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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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 transcription 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 gene
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 bromodomain-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 protein 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
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 available under aCC-BY-NC-ND 4.0 International license.
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 proteins (BRD2 and
106 BRD4) as YY1’s functional partners, co-mediating transcriptional activation of many
107 metabolic genes 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
5 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.
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
6 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.
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
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 available under aCC-BY-NC-ND 4.0 International license.
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
8 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.
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
9 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.
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
10 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.
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-
11 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.
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 bromodomains 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
12 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.
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
13 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.
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
14 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.
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 gene expression 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
15 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.
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 chromatin 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
16 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.
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
18 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.
426 References 427 1 Watson, P. A., Arora, V. K. & Sawyers, C. L. Emerging mechanisms of resistance to 428 androgen receptor inhibitors in prostate cancer. Nat Rev Cancer 15, 701-711, 429 doi:10.1038/nrc4016 (2015). 430 2 Shi, Y., Seto, E., Chang, L. S. & Shenk, T. Transcriptional Repression by Yy1, a Human 431 Gli-Kruppel-Related Protein, and Relief of Repression by Adenovirus E1a Protein. Cell 432 67, 377-388, doi:Doi 10.1016/0092-8674(91)90189-6 (1991). 433 3 Seto, E., Shi, Y. & Shenk, T. Yy1 Is an Initiator Sequence-Binding Protein That Directs 434 and Activates Transcription Invitro. Nature 354, 241-245, doi:DOI 10.1038/354241a0 435 (1991). 436 4 Gordon, S., Akopyan, G., Garban, H. & Bonavida, B. Transcription factor YY1: structure, 437 function, and therapeutic implications in cancer biology. Oncogene 25, 1125-1142, 438 doi:DOI 10.1038/sj.onc.1209080 (2006). 439 5 Donohoe, M. E. et al. Targeted disruption of mouse Yin Yang 1 transcription factor 440 results in peri-implantation lethality. Mol Cell Biol 19, 7237-7244 (1999). 441 6 Cairns, R. A., Harris, I. S. & Mak, T. W. Regulation of cancer cell metabolism. Nat Rev 442 Cancer 11, 85-95, doi:10.1038/nrc2981 (2011). 443 7 Elia, I., Schmieder, R., Christen, S. & Fendt, S. M. Organ-Specific Cancer Metabolism 444 and Its Potential for Therapy. Handb Exp Pharmacol 233, 321-353, 445 doi:10.1007/164_2015_10 (2016). 446 8 Lin, C. C. et al. Prostate Cancer Energetics and Biosynthesis. Adv Exp Med Biol 1210, 447 185-237, doi:10.1007/978-3-030-32656-2_10 (2019). 448 9 Wang, J. et al. Increased expression of glycolytic enzymes in prostate cancer tissues and 449 association with Gleason scores. Int J Clin Exp Patho 10, 11080-11089 (2017). 450 10 Mor, I., Cheung, E. C. & Vousden, K. H. Control of glycolysis through regulation of 451 PFK1: old friends and recent additions. Cold Spring Harb Symp Quant Biol 76, 211-216, 452 doi:10.1101/sqb.2011.76.010868 (2011). 453 11 Lang, L., Chemmalakuzhy, R., Shay, C. & Teng, Y. PFKP Signaling at a Glance: An 454 Emerging Mediator of Cancer Cell Metabolism. Reviews on Biomarker Studies of 455 Metabolic and Metabolism-Related Disorders 1134, 243-258, doi:10.1007/978-3-030- 456 12668-1_13 (2019). 457 12 Moon, J. S. et al. Kruppel-like Factor 4 (KLF4) Activates the Transcription of the Gene 458 for the Platelet Isoform of Phosphofructokinase (PFKP) in Breast Cancer. J Biol Chem 459 286, 23808-23816, doi:10.1074/jbc.M111.236737 (2011). 460 13 Li, X. B., Gu, J. D. & Zhou, Q. H. Review of aerobic glycolysis and its key enzymes - 461 new targets for lung cancer therapy. Thorac Cancer 6, 17-24, doi:10.1111/1759- 462 7714.12148 (2015). 463 14 Lee, J. H. et al. Stabilization of phosphofructokinase 1 platelet isoform by AKT promotes 464 tumorigenesis. Nature Communications 8, doi:ARTN 949 465 10.1038/s41467-017-00906-9 (2017). 466 15 Khachigian, L. M. The Yin and Yang of YY1 in tumor growth and suppression. Int J 467 Cancer 143, 460-465, doi:10.1002/ijc.31255 (2018). 468 16 Singh, D. et al. Gene expression correlates of clinical prostate cancer behavior. Cancer 469 Cell 1, 203-209, doi:Doi 10.1016/S1535-6108(02)00030-2 (2002). 470 17 Tomlins, S. A. et al. Integrative molecular concept modeling of prostate cancer 471 progression. Nat Genet 39, 41-51, doi:10.1038/ng1935 (2007).
19 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.
472 18 Bushmeyer, S., Park, K. & Atchison, M. L. Characterization of Functional Domains 473 within the Multifunctional Transcription Factor, Yy1. J Biol Chem 270, 30213-30220 474 (1995). 475 19 Alexander, K. E. & Rizkallah, R. Aurora A Phosphorylation of YY1 during Mitosis 476 Inactivates its DNA Binding Activity. Scientific reports 7, doi:ARTN 10084 477 10.1038/s41598-017-10935-5 (2017). 478 20 Moon, J. S. et al. Kruppel-like factor 4 (KLF4) activates the transcription of the gene for 479 the platelet isoform of phosphofructokinase (PFKP) in breast cancer. J Biol Chem 286, 480 23808-23816, doi:10.1074/jbc.M111.236737 (2011). 481 21 Hydederuyscher, R. P., Jennings, E. & Shenk, T. DNA-Binding Sites for the 482 Transcriptional Activator/Repressor Yy1. Nucleic Acids Res 23, 4457-4465, doi:DOI 483 10.1093/nar/23.21.4457 (1995). 484 22 Varambally, S. et al. Genomic Loss of microRNA-101 Leads to Overexpression of 485 Histone Methyltransferase EZH2 in Cancer. Science 322, 1695-1699, 486 doi:10.1126/science.1165395 (2008). 487 23 Roux, K. J., Kim, D. I., Burke, B. & May, D. G. BioID: A Screen for Protein-Protein 488 Interactions. Curr Protoc Protein Sci 91, 19 23 11-19 23 15, doi:10.1002/cpps.51 (2018). 489 24 Roux, K. J., Kim, D. I. & Burke, B. BioID: a screen for protein-protein interactions. Curr 490 Protoc Protein Sci 74, 19 23 11-19 23 14, doi:10.1002/0471140864.ps1923s74 (2013). 491 25 Wu, S. et al. A YY1-INO80 complex regulates genomic stability through homologous 492 recombination-based repair. Nat Struct Mol Biol 14, 1165-1172, doi:10.1038/nsmb1332 493 (2007). 494 26 Itzen, F., Greifenberg, A. K., Bosken, C. A. & Geyer, M. Brd4 activates P-TEFb for 495 RNA polymerase II CTD phosphorylation. Nucleic Acids Res 42, 7577-7587, 496 doi:10.1093/nar/gku449 (2014). 497 27 Guo, Y. et al. CRISPR-mediated deletion of prostate cancer risk-associated CTCF loop 498 anchors identifies repressive chromatin loops. Genome Biol 19, doi:ARTN 160 499 10.1186/s13059-018-1531-0 (2018). 500 28 Deng, Z. et al. Yin Yang 1 regulates the transcriptional activity of androgen receptor. 501 Oncogene 28, 3746-3757, doi:10.1038/onc.2009.231 (2009). 502 29 Blattler, S. M. et al. Defective Mitochondrial Morphology and Bioenergetic Function in 503 Mice Lacking the Transcription Factor Yin Yang 1 in Skeletal Muscle. Mol Cell Biol 32, 504 3333-3346, doi:10.1128/Mcb.00337-12 (2012). 505 30 Kleiman, E., Jia, H. Q., Loguercio, S., Su, A. I. & Feeney, A. J. YY1 plays an essential 506 role at all stages of B-cell differentiation. P Natl Acad Sci USA 113, E3911-E3920, 507 doi:10.1073/pnas.1606297113 (2016). 508 31 Wu, S. R. et al. Transcription Factor YY1 Promotes Cell Proliferation by Directly 509 Activating the Pentose Phosphate Pathway. Cancer Res 78, 4549-4562, 510 doi:10.1158/0008-5472.Can-17-4047 (2018). 511 32 Allouche, A. et al. The combined immunodetection of AP-2 alpha and YY1 transcription 512 factors is associated with ERBB2 gene overexpression in primary breast tumors. Breast 513 Cancer Res 10, doi:ARTN R9 514 10.1186/bcr1851 (2008). 515 33 Zhang, J. J. et al. Yin Yang-1 increases apoptosis through Bax activation in pancreatic 516 cancer cells. Oncotarget 7, 28498-28509, doi:10.18632/oncotarget.8654 (2016).
20 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.
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
21 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.
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
24 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.
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