bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

1 Title

2 YY1 cistrome analysis uncovers an essential requirement of the YY1:BRD4-PFKP

3 regulatory axis for promoting tumorigenesis of castration-resistant prostate

4 cancer

5 Authors

6 Chenxi Xu1,2, Yi-Hsuan Tsai1, Phillip Galbo3, Weida Gong1, Aaron J. Storey4, Yuemei 7 Xu5,6, Stephanie D. Byrum4, Young E. Whang1,7,8, Joel S. Parker1,9, Samuel G. 8 Mackintosh4, Ricky D. Edmondson4, Alan J. Tackett4, Jiaoti Huang5, Deyou Zheng3,10, H. 9 Shelton Earp1,7,11, Gang Greg Wang1,2, Ling Cai1,9* 10 1Lineberger Comprehensive Cancer Center, University of North Carolina at Chapel Hill 11 School of Medicine, Chapel Hill, NC 27599, USA 12 2Department of Biochemistry and Biophysics, University of North Carolina at Chapel Hill 13 School of Medicine, Chapel Hill, NC 27599, USA 14 3Department of Genetics, Albert Einstein College of Medicine, Bronx, NY 10461, USA 15 4Department of Biochemistry and Molecular Biology, University of Arkansas for Medical 16 Sciences, Little Rock, AR 72205, USA 17 5Department of Pathology, Duke University School of Medicine, Durham, NC 27710, 18 USA 19 6Department of Pathology, Nanjing Drum Tower Hospital and The Affiliated Hospital of 20 Nanjing University Medical School, Nanjing, China 210008 21 7Department of Medicine, University of North Carolina at Chapel Hill School of Medicine, 22 Chapel Hill, NC, 27599, USA 23 8Department of Pathology and Laboratory Medicine, University of North Carolina at 24 Chapel Hill School of Medicine, Chapel Hill, NC, 27599, USA 25 9Department of Genetics, University of North Carolina at Chapel Hill School of Medicine, 26 Chapel Hill, NC 27599, USA 27 10Department of Neurology and Department of Neuroscience, Albert Einstein College of 28 Medicine, Bronx, NY 10461, USA 29 11Department of Pharmacology, University of North Carolina at Chapel Hill School of 30 Medicine, Chapel Hill, NC, 27599, USA 31 32 *Correspondence to: [email protected] 33

34

1 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

35 Abstract (208 words)

36 Castration-resistant prostate cancer (CRPC) is a terminal disease, demanding a better

37 understanding of its pathogenesis. Targeted therapy needs to be developed for CRPC

38 due to its heterogeneity and resistance to current treatments. Here, through cistrome

39 study of YY1, a factor significantly overexpressed during prostate cancer

40 progression, we identify a YY1-PFKP axis to be essential for CRPC tumorigenesis.

41 Depletion of YY1 in independent CRPC models dramatically reduced tumor cell growth

42 in vitro and delayed oncogenic progression in vivo. Importantly, YY1 functions as a

43 master regulator of prostate tumor metabolism including the Warburg effect and

44 mitochondria respiration. Loss-of-function and rescue studies further reveals a

45 mechanistic underpinning in which YY1 directly binds and trans-activates PFKP, a

46 encoding the rate-limiting enzyme for glycolysis, significantly contributing to the YY1-

47 enforced oncogenic phenotypes such as enhanced tumor cell glycolysis and malignant

48 growth. Additionally, a vast majority of gene-regulatory element in advanced prostate

49 cancer cells are bound by YY1, lending a support for its role as a master regulator of

50 prostate cancer progression. YY1 interactome studies point to -containing

51 coactivators in prostate cancer, which act as functional partners of YY1 to potentiate

52 YY1-related target gene activation. Altogether, this study unveils an unexplored

53 YY1:BRD4-PFKP oncogenic axis operating in advanced prostate cancer with

54 implications for therapy.

55

56 Key words 57 prostate cancer, transcription, YY1, PFKP, metabolism, BRD4, histone 58

2 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

59 Introduction

60 Prostate cancer is the second leading cause of cancer-related death for men in the

61 United States. Standard treatment of prostate cancer with anti-androgen agents fails

62 inevitably due to development of therapy resistance and castration-resistant prostate

63 cancer (CRPC), a terminal disease1. Therefore, mechanistic understanding of CRPC

64 pathogenesis, as well as design of novel means to specially target CRPC vulnerabilities,

65 would greatly benefit clinical outcome of the affected patients.

66 Yin Yang 1 (YY1), a ubiquitously expressed transcription factor, was previously

67 shown to have dual roles in both gene activation and repression2,3. It belongs to the

68 GLI-Kruppel zinc finger family and carries four conserved C2H2 zinc fingers4. As

69 a multifunctional protein, YY1 is involved in various biological and physiological

70 processes including cell proliferation, lineage specification and embryonic development5.

71 Emerging evidence indicates that YY1 also play important roles in malignant diseases

72 including cancer. Our analysis of paired normal and tumor patient samples identified

73 YY1 to be significantly overexpressed during prostate cancer progression. However,

74 YY1’s function and regulated cistrome in CRPC are not studied to date.

75 Aerobic glycolysis, known as the Warburg effect, is essential for cancer to

76 acquire energy and metabolize nutrients for synthesis of macromolecular precursors, in

77 order to sustain high rates of cell proliferation6. It has been reported that primary

78 prostate cancers are metabolically different from many other solid tumors, due to their

79 enhanced reliance on oxidative phosphorylation (OXPHOS) in mitochondria, leading to

80 a modest level of glucose uptake7,8. Other studies, however, have shown increased

81 glycolysis or the Warburg effect also correlated with disease progression and poor

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82 prognosis among the advanced prostate cancers8. Thus, the exact molecular

83 mechanism underlying glycolysis regulation in advanced prostate tumors remains

84 elusive, although rising evidence points to possible deregulation of glycolytic enzymes9.

85 During glycolysis, phosphofructokinase 1 (PFK1) plays a critical role through catalyzing

86 fructose 6-phosphate (F6P) to fructose 1,6-biphosphate (F1,6BP), one of the rate-

87 limiting steps in glycolysis10. PFK1 has 3 isoforms, namely, PFKP (Phosphofructokinase,

88 Platelet), PFKM (Phosphofructokinase, Muscle) and PFKL (Phosphofructokinase, Liver).

89 While all isozymes are expressed in many tissues, PFKP and PFKM are mainly present

90 in platelet and muscle, respectively, whereas PFKL is predominant in liver and kidney11.

91 PFKP has recently been also shown to be prevalent in breast cancer, lung cancer and

92 glioblastoma12-14. The role of PFKP and its regulation in CRPC remains unexplored.

93 Here, we report YY1 to be significantly elevated among patient-derived CRPC

94 samples, relative to benign prostate controls, and to be essential for CRPC

95 tumorigenesis in multiple in vitro and in vivo CRPC models. We also use integrative

96 genomics approach with RNA-seq and ChIP-seq to determine the YY1-regulated

97 cistrome in CRPC. Strikingly, we found that YY1 binds a vast majority of gene-

98 regulatory elements (demarcated by H3K27ac) and potentiates various gene-

99 expression programs related to metabolic pathways such as mitochondria respiration

100 and the Warburg effect, thereby profoundly affecting metabolism of advanced prostate

101 tumor cells. A detailed loss-of-function and rescue study points to PFKP, a rate-limiting

102 glycolysis enzyme, directly bound and activated by YY1. Oncogenic actions of YY1 is at

103 least partially achieved via PFKP which significantly enhances tumor cell glycolysis,

104 proliferation, soft agar-based colony formation and xenografted tumor growth in mice.

4 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

105 Proteomics-based YY1 interactome studies identify bromodomain (BRD2 and

106 BRD4) as YY1’s functional partners, co-mediating transcriptional activation of many

107 metabolic including PFKP. Taken together, this study shows YY1 as a master

108 regulator of prostate tumorigenesis, unveils a previously unknown oncogenic axis

109 involving YY1:BRD4-PFKP, and elucidates the molecular mechanism underlying altered

110 metabolism of CRPC, implicative of new therapeutic strategies for treatment of lethal

111 CRPCs.

112

113 Results

114 YY1 shows significant upregulation among primary samples of prostate cancer

115 patients.

116 YY1 was previously reported to be highly expressed in breast and colorectal cancer15.

117 To assess relevance of YY1 in prostate cancer, we first examined the publicly available

118 prostate cancer datasets16,17 and found the YY1 mRNA levels to be significantly

119 elevated in tumors compared to adjacent benign tissues (Fig. 1a-b). Next, we performed

120 immunohistochemical (IHC) staining with thirteen paired tumor and normal tissues from

121 prostate cancer patients. We observed that the YY1 protein level was significantly

122 increased in nuclei of tumors, compared to their respective adjacent benign controls

123 (Fig. 1c-d and Supplementary Fig.1; P = 0.0071). By immunoblots, we further verified

124 upregulation of YY1 in prostate tumor relative to paired benign tissues (Fig. 1e).

125 Furthermore, we performed YY1 IHC staining with tissue microarrays that contained a

126 larger panel of benign prostates and samples representing different stages of prostate

127 tumors, including primary adenocarcinoma and CRPC, and found a significantly higher

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128 level of nuclear YY1 in CRPC, compared to normal controls (P = 0.0170) and

129 adenocarcinomas (P = 0.0339), as demonstrated by representative IHC images (Fig. 1f)

130 and quantitative analysis (Fig. 1g). Overall, these results lend a support for a

131 physiological involvement of YY1 in advanced prostate cancer pathogenesis, including

132 CRPC.

133

134 YY1 promotes the growth and colony formation of prostate cancer cells in vitro,

135 as well as tumorigenesis in xenografted animal models.

136 Next, we sought to determine the role for YY1 in prostate cancer tumorigenesis. Using

137 two independently validated YY1-targeting shRNAs (sh#94 and sh#98), we performed

138 YY1 knockdown (KD) in two androgen-independent CRPC models, 22Rv1 and C4-2

139 cells (Fig. 2a-f). YY1 KD significantly decreased tumor cell proliferation in liquid culture

140 (Fig. 2b, 2e) and colony formation in soft agar, a surrogate assay of transformation (Fig.

141 2c, 2f). Similar phenotypes were observed post-KD of YY1 in LNCaP cells, a prostate

142 cancer model showing androgen dependency (Supplementary Fig. 2a-b). Furthermore,

143 re-introduction of an shRNA-resistant YY1 into 22Rv1 cells with endogenous YY1

144 depleted was able to restore both tumor cell growth and colony formation, ruling out

145 potential off-target effects of the used shRNA (Fig. 2g-i). In accordance with shRNA-

146 mediated YY1 KD, YY1 depletion via two independent sgRNAs through a

147 CRISPR/Cas9 system, or via siRNA, all led to decreased cell proliferation (Fig. 2j-k,

148 Supplementary Fig. 2c-d). To further determine whether YY1 is important for CRPC

149 tumorigenesis in vivo, we subcutaneously xenografted the 22Rv1 cells, stably

150 transduced either with control or YY1-targeting shRNAs, into castrated

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151 NOD/scid/gamma (NSG) mice. 22Rv1 xenografts in the YY1 KD cohort grew

152 significantly slower relative to controls (Fig. 2l-m). Additionally, we validated YY1 KD in

153 the tumor xenografts (Fig. 2n). Altogether, we conclude that YY1 is crucial for CRPC

154 growth in vitro and in vivo.

155

156 YY1 directly binds a set of tumor metabolism-related genes, potentiating their

157 transcription.

158 To gain insight into molecular mechanisms underlying the YY1-mediated CRPC

159 tumorigenesis, we profiled 22Rv1 cell transcriptomes by RNA-seq, which revealed

160 differentially expressed genes (DEGs) caused by YY1 KD (Fig. 3a and Supplementary

161 Table 1). Gene Set Enrichment Analysis (GSEA) showed that genes upregulated by

162 YY1 are enriched in energy metabolism pathways including glycolysis (Fig. 3b, upper

163 panels), oncogenes involved in prostate cancer (Fig. 3b, left/bottom), and, as expected,

164 the YY1 targets (Fig. 3b, right/bottom). Notably, a set of metabolic enzymes involved in

165 glycolysis were downregulated upon YY1 depletion (Fig. 3c). To validate this regulatory

166 function of YY1, we additionally performed RNA-seq post-KD of YY1 in C4-2 cells,

167 another CRPC model, and subsequent GO and GSEA analyses revealed similar striking

168 enrichments of metabolic pathways among genes positively controlled by YY1

169 (Supplementary Fig. 3a-d, and Supplementary Table 2). Importantly, we identified the

170 DEGs common to both independent CRPC models after YY1 ablation, hereafter termed

171 “the YY1 signature genes in CRPC” (Fig. 3d and Supplementary Table 3), which

172 included a number of metabolism-associated genes such as PFKP, ALDOC, OGDHL

173 and NDUFA4L2. Using qRT-PCR, we further confirmed downregulation of these

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174 metabolic genes upon YY1 loss, relative to control, in both 22Rv1 and C4-2 cells, with

175 the change in PFKP expression being most prominent (Fig. 3e-f).

176 We next performed ChIP-seq to determine genome-wide YY1 binding sites in

177 22Rv1 cells that were ligand-starved followed by treatment with vehicle (DHT-) or AR

178 agonist (DHT+) (Supplementary Fig. 3e). YY1 binding patterns are highly similar

179 between vehicle- and DHT-treated cells (data not shown); thus, we chose the YY1

180 ChIP-seq peaks called from the vehicle-treated cells for further analysis, as this

181 condition more closely resembles CRPC. Genomic localization analysis showed

182 approximately 25% of the YY1 peaks at promoters, ~36% in the gene body and the rest

183 (~39%) at putative intergenic and distal enhancers (Fig. 3g). As expected, YY1 motif

184 was among the most enriched motifs identified within the YY1 peaks (Fig. 3h). Also,

185 YY1 peaks were found at almost all of the YY1-upregulated genes defined by RNA-seq,

186 including metabolic genes PFKP, ALDOC, ENO2 and NDUFA4L2 (Fig. 3i). ChIP-qPCR

187 of YY1 further validated its strong enrichment at metabolic gene promoters

188 (Supplementary Fig. 3f). Taken together, our genome-wide-profiling integration

189 analyses lend a strong support for a direct involvement of YY1 in upregulation of genes

190 related to cell metabolism in prostate cancer.

191

192 YY1 potentiates prostate tumor cell glycolysis via PFKP.

193 Next, we sought to assess whether YY1 regulates prostate cancer cell metabolism and

194 measured oxygen consumption rate (OCR) in both 22Rv1 and C4-2 cells post-KD of

195 YY1 versus control. Indeed, YY1 depletion led to the significantly reduced levels of

196 basal OCR and maximal respiratory capacity (Fig. 4a-b). To explore how YY1 regulates

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197 the Warburg effect of prostate cancer, we further measured the basal extracellular

198 acidification rate (ECAR), a key marker of glycolysis, and observed it to be dramatically

199 decreased after YY1 loss in three independent prostate tumor models, 22Rv1, C4-2 and

200 LNCaP cells (Fig. 4c-d, Supplementary Fig.4a). In agreement with these metabolic

201 changes caused by YY1 loss, overexpression of wildtype (WT) YY1, but not its mutant

202 form with the N-terminal domain deleted, which was previously reported to be

203 transactivation-defective18, was able to further enhance glycolysis of 22Rv1 and C4-2

204 cells (Fig. 4e-4g and Supplementary Fig.4b). Such a requirement of YY1’s

205 transactivation domain for promoting tumor cell glycolysis is in line with a role for YY1 in

206 transactivation of glycolysis-related genes revealed by RNA-seq.

207 Next, we aimed to determine which YY1’s downstream metabolic gene target(s)

208 mediates glycolysis in prostate tumor. Among the commonly YY1-upregulated signature

209 transcripts PFKP, ALDOC and ENO2 (Fig. 3c), we found that overexpression of ENO2

210 or ALDOC failed to significantly rescue the glycolysis defects caused by YY1 depletion

211 (Supplementary Fig. 4c-f). In contrast, the restored expression of PFKP largely rescued

212 YY1 loss-related defects (Fig. 4h-i). Consistent with this finding, KD of PFKP using

213 either of two independently validated hairpins (sh#75 and sh#77) significantly

214 diminished the rate of glycolysis in both 22Rv1 and C4-2 cells (Fig. 4j-k); likewise,

215 depletion of PFKP almost completely abolished the increased glycolysis caused by YY1

216 overexpression (Fig. 4l-m).

217 To this end, we show that YY1 exerts an essential role in potentiating prostate

218 cancer cell glycolysis, an event heavily relying on upregulation of PFKP expression.

219

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220 PFKP is a direct onco-target of YY1 in prostate cancer.

221 We next examined how YY1 regulates PFKP expression. First, the Volcano plots based

222 on RNA-seq profiles of 22Rv1 (Fig. 5a) and C4-2 (Fig. 5b) cells both pointed to PFKP

223 among the most altered transcripts upon YY1 depletion. Consistently, YY1 ablation in

224 these CRPC cells led to a significant decrease of PFKP protein levels, relative to control

225 (Fig. 5c). Meanwhile, rescue of YY1 loss by an exogenous YY1WT restored the PFKP

226 protein level in these cells (Fig. 5d, middle vs left lanes), an effect not seen with

227 YY1S365D, a DNA-binding-defective mutant19 (Fig. 5d, right lanes). This indicates that

228 PFKP induction by YY1 is DNA-binding-dependent, in agreement with our YY1 ChIP-

229 seq results showing a direct binding of YY1 to the PFKP promoter (Fig. 5e). Indeed,

230 YY1WT, but not its YY1S365D mutant, increased transcription activity from a luciferase-

231 based reporter that carried either a 2Kb-long20 (Fig. 5f) or 575bp-long region upstream

232 of PFKP’s transcriptional start site (TSS; Fig. 5g; based on our ChIP-seq data in Fig. 5e).

233 Furthermore, there are four YY1 core binding motifs, CCAT21, within the YY1 binding

234 peaks at the PFKP promoter (i.e., -575 to +37 bp off TSS; Supplementary Fig. 5).

235 Systematic mutagenesis of these four YY1-binding motifs (Fig. 5h; mutation of CCAT to

236 CGGT) revealed the first and third CCAT motifs, but not the second and fourth ones, to

237 be essential for the PFKP promoter-driven transactivation activity in both 22Rv1 and

238 C4-2 cells (Fig. 5i-j). Additionally, relative to the single motif mutation, compound

239 mutation of the first and third YY1 motifs further decreased the PFKP promoter activity

240 (Fig. 5i-j; last panels). Thus, YY1 transactivates PFKP through directly binding its

241 promoter, particularly through the first and third conserved CCAT motifs.

242

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243 PFKP is critically involved in prostate cancer tumorigenesis in vitro and in vivo.

244 Since PFKP functions as a key rate-limiting enzyme in glycolysis, we next examined its

245 involvement in prostate cancer tumorigenesis, which was previously unexplored. First,

246 examination of PFKP across different transcriptome datasets of prostate cancer

247 samples17,22 showed its significantly higher expression in prostate tumors, compared to

248 benign tissues (Fig. 6a-b). To further determine the role for PFKP in prostate

249 oncogenesis, we depleted PFKP in both 22Rv1 and C4-2 cells (Supplementary Fig. 6a-

250 b) and observed the significantly decreased levels of in vitro proliferation (Fig. 6c) and

251 soft agar-based growth (Fig. 6d), relative to mock. Importantly, overexpression of PFKP

252 in the YY1-depleted 22Rv1 and C4-2 cells partially but significantly rescued the

253 ameliorated proliferation phenotype caused by YY1 loss (Fig. 6e), consistent to PFKP’s

254 rescue effects in the tumor cell metabolic assays (Fig. 4i). In the 22Rv1 cell xenografted

255 mouse model, PFKP loss also dramatically decreased tumor growth in vivo (Fig. 6f-h).

256 Thus, PFKP, a downstream target of YY1, is essential for prostate cancer progression.

257

258 Bromodomain-containing protein acts as cofactor of YY1, critically contributing

259 to the YY1-related transactivation of metabolic genes in prostate cancer

260 To gain further insight into the gene-activation mechanism underlying the YY1-mediated

261 CRPC tumorigenesis, we examined into the YY1 interactome by employing a proximity

262 labeling-based BioID approach23,24 (Supplementary Fig. 7a), and a subsequent mass

263 spectrometry-based proteomics analyses identified INO80, YY2 and BRD2 among the

264 most significantly enriched hits as YY1-asscociated factors (Fig. 7a). INO80, a known

265 YY1-interacting proteins25, was identified, which validated our method. By co-

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266 immunoprecipitation (CoIP), we further verified the interaction between YY1 and BRD2

267 or a BRD2-related bromodomain protein BRD4 in 22Rv1 cells (Fig. 7b) and C4-2 cells

268 (Supplementary Fig. 7b). We further mapped that the of BRD4, which

269 are known to be highly conserved among all BRD family members, were required for

270 interaction with YY1 (Fig. 7c). Acetylated histones such as H3K27ac is known to provide

271 a platform for tethering the BRD4-pTEFb complexes, which in turn boost the release of

272 RNA Pol-II into a productive elongation phase26. In agreement, we found a striking

273 overlap between YY1 ChIP-seq peaks with those of H3K27ac in 22Rv1 cells27—about

274 90% of H3K27ac ChIP-seq peaks are co-localized with YY1 (Fig. 7d). Consistently,

275 BRD4 ChIP-seq in 22Rv1 cells revealed significant binding at the YY1 promoter peaks,

276 nearly as strong as YY1, and at most of the YY1 non-promoter peaks (Fig. 7e), as

277 exemplified by those at metabolic genes such as PFKP and ALDOC (Fig. 7f and

278 Supplementary Fig. 7c). In addition, treatment of 22Rv1 cells with JQ1, an inhibitor of

279 bromodomain proteins BRD2 and BRD4, dramatically decreased overall expression of

280 our defined YY1 signature genes including the metabolism-related ones (Fig. 7g). Using

281 RT-qPCR, we further validated the inhibitory effect of JQ1 on metabolic genes such as

282 PFKP (Fig. 7h). Collectively, the YY1-mediated potentiation of a metabolic gene-

283 expression program is enhanced by its coactivators, bromodomain-containing proteins

284 (BRD2/4), in prostate cancer (see a model in Fig. 7i).

285

286 Discussion

287 Metabolic reprogramming towards aerobic glycolysis seen in cancer, initially discovered

288 by Otto Warburg, is now appreciated to be a hallmark of tumor, especially advanced

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289 ones, to gain survival and growth advantages. Unlike many other solid cancer types,

290 prostate cancer exhibits a unique context- and stage-dependent alteration in

291 metabolism7. It is generally viewed that, at its early stage, prostate cancer relies on the

292 increased oxygen consumption, as well as aerobic glycolysis, to support enhanced cell

293 proliferation; the Warburg effect becomes increasingly more pronounced during

294 progression into aggressive, late-stage prostate tumor8. However, the molecular

295 underpinning of metabolic reprogramming seen in advanced prostate tumor remains

296 unclear.

297 Although YY1 was previously reported to interact with AR and regulate

298 expression of prostate tumor marker genes such as PSA28, there is a general lack of

299 understanding of the role for YY1 in prostate oncogenesis, especially as prostate cancer

300 advances to CRPC. In this report, we show that YY1 plays a pivotal role in prostate

301 cancer progression across independent tumor models such as AR-dependent and

302 CRPC cancer cell lines and in vivo xenografted animal models. Furthermore, we

303 demonstrate that, via direct binding and transactivation of downstream metabolic genes,

304 YY1 is paramount for potentiating metabolism-related programs including mitochondria

305 respiration and glycolysis in these prostate cancer models. This finding is in agreement

306 with what was reported in other biological contexts— for example, skeletal muscle-

307 specific knock-out of YY1 in mice exhibited defective mitochondria morphology and

308 functions29; YY1 also activates mitochondria bioenergetics-related genes in B cells30

309 and alters tumor cell metabolism in colon cancer by activating G6PD and the pentose

310 phosphate pathway31. In this study, we demonstrated that PFKP, a key metabolic gene

311 whose expression is profoundly affected by YY1 via a direct promoter binding, is

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312 essential for YY1-mediated tumor cell glycolysis. Altogether, our findings unveil a

313 previously unexplored axis involving YY1-PFKP, which acts in prostate cancer to

314 sustain aggressive cancer phenotypes. We also favor a view that YY1 likely acts as a

315 master regulator of cancer cell metabolism in many tumor types for sustaining their

316 needs in energy and metabolism controls (through directly activating mitochondrial

317 and/or glycolytic pathways) during progression into advanced diseases. Of note, YY1

318 was reported to mediate tumorigenesis such as breast cancer and melanoma as well,

319 indicating a multifaceted role of YY1 in oncogenesis 32-35. Additionally, our study sheds

320 light on potential strategies of targeting metabolism alterations in prostate cancer. By

321 Biotin followed by mass spectrometry, we identify BRD4 family proteins (BRD2 and

322 BRD4) as functional partners of YY1, providing a mechanistic explanation for the YY1-

323 induced transcriptional potentiation of metabolic programs seen in advanced CRPC.

324 Importantly, this pathway is druggable by the bromodomain inhibitors implicating a

325 promising therapy of CRPC.

326 Equally intriguing is that, besides a transcriptional regulator via direct binding to

327 consensus CCAT motifs (such as that seen at the YY1-targeted PFKP promoter), YY1

328 was recently shown to function as a three-dimensional genome ‘organizer’ through

329 interactions with additional factors (such as CTCF) for mediating formation of looping

330 between enhancers and promoters36,37. How YY1 regulates 3D genome structure of

331 prostate cancer remains an open area for future studies but the current data implicates

332 a direct role in an important node in altering CRPC metabolism. CRPC is a

333 heterogeneous and lethal disease among men with limited therapeutic options, and the

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334 gained understanding of CRPC pathogenesis due to this work shall aid in its improved

335 targeted therapies.

336

337 Materials and Methods

338 Analysis of public prostate cancer datasets. Public datasets are

339 from the Singh et al. (2002) study (GSE68907), the Tomlins et al. (2007) study

340 (GSE6099), the Varambally et al. (2008) study (GSE3325). Gene expression data

341 available for the gene of interest were extracted, log2 transformed for each sample.

342 These summarized values were tested for association with sample type (such as benign,

343 primary or metastatic) by ANOVA.

344 Tissue Microarray (TMA). TMAs, produced by the Duke Pathology department, were

345 subjected to YY1 immunohistochemistry (IHC) staining and evaluated in a blinded

346 fashion by the pathologists. Scoring was assessed on the basis of staining intensity

347 from 0 (no staining) to 3 (strong) and percentage of tumor cell expression (1 to 100%),

348 creating a composite score from 0 to 300.

349 RNA-seq. RNA was prepared as described before38, using 2 million of the 22Rv1 or C4-

350 2 cells stably transduced with shRNAs. Then, complementary DNA was generated,

351 amplified and subjected for library construction using TruSeq RNA Library Preparation

352 Kit v2 (Illumina; catalog# RS-122-2002). The multiplexed RNA-Seq libraries were

353 subject to deep sequencing using the Illumina Hi-Seq 2000/2500 platform according to

354 the manufacturer’s instructions.

355 ChIP-seq. YY1 ChIP-seq was carried out as before38. Briefly, 22Rv1 cells were first

356 cultured under ligand-starved conditions for three days, followed by a 6-hour drug

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357 treatment with vehicle or 10nM of DHT. Cells were cross-linked with 1% formaldehyde

358 at room temperature for 10 minutes, followed by addition of glycine to stop crosslinking.

359 After washing, lysis and sonication, cell fractions were incubated with

360 antibody-conjugated Dynabeads (Invitrogen) overnight at 4 degree. Chromatin bound

361 beads were then subject to extensive washing and elution. Eluted chromatin was de-

362 crosslinked overnight at 65 degree, followed by protein digestion with proteinase K and

363 DNA purification with QIAGEN PCR purification kit. The obtained ChIP DNA samples

364 were submitted to the UNC-Chapel Hill High-Throughput Sequencing Facility (HTSF) for

365 preparation of multiplexed libraries and deep sequencing with an Illumina High-Seq

366 2000/2500 platform according to the manufacturer’s instructions. H3K27ac and BRD4

367 ChIP-seq datasets in 22Rv1 cells were downloaded from the ENCODE project

368 (ENCODE Project Consortium, 2011) and published paper38, respectively.

369 In vivo tumor growth in xenograft models. All animal experiments were approved by

370 and performed in accord with the guidelines of the Institutional Animal Care and Use

371 Committee (IACUC) at UNC. One million of 22Rv1 cells with stable transduction of

372 shRNA or control empty vector were suspended in 100ul of PBS with 50% Matrigel (BD

373 Biosciences) and then subcutaneously (s.c.) injected into the dorsal flanks of castrated

374 NOD/SCID/gamma-null (NSG) mice bilaterally (carried out by the Animal Studies Core,

375 UNC at Chapel Hill Cancer Center). Tumor growth was monitored twice a week and the

376 tumor volume calculated.

377 Statistical analysis. Unless specifically indicated, the unpaired two-tail Student's t test

378 was used for experiments comparing two sets of data. Data represent mean ± SEM

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379 from three independent experiments. *, **, and *** denote P values < 0.05, 0.01, and

380 0.001, respectively. NS denotes not significant.

381 Data Availability. RNA-seq and ChIP-seq reads have been deposited to Gene

382 Expression Omnibus (GEO) under accession number GSE153640. The matched input

383 data of 22Rv1 cells (both EtOH and DHT-treated) were previously published by us

384 (available from NCBI GEO #GSE94013) and used herein for normalization of 22Rv1

385 YY1 ChIP-seq datasets.

386

387 Acknowledgments

388 We thank UNC’s facilities, including Imaging Core, High-throughput Sequencing Facility

389 (HTSF), Bioinformatics Core, Tissue Procurement Facility, Translational Pathology

390 Laboratory, Tissue Culture Facility and Animal Studies Core for their professional

391 assistance of this work. We graciously thank Drs. Kyung-Sup Kim and Hyun Sil Kim for

392 providing reagents and the Cai, Wang and Earp laboratory members for discussion and

393 technical support. We also acknowledge proteomics support from the University of

394 Arkansas for Medical Sciences Proteomics Core Facility, the IDeA National Resource

395 for Proteomics, the Translational Research Institute through the National Center for

396 Advancing Translational Sciences of the National Institutes of Health, and the Center for

397 Translational Pediatric Research Bioinformatics Core Resource with support through

398 TL1TR003109, P20GM121293, P20GM103625, P20GM103429, S10OD018445,

399 UL1TR003107, and R01CA236209. This work was supported in part by UNC

400 Lineberger Cancer Center UCRF Stimulus Initiative Grant (to L.C.). The cores affiliated

401 to UNC Cancer Center are supported in part by the UNC Lineberger Comprehensive

17 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

402 Cancer Center Core Support Grant P30-CA016086. G.G.W. is an American Cancer

403 Society (ACS) Research Scholar and a Leukemia and Lymphoma Society (LLS)

404 Scholar.

405

406 Declaration of interests

407 J.H. is a consultant for or owns shares in the following companies: Kingmed,

408 MoreHealth, OptraScan, Genetron, Omnitura, Vetonco, York Biotechnology, Genecode

409 and Sisu Pharma. G.G.W. is an inventor of a patent application filed by University of

410 North Carolina at Chapel Hill. G.G.W. received research fund from the

411 Deerfield Management/Pinnacle Hill Company.

412 413 Author contributions 414 415 C.X. performed most of the experiments. H.S.E., G.G.W. and L.C. conceived the project, 416 organized and led the study. Y.-H.T. and W.G. conducted analysis of RNA-seq and 417 public cancer datasets under the supervision of J.S.P., G.G.W. and L.C., A.J.S., S.G.M., 418 R.D.E., and S.D.B. performed proteomics analyses using mass spectrometry. A.J.T. 419 supervised the proteomics analysis. P.G analyzed ChIP-seq data under the supervision 420 of D.Z., G.G.W. and L.C. Y.X. and J.H. provided critical guidance and helps on IHC and 421 TMA. C.X., Y.W., J.S.P., J.H., D.Z., H.S.E., G.G.W. and L.C. interpreted the data. L.C. 422 conceived the idea, supervised the work and designed the research. C.X., G.G.W. and 423 L.C. wrote the manuscript with input from other authors. 424

425

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517 34 Baritaki, S. et al. YY1 Over-Expression in Human Brain Gliomas and Meningiomas 518 Correlates with TGF-1, IGF-1 and FGF-2 mRNA Levels. Cancer Invest 27, 184-192, 519 doi:Pii 908984693 520 10.1080/07357900802210760 (2009). 521 35 Chinnappan, D. et al. Transcription factor YY1 expression in human gastrointestinal 522 cancer cells. Int J Oncol 34, 1417-1423, doi:10.3892/ijo_00000270 (2009). 523 36 Weintraub, A. S. et al. YY1 Is a Structural Regulator of Enhancer-Promoter Loops. Cell 524 171, 1573-+, doi:10.1016/j.cell.2017.11.008 (2017). 525 37 Beagan, J. A. et al. YY1 and CTCF orchestrate a 3D chromatin looping switch during 526 early neural lineage commitment. Genome Res 27, 1139-1152, 527 doi:10.1101/gr.215160.116 (2017). 528 38 Cai, L. et al. ZFX Mediates Non-canonical Oncogenic Functions of the Androgen 529 Receptor Splice Variant 7 in Castrate-Resistant Prostate Cancer. Mol Cell 72, 341-+, 530 doi:10.1016/j.molcel.2018.08.029 (2018). 531

532

533

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534 Figure legends.

535 Figure 1. YY1 shows significantly higher expression among samples of prostate

536 cancer patients, when compared to benign tissues.

537 (a-b) Boxplots showing overall YY1 mRNA levels among patient cohorts reported in

538 (Singh et al., 2002) (panel a) and (Tomlins et al., 2007) (b), relative to their respective

539 normal controls.

540 (c-d) Representative images (panel d; 10x) and quantification (panel c) of YY1

541 immunohistochemistry (IHC) staining of thirteen paired normal and tumor tissues from

542 prostate cancer patients.

543 (e) Immunoblotting for YY1 using total lysates of the paired normal/benign (B) and

544 tumor (T) tissues from prostate cancer patients. b-actin acts as a loading control.

545 (f-g) Representative images (panel f) and quantification (panel g) of YY1 IHC staining

546 by using tissue microarrays (TMA) that contains samples of benign prostates (n=136),

547 primary prostate adenocarcinoma (n=138) and CRPC (n=55).

548

549 Figure 2. YY1 is required for malignant growth of prostate tumor cells in vitro and

550 in vivo.

551 (a-f) Immunoblotting for YY1 (panels a and d), measurement of proliferation (b and e)

552 and assessment of soft agar-based growth (c and f; with representative images shown)

553 after shRNA-mediated stable knockdown of YY1 (KD by sh#94 or sh#98) in either

554 22Rv1 (a-c) or C4-2 (d-f) cells, relative to transduction of empty vector (shEV). **P<0.01.

555 (g-i) Immunoblotting for YY1 (panel g), measurement of proliferation (h) and

556 assessment of soft agar-based growth (i; with representative images shown) post-

22 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

557 rescue of YY1 (using an exogenous HA-tagged YY1) in 22Rv1 cells with endogenous

558 YY1 knocked down (by sh#94). **P<0.01.

559 (j-k) Immunoblotting for YY1 (panel j) and measurement of proliferation (k) after the

560 CRISPR/cas9-mediated knockout of YY1 (sgYY#1 or sgYY#2) in 22Rv1 cells, relative to

561 transduction of empty vector (sgEV). **P<0.01.

562 (l-m) Summary of changes in the tumor volume (panel l), following subcutaneous

563 transplantation of stable shEV- (balck) or shYY1-expressing (red) 22Rv1 cells into

564 castrated NSG mice (n = 6 per group). Data presented are mean ± SEM (n = 6 mice for

565 each group). Statistical significance was determined by two-way ANOVA (** P< 0.005,

566 *** P< 0.0005, **** P< 0.0001). The image of representative tumors is shown in m.

567 (n) Immunoblotting for YY1 in the 22Rv1 tumor xenografts isolated from the indicated

568 NSG cohort (shEV or shYY1).

569

570 Figure 3. YY1 directly binds to and positively regulates metabolic genes in

571 prostate tumor.

572 (a) Heatmap showing the expression levels of differentially expressed genes (DEGs)

573 identified due to YY1 KD relative to mock (shEV) in 22Rv1 cells, with two biological

574 replicates (Rep 1 and 2) per group. Threshold of DEG is set at the adjusted DESeq P

575 value (padj) less than 0.01 and fold-change (FC) over 1.5 for transcripts with mean tag

576 counts of at least 10.

577 (b) GSEA revealing that, relative to mock, YY1 KD is positively correlated with

578 downregulation of the indicated genesets related to energy metabolism, glycolysis or

23 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

579 prostate cancer, and negatively correlated with upregulation of the indicated YY1-

580 repressed geneset (bottom/right) in 22Rv1 cells.

581 (c) Heatmap showing expression of the indicated glycolysis-related genes in 22Rv1

582 cells after YY1 KD, relative to mock.

583 (d) Venn diagram showing overlap between the YY1-upregulated genes as identified by

584 RNA-seq in 22Rv1 (left) and C4-2 (right) cells. Threshold of DEG is set at the adjusted

585 DESeq P value (padj) less than 0.01 and fold-change (FC) over 1.5 for transcripts with

586 mean tag counts of at least 10.

587 (e-f) RT-qPCR of YY1 (far left) and the indicated metabolic gene in 22Rv1 (e) and C4-2

588 (f) cells post-KD of YY1, compared with mock. Y-axis shows averaged fold-change ±SD

589 of three independent experiments after normalization to beta-Actin and then to mock-

590 treated. ** P <0.01, *** P <0.001.

591 (g) Pie chart showing genomic distribution of the called YY1 peaks in 22Rv1 cells.

592 (h) Motif search analysis revealing the most enriched motifs at the called YY1 peaks

593 found at gene promoters.

594 (i) IGV browser views of chromatin input and YY1 ChIP-seq peaks (depth normalized)

595 at the indicated metabolic pathway genes in 22Rv1 cells, ligand-starved followed by

596 treatment with vehicle (DHT-) or dihydrotestosterone (DHT+).

597

598 Figure 4. YY1 potentiates prostate tumor cell glycolysis via PFKP.

599 (a-b) Measurement of oxygen consumption rate (OCR) and mitochondrial bioenergetics

600 in 22Rv1 (panel a) and C4-2 (b) cells post-KD of YY1, compared to mock (shEV), by

601 using the Seahorse XF-24 extracellular flux analyzer. Injection of compounds during the

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602 assay is highlighted in left panels. Data quantifications are shown as mean±SEM in right

603 panels. ** P<0.01, *** P <0.001.

604 (c-d) Measurement of extra cellular acidification rate (ECAR) in 22Rv1 (panel c) and

605 C4-2 cells (d) post-KD of YY1, compared to mock (shEV). Injection of compounds

606 during the assay is highlighted in left panels. Data quantifications are shown as

607 mean±SEM in right panels. ** P<0.01, *** P <0.001.

608 (e-g) Immunoblotting for YY1 (panel e; anti-HA immunoblots in 22Rv1 cells), as well as

609 ECAR measurements in 22Rv1 (f) or C4-2 (g) cells post-transduction of the indicated

610 HA-tagged YY1, either WT or with a N-terminal transactivation domain deleted (Δ1-

611 100aa). * P<0.05; ** P <0.01; NS, not significant.

612 (h-i) Endogenous YY1 and anti-HA immunoblotting (panel h) and ECAR measurements

613 (i) post-transduction of an HA-tagged PFKP (h, lane 3), relative to mock (h, lane 2), into

614 the YY1-depleted 22Rv1 cells (lanes 2-3). The shEV-transduced cells (lane 1) serve as

615 control. * P<0.05; ** P <0.01; NS, not significant.

616 (j-k) ECAR measurements in 22Rv1 (panel j) or C4-2 (k) cells post-KD of PFKP (sh#75

617 or sh#77), compared to mock (shEV). * P<0.05; ** P <0.01; *** P <0.001.

618 (l-m) YY1 and PFKP immunoblotting (panel l) and ECAR measurements (m) post-

619 transduction of an HA-tagged YY1 (l, lanes 2 and 4), relative to vector mock (l, lanes 1

620 and 3), into 22Rv1 cells with stable expression of either shEV (lanes 1-2) or YY1-

621 targeting shRNA (lanes 3-4). * P<0.05; ** P <0.01; NS, not significant.

622

623 Figure 5. PFKP is a direct onco-target of YY1 in prostate cancer.

25 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

624 (a-b) Volcano plots based on RNA-seq profiles highlight PFKP among the most altered

625 transcripts upon YY1 depletion in 22Rv1 (a) and C4-2 (b) cells.

626 (c) YY1 and PFKP immunoblots post-KD of YY1 in 22Rv1 (left) and C4-2 (right) cells.

627 (d) YY1 and PFKP immunoblots post-transduction of an HA-tagged YY1WT or YY1S365D,

628 a DNA-binding-defective mutant, into 22Rv1 (left) and C4-2 (right) cells.

629 (e) IGV views of the YY1 ChIP-seq profile on the proximal promoter region of PFKP.

630 (f-g) Relative luciferase activities from a reporter that carries promoter region of PFKP,

631 either from -2,008 to +7 (f) or -575 to +37 (g), in 22Rv1 cells expressed with HA-tagged

632 YY1WT or YY1S365D. Y-axis shows averaged fold-change ±SD of three independent

633 experiments after normalization to internal luciferase controls and then to mock samples

634 (EV- expressing). ** P<0.01.

635 (h) Scheme showing mutations (mut) of putative YY1-binding sites within the PFKP

636 promoter. Empty box denotes the mutated YY1-binding site.

637 (i-j) Relative luciferase activities from a PFKP promoter reporter, either WT or that

638 carrying the indicated mutation of putative YY1-binding sites (refer to panel h), following

639 overexpression of YY1WT into either 22Rv1 (i) or C4-2 cells (j). Y-axis shows averaged

640 fold-change ±SD of three independent experiments after normalization to internal

641 luciferase controls and then to EV-expressing control samples (far left). * P <0.05; ** P

642 <0.01; *** P <0.001.

643

644 Figure 6. PFKP is critically involved in prostate cancer tumorigenesis in vitro and

645 in vivo.

26 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

646 (a-b) Box plots showing the PFKP expression levels among samples from the indicated

647 patient cohorts reported in (Tomlins et al., 2007) (panel a) or (Varambally et al., 2008)

648 (b).

649 (c-d) Assays for in vitro proliferation (c) and soft agar-based growth (d) after PFKP

650 depletion (sh#75 or sh#77), compared to control (shEV), in 22Rv1 (left) and C4-2 cells

651 (right). * P <0.05; ** P <0.01; *** P <0.001.

652 (e) Assays for proliferation post-transduction of an exogenous PFKP (red), relative to

653 mock (blue), into the YY1-depleted 22Rv1 (left) and C4-2 cells (right), with non-depleted

654 cells acting as controls (shEV; gray). ** P <0.01.

655 (f-g) Summary of changes in the tumor volume (f), following subcutaneous

656 transplantation of stable shEV- (balck) or shPFKP-expressing (red) 22Rv1 cells into

657 castrated NSG mice (n = 6 per group). Statistical significance was determined by two-

658 way ANOVA (** P< 0.005, *** P< 0.0005). The image of representative tumors is shown

659 in g.

660 (h) Immunoblotting for PFKP in the 22Rv1 tumor xenografts isolated from the indicated

661 NSG cohort (shEV or shPFKP).

662

663 Figure 7. Bromodomain-containing protein acts as co-activator of YY1,

664 potentiating expression of glycolysis-related genes in prostate cancer cells.

665 (a) Summary of the top hits identified using BioID and 22Rv1 cells expressing YY1 with

666 a biotin ligase fused to its N-terminus, compared to control cells (EV).

667 (b) Co-immunoprecipitation (CoIP) for interaction between endogenous YY1 and

668 bromodomain-containing proteins, BRD2 (top) and BRD4 (bottom), in 22Rv1 cells.

27 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license.

669 (c) CoIP for interaction between the exogenously expressed BRD4 (Flag-tagged), either

670 WT or BD domains deleted (ΔBD), and HA-tagged YY1 in 293 cells. TCL, total cell

671 lysate.

672 (d) Venn diagram showing overlap between the called YY1 and H3K27ac ChIP-seq

673 peaks identified from the ligand-stripped 22Rv1 cells.

674 (e) Heatmap of the YY1 and BRD4 ChIP-Seq read densities at promoter (top) and non-

675 promoter (bottom) YY1 peaks in vehicle- (DHT-) or DHT-treated 22Rv1 cells. The YY1

676 peaks from vehicle and DHT were combined, sorted, and used to compute ChIP-seq

677 read densities within 5 kb of the YY1 peak centers.

678 (f) IGV views of the YY1 and BRD4 ChIP-seq profiles at the indicated glycolytic genes

679 in vehicle- (DHT-) or DHT-treated 22Rv1 cells.

680 (g) Heatmap showing overall expression changes fo the YY1 gene signature (defined in

681 Figure 3C) in 22Rv1 cells after drug treatment, either DHT alone, DHT plus JQ1, or

682 DHT plus AR antagonist MDV3100 (MDV). Color bar, mean of the log2FC compared to

683 mock.

684 (h) RT-qPCR of the indicated metabolic gene in 22Rv1 post-treatment of DMSO or JQ1

685 for 8 hours. Y-axis shows averaged fold-change ±SD of three independent experiments

686 after normalization to beta-Actin and then to mock-treated. ** P <0.01, *** P <0.001.

687 (i) A model illustrating the role for YY1:BRD4 in potentiating energy metabolism in

688 advanced prostate tumor.

689

28 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Xu et al Fig 1. available under aCC-BY-NC-ND 4.0 International license.

a b c

Singh et al Cohort Tomlins et al Cohort P = 0.0071 P < 0.01 P < 0.05 ● ● 20 ●● ●● 5 ●●● ● ● ● 0.5 ● ● ● ● ●● ●●●● ● ● ●● ● ● ● ● ● ● ●● ● ● ● 15 4 ●● ●●●● ●●● ●●● ●●● ● ●● ●● ● ●● ●●● ●●●●● ● ●●●●●● ● 0.0 ● ●●●●●● ● ●●●● ● ●● ● ● ● ●● 10 ● ● ● ●● 3 ● ● ●● ● ● ●● ●● ● ●● ●● ● ● ● ●● ● ●● ●● ●● ● ● ● ● ● −0.5 ●● 5 2 ● ● ●●● ●

1 ●● ● ● ● ● 0 −1.0

Log (normalized YY1 gene expression) Log (normalized Benign Tumor Normal Tumor YY1 gene expression) MeanCtr (Log2 Normal Tumor Relative YY1 IHC scores in tumor vs. normal Relative (n=13) (n=13)

d Patient 1 Patient 2 Benign (10x) Tumor (10x) Benign (10x) Tumor (10x)

e f Benign (negative) g

#1 #2 #3 #4 #5 #6 B T B T B T B T B T B T P = 0.0170 75kD YY1 50kD 200 P = 0.0339 Actin-b 37kD 150

AdenoCa (focal) CRPC (strong) 100 YY1 IHC scores

50

0 Benign AdenoCa CRPC (n=136) (n=138) (n=55) Xu et al Fig 2.

bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint a (which was not certified by peer review)b is the author/funder, who has granted bioRxiv ac license to display the preprint22Rv1 in perpetuity. It is made available under aCC-BY-NC-ND 4.0 shEVInternational license. sh-EV shYY1(#94) shYY1(#98) 22Rv1 ** 1.6

shEV shYY1(#94)shYY1(#98) 1.2 ** shYY1(#98)

YY1 0.8

0.4 shYY1(#94)

Relative cell growth Actin-b 0.0 0 2 4 6 Days in culture d e f C4-2 sh-EV shYY1(#94) shYY1(#98) C4-2 1.6 shEV **

1.2 shYY1(#98) shEV shYY1(#94)shYY1(#98) ** 0.8 YY1 0.4 shYY1(#94) Relative cell growth Actin-b 0.0 0 2 4 6 Days in culture g h i 22Rv1 shEV shEV shYY1 shYY1+YY1 rescue shYY1 +mock 1.8 shYY1 +YY1

1.2 YY1 shEV sh shYY1+YY1 rescue

0.6 ** YY1 ** Relative cell growth Actin-b 0.0 0 2 4 6 Days in culture j k l sgEV 1600 1.6 sgYY1#1 ** 22Rv1 sgYY1#2 1400 sgEV sgYY1#1 sgYY1#2 1.2 1200 shEV shYY1 ** 1000 YY1 0.8 800 **** 600 *** 0.4 400 **

Actin-b volume (mm3) Tumor Relative cell growth 200 ** *** 0.0 0 0 2 4 6 0 10 20 30 40 Days in culture Days post-xenograft m n shEV shYY1

shEV YY1

shYY1 Actin-b bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made available under aCC-BY-NC-ND 4.0 International license. Xu et al Fig 3.

a shEV shYY1 b REACTOME_INTEGRATION_OF_ENERGY_METABOLISM KEGG_GLYCOLYSIS_GLUCONEOGENESIS c Rep1 Rep2 Rep1 Rep2 KEGG_GLYCOLYSIS_GLUCONEOGENESIS YY1 KD mock Rep1 Rep2 Rep1 Rep2 NES=-1.72 NES=-1.71 ALDH3B1 p=0.0 p=0.0 FDR q=0.067 ENO3 FDR q=0.070 ALDH2

Enrichment score(ES) LDHA Enrichment score(ES) ENO2 PFKP YY1_KD EV YY1_KD EV ACSS1 PFKM YEGNASUBRAMANIAN_PROSTATE_CANCER DE_YY1_TARGETS_DN ALDOC GCK GAPDH PGAM2 NES=-1.48 NES=-1.80 p=0.0 d p=0.0 FDR q=0.19 padj<0.01, |log2FC| > 0.58 FDR q=0.040 & BaseMean > 10 22Rv1 C4-2 Enrichment score(ES) Enrichment score(ES)

−1 0 1 YY1 siganture genes in prostate tumor YY1_KD EV YY1_KD EV Z−Score (PFKP, ALDOC, e f g OGDHL, NDUFA4L2) mock 22Rv1 mock 1.5 C4-2 h YY1 KD 1.5 YY1 KD YY1 (DHT-) *** *** ** *** ** *** *** *** ** *** *** *** *** ** *** ** 1.0 intergenic genebody Motif search (promoters) 1.0 18.9% SP1 1e-378 RN A expression RN A expression 35.9% 0.5 0.5 19.7% ELK1 1e-225

0.0 0.0 25.5%

Relative m distal Relative m YY1 1e-181 YY1 YY1 PFKL PFKLENO1ENO2 promoter PFKP ENO1ENO2 PFKP OGDHL ALDOCOGDHL ALDOC NDUFA4L2 NDUFA4L2 i Input [0 - 3] 5 kb [0 - 4] 1 kb DHT- YY1 [0 - 5] [0 - 8] Input [0 - 3] [0 - 4] DHT+ YY1 [0 - 5] [0 - 8] PFKP ENO2 0.5 kb Input [0 - 4] 0.5 kb [0 - 4] DHT- [0 - 15] YY1 [0 - 4] Input [0 - 4] [0 - 4] DHT+ YY1 [0 - 4] [0 - 15] ALDOC NDUFA4L2 Xu et al Fig 4. a bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596 ; this version b posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made shEV available under aCC-BY-NC-ND 4.0 InternationalshEV license. Maximal respiration C4-2 shYY1#94 Maximal respiration 22Rv1 shYY1#94 1000 ** shYY1#98 Rotenone 1500 ** 1000 shYY1#98 Rotenone ** 1500 ** 800 Basal Oligomycin Basal 800 respiration respiration Oligomycin 1000 600 1000 FCCP ** 600 *** ** ***

FCCP on sumption rates 400 400 on sumption rates 500 500 OCR (pmol/min) 200 200 OCR (pmol/min) Oxygen c 0 0 0 Oxygen c 0 0 50 100 0 50 100 V V V V Time (min) E E E E sh 1#94 1#98sh 1#94 1#98 Time (min) sh 1#94 1#98sh 1#94 1#98 YY YY YY YY YY YY YY YY h h h h h h h h c s s s s d s s s s Maximal glycolytic shEV shEV Maximal glycolytic capacity C4-2 shYY1#94 22Rv1 shYY1#94 250 capacity 80 *** shYY1#98 2-DG *** shYY1#98 250 Glycolysis *** 80 2-DG Glycolysis *** 200 60 ca tion rate *** ca tion rate Oligomycin *** 200 ** Oligomycin 150 60 *** Glucose 40 150 ac idifi

Glucose ac idifi 40 100 100

20 CA R (mpH/min) CA R (mpH/min) 20 50 E E 50 0 0 0 0 50 100 0 Extra cellular

Extra cellular V V 0 50 100 E E V V Time (min) h 1#94 1#98 h 1#94 1#98 E E s s Time (min) sh 1#94 1#98sh 1#94 1#98 YY YY YY YY h h h h YY YY YY YY s s s s sh sh sh sh

C4-2 e f g 2-DG 2-DG 22Rv1 250 + mock + mock Oligomycin 100 * ** + YY1 Oligomycin + YY1 200 * * + YY1-D1-100aa 80 Glucose ** * + YY1-D1-100aa mock HA-YY1 (WT)HA-YY1 (del1-100aa) Glucose* * * 75kD 150 NS 60 * * * NS HA-YY1 ** * ** * * NS 100 40 * 50kD NS

CA R (mpH/min) NS CA R (mpH/min) E 50 NS E 20

Actin-b 0 0 0 50 100 0 50 100 Time (min) Time (min) h i j 22Rv1 100 2-DG 22Rv1 2-DG shEV shEV 100 Oligomycin shPFKP#77 shYY1#94 + mock 80 * ** ** * * shYY1#94 + PFKP Oligomycin ** shPFKP#75 * shEV shYY1+mockshYY1+HA-PFKP * * ** Glucose 60 Glucose ** ** HA-PFKP 75kD 50 *** ** * * 40 *** ***

75kD CA R (mpH/min)

YY1 CA R (mpH/min) * E NS NS E 20 * * * 0 Actin-b 0 50 100 0 Time (min) 0 50 100 Time (min)

22Rv1 k 250 2-DG l shEV shPFKP#75 m 2-DG C4-2 Oligomycin + mock 80 +shEV *** *** *** shEV ** ***** + YY1 200 Oligomycin shPFKP#77 *** *** ** + mock Glucose shPFKP#75 +mock +YY1 +mock +YY1 +shPFKP#75 100kD 60 Glucose + YY1 150 *** ****** PFKP ** ** ** 40 * 75kD NS 100 * NS NS NS

CA R (mpH/min) * NS NS * E * YY1 CA R (mpH/min) NS * 50 E 20 NS NS * * * NS Actin-b NS 0 0 NS 0 50 100 0 50 100 Time (min) Time (min) a Xu etalFig5. e h c DHT+ DHT- bioRxiv preprint (which wasnotcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade

mut =CGGT wt =CCAT shEV -log(adj P value) Input Input shYY1(#94) b YY1 YY1 20 40 60 80 22Rv1 0

shYY1(#98) -10 [0 -3] [0 -3] [0 -3] [0 -3]

shEV doi:

shYY1(#94) PFKP PFKP-mut1/3 https://doi.org/10.1101/2020.09.17.302596 PFKP-mut4 PFKP-mut3 PFKP-mut2 PFKP-mut1

shYY1(#98) -5 C4-2 PFKP wt Actin-b PFKP YY1 log2(Fold change) NR_160682 -152 22Rv1 YY1 0 d -123-50 mock sY1#4 +shYY1(#94) +shYY1(#94)

YY1(WT) available undera -2 22Rv1 100 bp

PFKP YY1(S365D) 5 Luc Luc Luc Luc Luc Luc mock

YY1(WT)

YY1(S365D) CC-BY-NC-ND 4.0Internationallicense 10 C4-2 Actin-b PFKP YY1 ; this versionpostedSeptember19,2020. i f

Relative luciferase activity Luciferase activity, 10 15 20 25

0 5 relartive to mock -log(adj P value) 10 15 20 0 5 20 40 60 80

EV mock 0 -10 vector YY1(WT)

wt ** YY1(S365D)

m 22Rv1 + YY1 u t1 * m (-2,008bp to+7bp) u **

t2 PFKP-promoter mock m u YY1(WT) *

t YY1(S365D) -5 3 ** m

u ** mut t4 . log2(Fold change) 1 NS /3 g The copyrightholderforthispreprint C4-2 j Relative luciferase activity Luciferase activity, 0 YY1 PFKP 10 15 20 relartive to mock 0 5 0 2 4 6 8

EV mock vector YY1(WT)

** YY1(S365D) wt C4-2 + YY1

m 5 u t1 * m (-575bp to+37bp) ** u PFKP-promoter mock t2 m YY1(WT) u YY1(S365D) * t3 ** m *** u 10 mut t4

1 NS /3 bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made Xu et al Fig 6.available under aCC-BY-NC-ND 4.0 International license.

a PFKP: Tomlin e tal cohort b PFKP: Varambally et al cohort 0.8 ●● P < 0.05 P < 0.05 ●● ● ● 0.4 ● ● ●● ●●● 0 1 ●● ● ● ●●● ●● ●●●●●● ●● ●●● 0.0 ● ●●●● ●●● ●●● ●●● ●● −1 ●●● ●●● ●● ●● ● ●

MedianCtr(Log2_GeneExp) −0.4 ● −2 ●●

normal tumor YY1 gene expression) MeanCtr (Log2 Benign Primary Metastatic c 1.6 22Rv1 shEV C4-2 shEV ** 1.6 **

1.2 1.2 ** shPFKP#77 ** shPFKP#77 0.8 0.8

0.4 shPFKP#75 0.4 shPFKP#75 Relative cell growth Relative cell growth 0.0 0.0 0 2 4 6 0 2 4 6 Days in culture Days in culture d shEV shPFKP#77 shPFKP#75 shEV shPFKP#77 shPFKP#75

e shEV shEV 1.6 shYY1 22Rv1 1.8 shYY1 C4-2 shYY1+PFKP shYY1+PFKP 1.2 1.2 0.8

0.6 ** 0.4 ** Relative cell growth Relative cell growth ** ** 0.0 0.0 0 2 4 6 0 2 4 6 Days in culture Days in culture f g 1000 22Rv1 shEV 800 shEV shPFKP 600 shPFKP 400 *** h shEV shPFKP

Tumor volume(mm3) 200 ** #1 #2 #3 #1 #2 #3 *** 100kD ** ** ** PFKP 0 75kD 0 10 20 30 40 Days post-xenograft 37kD Actin-b Xu et al Fig 7. bioRxiv preprint doi: https://doi.org/10.1101/2020.09.17.302596; this version posted September 19, 2020. The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity. It is made a available underb a CC-BY-NC-ND22Rv1 4.0 Internationalc licenseFLAG. EV BRD4-wt BRD4-ΔBD HA-YY1 + + + 75kD Total Spectrum # log2FC ( YY1 Input IgG BRD2 IP HA Protein ID 150kD vs mock) IP: mock YY1 BRD2 Flag YY1 3 246 6.95 75kD 250kD FLAG ZMYM4 0 36 6.77 YY1 YY2 2 136 6.68 75kD HA INO80 0 22 6.06 22Rv1 ETFA 0 20 5.92 ALB 1 37 5.81 TCL 250kD FLAG RFC5 0 15 5.50 Input IgG BRD4 IP BRD4 RNH1 0 13 5.30 250kD 37kD 4.60 75kD GAPDH BRD2 0 8 YY1

YY1 DHT- [0 - 0.8] 1 kb d H3K27ac f BRD4 DHT+ [0 - 0.8] [0 - 4] DHT- YY1 [0 - 4] 36,090 40,353 5,623 DHT+ PFKP

DHT- [0 - 0.8] 0.5 kb BRD4 DHT+ [0 - 0.8] [0 - 4] DHT- YY1 [0 - 4] YY1 BRD4 DHT+ e DHT- DHT+ DHT- DHT+ ALDOC

g

DHT+mock DHT+mock rep1 DHT+JQ1 rep2 DHT+JQ1 rep1 DHT+MDV rep2 DHT+MDV rep1 rep2 i logFC to MOCK BRD2/4 0.5 0 promoter −0.5 Ac −1 YY1 −1.5 target genes

mean log (gene_exp/MOCK) glucose YY1 signature genes (CCAT) F6P h PFKP DMSO 1.5 22Rv1 F-1,6-BP JQ1

*** ** *** ** *** ** 1.0 glycolysis RN A expression 0.5 CRPC progression non-promoter

Relative m 0.0

-2.5kb 0 2.5kb PFKP PFKM PFKL ENO1ENO2 ALDH2 ALDOC ALDH3B2