPR screen reveals a role for the BRD9 ... · 2 15 Summary 16 Regulatory T cells (Tregs) play a pivotal role in suppressing auto-reactive T cells 17 and maintaining