Interplay of BAF and MLL4 Promotes Cell Type-Specific Enhancer Activation

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bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

1 Interplay of BAF and MLL4 promotes cell type-specific enhancer activation

3 Young-Kwon Park1,4, Ji-Eun Lee1,4, Tommy O’Haren1, Kaitlin McKernan1, Zhijiang Yan2, Weidong Wang2,

4 Weiqun Peng3, and Kai Ge1,*

6 1Adipocyte Biology and Gene Regulation Section, National Institute of Diabetes and Digestive and Kidney

7 Diseases, National Institutes of Health (NIH), Bethesda, MD 20892

8 2Laboratory of Genetics and Genomics, National Institute on Aging, NIH, Baltimore, MD 21224

9 3Department of Physics and Department of Anatomy and Cell Biology, The George Washington

10 University, Washington, DC 20052

11 4These authors contributed equally

13 *To whom correspondence should be addressed. (Email: [email protected])

14 Abstract

15 Cell type-specific enhancers are activated by coordinated actions of lineage-determining transcription

16 factors (LDTFs) and chromatin regulators. The SWI/SNF complex BAF and the histone H3K4

17 methyltransferase MLL4 (KMT2D) are both implicated in enhancer activation. However, the interplay

18 between BAF and MLL4 in enhancer activation remains unclear. Using adipogenesis as a model system,

19 we identified BAF as the major SWI/SNF complex that colocalizes with MLL4 and LDTFs on active

20 enhancers and is required for cell differentiation. In contrast, the promoter enriched SWI/SNF complex

21 PBAF is dispensable for adipogenesis. By depleting BAF subunits SMARCA4 (BRG1) and SMARCB1

22 (SNF5) as well as MLL4 in cells, we showed that BAF and MLL4 reciprocally regulate each other’s

23 binding on active enhancers before and during adipogenesis. By focusing on enhancer activation by the

24 adipogenic transcription factor C/EBPβ without inducing cell differentiation, we provide direct evidence for

25 an interdependent relationship between BAF and MLL4 in activating cell type-specific enhancers.

26 Together, these findings reveal a positive feedback between BAF and MLL4 in promoting LDTF-

27 dependent activation of cell type-specific enhancers.

28 Introduction

29 Throughout development, chromatin architecture undergoes dynamic changes that are critical for

30 promoting appropriate cell type-specific enhancer activation and gene expression. These changes are

31 coordinated by the action of transcription factors (TFs) and epigenomic regulators, including ATP-

32 dependent chromatin remodeling complexes, which modulate chromatin accessibility and gene

33 expression through the mobilization of nucleosomes1,2. The large multi-subunit SWItch/Sucrose Non-

34 Fermentable (SWI/SNF) chromatin remodeling complexes have been shown to play a crucial role in

35 cellular and tissue development1. SWI/SNF mutations are also frequently associated with human

36 diseases, including over 20% of cancers and a variety of neurodevelopmental disorders3,4.

37 Mammalian SWI/SNF complexes exist in three different isoforms with distinct subunit

38 compositions: BRG1/BRM-associated factor (BAF), polybromo-associated BAF (PBAF), and non-

39 canonical GLTSCR1L-containing BAF (GBAF, also known as ncBAF)5,6. All three complexes contain an

40 ATPase catalytic subunit, either SMARCA4 (BRG1) or SMARCA2 (BRM), and an initial core, composed

41 of SMARCC1 (BAF155), SMARCC2 (BAF170), and SMARCD1/D2/D3 (BAF60A/B/C)5,7. The BAF

42 complex contains SMARCB1 (SNF5, INI1, or BAF47), SMARCE1 (BAF57), and SS18 and BAF-specific

43 subunits ARID1A/B (BAF250A/B) and DPF1/2/3 (Fig 1A). Like BAF, PBAF complex also consists of the

44 initial core, SMARCB1 and SMARCE1 as well as PBAF-specific subunits PHF10, ARID2 (BAF200),

45 BRD7, and PBRM1. The recently characterized GBAF is a smaller complex containing SMARCA4 or

46 SMARCA2, the initial core, SS18, and GBAF-specific subunits GLTSCR1/L and BRD95-7. These three

47 distinct complexes exhibit different genomic localization patterns. BAF primarily binds on enhancers8-10.

48 Conversely, PBAF is more localized on promoter regions, and GBAF binding sites are enriched with the

49 CTCF motif11,12.

50 Enhancers are cis-acting regulatory regions containing TF binding sites and are responsible for

51 facilitating cell type-specific gene expression through communication with promoters13. Enhancers are

52 marked by H3K4 mono-methylation (H3K4me1), which is primarily placed by the partially redundant H3K4

53 methyltransferases MLL3 (KMT2C) and MLL4 (KMT2D)14,15. Along with H3K4me1, active enhancers are

54 further marked with histone acetyltransferases CBP/p300-catalyzed H3K27 acetylation (H3K27ac)16,17.

55 MLL3 and MLL4 are required for enhancer activation and cell type-specific gene expression during cell

56 differentiation14. Specifically, MLL3/MLL4 controls enhancer activation by facilitating CBP/p300 binding on

57 enhancers18. MLL3 and MLL4 are essential for embryonic development as well as the development of

58 multiple tissues, including adipose and muscle14,19,20.

59 Of the three SWI/SNF complexes, BAF primarily interacts with enhancer regions. Recent studies

60 have demonstrated that BAF is required for enhancer activation8-10. Following the deletion of BAF

61 catalytic subunit SMARCA4 and core subunit SMARCB1, H3K27ac levels on enhancers are decreased,

62 and enhancers fail to be activated. The deletion of SMARCB1 results in the destabilization of the BAF

63 complex on the genome9. BAF-specific subunit ARID1A also plays a major role in BAF-mediated

64 enhancer activation. Particularly, the loss of ARID1A has been shown to diminish BAF complex

65 occupancy on enhancers21. SMARCA4 interacts with LDTFs including the adipogenic pioneer factor

66 C/EBPβ and is required for adipogenesis22-25. Although the BAF complex plays an essential role in

67 enhancer activation and development, many questions remain regarding how it interacts with other

68 epigenomic regulators during enhancer activation.

69 In this study, we evaluated the interrelationship between BAF and MLL4 on cell type-specific

70 enhancers using adipogenesis as a model system. Loss of SWI/SNF subunits in cells and mice provides

71 evidence that BAF, but not PBAF, is critical for enhancer activation, adipogenesis and adipocyte gene

72 expression. Given that MLL4 is similarly important for enhancer activation, cell differentiation and cell

73 type-specific gene expression, we investigated the relationship between BAF and MLL4 before and

74 during adipogenesis, revealing that BAF and MLL4 colocalize with LDTFs on active enhancers and

75 facilitate enhancer activation in an interdependent manner. We further uncover the reciprocal regulation

76 between BAF and MLL4 in adipogenic TF C/EBPβ-mediated enhancer activation.

77 Results

78 Expression and genomic binding of SWI/SNF subunits in adipogenesis

79 To explore the role of each SWI/SNF complex in cell differentiation and cell type-specific

80 enhancer activation, we used differentiation of preadipocytes towards adipocytes (adipogenesis) as a

81 model system. We first explored the expression profiles of SWI/SNF subunits at four key time points in

82 adipogenesis: actively growing preadipocytes (day -3, D-3), over-confluent preadipocytes before the

83 induction of differentiation (day 0, D0), immature adipocytes during differentiation (day 2, D2), and

84 adipocytes after differentiation (day 7, D7)18. By RNA-Seq analysis, we found that all SWI/SNF subunits

85 except Dpf3, a BAF-specific subunit, were expressed in adipogenesis. The expression levels of SWI/SNF

86 subunits were largely invariant from D-3 to D7. Among the two enzymatic subunits, Smarca4 was

87 expressed at significantly higher levels than Smarca2 throughout the differentiation (Fig 1B).

88 Next, we performed ChIP-Seq analyses to profile genomic localizations of BAF, PBAF, and GBAF

89 complexes before (D-3) and during (D2) adipogenesis. We chose the enzymatic subunit SMARCA4,

90 SS18, BAF-specific subunit ARID1A, PBAF-specific subunit ARID2 and GBAF-specific subunit BRD9.

91 Each subunit exhibited a differentiation stage-specific genomic binding (Fig 1-S1A). ChIP-Seq identified

92 distinct genomic distribution of each subunit in four types of regulatory elements: active enhancer (AE),

93 primed enhancer, promoter and other regions defined as in our previous report14. SMARCA4 binding sites

94 were distributed in all types of regulatory elements, but primarily on primed enhancers and AEs. While

95 SS18 and ARID1A binding sites were mainly located on AEs, PBAF-specific ARID2 was strongly enriched

96 on promoter regions especially during adipogenesis. GBAF-specific BRD9 binding sites were enriched on

97 AEs and other regions (Fig 1C). By motif analysis of the top 3,000 binding sites of each subunit, we found

98 that SMARCA4, SS18 and ARID1A binding regions were enriched with motifs of AP-1 family TFs Jdp2,

99 JunD and Jun in preadipocytes (D-3), but with motifs of adipogenic TFs such as C/EBPα, C/EBPβ, and

100 ATF4 during adipogenesis (D2). ARID2 and BRD9 binding regions were selectively enriched with motifs

101 of ETS family TFs and CTCF, respectively (Fig 1D). These results demonstrate the distinct targeting of

102 each complex in adipogenesis: BAF to enhancers, PBAF to promoters and GBAF to CTCF sites.

103 Further, we defined confident genomic binding regions of BAF (16,003), PBAF (2,063) and GBAF

104 (1,053) at D2 of adipogenesis by selecting overlapping binding sites between SMARCA4 and complex-

105 specific subunits (Fig 1E and Fig 1-S1B). Even though SS18 was reported as a common subunit in BAF

106 and GBAF complexes, the majority of SS18 genomic binding sites (20,257/22,118) overlapped with those

107 of BAF-specific ARID1A, and only a small fraction (791/22,118) overlapped with those of GBAF-specific

108 BRD9. Among 17,165 SWI/SNF-associated SMARCA4 binding regions, BAF (16,003) exhibited

109 substantially greater genomic occupancy than PBAF (2,063) and GBAF (1,053) during adipogenesis (Fig

110 1-S1B). Only a small subset (1,504/16,003, 9.4%) of BAF binding sites overlapped with those of PBAF or

111 GBAF (Fig 1F). These findings suggest that among the three SWI/SNF complexes, BAF is the major

112 regulator of enhancers in adipogenesis.

113

114 BAF, but not PBAF, is required for adipogenesis

115 To address the functions of SWI/SNF complexes in adipogenesis, we first depleted endogenous

116 SMARCA4, the catalytic ATPase subunit, using the auxin-inducible degron (AID) system26. Primary

117 preadipocytes were isolated from newborn pups of Smarca4AID/AID mice, which were generated using

118 CRISPR-mediated insertion of an AID tag in the C-terminus of endogenous Smarca4 alleles. Cells were

119 immortalized and infected with a retroviral vector expressing the Myc-tagged auxin receptor Tir1. In the

120 presence of auxin, Tir1 binds to the AID tag and induces proteasome-dependent degradation of

121 endogenous SMARCA4 (Fig 2A). As shown in Fig. 2B, auxin induced a rapid Tir1-dependent depletion of

122 endogenous SMARCA4 protein within 4h. Depletion of SMARCA4 prevented the appearance of lipid

123 droplets, indicating that SMARCA4 is essential for adipogenesis in cell culture (Fig 2C).

124 Next, we examined the role of SMARCB1 in adipogenesis. We generated a conditional knockout

125 (cKO) of SMARCB1 in preadipocytes by crossing Smarcb1f/f mice with PdgfRα-Cre mice. The PdgfRα-

126 Cre transgene is expressed in most adipocyte precursor cells27. Smarcb1 cKO mice (Smarcb1f/f;PdgfRα-

127 Cre) died shortly after birth, presumably due to severe defects in cranial development (Fig 2-S1A-D).

128 Immunohistochemical analysis revealed that deletion of Smarcb1 led to a severe reduction of

129 interscapular brown adipose tissue (BAT) in E18.5 embryos (Fig 2D and Fig 2-S1E). Consistent with this

130 phenotype, deletion of Smarcb1 reduced the expression of adipogenesis markers Pparg, Cebpa, and

131 Fabp4 as well as BAT markers Prdm16 and Ucp1, but not myogenesis markers (Fig 2-S1F). These

132 findings indicate that SMARCB1 is required for adipose tissue development in mice.

133 To understand how SMARCB1 regulates adipogenesis, we isolated and immortalized Smarcb1f/f

134 preadipocytes. Deletion of Smarcb1 in immortalized preadipocytes did not affect growth rates (Fig 2E).

135 Consistent with previous data9, deletion of Smarcb1 did not affect the expression of SMARCA4 nor the

136 interaction between SMARCA4 and core subunit SMARCC2 (Fig 2F and Fig 2-S1G). However, deletion

137 of Smarcb1 blocked adipogenesis (Fig 2G). We performed RNA-Seq analysis before (D-3) and during

138 (D2) adipogenesis. Using a 2.5-fold cut-off for differential expression, we defined 937 and 1,382 genes

139 that were up- and down-regulated from D-3 to D2 of differentiation (Fig 2-S1H). Among the 937 up-

140 regulated genes, 362 were induced in a SMARCB1-dependent manner. GO analysis showed that these

141 genes were strongly associated functionally with fat cell differentiation and lipid metabolism (Fig 2-S1I).

142 Deletion of Smarcb1 did not affect the induction of pioneer TF C/EBPβ or the expression of other

143 SWI/SNF subunits, but blocked the induction of adipocyte marker genes Pparg, Cebpa, and Fabp4 (Fig

144 2F, 2H and Fig 2-S1J), suggesting that SMARCB1 is required for the induction of adipocyte genes

145 downstream of C/EBPβ.

146 Because SMARCA4 and SMARCB1 are common subunits in BAF and PBAF, we wanted to

147 clarify the function of each complex in adipogenesis. To determine the role of BAF in adipogenesis, we

148 infected preadipocytes with lentiviral shRNA targeting BAF-specific subunit ARID1A. Knockdown of

149 ARID1A did not affect SMARCA4 and SMARCB1 expression (Fig 2I), but it severely impaired

150 adipogenesis (Fig 2J-K). To study the role of PBAF in adipogenesis, we generated a cKO of PBAF-

151 specific subunit PBRM1 in precursor cells of brown adipocytes by crossing Pbrm1f/f mice with Myf5-Cre

152 mice. Myf5-Cre is specifically expressed in somitic precursor cells for both BAT and skeletal muscle in the

153 interscapular region of mice14. Pbrm1f/f;Myf5-Cre (cKO) pups were born at the expected Mendelian ratio

154 and survived to adulthood without obvious developmental defects (Fig 2-S2A-B). The adipose tissues

155 isolated from cKO mice were similar in size and morphology to the control mice (Fig 2-S2C-D). Although

156 the Pbrm1 gene level was reduced by over 80% in the BAT of cKO mice, deletion of Pbrm1 did not

157 significantly affect the expression of adipogenesis markers Pparg, Cebpa, and Fabp4 or BAT marker

158 Ucp1 (Fig 2-S2E). To further understand the role of PBRM1 in adipogenesis, we induced differentiation of

159 primary brown preadipocytes isolated from cKO mice. Deletion of Pbrm1 by Myf5-Cre had little effects on

160 adipogenesis in culture (Fig 2-S2F-G). By RNA-Seq analysis at D7 of adipogenesis, we found that only a

161 small number of genes were increased (0.4%) or decreased (0.4%) over 2.5-fold in Pbrm1 cKO cells

162 compared with control cells (Fig 2-S2H). Differentially regulated genes were not associated with

163 adipocyte differentiation or lipid metabolism (Fig 2-S2I-J). Taken together, these findings suggest that

164 BAF, but not PBAF, is essential for adipogenesis.

165

166 BAF co-localizes with LDTFs and MLL4 on cell type-specific enhancers during adipogenesis

167 To assess the genomic binding of BAF, we first selected SMARCA4+ AEs (31,349) or promoters

168 (8,871), then examined changes in genomic binding of BAF subunits SS18 and ARID1A before (D-3) and

169 during (D2) adipogenesis. While genomic binding of the common ATPase subunit SMARCA4 was found

170 on both AEs and promoters, genomic bindings of SS18 and ARID1A were mainly localized on AEs and

171 increased from D-3 to D2 (Fig 3A). Motif analysis of the top 3,000 BAF-bound (SMARCA4+ SS18+) AEs

172 showed enrichment of AP-1 family TF motifs at D-3 and motifs of adipogenic TFs C/EBPs and ATF4 at

173 D2 (Fig 3B and Fig 3-S1A-C). These results indicate that BAF is enriched on cell type-specific enhancers

174 during adipogenesis.

175 To obtain insights into how LDTFs coordinate with BAF on AEs, we analyzed the genomic

176 localization of adipogenic TFs C/EBPβ, C/EBPα and PPARγ, which are sequentially induced to activate

177 adipocyte-specific genes in adipogenesis28,29. Among the 10,813 BAF-bound AEs during adipogenesis

178 (D2), BAF was already bound to 4,410 of them in preadipocytes (D-3) and maintained the binding at D2

179 (BAF-prebound AEs). In contrast, 6,403 AEs exhibited emergent BAF binding at D2 but not at D-3 (BAF-

180 de novo AEs) (Fig 3C). Consistent with motif analysis, BAF was highly co-localized with C/EBPβ, C/EBPα

181 and PPARγ on AEs at D2 of adipogenesis. Particularly, C/EBPβ binding was detected on BAF-prebound

182 AEs at D-3 but increased markedly from D-3 to D2 on BAF-de novo AEs (Fig 3D-E). Since MLL4 and

183 CBP also colocalize with LDTFs on cell type-specific AEs during adipogenesis18, we assessed the co-

184 occupancy among BAF, MLL4 and CBP. We found that MLL4 and CBP co-localized with BAF on both

185 BAF-prebound and BAF-de novo AEs. While MLL4 and CBP binding on BAF-prebound AEs remained

186 largely unchanged from D-3 to D2 of adipogenesis, their binding on BAF-de novo AEs was induced.

187 Accordingly, the levels of MLL3/MLL4-catalyzed H3K4me1 and CBP/p300-catalyzed H3K27ac also

188 increased on BAF-de novo AEs from D-3 to D2 (Fig 3D-E). Colocalization of BAF with LDTFs, MLL4 and

189 CBP on BAF-de novo AEs was also found on Pparg and Cebpa gene loci during adipogenesis (Fig 3-

190 S1D-E). ATAC-Seq analysis revealed that chromatin accessibility was highly correlated with BAF binding

191 intensities on AEs during adipogenesis. The induction of chromatin accessibility on BAF-de novo AEs

192 coincided with the increase in the binding of LDTFs, MLL4 and CBP on AEs (Fig 3D-E and Fig 3-S1D-E).

193 These results indicate that BAF colocalizes with LDTFs, MLL4 and CBP on cell type-specific enhancers

194 and promotes chromatin accessibility during differentiation.

195 At D2 of adipogenesis, about 40% (4,362/10,813) of BAF-bound AEs were occupied by MLL4

196 (Fig 3-S2A). Differential motif analysis of BAF-bound AEs revealed that motifs of LDTFs PPARγ and

197 C/EBPs were enriched on MLL4+ AEs while motifs of the AP-1 family TFs were enriched on MLL4- AEs at

198 D2 (Fig 3-S2B). Heat maps and average profiles also showed that from D-3 to D2 of adipogenesis, the

199 binding of LDTFs and BAF subunits (SMARCA4, SS18 and ARID1A) was more significantly increased on

200 MLL4+ than MLL4- AEs. Particularly, levels of CBP and H3K27ac were only induced on MLL4+ AEs (Fig 3-

201 S2C and D). D2 MLL4- AEs were already accessible before adipogenesis (D-3). In contrast, chromatin

202 accessibility on D2 MLL4+ AEs was dramatically increased from D-3 to D2 (Fig 3-S2C and D). These

203 results suggest the involvement of MLL4 in enhancer activation by BAF.

204 To test the possibility of physical interaction between BAF and MLL4, we identified proteins

205 associated with endogenous MLL4 and UTX, a subunit of the MLL4 complex, in mouse embryonic stem

206 cells (ESCs) by mass spectrometry. Consistent with previous results30, while immunoprecipitation with an

207 anti-UTX antibody pulled down both MLL3 and MLL4 as well as other components of MLL3/MLL4

208 complexes, MLL3 was not co-immunoprecipitated by an anti-MLL4 antibody. Notably, several SWI/SNF

209 subunits, including SMARCA4, SMARCC1, SMARCD1, SMARCB1 and SMARCE1, were also pulled

210 down by anti-MLL4 and/or anti-UTX antibody from ESC nuclear extracts (Fig 3-S3A). We also confirmed

211 that endogenous SMARCA4 and SMARCB1 are associated with endogenous MLL4 and UTX, but not the

212 MLL1 complex subunit Menin, in HEK293T cells and preadipocytes at D2 of adipogenesis (Fig 3-S3B-C).

213 Together, these findings suggest that BAF cooperates with MLL4 to activate cell type-specific enhancers

214 during differentiation.

215

216 Reciprocal regulation between BAF and MLL4 on active enhancers during adipogenesis

217 To study the relationship between BAF and MLL4 on cell type-specific enhancers, we assessed

218 their genomic binding on adipogenic enhancers at D2 of differentiation. Adipogenic enhancers were

219 defined as AEs that are bound by C/EBPs or PPARγ (Fig 4A). This set of enhancers are mostly de novo.

220 We first asked whether MLL4 is required for BAF recruitment to adipogenic enhancers during

221 differentiation. To eliminate the compensatory effect of MLL3, we deleted Mll4 in Mll3−/−Mll4f/f cells14.

222 ChIP-Seq revealed that deletion of Mll4 prevented genomic binding of SMARCA4 and SS18 on

223 adipogenic enhancers. Consistently, ATAC-Seq showed that chromatin accessibility at these enhancers

224 was also significantly reduced in Mll4 KO cells (Fig 4A-B). Reduced binding of BAF on adipogenic

225 enhancers in Mll4 KO cells was confirmed on Pparg and Fabp4 gene loci (Fig 4-S1A-B).

226 We next evaluated whether BAF contributes to the genomic binding of MLL4 on adipogenic

227 enhancers during differentiation. ChIP-Seq and ATAC-Seq revealed that Smarcb1 deletion in

228 preadipocytes reduced genomic binding of SMARCA4, SS18 and ARID1A, as well as chromatin

229 accessibility, on adipogenic enhancers at D2 of adipogenesis (Fig 4C and 4E). Moreover, deletion of

230 Smarcb1 reduced binding of MLL4 and CBP on adipogenic enhancers, indicating that BAF is required for

231 the activation of cell type-specific enhancers (Fig 4D-E). H3K4me1 and H3K27ac levels also decreased

232 on adipogenic enhancers in Smarcb1 KO cells, although global levels of these enhancer marks were

233 unchanged (Fig 4F). Reduced binding of MLL4 on adipogenic enhancers in Smarcb1 KO cells was

234 confirmed on the Pparg gene locus (Fig 4-S1C). Together, these findings reveal reciprocal regulation

235 between BAF and MLL4 on adipogenic enhancers and suggest that BAF and MLL4 coordinate to activate

236 cell type-specific enhancers during differentiation.

237

238 BAF and MLL4 reciprocally promote each other’s binding to maintain active enhancers in

239 preadipocytes

240 Because we observed interdependent genomic binding of BAF and MLL4 during adipogenesis,

241 we next wondered whether such a relationship is also present in undifferentiated preadipocytes. To

242 examine the genomic association of BAF and MLL4 on AEs in undifferentiated cells, we identified 6,304

243 BAF+ MLL4+ AEs that were shared by Mll3-/-Mll4f/f and Smarcb1f/f preadipocytes. Deletion of Mll4 in Mll3-/-

244 Mll4f/f preadipocytes reduced ARID1A binding and H3K27ac levels, but not SMARCA4 binding, on BAF+

245 MLL4+ AEs in preadipocytes (Fig 5A-B). Next, we investigated whether BAF is required for maintaining

246 MLL4 binding on AEs in preadipocytes. While deletion of Smarcb1 had little effect on maintaining

247 genomic binding of SMARCA4 or chromatin accessibility, it reduced MLL4 binding on BAF+ MLL4+ AEs

248 (Fig 5C-D). Decreased MLL4 binding on BAF+ MLL4+ AEs was also observed in SMARCA4-depleted

249 preadipocytes in addition to widespread reduction in chromatin accessibility (Fig 5E-F). Together, these

250 data suggest a reciprocal regulation between BAF and MLL4 in promoting their bindings on AEs in

251 undifferentiated cells.

252

253 MLL4 is required for BAF binding on C/EBPβ-activated enhancers

254 Interdependent genomic binding of BAF and MLL4 on AEs during adipogenesis could be due to

255 indirect consequences of differentiation defect and failed induction of LDTFs. To evaluate the primary

256 effect of MLL4 on LDTF-activated enhancers, we used Mll3−/−Mll4f/f preadipocytes expressing ectopic

257 C/EBPβ, which is a pioneer factor that activates a subset of adipogenic enhancers in preadipocytes

258 without inducing differentiation14,25. Neither ectopic expression of C/EBPβ nor deletion of Mll4 affected

259 expression levels of BAF subunits SMARCA4, SS18 and ARID1A in preadipocytes (Fig 6A). We identified

260 571 C/EBPβ-activated enhancers that were bound by MLL4 and BAF subunits SMARCA4 and ARID1A

261 (see Materials and Methods). Among these enhancers, 324 displayed ectopic C/EBPβ-induced de novo

262 BAF binding (BAF-de novo), whereas the remaining 247 were pre-marked by BAF in control cells before

263 ectopic expression of C/EBPβ (BAF-prebound) (Fig 6B). As expected, motifs of C/EBPs were enriched on

264 both BAF-de novo and BAF-prebound C/EBPβ-activated enhancers (Fig 6C). Consistent with our

265 previous finding, deletion of Mll4 markedly decreased H3K27ac levels on C/EBPβ-activated enhancers.

266 Importantly, deletion of Mll4 not only prevented C/EBPβ-induced de novo BAF binding (as measured by

267 SMARCA4 and ARID1A), but also compromised genomic binding of SMARCA4 and ARID1A on BAF-

268 prebound enhancers, despite little changes in C/EBPβ binding (Fig 6D and E). These results indicate that

269 MLL4 is required for genomic binding of BAF on C/EBPβ-activated enhancers and suggest a direct role of

270 MLL4 in mediating BAF localization on enhancers.

271

272 BAF regulates MLL4 binding on C/EBPβ-activated enhancers

273 We next asked whether BAF is required for MLL4 binding on C/EBPβ-activated enhancers. For

274 this purpose, we infected Smarca4AID/AID preadipocytes with retroviruses expressing C/EBPβ followed by

275 retroviruses expressing Tir1. Cells were then treated with auxin for 4h to deplete endogenous SMARCA4

276 protein. Western blot showed that acute depletion of SMARCA4 did not affect the expression levels of

277 ectopic C/EBPβ in preadipocytes (Fig 7A). Among 449 C/EBPβ-activated enhancers that we identified,

278 C/EBPβ binding was unchanged at 220 AEs (SMARCA4-independent), while C/EBPβ occupancy was

279 reduced at the remaining 229 AEs (SMARCA4-dependent) upon SMARCA4 depletion (Fig 7B). Only

280 SMARCA4-independent C/EBPβ binding AEs were enriched with C/EBP motifs while SMARCA4-

281 dependent ones were enriched with motifs of AP1 family TFs, suggesting that SMARCA4-independent

282 C/EBPβ binding on AEs is direct while SMARCA4-dependent C/EBPβ binding is through tethering to

283 AP1-bound AEs (Fig 7C). To assess the direct effect of SMARCA4 on MLL4 recruitment to C/EBPβ-

284 activated enhancers, we focused on SMARCA4-independent AEs. Despite intact C/EBPβ binding,

285 SMARCA4 depletion led to decreased MLL4 binding and chromatin accessibility on these AEs (Fig 7D-E).

286 These data demonstrate that SMARCA4 is required for MLL4 binding on C/EBPβ-activated enhancers.

287 Together, our data suggest an interdependent relationship between BAF and MLL4 in pioneer LDTF-

288 mediated enhancer activation (Fig 7F).

289 Discussion

290 Using adipogenesis as a model system, we provide several lines of evidence to support that BAF

291 cooperates with MLL4 to promote LDTF-dependent enhancer activation and cell type-specific gene

292 expression during cell differentiation. We found that BAF, similar to MLL4, is required for adipogenesis by

293 using conditional knockout mice and derived cells. BAF, but not PBAF, colocalizes with LDTFs and MLL4

294 on cell type-specific enhancers. Similar to MLL4, BAF is essential for enhancer activation and cell type-

295 specific gene induction during adipogenesis. By depleting BAF subunits SMARCA4 and SMARCB1 as

296 well as MLL4 in cells, we demonstrated that BAF and MLL4 reciprocally regulate one another’s binding on

297 AEs before and during adipogenesis. Finally, by evaluating pioneer factor C/EBPβ-activated enhancers

298 without inducing differentiation, we showed direct evidence for an interdependent relationship between

299 BAF and MLL4 on activating cell type-specific enhancers.

300

301 Function of SWI/SNF complexes in adipogenesis

302 Several SWI/SNF subunits have been shown to regulate adipogenesis in culture28. Ectopic

303 expression of dominant-negative SMARCA4 interferes with PPARγ-, C/EBPα- and C/EBPβ-stimulated

304 adipogenesis in fibroblasts24. Knockdown of Smarcb1 in 3T3-L1 preadipocytes inhibits adipogenesis31.

305 Using the AID system, we demonstrated that endogenous SMARCA4 is essential for adipogenesis. We

306 showed the essential role of SMARCB1 in adipogenesis of SV40T-immortalized preadipocytes.

307 Immortalization by SV40T, which inactivates tumor suppressors p53 and Rb32, prevents growth defects

308 caused by Smarcb1 deletion33,34, and enables the study of SMARCB1 in preadipocyte differentiation.

309 Further, we showed that SMARCB1 is required for adipose tissue development in mice. We also

310 observed that the BAF-specific subunit ARID1A is required for adipogenesis in preadipocytes. Together,

311 these findings demonstrate a critical role of BAF in adipogenesis. Our observations that the PBAF-

312 specific PBRM1 is dispensable for adipogenesis in vitro and in vivo provide an example of distinct

313 functions of BAF and PBAF in regulating cell differentiation and development. The role of GBAF in

314 adipogenesis remains to be determined.

315

316 Distinct genomic localization of BAF, PBAF and GBAF

317 Using adipogenesis as a model system for cell differentiation, we confirmed previous

318 observations on the distinct genomic localization of BAF, PBAF and GBAF in ESCs and in cancer cells:

319 BAF to AEs, PBAF to promoters and GBAF to CTCF binding sites11,12,35. Although SMARCA4 and SS18

320 were reported to physically associate with both BAF and GBAF complexes, we found that on AEs, the

321 vast majority of them are associated with BAF. We also showed that BAF is the major SWI/SNF complex

322 on AEs and that BAF binding sites dynamically change during adipogenesis. Motifs of AP-1 family TFs

323 are enriched on BAF-bound AEs in preadipocytes, consistent with a recent finding that AP-1 can recruit

324 BAF to establish accessible chromatin in fibroblasts36. Although GBAF is enriched on CTCF binding sites,

325 it also colocalizes with BAF on a subset of AEs during adipogenesis. The role of GBAF on AEs in cell

326 differentiation remains unclear.

327

328 Reciprocal regulation between BAF and MLL4 in enhancer activation

329 We identified 4,410 BAF-prebound AEs and 6,403 BAF-de novo AEs at D2 of adipogenesis (Fig

330 3D-E). While BAF-prebound enhancers were constitutively active, BAF-de novo enhancers were

331 activated from D-3 (preadipocytes) to D2 of adipogenesis. BAF-de novo AEs were more enriched with

332 MLL4 binding (63.4%; 4,065/6,403), compared with BAF-prebound ones (6.7%; 297/4,410) at D2 of

333 adipogenesis. In contrast to MLL4- AEs, MLL4+ ones showed markedly increased binding of BAF and

334 LDTFs, chromatin accessibility and enhancer activation during adipogenesis (Fig 3-S2). These

335 observations suggest that BAF cooperates with MLL4 to activate cell type-specific enhancers during cell

336 fate transition.

337 It has been reported that MLL3/MLL4-catalyzed H3K4me1 can directly recruit BAF complex to

338 enhancers and that knockout of MLL3/MLL4 in ESCs reduces SMARCA4 binding on enhancers37, which

339 imply that MLL3/MLL4 are necessary for BAF recruitment to enhancers. On the other hand, a more recent

340 study showed that BAF does not require pre-marked H3K4me1 for binding on enhancers and that ectopic

341 expression of SMARCA4 in SMARCA4/SMARCA2-deficient cancer cells establishes MLL3/MLL4 binding

342 on AEs bound by BAF38, suggesting that BAF plays a critical role in MLL3/MLL4 binding on enhancers.

343 Through the use of several gene knockout and protein depletion systems, we establish a bidirectional

344 relationship between BAF and MLL4 on AEs in preadipocytes and during adipogenesis, which rectifies

345 seemingly conflicting results from previous separate studies. Reciprocal regulation between BAF and

346 MLL4 is also supported by their physical association observed in this study and reported in the

347 literature39. A recent study further showed that UTX, a component of MLL3/MLL4 complex, physically and

348 functionally associates with SMARCA4 to maintain chromatin accessibility in myeloid cells40. Whether

349 BAF and MLL4 regulate each other on AEs through physical interaction of their components or enhancer

350 histone marks remains to be determined.

351

352 Interplay of BAF and MLL4 promotes pioneer factor-dependent enhancer activation

353 Pioneer factors such as OCT4 and C/EBPβ physically associate with SMARCA4 and other BAF

354 subunits in cells23,41. Pioneer factors recruit SMARCA4 to induce chromatin accessibility to facilitate

355 enhancer activation42. We reported previously that OCT4 and C/EBPβ physically associate with the MLL4

356 complex in cells and that ectopic C/EBPβ recruits MLL4 to activate a subset of C/EBPβ binding

357 adipogenic enhancers in undifferentiated cells14,20. It is worth noting that MLL4 loss does not affect

358 C/EBPβ binding on AEs. In contrast, acute depletion of SMARCA4 reduces C/EBPβ occupancy on AEs,

359 consistent with recent reports on the role of BAF in the binding of pioneer factors on enhancers42,43. Our

360 findings in the current study further suggest a model for reciprocal regulation between BAF and MLL4 on

361 C/EBPβ-activated enhancers. The pioneer factor C/EBPβ recruits BAF to remodel chromatin to enable

362 MLL4 binding on enhancers. Meanwhile, C/EBPβ recruits MLL4 to facilitate BAF binding on enhancers.

363 BAF and MLL4 may cooperate with C/EBPβ to maintain chromatin accessibility and active enhancer

364 landscape and facilitate other LDTFs binding (Fig 7F). Thus, BAF and MLL4 form a positive feedback

365 loop to activate enhancers and lineage-specific gene expression during cell differentiation.

366 Materials and Methods

367 Plasmids and antibodies

368 The retroviral plasmids pBABEneo-SV40LT and pWZLhygro-C/EBPβ have been described14. The

369 homemade antibodies anti-UTX44 and anti-MLL445 have been described. Anti-SMARCB1/SNF5/INI1 (A-5,

370 sc-166165), anti-SMARCC2/BAF170 (E-6, sc-17838X), anti-ARID2/BAF200 (E-3, sc-166117X), anti-

371 C/EBPβ (C-19, sc-150X), anti-C/EBPα (144AA, sc-61X) and anti-PPARγ (H-100, sc-7196X) were from

372 Santa Cruz. Anti-RbBP5 (A300-109A) was from Bethyl Laboratories. Anti-SMARCA4/BRG1 (ab110641)

373 and anti-H3K27ac (ab4729) were from Abcam. Anti-ARID1A/BAF250A (D2A8U, #12354), anti-SS18

374 (D6I4Z, #21792) and anti-CBP (D6C5, #7389) were from Cell Signaling. Anti-BRD9 (#61537) was from

375 Active Motif. Anti-H3K4me1 (13-0040) was from EpiCypher.

376

377 Mouse experiments

378 To generate Smarcb1f/f;PdgfRα-Cre mice, Smarcb1f/f mice46 obtained from Charles W.M. Roberts

379 (St. Jude Children's Research Hospital, Memphis, TN) were crossed with PdgfRα-Cre mice (Jackson

380 No.013148). Pbrm1f/f mice (Jackson No. 029049) were crossed with Myf5-Cre mice (Jackson No. 007893)

381 to get Pbrm1f/f;Myf5-Cre mice. Generation of Smarca4AID/AID mice will be described in a separate

382 manuscript. Histology and immunohistochemistry analyses of E18.5 embryos were done as described14.

383 Anti-Myosin (1:20 dilution, MF20; Developmental Studies Hybridoma Bank) and anti-UCP1 (1:400

384 dilution, ab10983; Abcam) were used. For fluorescent secondary antibodies, anti-mouse Alexa Fluor 488

385 and anti-rabbit Alexa Fluor 555 (Life Technologies, Carlsbad, CA, USA) were used. All mouse work was

386 approved by the Animal Care and Use Committee of NIDDK, NIH.

387

388 Isolation and immortalization of primary preadipocytes, virus infection and adipogenesis assay

389 Primary preadipocytes were isolated from interscapular BAT of newborn pups and were

390 immortalized with retroviral vectors expressing SV40T as described45. Adenoviral infection of

391 preadipocytes was done at 50 moi. Cells were routinely cultured in DMEM plus 10% FBS. For

392 adipogenesis assays, preadipocytes were plated at a density of 1 × 105 in each well of 6-well plates in

393 growth medium (DMEM plus 10% FBS) 4 days before induction of adipogenesis. At day 0, cells were fully

394 confluent and were treated with differentiation medium (DMEM plus 10% FBS, 0.1 μM insulin and 1 nM

395 T3) supplemented with 0.5 mM IBMX, 1 μM DEX, and 0.125 mM indomethacin. Two days later, cells

396 were changed to the differentiation medium. Cells were replenished with fresh medium at 2-day intervals.

397 Fully differentiated cells were either stained with Oil Red O or subjected to gene expression analyses.

398

399 The auxin-inducible degron (AID) system for SMARCA4 depletion

400 Smarca4AID/AID preadipocytes were isolated from interscapular BAT of Smarca4AID/AID newborn

401 pups and immortalized with SV40T. Immortalized Smarca4AID/AID preadipocytes were infected with

402 retroviral vector expressing the auxin receptor Tir1 of rice (pBabePuro-OsTir1-9Myc, Addgene #80074).

403 To induce degradation of SMARCA4, auxin (indole-3-acetic acid sodium salt, Sigma-Aldrich #I5148) was

404 added to the culture medium in a final concentration of 0.5 mM for indicated times.

405

406 shRNA knockdown

407 For shRNA transduction, individual shRNAs targeting mouse Arid1a (TRCN0000238303,

408 shArid1a-#1; TRCN0000238306, shArid1a-#5) were obtained from Sigma Mission shRNA library (Sigma-

409 Aldrich). Immortalized Smarcb1f/f preadipocytes were infected with lentiviral shRNA targeting Arid1a or

410 control virus alone for 24h. Infected cells were selected with puromycin (2 μg/mL) for 4 days before

411 performing further experiments.

412

413 Western blot and immunoprecipitation

414 Nuclear proteins were extracted using the nuclear extract preparation method as described

415 previously19. Briefly, cells were washed with cold PBS, resuspended in buffer A (10 mM HEPES, pH 7.9,

416 1.5 mM MgCl2, 10 mM KCl and 0.1% NP40) supplemented with protease inhibitors (Roche), 0.5 mM DTT

417 and 0.2 mM phenylmethylsulfonyl fluoride (PMSF), and incubated on ice for 10 min. After centrifugation at

418 1,000g, nuclei were resuspended in buffer C (20 mM HEPES, pH 7.9, 1.5 mM MgCl2, 420 mM NaCl, 0.2

419 mM EDTA and 25% glycerol) supplemented with protease inhibitors, 0.5 mM DTT and 0.2 mM PMSF.

420 Nuclear extracts were separated using 4–15% Tris-Glycine gradient gels (Bio-Rad Laboratories). For

421 Western blotting of MLL4, nuclear extracts were separated using 3–8% Tris-Acetate gradient gels

422 (Invitrogen). Total proteins on the gel were transferred to a PVDF membrane (Bio-Rad Laboratories). The

423 membranes were probed using specific antibodies.

424 For co-immunoprecipitation (IP), nuclear proteins were extracted from HEK293T and

425 preadipocytes, diluted with an IP Buffer (50 mM Tris-HCl, pH 7.5, 1 mM EDTA, 150 mM NaCl, and 1%

426 Triton X-100) supplemented with protease inhibitors, 1 mM DTT and 1 mM PMSF, and incubated with

427 anti-MLL4, anti-UTX, anti-SMARCA4 or anti-SMARCB1 antibodies overnight at 4 °C. Samples were

428 additionally incubated with Dynabeads Protein A (Life Technologies, 10008D) at 4 °C for 2 h. Beads were

429 washed three times with an IP buffer and the immunoprecipitates were eluted with 20 μLof 1x sample

430 buffer (NuPage LDS buffer, Thermo Scientific) including 100 mM DTT.

431

432 RNA isolation and qRT-PCR analysis

433 Total RNA was extracted using TRIzol (Life Technologies) and reverse transcribed using

434 ProtoScript II first-strand cDNA synthesis kit (NEB, E6560), following manufacturer's instructions.

435 Quantitative RT-PCR (qRT-PCR) was performed in duplicate with the Luna® Universal qPCR Master Mix

436 (NEB, M3003) using QuantStudio 5 Real-Time PCR System (Thermo Fisher). PCR amplification

437 parameters were 95 °C (3 min) and 40 cycles of 95 °C (15 s), 60 °C (60 s), and 72 °C (30 s). Primer

438 sequences are available upon request. Statistical significance was calculated using the two-tailed

439 unpaired t-test on two experimental conditions.

440

441 RNA-Seq library preparation

442 Total RNA was subjected to mRNA purification using the NEBNext Poly(A) mRNA Magnetic

443 Isolation Module (NEB, E7490). Isolated mRNAs were reverse-transcribed into double stranded cDNA

444 and subjected to sequencing library construction using the NEBNext Ultra™ II RNA Library Prep Kit for

445 Illumina (NEB, E7770) according to the manufacturer’s instructions. RNA libraries were sequenced on

446 Illumina HiSeq 3000.

447

448 ChIP and ChIP-Seq library preparation

449 Chromatin immunoprecipitation (ChIP) and ChIP-Seq were done as described47. Briefly, cells

450 were cross-linked with 1.5% formaldehyde for 10 min and quenched by 125 mM glycine for 10 min. 10

451 million fixed cells were resuspended in ice cold buffer containing 5mM PIPES, pH 7.5, 85mM KCl, 1%

452 NP-40 and protease inhibitors, incubated on ice for 15 min, and centrifuged at 500 g for 5 min at 4°C.

453 Nuclei were resuspended with 1mL buffer containing 50mM Tris-HCl, pH8.0, 10mM EDTA, 0.1% SDS

454 and protease inhibitors, and subjected to sonication. Sheared chromatin was clarified by centrifugation at

455 13,000 g for 10 min at 4°C. The supernatant was transferred to a new tube and further supplemented with

456 1% Triton-X100, 0.1% sodium deoxycholate and protease inhibitors. 2% of the mixture was set aside as

457 input and 20ng of spike-in chromatin (Active Motif, #53083) was added to the rest. For each ChIP, 4 - 10

458 µg of target antibodies and 2μg of spike-in antibody (anti-H2Av, Active Motif, #61686) were added and

459 incubated on a rotator at 4°C overnight. ChIP samples were added with 50μL prewashed protein A

460 Dynabeads (ThermoFisher) and incubated for 3h at 4°C. Beads were then collected on a magnetic rack

461 and washed twice with 1mL cold RIPA buffer, twice with 1mL cold RIPA buffer containing 300mM NaCl,

462 twice with 1mL cold LiCl buffer and once with PBS. Beads were then eluted with 100 μL buffer containing

463 0.1M NaHCO3, 1% SDS, and 20μg Proteinase K at 65°C overnight. Input sample volumes were adjusted

464 to 100μL with the elution buffer. Samples were then purified using QIAquick PCR purification kit (Qiagen)

465 and eluted in 30 μL 10mM Tris-HCl. For ChIP-Seq, entire ChIPed DNA or 300ng of the input DNA were

466 used to construct libraries using NEBNext® Ultra™ II DNA Library Prep kit with AMPure XP magnetic

467 beads (Beckman Coulter). Library quality and quantity were estimated with Bioanalyzer and Qubit assays.

468 The final libraries were sequenced on Illumina HiSeq 3000.

469

470 ATAC-Seq library preparation

471 The Assay for Transposase-Accessible Chromatin with high-throughput sequencing (ATAC-Seq)

472 was performed as described48. For each ATAC reaction, briefly, 5 × 104 freshly collected cells were

473 aliquoted into a new tube and spun down at 500 × g for 5 min at 4 °C. The cell pellet was incubated in

474 50 µL of ATAC-RSB buffer (10 mM Tris-HCl pH 7.4, 10 mM NaCl, 3 mM MgCl2) containing 0.1% NP-40,

475 0.1% Tween-20, and 0.01% digitonin (Promega) on ice for 3 min and was washed out with 1 mL of ATAC-

476 RSB buffer containing 0.1% Tween-20. Nuclei were collected by centrifugation at 500 × g for 10 min at

477 4 °C, then resuspended in 50 µL of transposition reaction buffer containing 25 µL 2x Tagment DNA buffer,

478 2.5 µL transposase (100 nM final; Illumina), 16.5 µL PBS, 0.5 µL 1% digitonin, 0.5 µL 10% Tween-20, and

479 5 µL H2O. The reaction was incubated for 30 min at 37 °C with mixing (1000 r.p.m.) and directly subjected

480 to DNA purification using the MinElute Reaction Cleanup Kit (Qiagen) according to the manufacturer’s

481 instructions. Eluted DNA was amplified with PCR using Nextera i7- and i5-index primers (Illumina).

482 Purification and size selection of the amplified DNA were carried out with AMPure XP magnetic beads

483 (Beckman Coulter) to remove primer dimers and >1,000 bp fragments. For purification, the ratio of

484 sample to beads was set to 1:1.8, whereas for size selection, the ratio was set to 1:0.55. Sequencing

485 libraries were analyzed with Qubit and sequenced on HiSeq3000.

486

487 Computational analysis

488 RNA-Seq data analysis

489 Raw sequencing data were aligned to the mouse genome mm9 using STAR software49. Reads

490 on exons were collected to calculate reads per kilobase per million (RPKM) as a measure of gene

491 expression level. Only genes with exonic reads of RPKM > 1 were considered expressed. For comparing

492 gene expression levels before (D-3) and during (D2) adipogenesis, or in control and SMARCB1-deficient

493 cells (Fig 2-S1), fold change cutoff of > 2.5 was used to identify differentially expressed genes. Gene

494 ontology (GO) analysis was done using DAVID with the whole mouse genome as background

495 (https://david.ncifcrf.gov).

496

497 ChIP-Seq peak calling

498 Raw sequencing data were aligned to the mouse genome mm9 and the drosophila genome dm6

499 using Bowtie2 (v2.3.2)50. To identify ChIP-enriched regions, SICER (v1.1) was used51. For ChIP-Seq of

500 histone modifications (H3K4me1 and H3K27ac), the window size of 200 bp, the gap size of 200 bp, and

501 the false discovery rate (FDR) threshold of 10-3 were used. For ChIP-Seq of non-histone factors, the

502 window size of 50 bp, the gap size of 50 bp, and the FDR threshold of 10-10 were used.

503

504 Genomic distribution of ChIP-Seq peaks

505 To define regulatory regions (Fig 1C), combination of genomic coordinates and histone

506 modification ChIP-Seq data were used. Promoter was defined as transcription start sites ± 1 kb.

507 Promoter-distal regions were further separated into active enhancers (H3K4me1+ H3K27ac+), primed

508 enhancers (H3K4me1+ H3K27ac-), and the other (H3K4me1-).

509

510 Motif analysis

511 To find enriched TF motifs in given genomic regions identified by ChIP-Seq, we utilized the

512 SeqPos motif tool in the Cistrome toolbox (http://cistrome.org/ap/)52. For ChIP-Seq data with more than

513 5,000 regions (Fig 1, 3, 3-S1), top 3,000 significant regions were used. For ChIP-Seq data with small

514 numbers (Fig 6 and 7), all regions were used. For differential motif analysis of given two regions (Fig 3-

515 S2), HOMER software was used (http://homer.ucsd.edu/homer/)53.

516

517 Heat maps and box plots

518 The heat map matrices were generated using in-house scripts with 50 bp resolution and

519 visualized in R using gplots package. Enhancers shown in the heat maps were ranked according to the

520 intensity of SMARCA4 at the center of 400 bp window in control cells (Fig 4 and 5) or that of C/EBPβ in

521 control cells expressing ectopic C/EBPβ (Fig 6 and 7). The ratio of normalized ChIP-Seq read counts in

522 SMARCB1-deficient and control cells (Fig 4E and 5D), those in MLL4-deficient and control cells (Fig 5B),

523 or those in SMARCA4-depleted and control cells (Fig 5F) in base 2 logarithm were plotted using box plot,

524 with outliers not shown. Wilcoxon signed rank test (one-sided) was used to determine statistical

525 differences in SMARCB1-deficient and control cells (Fig 4E and 5D), in MLL4-deficient and control cells

526 (Fig 5B), or in SMARCA4-depleted and control cells (Fig 5F).

527

528 ATAC-Seq data processing

529 Raw ATAC-Seq reads were processed using Kudaje lab’s ataqc pipelines

530 (https://github.com/kundajelab/atac_dnase_pipelines) which included adapter trimming, aligning to mouse

531 mm9 genome by Bowtie2, and peak calling by MACS2. For downstream analysis, we used filtered reads

532 that were retained after removing unmapped reads, duplicates and mitochondrial reads.

533 C/EBPβ-activated enhancers

534 To identify C/EBPβ-activated enhancers in preadipocytes (Fig 6 and 7), we first selected

535 C/EBPβ+ AEs (C/EBPβ+ H3K27ac+ promoter-distal regions) in ectopic C/EBPβ-expressed cells. We

536 further narrowed the focus down to SMARCA4+ ARID1A+ MLL4+ ones to better assess direct relationship

537 between BAF and MLL4. We compared H3K27ac enrichment intensities in control and ectopic C/EBPβ-

538 expressed cells. Enhancers with over 2-fold increased H3K27ac intensities in ectopic C/EBPβ-expressed

539 cells were considered as C/EBPβ-activated enhancers.

540

541 Accession numbers

542 All datasets described in this paper have been deposited in NCBI Gene Expression Omnibus

543 under access #GSE151115.

544

545 Acknowledgement

546 We thank Keji Zhao for Smarca4AID/AID mice, Charles W. M. Roberts for Smarcb1f/f mice, Ruth

547 Kopyto for assistance in schematic drawing, Guojia Xie for suggestions, NIDDK Genomics Core and

548 NHLBI DNA Sequencing and Genomics Core for next generation sequencing. This work was supported

549 by the Intramural Research Program of NIDDK, NIH to K.G.

550

551 Author Contributions

552 Conceptualization, Y.-K.P., J.-E.L., and K.G.; Methodology, Y.-K.P., J.-E.L., Z.Y., W.P., and W.W.;

553 Investigation, Y.-K.P., T. O., and K.M.; Software, Formal Analysis, and Data Curation, J.-E.L. and W.P.;

554 Writing – Original Draft, Y.-K.P., J.-E.L., and K.M.; Writing – Review & Editing, Y.-K.P., J.-E.L., K.M., and

555 K.G.; Project Administration and Funding Acquisition, K.G.

556

557 Declaration of Interests

558 The authors declare no competing interests.

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644 38. Pan, J. et al. The ATPase module of mammalian SWI/SNF family complexes mediates 645 subcomplex identity and catalytic activity-independent genomic targeting. Nat Genet 51, 646 618-626 (2019). 647 39. Lee, S. et al. Crucial roles for interactions between MLL3/4 and INI1 in nuclear receptor 648 transactivation. Mol Endocrinol 23, 610-9 (2009). 649 40. Gozdecka, M. et al. UTX-mediated enhancer and chromatin remodeling suppresses 650 myeloid leukemogenesis through noncatalytic inverse regulation of ETS and GATA 651 programs. Nat Genet 50, 883-894 (2018). 652 41. Ding, J., Xu, H., Faiola, F., Ma'ayan, A. & Wang, J. Oct4 links multiple epigenetic 653 pathways to the pluripotency network. Cell Res 22, 155-67 (2012). 654 42. King, H.W. & Klose, R.J. The pioneer factor OCT4 requires the chromatin remodeller 655 BRG1 to support gene regulatory element function in mouse embryonic stem cells. Elife 656 6(2017). 657 43. Xu, G. et al. ARID1A determines luminal identity and therapeutic response in estrogen- 658 receptor-positive breast cancer. Nat Genet 52, 198-207 (2020). 659 44. Hong, S. et al. Identification of JmjC domain-containing UTX and JMJD3 as histone H3 660 lysine 27 demethylases. Proc Natl Acad Sci U S A 104, 18439-44 (2007). 661 45. Cho, Y.W. et al. Histone Methylation Regulator PTIP Is Required for PPARgamma and 662 C/EBPalpha Expression and Adipogenesis. Cell Metab 10 , 27-39 (2009). 663 46. Roberts, C.W., Leroux, M.M., Fleming, M.D. & Orkin, S.H. Highly penetrant, rapid 664 tumorigenesis through conditional inversion of the tumor suppressor gene Snf5. Cancer 665 Cell 2, 415-25 (2002). 666 47. Park, Y.K. & Ge, K. Glucocorticoid Receptor Accelerates, but Is Dispensable for, 667 Adipogenesis. Mol Cell Biol 37(2017). 668 48. Corces, M.R. et al. Lineage-specific and single-cell chromatin accessibility charts human 669 hematopoiesis and leukemia evolution. Nat Genet 48, 1193-203 (2016). 670 49. Dobin, A. et al. STAR: ultrafast universal RNA-seq aligner. Bioinformatics 29, 15-21 671 (2013). 672 50. Langmead, B. & Salzberg, S.L. Fast gapped-read alignment with Bowtie 2. Nat Methods 673 9, 357-9 (2012). 674 51. Zang, C. et al. A clustering approach for identification of enriched domains from histone 675 modification ChIP-Seq data. Bioinformatics 25, 1952-8 (2009). 676 52. He, H.H. et al. Nucleosome dynamics define transcriptional enhancers. Nat Genet 42, 677 343-7 (2010). 678 53. Heinz, S. et al. Simple combinations of lineage-determining transcription factors prime 679 cis-regulatory elements required for macrophage and B cell identities. Mol Cell 38 , 576- 680 89 (2010). 681

682 Figure legends

683 Figure 1. Expression and genomic binding of SWI/SNF subunits in adipogenesis

684 (A) Schematic of subunits in SWI/SNF complexes BAF, PBAF and GBAF (ncBAF).

685 (B) Expression profiles of SWI/SNF subunits in adipogenesis. Published RNA-Seq data sets were used

686 (GSE74189)18. D-3, day -3. D2, day 2.

687 (C) Genomic distributions of SMARCA4 (BRG1), SS18, BAF-specific ARID1A, PBAF-specific ARID2 and

688 GBAF-specific BRD9 before (D-3) and during (D2) adipogenesis were determined by ChIP-Seq.

689 Promoters were defined as transcription start sites ± 1kb. Active enhancers were defined as H3K4me1+

690 H3K27ac+ promoter-distal regions. Primed enhancers were defined as H3K4me1+ H3K27ac- promoter-

691 distal regions. Numbers of binding sites are indicated.

692 (D) Motif analysis of SWI/SNF subunits binding regions at D-3 and D2 of adipogenesis. Top 3,000

693 significant binding regions were used.

694 (E) Venn diagrams depicting genomic binding of SMARCA4, SS18, ARID1A, ARID2 and BRD9 at D2 of

695 adipogenesis. BAF, PBAF, and GBAF binding sites were defined as SMARCA4+ SS18+ ARID1A+,

696 SMARCA4+ ARID2+, and SMARCA4+ BRD9+ regions, respectively.

697 (F) Venn diagram depicting genomic binding of BAF, PBAF and GBAF at D2 of adipogenesis.

698

699 Figure 1-Supple 1. Genomic binding of SWI/SNF subunits in adipogenesis

700 (A) Venn diagrams depicting genomic binding of SMARCA4, SS18, BAF-specific ARID1A, PBAF-specific

701 ARID2 and GBAF-specific BRD9 before (D-3) and during (D2) adipogenesis. Numbers of binding sites

702 are indicated.

703 (B) Heat maps for genomic binding of SMARCA4, SS18, ARID1A, ARID2 and BRD9 are shown around

704 SMARCA4 binding sites at D2 of adipogenesis. Regions associated with either BAF, PBAF or GBAF are

705 shown.

706

707 Figure 2. BAF subunits SMARCA4, SMARCB1 and ARID1A are required for adipogenesis

708 (A-C) Depletion of SMARCA4 by auxin-inducible degron (AID) system inhibits adipogenesis.

709 (A) A schematic illustration of the auxin-induced depletion of endogenous SMARCA4 protein in Tir1-

710 expressing Smarca4AID/AID cells. TIR1, transport inhibitor response 1; Cul1, Cullin-1; Rbx1, RING-box

711 protein 1; Skp1, S-phase kinase-associated protein 1.

712 (B) Western blot analyses using antibodies indicated on the left. RbBP5 was used as a loading control.

713 (C) Oil Red O staining at day 7 (D7) of adipogenesis. Cells were treated with auxin throughout the

714 differentiation.

715 (D) SMARCB1 is essential for adipogenesis in vivo. Sections of the interscapular area of E18.5 Smarcb1f/f

716 (f/f) and Smarcb1f/f;PdgfRα-Cre (cKO) embryos were stained with H&E (left panels) or with antibodies

717 recognizing brown adipose tissue (BAT) marker Ucp1 (green) and skeletal muscle marker Myosin (red)

718 (right panels). B, BAT; M, muscle. Scale bar = 1mm.

719 (E-H) SMARCB1 is essential for adipogenesis in culture. SV40T-immortalized Smarcb1f/f preadipocytes

720 were infected with adenoviral GFP or Cre, followed by adipogenesis assays.

721 (E) Deletion of Smarcb1 does not affect cell growth rates. 1 x 105 preadipocytes were plated and the

722 cumulative cell numbers were determined for 5 days.

723 (F) Before (D-3) and during (D2) adipogenesis, nuclear extracts were analyzed by Western blot using

724 indicated antibodies.

725 (G) Oil Red O staining at D7 of adipogenesis.

726 (H) Expression of Cebpb, Pparg, Cebpa and Fabp4 before (D-3) and during (D2) adipogenesis was

727 determined using RNA-Seq. RPKM values indicate gene expression levels.

728 (I-K) ARID1A is required for adipogenesis in culture. Preadipocytes were infected with lentiviral vectors

729 expressing control (shCtrl) or Arid1a knockdown shRNA (shArid1a), followed by adipogenesis assays.

730 (I) Western blot analysis of ARID1A in preadipocytes.

731 (J) Oil Red O staining at D7 of adipogenesis.

732 (K) qRT-PCR analysis of adipogenesis marker expression at indicated time points of adipogenesis.

733

734 Figure 2-Supple 1. SMARCB1 is required for adipogenesis in vivo and in culture

735 (A-D) Smarcb1f/f;PdgfRα-Cre (cKO) mice showed defects in skull bone and died shortly after birth.

736 (A) Genotype of progeny from the crossing between Smarcb1f/f (f/f) and Smarcb1f/+;PdgfRα-Cre mice at

737 embryonic day 18.5 (E18.5), post-natal day 0.5 (P0.5) and weaning (3 weeks of age). Dead pups are

738 indicated by an asterisk.

739 (B) Representative morphology of E18.5 embryos.

740 (C) Defective coronal sutures in cKO mice.

741 (D) H&E staining of the sagittal sections of head. Arrows indicate the skull bone.

742 (E-F) Impaired BAT development in cKO mice.

743 (E) Representative pictures of interscapular BAT.

744 (F) qRT-PCR analysis of Smarcb1, adipocyte-enriched genes Pparg, Cebpa, Fabp4, Prdm16 and Ucp1

745 as well as myogenesis marker genes Myogenin, MyHC and MCK in BAT isolated from E18.5 f/f (n = 5)

746 and cKO (n = 5) embryos. Quantitative PCR data is presented as means ± SEM. *** p<0.001. n.s., not

747 significant.

748 (G-J) SMARCB1 is required for adipogenesis in culture. Immortalized Smarcb1f/f preadipocytes were

749 infected with adenoviral GFP or Cre, followed by adipogenesis assays. Cells were collected at D-3 and

750 D2 of adipogenesis for RNA-Seq.

751 (G) Deletion of Smarcb1 does not affect the interaction between SMARCA4 and SMARCC2. Nuclear

752 extracts prepared at D2 of adipogenesis were immunoprecipitated (IP) with SMARCA4 or SMARCB1

753 antibody. Immunoprecipitates were analyzed by Western blot using antibodies indicated on the left.

754 (H) RNA-Seq analysis at D2 of adipogenesis. Pie chart depicts SMARCB1-dependent and -independent

755 up-regulated genes as well as down-regulated genes from D-3 to D2 of adipogenesis. The cut-off for

756 differential expression is 2.5-fold.

757 (I) Gene ontology (GO) analysis of 362 SMARCB1-dependent up-regulated genes defined in (H).

758 (J) Deletion of Smarcb1 does not affect the expression of other SWI/SNF subunits in adipogenesis.

759

760 Figure 2-Supple 2. PBAF-specific subunit PBRM1 is dispensable for adipogenesis in vivo and in

761 culture

762 (A-E) Characterization of adult (8 week-old) Pbrm1f/f;Myf5-Cre (cKO) mice. Pbrm1f/f (f/f) were crossed

763 with Pbrm1f/+;Myf5-Cre to obtain cKO mice.

764 (A) Genotyping results. The expected ratios of the four genotypes are 1:1:1:1.

765 (B) Representative pictures of 8-week-old f/f or cKO mice.

766 (C) The average adipose tissue weights in f/f (n = 5) and cKO (n = 5) mice are shown as % of body

767 weight. n.s., not significant.

768 (D) Pictures of interscapular BAT isolated from three f/f or cKO mice.

769 (E) Total RNA was extracted from BAT of f/f (n = 5) or cKO (n = 5) mice for qRT-PCR analysis of Pbrm1,

770 adipogenesis markers Pparg, Cebpα and Fabp4 and BAT marker Ucp1. Quantitative PCR data in all

771 figures are presented as means ± SEM. *** p<0.001. n.s., not significant.

772 (F-J) Pbrm1 is dispensable for adipogenesis in cell culture. Primary preadipocytes were isolated from f/f

773 and cKO pups at P0.5, followed by adipogenesis until D7.

774 (F) Pbrm1 deletion in cKO preadipocytes was confirmed by qPCR of genomic DNA. *** p<0.001.

775 (G) Oil Red O staining of differentiated cells.

776 (H) RNA-Seq analysis of gene expression in differentiated f/f (n = 3) or cKO (n = 3) cells at D7. Pie chart

777 depicts decreased or increased genes in cKO versus f/f cells. The cut-off is 2.5-fold.

778 (I) Gene ontology (GO) analysis of decreased or increased genes defined in (H).

779 (J) The list of representative adipocyte differentiation genes (upper panel) and significantly decreased or

780 increased genes in Pbrm1 KO cells (lower panel). RPKM values indicate gene expression levels.

781

782 Figure 3. BAF co-localizes with LDTFs and MLL4 on active enhancers during adipogenesis

783 (A) Average binding profiles of SMARCA4, SS18, and ARID1A around the center of SMARCA4+ active

784 enhancers (AEs) or promoters before (D-3) and during (D2) adipogenesis. Normalized read counts are

785 shown.

786 (B) Motif analysis of the top 3,000 SMARCA4+ SS18+ AEs.

787 (C) Pie chart depicting BAF-binding (SMARCA4+ SS18+) AEs at D2 of adipogenesis. Among the 10,813

788 BAF-binding AEs at D2, 6,403 are de novo BAF-binding AEs (BAF-de novo), while 4,410 AEs are already

789 bound by BAF (BAF-prebound) at D-3.

790 (D-E) BAF subunits SMARCA4, SS18 and ARID1A colocalize with LDTFs (C/EBPβ, C/EBPα, and

791 PPARγ), MLL4, CBP and open chromatin on AEs (H3K4me1+ H3K27ac+) at D2 of adipogenesis. The

792 local epigenetic environment exhibits coordinated changes during differentiation. Heat maps (D) and

793 average profiles (E) were aligned around the center of SMARCA4+ SS18+ AEs. Published ChIP-Seq data

794 sets for MLL4, CBP, C/EBPβ, C/EBPα, and PPARγ were used (GSE74189)18.

795

796 Figure 3-Supple 1. BAF co-localizes with LDTFs on active enhancers during adipogenesis

797 (A-C) Motif analysis of SMARCA4+ (A), SS18+ (B), or ARID1A+ (C) active enhancers (AEs) at D-3 and D2

798 of adipogenesis. Top 3,000 binding regions on AEs were used for motif analysis.

799 (D-E) Genome browser shot of ChIP-Seq data of BAF subunits (SMARCA4, SS18 and ARID1A), MLL4,

800 CBP, LDTFs (C/EBPβ, C/EBPα, and PPARγ), H3K4me1, and H3K27ac as well as ATAC-Seq data on

801 AEs of Pparg (D) or Cebpa (E) gene loci.

802

803 Figure 3-Supple 2. Markedly increased BAF binding and chromatin accessibility on MLL4+ active

804 enhancers during adipogenesis

805 (A) Among the 10,813 BAF-binding (SMARCA4+ SS18+) active enhancers (AEs) at D2 of adipogenesis,

806 4,362 are bound by MLL4.

807 (B) Differential motif analysis of MLL4+ or MLL4- AEs at D2 of adipogenesis using HOMER.

808 (C-D) Heat maps (C) and average profiles (D) around SMARCA4+ SS18+ AEs. Levels of CBP and

809 H3K27ac were only induced on MLL4+ AEs. Normalized read counts are shown.

810

811 Figure 3-Supple 3. SWI/SNF subunits associate with MLL4 complex in cells

812 (A) MLL4- and UTX-associated proteins identified by IP-mass spectrometry analysis. Nuclear extracts

813 prepared from mouse embryonic stem cells were subjected to immunoprecipitation with anti-MLL4 or anti-

814 UTX antibody. Peptide numbers for IP-enriched BAF and PBAF complex subunits as well as MLL4

815 complex subunits are shown.

816 (B-C) MLL4 complex associates with SMARCA4 and SMARCB1 in cells. Nuclear extracts from HEK293T

817 cells (B) or preadipocytes during (D2) adipogenesis (C) were subjected to immunoprecipitation with anti-

818 MLL4 or anti-UTX antibody. Immunoprecipitates were analyzed by Western blotting with antibodies

819 indicated on the left.

820 Figure 4. Reciprocal regulation between BAF and MLL4 on active enhancers during adipogenesis

821 Mll3-/-Mll4f/f or Smarcb1f/f preadipocytes were infected with adenoviruses expressing GFP or Cre, followed

822 by adipogenesis assays. Cells were collected before (D-3) and during (D2) adipogenesis for ChIP-Seq,

823 ATAC-Seq and Western blot analyses.

824 (A-B) Decreased SMARCA4 and SS18 binding and chromatin accessibility on adipogenic enhancers in

825 Mll4 KO (Cre) cells during adipogenesis. Adipogenic enhancers were defined as active enhancers bound

826 by either C/EBPβ, C/EBPα, or PPARγ at D2 of adipogenesis. Published ChIP-Seq data sets for C/EBPβ,

827 C/EBPα, and PPARγ were used (GSE74189) 18. Heat maps (A) and average profiles (B) are shown

828 around SMARCA4+ SS18+ MLL4+ adipogenic enhancers. Adipogenic enhancers shown in the heat maps

829 were ranked by the binding intensity of SMARCA4 at the center in D2 control (GFP) cells.

830 (C-F) Deletion of Smarcb1 reduces binding of BAF subunits (SMARCA4, SS18, and ARID1A) as well as

831 chromatin opening, and impairs MLL4 binding and activation of adipogenic enhancers. Adipogenic

832 enhancers at D2 of adipogenesis were re-defined using ChIP-Seq data sets obtained in Smarcb1f/f cells.

833 (C) Heat maps for ChIP-Seq of BAF subunits (SMARCA4, SS18, and ARID1A) and ATAC-Seq around

834 the center of SMARCA4+ SS18+ MLL4+ adipogenic enhancers.

835 (D) Heat maps for ChIP-Seq of MLL4, CBP, H3K4me1 and H3K27ac on adipogenic enhancers.

836 Adipogenic enhancers shown in the heat maps were ranked by the intensity of SMARCA4 at the center in

837 D2 control (GFP) cells.

838 (E) Fold changes of intensities between control (GFP) and Smarcb1 KO (Cre) cells on adipogenic

839 enhancers are shown in box plots. Statistical significance levels are indicated (Wilcoxon signed rank test,

840 one-sided).

841 (F) Western blot analyses of H3K4me1 and H3K27ac in control and Smarcb1 KO cells during

842 adipogenesis. Total histone H3 was used as a loading control.

843

844 Figure 4-Supple 1. Reciprocal regulation between BAF and MLL4 on active enhancers during

845 adipogenesis

846 (A-B) MLL4 is required for SMARCA4 and SS18 binding and chromatin opening on adipogenic

847 enhancers around Pparg (A) and Fabp4 (B) genes at D2 of adipogenesis.

848 (C) SMARCB1 is required for SMARCA4, MLL4, and CBP binding and activation of enhancers around the

849 Pparg gene at D2 of adipogenesis.

850

851 Figure 5. BAF and MLL4 reciprocally promote each other’s binding to maintain active enhancers

852 in preadipocytes

853 Mll3-/-Mll4f/f or Smarcb1f/f preadipocytes were infected with adenoviruses expressing GFP or Cre. Sub-

854 confluent cells were collected for ChIP-Seq and ATAC-Seq. BAF+ MLL4+ active enhancers (AEs) were

855 defined as those bound by SMARCA4, ARID1A and MLL4.

856 (A-B) Deletion of Mll4 reduces ARID1A binding on BAF+ MLL4+ AEs in preadipocytes.

857 (A) AEs shown in the heat maps were ranked by the intensity of SMARCA4 at the center in control (GFP)

858 cells.

859 (B) Fold changes of intensities between control and Mll4 KO (Cre) cells on AEs in preadipocytes are

860 shown in box plots. Statistical significance levels are indicated (Wilcoxon signed rank test, one-sided).

861 (C-D) SMARCB1 is required for maintaining MLL4 binding on BAF+ MLL4+ AEs in preadipocytes.

862 (C) AEs shown in the heat maps were ranked by the intensity of SMARCA4 at the center in control cells.

863 (D) Fold changes of intensities between control (GFP) and Smarcb1 KO (Cre) cells on AEs in

864 preadipocytes are shown in box plots. Statistical significance levels are indicated (Wilcoxon signed rank

865 test, one-sided).

866 (E-F) SMARCA4 is required for maintaining MLL4 binding on BAF+ MLL4+ AEs in preadipocytes.

867 Smarca4AID/AID preadipocytes were infected with retroviruses expressing Tir1 or vector (Vec) only,

868 followed by auxin (0.5 mM) treatment for 4h. Cells were then collected for ATAC-Seq and ChIP-Seq of

869 MLL4.

870 (E) AEs shown in the heat maps were ranked by the intensity of MLL4 at the center in control cells.

871 (F) Fold changes of intensities between SMARCA4-depleted (Tir1) and control (Vec) cells on AEs in

872 preadipocytes are shown in box plots. Statistical significance levels are indicated (Wilcoxon signed rank

873 test, one-sided).

874

875 Figure 6. MLL4 is required for BAF binding on C/EBPβ-activated enhancers

876 Mll3-/-Mll4f/f preadipocytes were infected with retroviruses expressing C/EBPβ or vector (Vec), and then

877 infected with adenoviruses expressing GFP or Cre. Sub-confluent cells were collected without inducing

878 adipogenesis for Western blot and ChIP-Seq analyses. Published ChIP-Seq data sets for C/EBPβ, MLL4,

879 H3K27ac were used (GSE50466)14.

880 (A) Western blot analyses of BAF subunits (SMARCA4, SS18, and ARID1A) and C/EBPβ expression in

881 control (GFP) and Mll4 KO (Cre) preadipocytes.

882 (B) Among the 571 C/EBPβ-activated enhancers in preadipocytes, BAF (SMARCA4 and ARID1A) binding

883 was induced on 324 AEs only after ectopic expression of C/EBPβ (BAF-de novo), while 247 AEs were

884 already bound by BAF before ectopic expression of C/EBPβ in preadipocytes (BAF-prebound).

885 (C) Motif analysis of BAF-de novo and BAF-prebound AEs.

886 (D-E) Deletion of Mll4 does not affect C/EBPβ binding but reduces BAF binding on C/EBPβ-activated

887 enhancers. Heat maps (D) and average profiles (E) around C/EBPβ-activated enhancers are shown.

888 Enhancers shown in the heat maps were ranked by the intensity of C/EBPβ at the center in control cells

889 expressing ectopic C/EBPβ.

890

891 Figure 7. BAF regulates MLL4 binding on C/EBPβ-activated enhancers

892 Smarca4AID/AID preadipocytes were infected with retroviruses expressing C/EBPβ or vector (Vec), and

893 then infected with retroviruses expressing Tir1. Sub-confluent cells were treated with auxin for 4h and

894 collected for Western blot, ChIP-Seq and ATAC-Seq analyses.

895 (A) Western blot analyses using antibodies indicated on the left.

896 (B) Among the 449 C/EBPβ-activated enhancers in preadipocytes, 220 exhibited SMARCA4-independent

897 C/EBPβ binding, while the remaining 229 showed SMARCA4-dependent C/EBPβ binding.

898 (C) Motif analysis of SMARCA4-independent and -dependent C/EBPβ-activated enhancers in

899 preadipocytes.

900 (D-E) Auxin-induced, Tir1-dependent acute depletion of SMARCA4 reduces MLL4 binding on C/EBPβ-

901 activated enhancers in preadipocytes. Heat maps (D) and average profiles (E) around C/EBPβ-activated

902 enhancers are shown. Enhancers shown in the heat maps were ranked by the intensity of C/EBPβ at the

903 center in control cells expressing ectopic C/EBPβ.

904 (F) A model depicting the interdependent relationship between BAF and MLL4 in promoting pioneer TF-

905 and LDTF-dependent cell type-specific enhancer activation.

A BAF Actl6a PBAF Actl6a GBAF Actl6a Actb Actb BCL7 BRD7 Actb BCL7 BCL7 DPF1/2/3 BRD9 SMARCA4/ PHF10 SMARCA4/ SMARCA4 SMARCA2 ARID1A/B SMARCA2 ARID2 GLTSCR1/L SS18 SS18 SMARCD1/2/3 SMARCD1/2/3 SMARCD1/2/3 SMARCB1 SMARCB1 SMAR SMAR SMAR SMAR SMAR SMAR CC1 CC2 SMARCE1 CC1 CC2 SMARCE1 CC1 CC1

B Expression profiles 80 > 400 D-3 D0 D2 D7 60

40 RPKM 20 ND 0

Core subunits ATPase module BAF-specific PBAF-specific GBAF-specific C Genomic distribution SMARCA4 SS18 ARID1A ARID2 BRD9 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 (65,912) (61,708) (43,559) (22,118) (45,627) (36,371) (22,306) (5,469) (7,082) (2,949) Active enhancer 3,215 Primed enhancer Promoter 32,574 31,349 30,367 17,018 30,816 24,967 3,796 1,431 7,603 1,221 Other

D Motif analysis: top 3,000 binding sites F ChIP-Seq (D2) SMARCA4 SS18 ARID1A SMARCA4 D-3 D2 D-3 D2 D-3 D2 BAF Motif P-value Motif P-value Motif P-value Motif P-value Motif P-value Motif P-value (16,003) PBAF JDP2 1E-07 C/EBPβ 4E-36 JUNB 5E-117 C/EBPα 2E-250 JDP2 4E-55 C/EBPα 3E-77 (2,063) BACH2 1E-07 C/EBPα 5E-32 JUN 1E-109 C/EBPβ 2E-234 FOS 5E-54 C/EBPβ 3E-61 FOS 1E-06 JUN 2E-31 FOS 6E-99 C/EBPδ 6E-203 JUND 3E-51 C/EBPδ 7E-49 BACH1 5E-06 JUND 8E-31 FOSL1 2E-98 C/EBPγ 1E-180 JUNB 2E-50 C/EBPγ 3E-47 JUN 1E-05 FOS 2E-30 JUND 1E-97 NFIL3 2E-71 FOSL1 2E-49 NFIL3 6E-23 NFE2 4E-05 JUNB 6E-30 BACH1 3E-86 HLF 6E-68 BACH1 5E-49 HLF 4E-20 GBAF JUNB 7E-05 FOSL1 2E-28 JDP2 2E-85 ATF4 4E-65 JUN 1E-48 ATF4 1E-18 (1,053) FOSL1 2E-04 C/EBPδ 1E-25 BACH2 2E-84 DBP 3E-48 NFE2 6E-41 TEF 5E-15

ARID2 BRD9 E ChIP-Seq (D2)

D-3 D2 D-3 D2 BAF (16,003) PBAF (2,063) GBAF (1,053)

Motif P-value Motif P-value Motif P-value Motif P-value SMARCA4 SMARCA4 SMARCA4 ARID1A (61,708) (61,708) (61,708) SIX5 1E-198 SIX5 1E-303 CTCF 7E-293 CTCF 4E-108 (36,371) ETS1 1E-193 BCL6 3E-302 FOSL1 7E-24 C/EBPδ 2E-20 ARID2 BRD9 BCL6 2E-193 ETS1 3E-299 JUNB 1E-23 C/EBPβ 4E-18 (5,469) (2,949) ELK3 3E-108 ELK1 6E-213 JUND 1E-21 C/EBPα 2E-16 16,003 2,063 1,053 ELK1 3E-104 ELK3 5E-200 JUN 2E-21 C/EBPγ 6E-15 ELF1 3E-97 GABPA 1E-194 FOS 2E-20 NFIC 4E-09 SS18 GABPA 3E-95 ELF1 7E-190 HIC1 8E-18 ZFP423 8E-09 (22,118) ETV3 8E-95 ELF2 3E-189 BACH1 7E-16 ATF4 3E-07

Figure 1. Expression and genomic binding of SWI/SNF subunits in adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A ChIP-Seq SMARCA4 SS18 ARID1A ARID2 BRD9

D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 (65,912) (61,708) (43,559) (22,118) (45,627) (36,371) (22,306) (5,469) (7,082) (2,949) 389 3,622 25,622 10,572 15,902

B ChIP-Seq (D2)

SMARCA4 SS18 ARID1A ARID2 BRD9

BAF (16,003) (17,165)

PBAF (2,063) GBAF (1,053) SWI/SNF-associated SWI/SNF-associated SMARCA4 -5 +5 Distance from SMARCA4 binding sites (kb)

Figure 1 - Supple 1. Genomic binding of SWI/SNF subunits in adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Smarca4AID/AID preadipocytes ACB Myc Vec Tir1 SMARCA4 AID Tir1 Auxin 0h 4h 0h 1h 4h Auxin - 250 + Auxin AID Vec Tir1 - 150 SMARCA4 SMARCA4 AID Tir1 - 150 Skp1 - 100 Ubiquitin Myc (Tir1) -75 Rbx1 RbBP5 -50 SMARCB1 Degradation by proteasome

Smarcb1f/f x Smarcb1f/+;PdgfRα-Cre Smarcb1f/f preadipocytes D E F H&E staining IHC D-3 D2 30 )

f/f cKO f/f cKO 5 GFP GFP Cre GFP Cre 25 Cre -50 20 SMARCB1 M M B M M 15 SMARCA4 B - 150 B 10 B B C/EBPβ -37 5 Ucp1 Cell number ( x 10 -75 0 RbBP5 Myosin Days 0135

G H RNA-Seq I shArid1a GFP Cre Cebpb Pparg Cebpa Fabp4 shCtrl #1 #5 200 40 250 3000 GFP ARID1A - 250 Cre 200 2000 150 SMARCA4 100 20 - 150

RPKM 100 1000 -50 50 SMARCB1 -75 0 0 0 0 RbBP5 D-3 D2 D-3 D2 D-3 D2 D-3 D2 J K shArid1a Pparg Cebpa Fabp4 16 12 50 shCtrl #1 #5 D0 D0 D0 40 12 D7 9 D7 D7 30 8 6 20 4 3 10 RNA levels (fold) 0 0 0

Figure 2. BAF subunits SMARCA4, SMARCB1 and ARID1A are required for adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B E18.5 C Defect of coronal sutures in cKO embryos Smarcb1f/f x Smarcb1f/+;PdgfRα-Cre f/f cKO E18.5 P0.5 3 weeks Genotype (54) (51) (64) f/f cKO Smarcb1f/f (f/f) 13 15 22 Smarcb1f/f;PdgfRα-Cre (cKO) 14 *3 0 Smarcb1f/+ 14 16 20 Smarcb1f/+;PdgfRα-Cre 13 17 22

D Skull bone defect in cKO embryos f/f cKO

E BAT (E18.5) F qRT-PCR (n=5)

2 f/f Brown adipocyte-enriched genes Myogenesis markers f/f cKO n.s. n.s. n.s. 1.5 *** *** *** *** *** *** 1 cKO 0.5

Relative RNA (fold) 0 Smarcb1 Pparg Cebpa Fabp4 Prdm16 Ucp1 Myogenin MyHc MCK Smarcb1f/f preadipocytes G IP-WB H RNA-Seq D-3  D2 SMARCB1-dependent IP 5% input IgG SMARCA4 SMARCB1 Up-regulated 362 12723 937 mild or GFP Cre GFP Cre GFP Cre GFP Cre 1382 no change Down-regulated 575 SMARCA4 - 150 SMARCB1-independent SMARCC2 I - 150 Gene group GO term Count P-Value -50 Up-regulated SMARCB1 lipid metabolic process 75 8.30E-26 (SMARCB1 fat cell differentiation 34 5.50E-24 -75 -dependent) organic acid metabolic process 60 1.50E-22 RbBP5 cellular lipid metabolic process 61 2.10E-22 brown fat cell differentiation 18 1.70E-21 triglyceride metabolic process 23 2.00E-20 J RNA-Seq 80 > 300 GFP (D-3) Cre (D-3) GFP (D2) Cre (D2) 60

40 RPKM 20 ND 0

Core subunits ATPase module BAF-specific PBAF-specific GBAF-specific

Figure 2 - Supple 1. SMARCB1 is required for adipogenesis in vivo and in culture bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A BC f/f cKO Pbrm1f/f x Pbrm1f/+;Myf5-Cre 2.0 f/f Genotype Number cKO n.s. n.s. (3 weeks) of pups 1.5 Pbrm1f/f (f/f) 28 1.0 Pbrm1f/f;Myf5-Cre (cKO) 28 n.s. 0.5

Pbrm1f/+ 23 % of body weight 0.0 Pbrm1f/+;Myf5-Cre 26 BAT ingWAT epiWAT

D BAT E qRT-PCR (n=5) f/f cKO 2 f/f cKO #1 1.5 *** n.s. n.s. n.s. n.s.

#2 1 0.5 #3 Relative RNA (fold) 0 Pbrm1 Pparg Cebpa Fabp4 Ucp1

Pbrm1f/f (f/f) and Pbrm1f/f;Myf5-Cre (cKO) primary brown preadipocytes

FJG RPKM Gene f/f cKO Ratio f/f cKO 1.5 Pparg 115 91 0.8 *** level Adipogenesis Cebpa 150 159 1.1 1 marker Adipoq 475 596 1.3 Fabp4 5140 6307 1.2 Pbrm1 0.5 Nr1h2 30 29 1.0 Nr1h3 17 16 1.0 Lipogenic TFs Relative 0 Srebf1 198 101 0.5 f/f cKO Srebf2 16 14 0.9 Mlxipl 20 13 0.7 H RNA-Seq (D7) Acly 109 56 0.5 Acaca 58 36 0.6 cKO vs f/f Lipogenesis Acacb 22 12 0.5 (2.5 fold cut-off) enzymes Fasn 136 109 0.8 Scd1 2345 2479 1.1 11,002 (99.2%) 40 (0.4%) Decreased Lpl 779 876 1.1 Mild or no Cd36 178 157 0.9 47 (0.4%) Increased Lipid uptake change Slc27a1 30 37 1.2 Slc27a4 10 9 0.9

I Thrsp 282 87 0.3 GO term – Decreased genes P-Value Itih4 231 65 0.3 Decreased Ifi202b 37 14 0.4 execution phase of apoptosis 6.30E-03 genes regulation of T cell proliferation 8.00E-03 Cidea 20 7 0.4 GO term – Increased genes P-Value Mdk 17 5 0.3 defense response 1.90E-17 Retn 50 167 3.3 response to external stimulus 6.00E-13 Increased Ccl6 6183.1 inflammatory response 2.40E-12 genes Ccl12 2105.3 leukocyte migration 5.50E-11 C1qa 153.8 regulation of immune system process 6.00E-10 Pck1 10 35 3.4

Figure 2 - Supple 2. PBAF-specific subunit PBRM1 is dispensable for adipogenesis in vivo and in culture bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B Motif analysis (SMARCA4  SS18 AE) C  SMARCA4 SS18 ARID1A SMARCA4 SS18 AE  30 D-3 D2 D-3 (D2) 20 D2 20 20 Motif P-value Motif P-value

AE BAF-prebound 10 10 JUNB 4E-105 C/EBPα 1E-103 (31,349) 10 (4,410) SMARCA4 JUN 6E-104 C/EBPβ 8E-102 24,967 0 0 0 FOSL1 7E-102 C/EBPδ 1E-84 30 JUND 7E-95 C/EBPγ 6E-77  20 20 FOS 3E-94 NFIL3 9E-41 20 BACH2 6E-87 HLF 5E-39 JDP2 6E-80 ATF4 5E-34 10 10 (8,871) 10 BACH1 2E-75 DBP 3E-32 BAF-de novo promoter SMARCA4 NFE2L2 5E-47 NFIC 6E-28 (6,403) 0 0 0 -2.5 +2.5 -2.5 +2.5 -2.5 +2.5 MAFF 3E-19 TEF 1E-24 Distance from D2 SMARCA4 binding sites (kb)

D SMARCA4 SS18 AE (D2) SMARCA4 SS18ARID1A MLL4 CBP C/EBPβ C/EBPα PPARγ H3K4me1 H3K27ac ATAC D-3 D2 D-3 D2D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 BAF- (6,403) de novo BAF- (4,410) prebound -5 +5 Distance from SMARCA4  SS18 AE (kb)

Average profiles E D-3 D2 SMARCA4SS18 ARID1A MLL4 CBP C/EBPβ C/EBPαPPARγ H3K4me1 H3K27ac ATAC 30 80 40 80 30 60 60 40 4 8 10 20 40 20 40 15 30 30 20 2 4 5

BAF- 10 (6,403) de novo 0 0 0 0 0 0 0 0 0 0 0 30 80 40 80 30 60 60 40 4 8 10 20 40 20 40 15 30 30 20 2 4 5

BAF- 10 (4,410) prebound 0 0 0 0 0 0 0 0 0 0 0 -2 +2 Distance from SMARCA4  SS18 AE (kb)

Figure 3. BAF co-localizes with LDTFs and MLL4 on active enhancers during adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A Motif analysis: SMARCA4  AE B Motif analysis: SS18  AE C Motif analysis: ARID1A AE D-3 D2 D-3 D2 D-3 D2

Motif P-value Motif P-value Motif P-value Motif P-value Motif P-value Motif P-value

JUN 7E-105 C/EBPβ 2E-87 JUN 3E-149 C/EBPα 2E-275 FOS 1E-53 C/EBPα 2E-71 JUNB 7E-103 C/EBPα 5E-87 JUNB 4E-143 C/EBPβ 3E-246 JDP2 3E-53 C/EBPβ 1E-56 FOSL1 6E-101 C/EBPδ 7E-77 FOS 1E-139 C/EBPδ 4E-214 JUNB 3E-49 C/EBPδ 4E-48 FOS 2E-94 C/EBPγ 9E-77 FOSL1 2E-126 C/EBPγ 2E-188 FOSL1 1E-48 C/EBPγ 2E-43 JUND 2E-93 NFIL3 5E-37 JUND 6E-126 JUND 6E-81 JUND 2E-48 NFIL3 2E-23 BACH2 9E-86 HLF 7E-36 JDP2 7E-115 NFIL3 5E-76 BACH1 2E-48 ATF4 1E-18 JDP2 2E-80 ATF4 2E-32 BACH1 2E-107 JUN 2E-75 JUN 2E-46 HLF 8E-18 BACH1 2E-77 DBP 1E-28 BACH2 4E-97 NFIC 2E-69 BACH2 2E-39 DBP 2E-14 NFE2L2 1E-44 NFIX 3E-25 MAF 4E-60 JUNB 5E-68 NFE2 6E-39 NFIX 3E-12 MAFF 6E-18 NFIC 3E-24 NFE2L2 2E-55 NFIA 6E-67 BATF 4E-21 EBF 4E-11

D Pparg locus E Cebpa locus

D-3 D-3 SMARCA4 SMARCA4 D2 D2 D-3 D-3 SS18 SS18 D2 D2 D-3 D-3 ARID1A ARID1A D2 D2 D-3 D-3 MLL4 MLL4 D2 D2 D-3 D-3 CBP CBP D2 D2 D-3 D-3 C/EBPβ C/EBPβ D2 D2 D-3 D-3 C/EBPα C/EBPα D2 D2 D-3 D-3 PPARγ PPARγ D2 D2 D-3 D-3 H3K4me1 H3K4me1 D2 D2 D-3 D-3 H3K27ac H3K27ac D2 D2 D-3 D-3 ATAC ATAC D2 D2

Pparg1 Cebpa Pparg2

Figure 3 - Supple 1. BAF colocalizes with LDTFs on active enhancers during adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A B Differential motif analysis (SMARCA4 SS18 AE, D2)  SMARCA4 SS18 AE MLL4 MLL4 (D2) MotifLogo P-value MotifLogo P-value  MLL4 (4,362) PPARγ 1E-34 FRA1 1E-197 C/EBP:AP1 1E-33 JUNB 1E-188 C/EBP 1E-32 BATF 1E-188 RXR 1E-29 ATF3 1E-186 PPARα 1E-27 cJUN 1E-181 MLL4  6,451) ATF4 1E-23 FRA2 1E-176 NFIL3 1E-22 FOSL2 1E-168

C SMARCA4 SS18 AE (D2) SMARCA4 SS18 ARID1A MLL4 CBP C/EBPβ C/EBPα PPARγ H3K4me1 H3K27ac ATAC

 D-3 D2 D-3 D2D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 D-3 D2 MLL4 (4,362)  MLL4 (6,451)

-5 +5 Distance from SMARCA4 SS18 AE (kb)

D Average profiles D-3 D2 SMARCA4 SS18 ARID1A CBP C/EBPβ C/EBPα PPARγ H3K4me1 H3K27ac ATAC 40 80 40 40 50 80 80 5 10 10 

20 40 20 20 25 40 40 5 5 MLL4 (4,362) 0 0 0 0 0 0 0 0 0 0 40 80 40 40 50 80 80 5 10 10 

20 40 20 20 25 40 40 5 5 MLL4 (6,451) 0 0 0 0 0 0 0 0 0 0 -2 +2 Distance from SMARCA4 SS18 AE (kb)

Figure 3 - Supple 2. Markedly increased BAF binding and chromatin accessibility on MLL4+ active enhancers during adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

A α-MLL4 IP α-UTX IP Protein Peptides Protein Peptides

MLL4 89 MLL4 70 MLL3 64 UTX 32 UTX 30 PTIP 19 PTIP 16 ASH2L 15 ASH2L 13 NCOA6 9 NCOA6 12 WDR5 5 WDR5 6 PA1 4 PA1 5 DPY30 2 DPY30 1 53BP1 1 53BP1 15

BRG1 / SMARCA4 1 BRG1 / SMARCA4 7 BAF155 / SMARCC1 6 BAF155 / SMARCC1 15 BAF60A / SMARCD1 1 BAF60A / SMARCD1 3 SNF5 / BAF47 / SMARCB1 2 BAF57 / SMARCE1 4 BAF200 / ARID2 5 BAF180 / PBRM1 7 BRD7 4

B HEK293T cells C D2 of adipogenesis IP IP

kDa kDa

SMARCA4 SMARCA4 - 150 - 150 -50 -50 SMARCB1 SMARCB1

MLL4 MLL4

- 250 - 250 - 150 - 150 UTX UTX -75 -75 RbBP5 RbBP5

-75 -75 Menin Menin

Figure 3 - Supple 3. SWI/SNF subunits associate with MLL4 complex in cells bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Ad-Cre D-3 D2 Mll3-/-Mll4f/f preadipocytes ChIP-Seq, ATAC-Seq A Heat maps (SMARCA4 SS18 MLL4 adipogenic enhancers) D2 MLL4 SMARCA4 SS18 ATAC GFP D-3 D2 D-3 D2 D-3 D2 D-3 D2 GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre 2,158

-5 +5 Distance from SMARCA4 SS18 MLL4  adipogenic enhancers (kb) B Average profiles MLL4 SMARCA4 SS18 ATAC 150 40 80 10 GFP, D-3 100 Cre, D-3 20 40 5 GFP, D2 50 Cre, D2 0 0 0 0 -2kb +2kb Normalized read count Distance from SMARCA4 SS18 MLL4 adipogenic enhancers (kb)

Ad-Cre D-3 D2 Smarcb1f/f preadipocytes ChIP-Seq, ATAC-Seq C Heat maps (SMARCA4 SS18 MLL4 adipogenic enhancers) D2 SMARCA4 SS18 ARID1A ATAC D-3 D2 D-3 D2 D-3 D2 D-3 D2 GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre 3,530

-5 +5 Distance from SMARCA4 SS18 MLL4 adipogenic enhancers (kb)

D MLL4 CBP H3K4me1 H3K27ac D-3 D2 D-3 D2 D-3 D2 D-3 D2 GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre GFP Cre 3,530

-5 +5 Distance from SMARCA4 SS18 MLL4 adipogenic enhancers (kb)

E Relative intensity F D-3 D2 8 *** GFP Cre GFP Cre *** 4 H3K4me1 *** *** SMARCA4 MLL4 *** *** *** *** SS18 CBP 0 H3K27ac ARID1A H3K4me1 fold change 2 -4 ATAC H3K27ac

(D2, Cre/GFP) H3 Log

-8 *** p<2.2E-16 Figure 4. Reciprocal regulation between BAF and MLL4 on active enhancers during adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Mll3-/-Mll4f/f preadipocytes Smarcb1f/f preadipocytes A Pparg locus C Pparg locus GFP GFP D-3 D-3 Cre Cre GFP GFP D2

D2 SMARCA4 Cre

SMARCA4 Cre GFP GFP D-3 D-3 Cre Cre

SS18 GFP D2

SS18 GFP D2 Cre Cre GFP GFP D-3 D-3 Cre Cre GFP

GFP ARID1A D2 ATAC D2 Cre Cre GFP D-3 Pparg1 Cre Pparg2 GFP MLL4 D2 Cre GFP B Fabp4 locus D-3 Cre

GFP CBP GFP D-3 D2 Cre Cre GFP GFP D2 D-3

SMARCA4 Cre Cre GFP GFP D-3 D2 Cre H3K4me1 Cre GFP

SS18 GFP D2 D-3 Cre Cre GFP GFP D2

D-3 H3K27ac Cre Cre GFP GFP ATAC D2 D-3 Cre Cre GFP Fabp4 Fabp12 ATAC D2 Cre

Pparg1 Pparg2

Figure 4 - Supple 1. Reciprocal regulation between BAF and MLL4 on active enhancers during adipogenesis bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

Mll3-/-Mll4f/f preadipocytes A Heat maps (BAF  MLL4 AE) B Relative intensity

SMARCA4 ARID1A ATAC H3K27ac Mll3-/-Mll4f/f 4 Cre  2 *** SMARCA4 *** *** ARID1A 0

fold change ATAC 2 (Cre/GFP) 6,304 -2 H3K27ac Log -4 -2 +2 *** p<2.2E-16 Distance from SMARCA4 binding sites (kb)

Smarcb1f/f preadipocytes Smarca4AID/AID preadipocytes C Heat maps (BAF  MLL4 AE) E Heat maps (BAF  MLL4 AE) SMARCA4 ARID1A ATAC MLL4 H3K4me1 H3K27ac ATAC MLL4

Cre  Tir1  6,304 6,304

-2 +2 -2 +2 Distance from SMARCA4 binding sites (kb) Distance from MLL4 binding sites (kb)

D Relative intensityF Relative intensity Smarcb1f/f Smarca4AID/AID 4 4

2 *** 2 *** *** *** SMARCA4 MLL4 *** *** ATAC 0 ARID1A H3K4me1 0 fold change fold change MLL4 2 2 (Tir1/Vec)

(Cre/GFP) ATAC H3K27ac -2 -2 Log Log -4 -4 *** p<2.2E-16 *** p<2.2E-16

Figure 5. BAF and MLL4 reciprocally promote each other’s binding to maintain active enhancers in preadipocytes bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

C/EBPβ Ad-Cre Mll3-/-Mll4f/f preadipocytes ChIP-Seq

Motif analysis A Vec C/EBPβ B C BAF-de novo BAF-prebound C/EBPβ-activated GFP Cre GFP Cre (324) (247) enhancers (571) -37 C/EBPβ Motif P-value Motif P-value BAF-de novo SMARCA4 (324) C/EBPβ 1E-27 C/EBPα 3E-14 - 150 C/EBPα 6E-27 C/EBPδ 2E-09 SS18 -50 C/EBPδ 1E-26 C/EBPβ 2E-09 C/EBPγ 3E-26 RUNX1 2E-06 ARID1A - 250 DBP 1E-12 RUNX2 4E-05 -75 BAF-prebound RbBP5 (247) NFIL3 1E-12 TEAD3 6E-05

D Heat maps (C/EBPβ-activated enhancers) C/EBPβ MLL4 SMARCA4 ARID1A H3K27ac C/EBPβ         Cre    BAF- (324) de novo BAF- (247) prebound -5 +5 Distance from C/EBPβ-activated enhancers (kb)

E Average profiles C/EBPβ MLL4 SMARCA4 ARID1A H3K27ac 60 90 10 15 6 60 10 30 5 3 BAF- (324) 30 5 de novo Vec/GFP 0 0 0 0 0 Vec/Cre 60 90 10 15 6 C/EBPβ/GFP C/EBPβ/Cre 60 10 30 5 3 BAF- (247) 30 5 prebound 0 0 0 0 0 -2 +2 Distance from C/EBPβ-activated enhancers (kb)

Figure 6. MLL4 is required for BAF binding on C/EBPβ-activated enhancers bioRxiv preprint doi: https://doi.org/10.1101/2020.06.24.168146; this version posted June 25, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder. All rights reserved. No reuse allowed without permission.

C/EBPβ Auxin (4h) Smarca4AID/AID preadipocytes ChIP-Seq, ATAC-Seq ABC Motif analysis Vec C/EBPβ C/EBPβ-activated SMARCA4 SMARCA4 -independent -dependent Tir1  enhancers (449) (220) (229) C/EBPβ -37 SMARCA4-independent Motif P-value Motif P-value - 250 (220) AID C/EBPδ 7E-30 JUN 1E-22 - 250 C/EBPα 3E-22 FOSL1 2E-20 SMARCA4 C/EBPβ 1E-20 JUNB 3E-20 - 100 Myc (Tir1) C/EBPγ 1E-19 FOS 4E-19 SMARCA4-dependent ATF4 4E-10 JUND 2E-16 -75 (229) RbBP5

D Heat maps (C/EBPβ-activated enhancers) C/EBPβ ATAC MLL4 H3K27ac C/EBPβ      Tir1   SMARCA4 -independent (220)

SMARCA4 -dependent (229)

-5 +5 Distance from C/EBPβ-activated enhancers (kb)

E Average profiles C/EBPβ ATAC MLL4 H3K27ac 40 5 50 2 ndent 20 2.5 25 1 (220) Vec/Vec SMARCA4

-indepe 0 0 0 0 Vec/Tir1 40 5 50 C/EBPβ/Vec 2 C/EBPβ/Tir1

ndent 20 2.5 25 1 (229) SMARCA4 -depe 0 0 0 0 -2 +2 Distance from C/EBPβ-activated enhancers (kb) F Enhancer priming Enhancer activation

me H3K4me1 BAF MLL4 p300 ac H3K27ac BAF MLL4

me me me me me me ac OFF ac ON LDTF Pioneer Pioneer

Enhancer TSS Enhancer TSS

Figure 7. BAF regulates MLL4 binding on C/EBPβ-activated enhancers