H3.3K4M Destabilizes Enhancer Epigenomic Writers MLL3/4 and Impairs Adipose Tissue

bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. The copyright holder for this preprint (which was not certified by peer review) is the author/funder.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 H3.3K4M destabilizes enhancer epigenomic writers MLL3/4 and impairs adipose tissue

2 development

4 Younghoon Jang1, Chaochen Wang1, Aaron Broun1, Young-Kwon Park1, Lenan Zhuang1, Ji-Eun Lee1, 5 Eugene Froimchuk1, Chengyu Liu2, and Kai Ge1 6

7 1Adipocyte Biology and Gene Regulation Section, Laboratory of Endocrinology and Receptor Biology,

8 National Institute of Diabetes and Digestive and Kidney Diseases, National Institutes of Health, Bethesda,

9 MD 20892

10 2Transgenic Core, National Heart, Lung, and Blood Institute, NIH, Bethesda, MD 20892

12 *Correspondence: [email protected]

13 Abstract

14 Histone H3K4 mono-methyltransferases MLL3 and MLL4 (MLL3/4) are required for enhancer activation

15 during cell differentiation, though the mechanism is incompletely understood. To address MLL3/4

16 enzymatic activity in enhancer regulation, we have generated two mouse lines: one expressing H3.3K4M,

17 a lysine-4-to-methionine (K4M) mutation of histone H3.3 that inhibits H3K4 methylation, and the other

18 carrying conditional double knockout of MLL3/4 enzymatic SET domains. Expression of H3.3K4M in

19 lineage-specific precursor cells depletes H3K4 methylation and prevents adipogenesis and adipose tissue

20 development. Mechanistically, H3.3K4M prevents enhancer activation in adipogenesis by destabilizing

21 MLL3/4 proteins but not other Set1-like H3K4 methyltransferases. Notably, deletion of the enzymatic SET

22 domain of MLL3/4 in lineage-specific precursor cells mimics H3.3K4M expression and prevents adipose

23 tissue development. Interestingly, destabilization of MLL3/4 by H3.3K4M in adipocytes does not affect

24 adipose tissue maintenance and function. Together, our findings indicate that H3.3K4M destabilizes

25 enhancer epigenomic writers MLL3/4 and impairs adipose tissue development.

26 Introduction

27 During cell differentiation, transcriptional enhancers are bound by lineage-determining transcription

28 factors (LDTFs) and play a key role in regulating cell type-specific gene expression. Cell type-specific

29 enhancers are marked by specific epigenomic features 1. Histone 3 lysine 4 (H3K4) mono-methylation

30 (H3K4me1) is the predominant mark of a primed enhancer state. Histone 3 lysine 27 acetylation

31 (H3K27ac) by H3K27 acetyltransferases CBP/p300 further follows H3K4me1 to mark an active enhancer

32 state 2. There are six mammalian Set1-like H3K4 methyltransferases, each containing a catalytic SET

33 domain that enables the deposition of methyl marks on H3K4: MLL1 (or KMT2A), MLL2 (or KMT2B),

34 MLL3 (or KMT2C), MLL4 (or KMT2D), SET1A (or KMT2F), and SET1B (or KMT2G) 3. Among them,

35 MLL4 is a major mammalian H3K4 mono-methyltransferase with partial functional redundancy with MLL3.

36 MLL3 and MLL4 (MLL3/4) are required for CBP/p300-mediated enhancer activation in cell differentiation

37 and cell fate transition 4-6. Deletion of Mll3/4 genes depletes H3K4me1 in cells and prevents the

38 enrichment of CBP/p300-mediated H3K27ac, epigenome reader BRD4, Mediator coactivator complex,

39 and RNA Polymerase II on enhancers. Consequently, Mll3/4 deletion prevents enhancer RNA production,

40 cell type-specific gene induction and cell differentiation 7. However, the role of H3K4me1 in enhancer

41 regulation, cell differentiation and function in vivo is poorly understood.

42 Adipogenesis and adipose tissue are useful model systems for studying cell differentiation as well

43 as tissue development and function. Adipogenesis is mainly controlled by a cascade of sequentially

44 expressed transcription factors (TFs) 8. Although many TFs have been implicated in adipogenesis, PPARγ

45 and C/EBPα are primary drivers of the induction of thousands of adipocyte genes 9,10. Adipose tissues,

46 including white adipose tissue (WAT) and brown adipose tissue (BAT), are dynamic endocrine organs

47 that regulate thermogenesis, energy metabolism and homeostasis 11. The study of adipogenesis and

48 adipose tissue in vivo requires the isolation of tissues of the adipose lineage at particular developmental

49 and functional stages. Myogenic factor 5 (Myf5) promoter-driven Cre (Myf5-Cre) allows factors to be

50 expressed or deleted in mouse preadipocytes, enabling a focus on adipogenesis. Conversely,

51 Adiponectin promoter-driven Cre (Adiponectin-Cre) is generally expressed in differentiated adipocytes but

52 not precursor cells, permitting study of adipocyte function 12. By crossing Mll4 conditional knockout (KO)

53 mice with Myf5-Cre or Adiponectin-Cre mice, we have shown that MLL4 is required for adipose tissue

54 development but largely dispensable for adipose tissue maintenance 4,5. However, the roles of MLL3/4

55 enzymatic activities and MLL3/4-mediated H3K4me1 in adipose tissue development and function are

56 unclear.

57 By tissue-specific ectopic expression of a histone H3.3 lysine-to-methionine mutant (H3.3K4M) in

58 mice, we show that depletion of H3K4 methylation by H3.3K4M inhibits adipose tissue development. By

59 tissue-specific deletion of the enzymatic SET domain of MLL3/4 in mice, we further show that the SET

60 domain is required for adipose tissue development. Mechanistically, expression of H3.3K4M or deletion of

61 the SET domain prevents MLL3/4-mediated enhancer activation in adipogenesis by destabilizing MLL3/4

62 proteins. Interestingly, H3.3K4M does not affect adipose tissue maintenance nor the thermogenic function

63 of BAT.

64 Results

65 Histone H3.3K4M and H3.3K36M mutations impair adipogenesis

66 Previous studies reported that ectopic expression of histone H3.3 lysine-to-methionine (K-to-M) mutant

67 specifically depletes endogenous lysine methylation in cells 13,14. To understand the role of site-specific

68 histone methylation in adipogenesis, we used retroviruses to stably express wild type (WT) or K-to-M

69 mutant (K4M, K9M, K27M or K36M) of histone H3.3 in brown preadipocytes. The expression levels of

70 FLAG-tagged H3.3 were much lower than that of endogenous H3 (Figure 1a). Consistent with previous

71 reports 13,14, ectopic expression of H3.3K4M selectively decreased global H3K4me1, H3K4me2, and

72 H3K4me3 levels, while H3.3K9M and H3.3K27M selectively decreased global levels of H3K9me2 and

73 H3K27me3, respectively. Ectopic expression of H3.3K36M selectively depleted global H3K36me2 and

74 moderately increased H3K27me3 levels (Figure 1a and Supplementary Fig S1a). Cells were induced to

75 undergo adipogenesis. As shown in Figure 1b-c, H3.3K4M-expressing (K4M) cells showed severe

76 defects in adipogenesis and associated expression of adipogenesis markers Pparg, Cebpa and Fabp4.

77 Consistent with our recent report 29, H3.3K36M-expressing cells also showed defects in adipogenesis.

78 These results indicate that histone H3.3K4M and H3.3K36M mutations impair adipogenesis.

79 H3.3K4M inhibits adipose tissue and muscle development

80 Next, we investigated whether H3.3K4M affects adipose tissue development in vivo. We generated a

81 conditional H3.3K4M transgenic mouse line, lox-STOP-lox-H3.3K4M (LSL-K4M) for tissue-specific

82 expression of H3.3K4M. The insertion of 4 copies of SV40 stop signals (STOP) flanked by two loxP sites

83 prevents the CAG promoter-driven expression of FLAG-tagged H3.3K4M in the absence of Cre (Figure

84 2a-b). We crossed LSL-K4M mice with Myf5-Cre mice to induce H3.3K4M expression specifically in

85 somitic precursor cells of brown adipose tissue (BAT) and skeletal muscle 4. No LSL-K4M;Myf5-Cre mice

86 were observed at the weaning age. Newborn (P0) LSL-K4M;Myf5-Cre pups were obtained but died

87 immediately after birth from breathing malfunctions due to deficient muscle groups in the rib cage (Figure

88 2c-d). Immunohistochemical analysis of cervical regions of E18.5 LSL-K4M;Myf5-Cre embryos revealed

89 marked reduction in BAT and back muscle mass (Figure 2e-f), indicating that expressing H3.3K4M in

90 progenitor cells prevents adipose tissue and muscle development in vivo.

91 H3.3K4M destabilizes MLL3/4 proteins in adipogenesis

92 To confirm that H3.3K4M prevents adipogenesis in a cell autonomous manner, we crossed LSL-K4M with

93 Cre-ER mice to obtain primary LSL-K4M;Cre-ER brown preadipocytes. After immortalization, cells were

94 treated with 4-hydroxytamoxifen (4OHT) to delete the STOP cassette and induce FLAG-tagged H3.3K4M

95 expression. As expected, induction of H3.3K4M expression decreased global levels of endogenous

96 H3K4me1/2/3. Interestingly, H3.3K4M expression also decreased H3K27ac levels (Figure 3a). Consistent

97 with our previous findings observed in embryonic stem (ES) cells 15, expressing H3.3K4M destabilized

98 endogenous MLL3/4 as well as the MLL3/4-associated protein UTX in cells. However, H3.3K4M did not

99 affect protein levels of other members of the mammalian Set1-like H3K4 methyltransferase family,

100 including SET1A, SET1B, and MLL1 16 (Figure 3b).

101 Ectopic expression of H3.3K4M had little effect on cell proliferation (Figure 3c), but prevented

102 adipogenesis and the induction of adipocyte marker genes such as Pparg, Cebpa and Fabp4 as well as

103 BAT-specific marker gene Ucp1 (Figure 3d-e). We confirmed these H3.3K4M-driven adipogenesis defects

104 using independent brown preadipocyte lines stably expressing WT H3.3 or H3.3K4M (Supplementary Fig

105 S1). To investigate how H3.3K4M inhibits adipogenesis, we further performed RNA-seq analysis of LSL-

106 K4M;Cre-ER preadipocytes treated with or without 4OHT. Using a 2.5-fold cut-off for differential gene

107 expression from RNA-seq analysis, we defined genes up-regulated (605/5.0%) or down-regulated

108 (709/5.8%) by H3.3K4M at D7 of differentiation (Figure 3f-g). Gene ontology (GO) analysis showed that

109 down-regulated genes were strongly associated functionally with fat cell differentiation and lipid

110 metabolism (Figure 3h). Because MLL3/4 are essential for adipogenesis 4, these data suggest that

111 H3.3K4M inhibits adipogenesis at least in part by destabilizing MLL3/4 proteins.

112 H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis

113 Next, we investigated whether H3.3K4M affects MLL3/4-mediated enhancer activation in adipogenesis.

114 We performed ChIP-seq analyses of enhancer marks H3K4me1 and H3K27ac in LSL-K4M;Cre-ER cells

115 treated with or without 4OHT at D4 of adipogenesis. Since ChIP-seq analysis did not consider the global

116 differences between samples, we used histone Western blot data as a normalization control for more

117 accurate quantitative analysis of H3K4me1 and H3K27ac (Supplementary Fig S2). By comparing with the

118 published MLL4 ChIP-seq data during adipogenesis 4,6, we identified 6,686 MLL4+ active enhancers

119 during adipogenesis. 4OHT-induced H3.3K4M expression prevented H3K4me1 and H3K27ac

120 accumulation on MLL4+ active enhancers during adipogenesis (Figure 4a-b). Similar results were

121 observed on Pparg and Cebpa loci (Figure 4c). Further, ChIP-qPCR analyses revealed that, on

122 representative MLL4+ active enhancers (e1-e5) located on gene loci of master adipogenic regulators

123 PPARγ and CEBPα 7, H3.3K4M markedly reduced the occupancy of MLL4, MLL3/MLL4-mediated

124 H3K4me1, CBP/p300-mediated H3K27ac, BRD4, the MED1 subunit of the Mediator coactivator complex,

125 and Pol II (Figure 4c-d). H3.3K4M also decreased eRNA production from MLL4+ adipogenic enhancers

126 (Figure 4e). Together, these results suggest that H3.3K4M prevents MLL3/4-mediated enhancer

127 activation in adipogenesis.

128 Deletion of the enzymatic SET domain of MLL3/4 prevents adipose tissue and muscle

129 development

130 MLL3 and MLL4 are partially redundant and are major H3K4 mono-methyltransferases on enhancers in

131 cells 4. In a separate attempt to investigate the functional role of MLL3/4 enzymatic activity in

132 differentiation and development in vivo, we used two conditional KO mouse lines targeting the enzymatic

133 SET domain of MLL3 and MLL4 (Mll3f/f and Mll4SETf/f, Figure 5a-b). In the Mll3f/f mice, exons 57 and 58,

134 which encode critical amino acids of the SET domain, were flanked by two loxP sites 17. In the Mll4SETf/f

135 mice, exons 50 and 51, which encode the entire SET domain, were flanked by two loxP sites. Cre-

136 mediated deletion of these exons would result in the production of enzyme-dead MLL3/4.

137 We first crossed Mll4SETf/f with Myf5-Cre mice. The resulting Mll4SETf/f;Myf5-Cre mice survived

138 until birth. E17.5~18.5 Mll4SETf/f;Myf5-Cre embryos were unable to breathe and died immediately after

139 isolation, displaying an abnormal hunched posture and severe reduction of back muscles (Supplementary

140 Fig S3a-c). These embryos showed only a moderate decrease of BAT mass compared to WT, possibly

141 due to a compensatory effect of MLL3 (Supplementary Fig S3c). To eliminate the compensatory effect,

142 we crossed Mll4SETf/f with Mll3f/f and Myf5-Cre mice to delete both Mll3 and Mll4 genes in progenitor cells

143 of BAT and muscle lineages. The resulting E18.5 Mll3f/f;Mll4SETf/f;Myf5-Cre (conditional double KO, DKO)

144 embryos were unable to breathe and died immediately after isolation. These embryos showed profound

145 reduction of BAT as well as muscle mass (Figure 5c-e). These data indicate that deletion of the enzymatic

146 SET domain of MLL3/4 prevents adipose tissue and muscle development.

147 Deletion of the enzymatic SET domain inhibits adipogenesis by destabilizing MLL3/4

148 To confirm that deletion of the enzymatic SET domain of MLL3/4 prevents adipogenesis in a cell

149 autonomous manner, we crossed Mll3f/f;Mll4SETf/f mice with Cre-ER mice to obtain primary

150 Mll3f/f;Mll4SETf/f;Cre-ER brown preadipocytes. After immortalization, cells were treated with 4OHT to

151 delete exons encoding the SET domain of MLL3/4. Consistent with our previous finding that MLL3 and

152 MLL4 are major H3K4 mono- and di-methyltransferases in cells 4,5, deletion of the enzymatic SET domain

153 in preadipocytes decreased global levels of endogenous H3K4me1/2 but not H3K4me3 (Figure 6a). In

154 addition, deleting the enzymatic SET domain destabilized endogenous MLL3/4 and reduced protein levels

155 of MLL3/4-associated UTX in cells (Figure 6b), which is consistent with a recent report that deleting the

156 SET domain destabilizes MLL3/4 in ES cells 18.

157 Deletion of the enzymatic SET domain of MLL3/4 had little effect on cell proliferation (Figure 6c),

158 but prevented adipogenesis and the induction of Pparg, Cebpa, Fabp4, and Ucp1 (Figure 6d-e). RNA-seq

159 analysis at D7 of differentiation confirmed the deletion of target exons of both Mll3 and Mll4 loci (Figure

160 6f). We further performed RNA-seq analysis of Mll3f/f;Mll4SETf/f;Cre-ER preadipocytes treated with or

161 without 4OHT. Using a 2.5-fold cut-off for differential expression from RNA-seq analysis, we defined

162 genes up-regulated (623/5.1%) or down-regulated (890/7.3%) by deletion of the enzymatic SET domain

163 at D7 of differentiation (Figure 6g-h). GO analysis showed that down-regulated genes were strongly

164 functionally associated with fat cell differentiation and lipid metabolism (Figure 6i). These data suggest

165 that deletion of the enzymatic SET domain inhibits adipogenesis by destabilizing MLL3/4.

166 H3.3K4M expression mimics MLL3/4 SET domain deletion in preventing adipogenesis

167 Next, we generated scatter plots of gene expression changes using RNA-seq data from cells with deletion

168 of MLL3/4 SET domain (DKO) and cells with expression of H3.3K4M (K4M) (Figure 7a). A combined

169 scatter plot revealed that down- or up-regulated genes were highly correlated between DKO and K4M

170 cells (Figure 7b). We found that the majority of down or up-regulated genes at D7 of differentiation were

171 shared by DKO and K4M (Figure 7c-d). The shared down-regulated genes were highly associated

172 functionally with fat cell differentiation and lipid metabolism whereas shared up-regulated genes were

173 highly associated with cell proliferation and development (Figure 7e-f). These data suggest that ectopic

174 H3.3K4M mimics the effect of Mll3/4 SET domain deletion in preventing adipogenesis.

175 H3.3K4M does not affect adipose tissue maintenance and function

176 We also investigated the role of H3K4 methylation in adipose tissue maintenance and function in mice.

177 For this purpose, we crossed LSL-K4M mice with Adipoq-Cre mice to achieve adipocyte-selective

178 expression of H3.3K4M in vivo. Histone Western blot of BAT from LSL-K4M;Adipoq-Cre mice showed that

179 ectopic expression of H3.3K4M depleted endogenous H3K4me1/2/3 and MLL3/4 levels in BAT (Figure

180 8a-b). LSL-K4M;Adipoq-Cre mice did not show any discernable differences in body weight, fat/lean mass,

181 or adipose tissue mass relative to control (LSL-K4M) mice at 8-10 weeks of age (Figure 8c-e). In the BAT,

182 induction of H3.3K4M expression was successful in LSL-K4M;Adipoq-Cre mice, but the expression levels

183 of adipocyte identity genes Pparg, Cebpa, and Fabp4 were similar between LSL-K4M;Adipoq-Cre and

184 control mice (Figure 8f). This phenotype is consistent with what we have reported for the

185 Mll4SETf/f;Adipoq-Cre mice, in which MLL4 is dispensable for the maintenance of differentiated

186 adipocytes in vivo 5.

187 We further asked about the functional consequence of ectopic H3.3K4M expression in adipocytes

188 in vivo. For this purpose, we acutely exposed the mice to environmental cold (6°C) up to 6 h. LSL-

189 K4M;Adipoq-Cre mice maintained normal body temperatures, were cold tolerant and behaved similarly as

190 control mice in the cold tolerance test (Figure 8g). After cold exposure, expression levels of

191 thermogenesis genes Ucp1, Dio2, and Elovl3 were similarly induced in BAT of LSL-K4M;Adipoq-Cre and

192 control mice (Figure 8h). Together, our data indicate that while H3.3K4M prevents adipose tissue

193 development, it does not affect the maintenance and function of adipose tissues. Our data also suggest

194 that H3K4 methylation is dispensable for the maintenance and function of differentiated adipocytes.

195 Discussion

196 Site-specific histone methylations are generally correlated with gene activation or gene repression. To

197 investigate the role of site-specific histone methylations in cell differentiation and development, we

198 screened several K-to-M mutants of H3.3 and found that H3.3K4M and H3.3K36M mutations impair

199 adipogenesis in cell culture. Mechanistically, H3.3K4M destabilizes MLL3/4 proteins but not other

200 members of the mammalian Set1-like H3K4 methyltransferase family, including SET1A, SET1B, and

201 MLL1. Consequently, H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis. Using

202 tissue-specific expression of H3.3K4M in mice, we next showed that H3.3K4M inhibits adipose tissue and

203 muscle development. However, H3.3K4M does not affect adipose tissue maintenance nor the

204 thermogenic function of BAT. Using tissue-specific deletion of the enzymatic SET domains of MLL3/4 in

205 mice, we demonstrated that the SET domains are required for adipose tissue and muscle development.

206 Mechanistically, deletion of the SET domains destabilizes MLL3/4 proteins. Notably, H3.3K4M expression

207 mimics MLL3/4 SET domain deletion in preventing adipogenesis. Together, our findings suggest that

208 H3.3K4M destabilizes enhancer epigenomic writers MLL3/4 and impairs adipose tissue development

209 (Figure 4f).

210 Consistent with our observation in preadipocytes, it was shown recently that MLL3/4 proteins with

211 SET domain deletion are unstable in ES cells 18. However, another recent report contradictorily showed

212 no MLL3/4 protein stability defects in ES cells with MLL3/4 SET domain deletion 19. To find out what

213 caused such discrepancies, we examined the different designs for SET domain deletion on MLL3/4

214 proteins in these three studies. We showed that deleting exons 57 and 58 results in a destabilized MLL3

215 protein in cells (Figure 6b). In contrast, Rickels et al. deleted exons 56, 57 and 58 without affecting MLL3

216 protein stability in cells 19. The full-length mouse MLL4 protein has 5588aa. Dorighi et al. showed that a

217 truncated MLL4 protein containing amino acids 1-5482 is unstable in ES cells 18. In contrast, Rickels et al.

218 showed that a more severely truncated MLL4 protein containing amino acids 1-5402 is stable in ES cells

219 19. Future studies are needed to verify whether the SET domain is required for MLL3/4 protein stability in

220 cells. Consistent with our previous report that ectopic expression of H3.3K4M reduces endogenous levels

221 of MLL3/4 and MLL3/4-associated UTX protein in ES cells 15, we now show that H3.3K4M destabilizes

222 MLL3/4 and UTX proteins in preadipocytes and BAT. However, it remains to be determined

223 mechanistically how H3.3K4M and the SET domains regulate MLL3/4 protein stability.

224 Although H3K4me1 is the predominant mark of a primed enhancer state, it is unclear whether

225 H3K4me1 affects or simply correlates with enhancer activation in cell differentiation and development.

226 Interestingly, the latest two studies from one group reported that MLL3/4-dependent H3K4me1 has an

227 active role at enhancers by facilitating binding of the chromatin remodeler SWI/SNF complex and

228 recruitment of the chromatin organization regulator cohesin complex to orchestrate long-range chromatin

229 interactions in ES cells 20,21. These findings imply that H3K4me1 is not simply a correlative outcome in

230 enhancer regulation. Conversely, using CRISPR to generate catalytically inactive MLL3/4, another recent

231 study surprisingly reported that MLL3/4 proteins, rather than MLL3/4-mediated H3K4me1, are required for

232 enhancer activation and gene transcription in undifferentiated ES cells. However, the role of MLL3/4-

233 mediated H3K4me1 in ES cell differentiation was not elucidated 18. Thus, despite various efforts to

234 uncover the role of H3K4me1 in enhancer function, it is still unclear whether H3K4me1 controls enhancer

235 activation in cell differentiation. Therefore, future work will be needed to clarify the role of MLL3/4-

236 mediated H3K4me1 in enhancer function during cellular differentiation and animal development.

237 Methods

238 Plasmids, Antibodies and Chemicals

239 The retroviral pQCXIP plasmids expressing FLAG-tagged wild type (WT) or mutants of histone H3.3

240 including K4M, K9M, K27M and K36 were described previously 14. The following homemade antibodies

241 have been described: anti-MLL4#3 22, anti-MLL3#3 16 and anti-UTX 23. Anti-RbBP5 (A300-109A), anti-

242 BRD4 (A301-985A100) and anti-MED1 (A300-793A) were from Bethyl Laboratories. Anti-SET1A/B

243 antibodies were described previously 24. Anti-H3 (ab1791), anti-H3K4me1 (ab8895), anti-H3K4me2

244 (ab7766), anti-H3K27ac (ab4729) and H3K36me3 (ab9050) were from Abcam. Anti-MLL1N (A700-010),

245 anti-MLL1C (A300-374A), anti-Pol II (17-672), anti-H3K4me3 (07-473), anti-H3K9me2 (17-648), anti-

246 H3K27me3 (07-449) and anti-H3K36me2 (07-369) were from Millipore. Anti-FLAG-M2 (F3165) and (Z)-4-

247 Hydroxytamoxifen (4OHT) (H7904) were from Sigma.

248 Generation of Mouse Strains

249 To generate LSL-K4M transgenic mice, H3.3K4M and a 3’ FLAG tag (H3.3K4M-FLAG) were fused

250 downstream of CAG promoter with a loxP-STOP-loxP cassette in the middle of the pBT346.6 plasmid

251 (AST-3029, Applied StemCell) (Figure 2a). H3.3K4M-FLAG of pQCXIP-H3.3K4M was subcloned into the

252 pBT346.6; after confirmation by DNA sequencing, the plasmid was linearized by SpeI and ScaI, gel

253 purified and injected into zygotes harvested from C57BL/6 mice. Founder mice were identified by

254 genotyping. For genotyping the LSL-K4M alleles, PCR was done using the following primers: 5’-

255 CTAGCTGCAGCTCGAGTGAACCATGGC-3’ and 5’-TTCGCGGCCGCGAATTCCTAGGCGTAGTCG-3’.

256 PCR amplified 524 bp from the LSL-K4M alleles.

257 Mll3f/f mice were obtained from Jae W. Lee 17 (Figure 5a, left panel). To generate Mll4SET

258 conditional KO mice, the loxP/FRT-flanked neomycin cassette was inserted at the 3’ end of exon 51 and

259 the single loxP site was inserted at the 5’ end of exon 50 (Figure 5a, right panel). We electroporated the

260 linearized targeted construct, which includes exons 50–51, into WT ES cells. After selection with G418,

261 surviving clones were expanded for PCR genotyping to identify Mll4SETfloxneo/+ ES cells, which were

262 further micro-injected into mouse blastocysts following standard procedures. Mice bearing germline

263 transmission (Mll4SETfloxneo/+) were crossed with FLP1 mice (Jackson no. 003946) to generate Mll4SETf/+

264 mice. For genotyping the Mll3 and Mll4SET alleles, PCRs were done using the following primers: Mll3 (5’-

265 GTCATCGGTGTGGTCTGAATGA-3’ and 5’-AACCGGAAGGAGAAGCTTTATGA-3’) and Mll4SET (5’-

266 CAGTTGAGCTAGTCAAGTGATT-3’ and 5’-TTCAATGTGGAGGGGAGTGACAG-3’). PCR amplified 174

267 bp from the wild-type Mll3 and 208 bp from the Mll3 floxed allele, or 277 bp from the wild-type Mll4 and

268 346 bp from the Mll4SET floxed allele.

269 LSL-K4M mice and Mll3f/f;Mll4SETf/f mice were crossed with Myf5-Cre (Jackson no. 007893),

270 Cre-ER (Jackson no. 008463), or Adipoq-Cre (Jackson no. 028020) to generate LSL-K4M;Myf5-Cre, LSL-

271 K4M;Cre-ER, LSL-K4M;Adipoq-Cre, Mll4SETf/f;Myf5-Cre, Mll3f/f;Mll4SETf/f;Myf5-Cre, or

272 Mll3f/f;Mll4SETf/f;Cre-ER mice.

273 Histology and Immunohistochemistry

274 E18.5 embryos were isolated and fixed in 4% paraformaldehyde, dehydrated in ethanol and embedded in

275 paraffin for sectioning. Paraffin sections were stained with routine H&E or subjected to

276 immunohistochemistry using anti-Ucp1 (ab10983; Abcam) and anti-Myosin (MF20; Developmental

277 Studies Hybridoma Bank) antibodies as described 4.

278 Immortalization of Primary Brown Preadipocytes and Adipogenesis

279 Primary brown preadipocytes were isolated from interscapular BAT of newborn LSL-K4M;Cre-ER or

280 Mll3f/f;Mll4SETf/f;Cre-ER pups, and immortalized by SV40T. Isolation, immortalization and adipogenesis

281 assays of brown preadipocytes were done as described 25,26.

282 Western Blot and qRT-PCR

283 Western blot of histone modifications using acid extracts or of nuclear proteins using nuclear extracts

284 were done as described 15. Total RNA was extracted using TRIzol (Invitrogen) and reverse transcribed

285 using ProtoScript II first-strand cDNA synthesis kit (NEB), following the manufacturers' instructions. qRT-

286 PCR was done using the following SYBR green primers: H3.3K4M (forward, 5’-

287 AACCTGTGTGCCATCCACG-3’, and reverse, 5’- CGACTTGTCATCGTCGTCCTT-3’), Mll3 (forward, 5’-

288 GATTGACGCCACACTCACAG-3’, and reverse, 5’-TTTCTGTATCCTCCGGTTGG-3’), and Mll4SET

289 (forward, 5’-GGGTGGAGAGCTGTCAGAATTATT-3’, and reverse, 5’-CATGAGCGGTAACTCCATCAGA-

290 3’). SYBR green primers for other genes were described previously 27.

291 RNA-Seq and ChIP-seq

292 RNA-seq and ChIP-seq were performed as described in detail previously with the use of Illumina HiSeq

293 2500 4,6,7. For RNA-seq, we purified mRNAs using Dynabeads mRNA purification kit (Invitrogen) then

294 synthesized double-stranded cDNAs using SuperScript Double-stranded cDNA Synthesis kit (Invitrogen),

295 following the manufacturers' instructions. For ChIP-seq, we collected ChIP-DNA using Dynabeads Protein

296 A (Invitrogen) then purified DNA using QIAquick PCR Purification Kit (QIAGEN), following the

297 manufacturers' instructions. Library construction for RNA-seq and ChIP-seq was completed using

298 NEBNext Ultra II DNA Library Prep Kit (NEB), following the manufacturers' instructions.

299 Computational Analysis and Data Availability

300 For differentially expressed genes from RNA-seq data, gene ontology (GO) analysis was carried out

301 using DAVID (https://david.ncifcrf.gov). To identify H3K4me1 or H3K27ac enriched regions at D4 of

302 adipogenesis in LSL-K4M;CreER brown preadipocytes, we used ‘SICER’ method 28 with window size of

303 200 bp and with an estimated false discovery rate (FDR) threshold of 10−3. MLL4 ChIP-Seq data at D2 of

304 adipogenesis was downloaded (GSE74189) 4 and the window size was chosen to be 50 bp. To define

305 MLL4+ active enhancers during adipogenesis, we compared 13,871 MLL4 binding sites at D2 with 64,757

306 active enhancers (H3K4me1+H3K27ac+ at D4). 6,686 MLL4+ (10.3%, 6,686/64,757) sites were located on

307 active enhancers. Heat maps were generated with 50 bp resolution and ranked according to the intensity

308 of 6,686 MLL4+ active enhancers at the center. Average profiles were plotted using the number of ChIP-

309 Seq reads from the center of 6,686 MLL4+ active enhancers to 10 kb on both sides. H3K4me1 and

310 H3K27ac ChIP-seq signal intensity was normalized by histone Western blot for baseline modification

311 levels to achieve more accurate quantitative analysis.

312 All datasets described in the paper have been deposited in NCBI Gene Expression Omnibus

313 under accession number GSE110972.

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385 Acknowledgements

386 We thank Victoria Noe-Kim for helping computational analyses. This work was supported by the

387 Intramural Research Program of NIDDK, NIH to KG.

388 Author contributions

389 YJ and KG conceived and designed the experiments. Y.J., C.W., Y-K.P., L.Z., J-E.L., A.B. and, E.F.

390 performed experiments. Y.J., C.W., Y-K.P., L.Z., J-E.L. and, K.G. analyzed the data. Y.J., and C.L.

391 generated LSL-K4M mice and C.W., and C.L. generated Mll4SETf/f mice. Y.J. performed computational

392 analyses. Y.J., A.B., and K.G. wrote the manuscript. K.G. supervised all the experiments.

393 Additional information

394 Supplementary Information:

395 Competing interests: We have no competing financial interests.

396 Figure legends

397 Figure 1. Histone H3.3K4M and H3.3K36M mutations impair adipogenesis

398 Immortalized brown preadipocytes were infected with retroviral vector (Vec) expressing FLAG-tagged wild

399 type (WT) or K-to-M mutant histone H3.3, followed by adipogenesis assay.

400 (a) Histone extracts from preadipocytes were subjected to Western blot analyses using antibodies

401 indicated on the left. Long exposure of histone H3 Western blot reveals the relative levels of ectopic H3.3

402 and endogenous H3. (b) 6 days after induction of differentiation, cells were stained with Oil Red O. Upper

403 panels, stained dishes; lower panels, representative fields under microscope. (c) qRT-PCR of Pparg,

404 Cebpa and Fabp4 expression at day 0 (D0) and day 6 (D6) of adipogenesis. Quantitative PCR data in all

405 figures except Figure 8 are presented as means ± SD.

406 Figure 2. H3.3K4M prevents adipose tissue and muscle development

407 (a) Schematic of lox-STOP-lox-H3.3K4M (LSL-K4M) transgene and breeding scheme. The LSL-K4M

408 transgene consists of the following elements from 5’ to 3’: a CAG promoter, quadruple copies of SV40

409 stop signals flanked by two loxP sites, H3.3K4M with C-terminal FLAG and HA tags, and polyadenylation

410 signal. LSL-K4M transgenic mice were crossed with Myf5-Cre to generate mice expressing ectopic

411 H3.3K4M in brown adipose tissue (BAT) and muscle. The locations of PCR genotyping primers P1 and

412 P2 are indicated by arrows. (b) PCR genotyping of LSL-K4M transgenic mice. (c) Genotype of progeny

413 from crossing between LSL-K4M and Myf5-Cre at 3 weeks age, new born pups (P0) and E18.5 embryos.

414 LSL-K4M;Myf5-Cre mice died soon after birth from breathing malfunction due to defects in muscles of the

415 rib cage. (d) Representative morphology of P0 pups. (e) Representative morphology of E18.5 embryos. (f)

416 Histological analysis of E18.5 embryos. Sagittal sections of cervical/thoracic area were stained with H&E

417 (left panels) or with antibodies against the BAT (B) marker UCP1 (green) and the muscle (M) marker

418 Myosin (red) (right panels). Scale bar = 80 µm.

419 Figure 3. H3.3K4M destabilizes MLL3/4 proteins in adipogenesis

420 Immortalized LSL-K4M;Cre-ER brown preadipocytes were treated with 4-hydroxytamoxifen (4OHT) to

421 induce ectopic H3.3K4M expression, followed by adipogenesis assay.

422 (a–b) H3.3K4M destabilizes MLL3/4 proteins. Histone extracts (a) or nuclear extracts (b) were analyzed

423 by Western blot using antibodies indicated on the left.

424 (c-e) H3.3K4M prevents adipogenesis and induction of adipocyte genes. (c) Cell growth rates. 5 x 105

425 preadipocytes were plated at D0 and the cumulative cell numbers were determined every day for 5 days.

426 (d) Oil red O staining at day 7 (D7) of adipogenesis. (e) qRT-PCR of H3.3K4M-FLAG, Pparg, Cebpa,

427 Fabp4 and Ucp1 expression at D0 and D7 of adipogenesis.

428 (f-h) RNA-seq analyses were performed at D7 of adipogenesis. (f-g) Identification (f) and heat map (g) of

429 down- or up-regulated genes in H3.3K4M-expressing cells. The cut-off for differential expression is 2.5-

430 fold. (h) Gene ontology (GO) analysis of gene groups defined in (f).

431 Figure 4. H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis

432 4OHT-treated LSL-K4M;Cre-ER brown preadipocytes were collected at D4 of adipogenesis for ChIP-seq

433 of H3K4me1 and H3K27ac (a-b), ChIP of MLL4, H3K4me1, H3K27ac, BRD4, MED1 and Pol II, and qRT-

434 PCR of eRNAs (c-e).

435 (a-b) Heat maps (a) and average profiles (b) around MLL4+ active enhancers during adipogenesis. (c)

436 Genome browser view of H3K4me1 and H3K27ac on Pparg and Cebpa gene loci during adipogenesis

437 with schematic of genomic locations of representative MLL4+ active enhancers (e1-e5). MLL4 binding

438 data were obtained from 4. (d) ChIP-qPCR analyses of indicated factors are shown on enhancers e1-e5 at

439 D0 and D4 of adipogenesis. An enhancer of constitutively expressed gene Jak1 was chosen as negative

440 control (n). (e) qRT-PCR of eRNA transcription on enhancers e1-e5 at D0 and D4 of adipogenesis. (f)

441 Proposed model showing that H3.3K4M prevents enhancer activation in adipogenesis by destabilizing

442 MLL3/4.

443 Figure 5. Deletion of the enzymatic SET domain of MLL3/4 prevents adipose tissue and muscle

444 development

445 (a) Conditional KO mouse lines targeting the SET domain of MLL3 and MLL4. Schematics of WT allele,

446 targeted allele, conditional KO (flox) allele and KO allele are shown in upper panels. Deletion of neomycin

447 selection cassette by FLP recombinase generates the flox allele. The locations of PCR genotyping

448 primers P1-P4 are indicated by arrows. Expected protein domains and molecular weights are shown in

449 bottom panels. (b) PCR genotyping of flox and WT alleles using P1-P4 primers. (c) Genotypes of progeny

450 at E18.5 from crossing between Mll3f/f;Mll4SETf/f (f/f;f/f) and Mll3f/+;Mll4SETf/+;Myf5-Cre.

451 Mll3f/+;Mll4SETf/f;Myf5-Cre and Mll3f/f;Mll4SETf/f;Myf5-Cre (conditional double KO, DKO) mice died

452 immediately after cesarean section from breathing malfunction due to defects in muscles of the rib cage.

453 (d) Representative morphology of E18.5 embryos. (e) Histological analysis of E18.5 embryos. Sagittal

454 sections of cervical/thoracic area were stained with H&E (upper panels) or with antibodies against the

455 BAT (B) marker UCP1 (green) and the muscle (M) marker Myosin (red) (lower panels). Scale bar = 80 µm.

456 Figure 6. Deletion of the enzymatic SET domain inhibits adipogenesis by destabilizing MLL3/4

457 Immortalized Mll3f/f;Mll4SETf/f;Cre-ER brown preadipocytes were treated with 4OHT to induce deletion of

458 the exons encoding the SET domain of MLL3 and MLL4 proteins, followed by adipogenesis assay.

459 (a–b) Deletion of the enzymatic SET domain destabilizes MLL3/4 proteins. Histone extracts (a) or nuclear

460 extracts (b) from preadipocytes were analyzed by Western blot using antibodies indicated on the left.

461 (c-e) Deletion of the SET domain of MLL3/4 prevents adipogenesis. (c) Cell growth rates. (d) Oil red O

462 staining at D7 of adipogenesis. (e) qRT-PCR of Mll3, Mll4SET, Pparg, Cebpa, Fabp4 and Ucp1

463 expression at D0 and D7 of adipogenesis.

464 (f-i) RNA-seq analyses were performed at D7 of adipogenesis. (f) Genome browser views of RNA-Seq

465 analysis on Mll3 and Mll4 loci. The targeted exons are highlighted in red boxes. (g-h) Schematic of

466 identification (f) and heat map (h) of down- or up-regulated genes in double KO (DKO) cells. The cut-off

467 for differential expression is 2.5-fold. (i) GO analysis of gene groups defined in (g).

468 Figure 7. H3.3K4M expression mimics MLL3/4 SET domain deletion in preventing adipogenesis

469 (a) Scatter plots of down- or up-regulated genes in H3.3K4M-expressing or MLL3/4 SET domain DKO

470 cells compared to the respective control (Ctrl) cells. RNA-seq data were from Figures 3 and 6. The cut-off

471 for differential expression is 2.5-fold. Down- or up-regulated genes are depicted in green and red,

472 respectively. (b) Combined scatter plot showing correlation of fold changes of gene expression by

473 H3.3K4M expression and by MLL3/4 SET domain DKO. (c-d) Venn diagram showing the overlap of

474 down- or up-regulated genes in H3.3K4M-expressing and MLL3/4 SET domain DKO cells. (e-f) GO

475 analysis of gene groups defined in (c-d).

476 Figure 8. H3.3K4M does not affect adipose tissue maintenance and function

477 All data were from 8 to 10-week-old male mice fed with regular diet.

478 (a-b) Histone extracts (a) or nuclear extracts (b) from BAT were analyzed by Western blot using

479 antibodies indicated on the left. (c) Representative picture of BAT, inguinal WAT (iWAT), epididymal WAT

480 (eWAT), and liver. (d) Fat mass, lean mass, and total body weight were measured by MRI (n=9 per

481 group). (e) The average tissue weights are presented as % of body weight (n=6 per group). (f) qRT-PCR

482 of H3.3K4M, Pparg, Cebpa, and Fabp4 expression in BAT (n=6 per group). (g-h) Cold tolerance test.

483 Mice were housed at room temperature (RT, 22°C) and then in a cold room (6°C) for 6h (n=6 per group).

484 (g) Body temperatures. (h) qRT-PCR of gene expression in BAT after 6h. All values in Figure 8 are

485 presented as mean ± S.E.M.

486

Figure 1. Histone H3.3K4M and H3.3K36M mutations impair adipogenesis

Immortalized brown preadipocytes expressing H3.3WT, K4M, K9M, K27M or K36M adipocytes a b H3.3 H3.3 Vec WT K4M K9M K27M K36M Vec WT K4M K9M K27M K36M kDa FLAG 20

H3K4me1 15

H3K4me2 15

H3K9me2 15

H3K27me3 15

H3K36me2 15

short 15 H3 20 H3.3-FLAG long endogenous H3 15

c Pparg Cebpa Fabp4 24 20 800 D0 18 D6 15 600

12 10 400

6 5 200 mRNA level (fold) level mRNA 0 0 0 bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 2. H3.3K4M prevents adipose tissue and muscle development

a LSL-K4M transgene and breeding scheme

X loxP loxP FLAG/HA CAG promoter STOP H3.3K4M PolyA P1 P2 Myf5-Cre

CAG promoter H3.3K4M PolyA

b c d P0 LSL-K4M X Myf5-Cre WT LSL-K4M bp H3.3K4M Genotype 3 weeks P0 E18.5 524 (P1+P2) WT 20 14 10 * internal 604 Myf5-Cre 17 16 12 control LSL-K4M 19 10 14 * LSL-K4M;Myf5-Cre 0 5* 10 * LSL-K4M;Myf5-Cre

e E18.5 f H&E IHC

LSL-K4M LSL-K4M;Myf5-Cre LSL-K4M LSL-K4M;Myf5-Cre LSL-K4M LSL-K4M;Myf5-Cre Ucp1 Myosin M

B B

B M M B M B bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 3. H3.3K4M destabilizes MLL3/4 proteins in adipogenesis

D0 D7 4OHT LSL-K4M;Cre-ER brown preadipocytes adipocytes

a Histone extracts b Nuclear extracts c d 4OHT – + 4OHT – + – + 30 4OHT kDa kDa SET1A 250 – 4OHT FLAG )

20 5 25 + 4OHT SET1B H3K4me1 15 250 20 15 H3K4me2 15 MLL1N 250 10 MLL1C H3K4me3 15 150 5 cell number number (x10 cell H3K27ac 15 MLL3 0 D0 D1 D2 D3 D4 D5 H3 15 250 MLL4

250 UTX 150 75 RbBP5 e qRT-PCR H3.3K4M Pparg Cebpa Fabp4 Ucp1 1500 60 45 600 15000 – 4OHT +4OHT 1000 40 30 400 10000

500 20 15 200 5000 mRNA level (fold) level mRNA 0 0 0 0 0 D0 D7 D0 D7 D0 D7 D0 D7 D0 D7

RNA-seq (D7) f 709 (5.8%) h down-regulated 605 (5.0%) Gene group GO term P-Value up-regulated organic acid metabolic process 7.8E-48 carboxylic acid metabolic process 2.7E-46 oxoacid metabolic process 6.0E-46 lipid metabolic process 2.7E-44 monocarboxylic acid metabolic process 9.2E-43 10,865 (89.2 %) down- mild or no change regulated fatty acid metabolic process 2.0E-39 cellular lipid metabolic process 2.2E-38 oxidation-reduction process 7.8E-33 g brown fat cell differentiation 5.6E-29 – 4OHT + 4OHT log2RPKM Ucp1 8 single-organism catabolic process 1.2E-25 Pparg organ morphogenesis 4.5E-18 6 locomotion 6.1E-17 Fabp4

regulated cell development 2.4E-16 - Cebpa 4 cell proliferation 2.2E-15 down up- circulatory system development 2.9E-15 2 regulated cardiovascular system development 2.9E-15 regulation of cell proliferation 3.1E-15 0 negative regulation of cell proliferation 5.7E-15 regulated - movement of cell or subcellular component 9.3E-15

up -2 cell surface receptor signaling pathway 2.1E-14 bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 4. H3.3K4M prevents MLL3/4-mediated enhancer activation in adipogenesis

a Heat maps b Average plots

H3K4me1 H3K27ac – 4OHT + 4OHT – 4OHT + 4OHT H3K4me1 H3K27ac 8 8

6 6 – 4OHT 4 4 + 4OHT 2 2

Normalize read read count Normalize 0 0 –10 0 +10 –10 0 +10 + –10 0 +10 –10 0 +10 –10 0 +10 –10 0 +10 Distance from MLL4 active enhancers (kb) Distance from MLL4+ active enhancers (kb)

c 50 kb 20 kb

MLL4

– 4OHT

+ 4OHT H3K4me1

– 4OHT

+ 4OHT H3K27ac

Pparg1 e1 e2 e3 Cebpa e4 e5 Pparg2

d ChIP-qPCR

0.09 MLL4 H3K4me1 H3K27ac D0 1.5 6 D4 0.06 1 4

0.03 0.5 2

0 0 0 0.15 BRD4 0.06 MED1 0.03 Pol II % of %input 0.1 0.04 0.02

0.05 0.02 0.01

0 0 0 4OHT - + - + - + - + - + - + 4OHT - + - + - + - + - + - + 4OHT - + - + - + - + - + - + e1 e2 e3 e4 e5 n e1 e2 e3 e4 e5 n e1 e2 e3 e4 e5 n

H3.3K4M e qRT-PCR f Destabilization 24 eRNAs D0 D4 MLL3/4 MLL3/4 16 CBP/ p300 H3K4me1  8  H3K27ac on x BRD4 MED expression expression (fold)

0 PPAR CEBP 4OHT - + - + - + - + - + - + Pol II e1 e2 e3 e4 e5 n eRNA Enhancer priming by MLL3/4 Enhancer activation bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 5. Deletion of the enzymatic SET domain of MLL3/4 prevents adipose tissue and muscle development a Mll3 Mll4SET

55 56 57 58 59 48 49 50 51 52 53 54 55 WT allele UTR UTR

P1 P2 P3 P4 targeted allele Neo Neo

flox allele

KO allele FRT LoxP

MLL3 protein MLL4 protein

WT 4904 aa (540 kDa) WT 5588 aa (600 kDa)

KO 4843 aa (532 kDa) KO 5316 aa (569 kDa) in frame deletion frameshift after 5312 aa

PHD HMG-I FYRN FYRC SET b c f/f f/+ +/+ bp Mll3 f/f ;Mll4SET f/f X Mll3 f/+ ;Mll4SET f/+ ;Myf5-Cre Mll3 208 Genotype Number of Survival after (P1+P2) 174 (E18.5) embryos isolation Mll3 f/+ ;Mll4SET f/+ 13 13 Mll4SET 346 (P3+P4) 277 Mll3 f/f ;Mll4SET f/+ 5 5 Mll3 f/+ ;Mll4SET f/f 9 9 Mll3 f/f ;Mll4SET f/f (f/f;f/f) 11 11 d E18.5 f/f;f/f DKO Mll3 f/+ ;Mll4SET f/+ ;Myf5-Cre 5 5 Mll3 f/f ;Mll4SET f/+ ;Myf5-Cre 4 4 Mll3 f/+ ;Mll4SET f/f ;Myf5-Cre 15 0 Mll3 f/f ;Mll4SET f/f ;Myf5-Cre (DKO) 5 0

e H&E IHC f/f;f/f DKO f/f;f/f DKO

Ucp1 Myosin M

B B B

B bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 6. Deletion of the enzymatic SET domain inhibits adipogenesis by destabilizing MLL3/4 D0 D7 4OHT Mll3 f/f;Mll4SET f/f;Cre-ER brown preadipocytes adipocytes

a histone extracts b nuclear extracts c d 4OHT – + 4OHT – + 4OHT – + – + 25 kDa – 4OHT ) H3K4me1 15 MLL3 5 20 kDa + 4OHT H3K4me2 15 250 15

H3K4me3 15 MLL4 10

H3 15 250 5 cell number number (x10 cell UTX 150 75 0 RbBP5 D0 D1 D2 D3 D4 D5 Exposure: short long e qRT-PCR Mll3 Mll4SET Pparg Cebpa Fabp4 Ucp1 2.4 1.5 60 45 600 450 – 4OHT + 4OHT 1.6 1 40 30 400 300

0.8 0.5 20 15 200 150 mRNA level (fold) level mRNA

0 0 0 0 0 0 D0 D7 D0 D7 D0 D7 D0 D7 D0 D7 D0 D7 RNA-seq (D7) f 2 kb 2 kb – 4OHT

+ 4OHT

Exon 58 57 Mll3 Exon 51 50 Mll4

g 890 (7.3%) i down-regulated Gene group GO term P-Value 623 (5.1%) monocarboxylic acid metabolic process 3.5E-48 up-regulated carboxylic acid metabolic process 4.0E-44 organic acid metabolic process 4.3E-44 oxoacid metabolic process 9.3E-44

10,1671 (87.6 %) down- lipid metabolic process 6.1E-40 mild or no change regulated cellular lipid metabolic process 2.6E-35 fatty acid metabolic process 2.0E-34 oxidation-reduction process 2.9E-27 h regulation of lipid metabolic process 2.0E-26 log2RPKM Fabp4 – 4OHT + 4OHT 8 fat cell differentiation 1.7E-24 Pparg cell development 2.4E-16 Ucp1 6 Cebpa nervous system development 8.2E-16 neurogenesis 3.4E-14 regulated - 4 cell projection organization 5.4E-14

down neuron differentiation 8.9E-14 2 up- regulated neuron projection development 9.5E-14 generation of neurons 1.8E-13 0 locomotion 2.1E-13

regulated neuron development 3.9E-13 - -2

up movement of cell or subcellular component 7.5E-13 bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 7. H3.3K4M expression mimics MLL3/4 SET domain deletion in preventing adipogenesis

RNA-seq (D7)

a b up-regulated by DKO or K4M only up-regulated by DKO and K4M up-regulated by DKO or K4M mild or no change mild or no change down-regulated by DKO or K4M only down-regulated by DKO or K4M down-regulated by DKO and K4M 18 1212 1212 DKO K4M 1015 99 99 Fabp4 125

Fabp4 09 66 66 Cebpa Cebpa RPKM(K4M) RPKM(DKO) 2 2

RPKM(DKO/Ctrl) -56 Pparg Cebpa Pparg 2

33 log 33 log Fabp4 Pparg log -103 0 0 0 0 0 0 0 3 6 9 12 0 3 6 9 12 0 -103 -65 90 125 1510 18

log2RPKM(Ctrl) log2RPKM(Ctrl) log2RPKM(K4M/Ctrl) c d down-regulated by DKO down-regulated by K4M up-regulated by DKO up-regulated by K4M 890 709 623 605

379 511 198 286 337 268

e f

Gene group GO term P-Value Gene group GO term P-Value organic acid metabolic process 2.9E-45 cell proliferation 7.1E-12 carboxylic acid metabolic process 3.6E-44 cell development 1.3E-11 oxoacid metabolic process 7.0E-44 negative regulation of cell proliferation 2.6E-11 lipid metabolic process 1.1E-43 organ morphogenesis 4.6E-11 down- monocarboxylic acid metabolic process 2.2E-43 up-regulated by Locomotion 9.7E-11 regulated by DKO and K4M DKO and K4M cellular lipid metabolic process 2.8E-40 regulation of cell proliferation 1.7E-10 fatty acid metabolic process 8.3E-39 movement of cell or subcellular component 3.9E-10 brown fat cell differentiation 1.1E-28 positive regulation of cell communication 9.9E-10 fat cell differentiation 1.5E-27 positive regulation of signaling 1.2E-09 oxidation-reduction process 3.5E-26 nervous system development 1.2E-09 bioRxiv preprint doi: https://doi.org/10.1101/301986; this version posted April 16, 2018. 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.

Figure 8. H3.3K4M does not affect adipose tissue maintenance and function

a BAT, histone extracts b BAT, nuclear extracts c LSL-K4M; LSL-K4M; LSL-K4M; LSL-K4M LSL-K4M LSL-K4M Adipoq-Cre Adipoq-Cre Adipoq-Cre kDa 20 BAT FLAG MLL3 H3K4me1 15 kDa iWAT 250 H3K4me2 15 MLL4 eWAT H3K4me3 15 250 H3 15 Liver UTX 150 75 RbBP5 LSL-K4M LSL-K4M;Adipoq-Cre d Body composition e f qRT-PCR H3.3K4M Pparg Cebpa Fabp4 30 8 150 1.5 1.5 1.5 25 6 20 100 1 1 1 15 4

Weight (g) Weight 10 50 0.5 0.5 0.5 2

5 (fold) level mRNA % of body of %body weight 0 0 0 0 0 0 Fat Lean Total BAT iWAT eWAT Liver

Cold tolerance g h qRT-PCR

H3.3K4M Ucp1 Dio2 Elovl3 40 150 3.6 30 16 C) ° 14 38 12 100 2.4 20 36 10 8 34 50 1.2 10 6 LSL-K4M 4

32 (fold) level mRNA 2 body body ( temperature LSL-K4M;Adipoq-Cre 30 0 0 0 0 0h 1h 2h 3h 4h 5h 6h RT 6°C RT 6°C RT 6°C RT 6°C