bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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 The copyright holder for this preprint (which was not certified by peer review) is the author/funder, who has granted bioRxiv a license to display the preprint in perpetuity.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.

Higher-order chromatin organization defines Progesterone and PAX2 binding to regulate estradiol-primed endometrial cancer expression

Alejandro La Greca 1, Nicolas´ Bellora 3,6‡, Francois Le Dily,2,5‡, Rodrigo Jara 1, Javier Quilez Oliete 2, Jose´ Luis Villanueva 2, Enrique Vidal 2, Gabriela Merino 4, Cristobal´ Fresno 4, Inti Tarifa Rieschle 1, Griselda Vallejo 1,6, Guillermo P. Vicent 2, Elmer Fernandez´ 4,6, Miguel Beato 2,5, Patricia Saragueta¨ 1,6∗ 1Biology and Experimental Medicine Institute, IBYME-CONICET, Buenos Aires, Argentina. 2Centre for Genomic Regulation (CRG), Barcelona Institute for Science and Technology, Barcelona, Spain. 3Biodiversity and Environment Investigations Institute (INBIOMA), Bariloche, Argentina. 4Bioscience Data Mining Group, C´ordoba University, C´ordoba, Argentina. 5Universitat Pompeu Fabra (UPF), Barcelona, Spain. 6Consejo Nacional de Investigaciones Cient´ıficasy T´ecnicas(CONICET), Buenos Aires, Argentina ‡Equal contributors ∗Corresponding author: [email protected] Running title: Endometrial hormone-dependent PR gene regulation.

Abstract Estrogen (E2) and Progesterone (Pg), via their specific receptors (ER and PR respectively), are major determinants in the development and progression of endometrial malignancies. Here, we have studied how E2 and the synthetic progestin R5020 affect genomic functions in Ishikawa endometrial cancer cells. Using ChIPseq in cells exposed to the corresponding hormones, we identified cell specific binding sites for ER (ERbs) and PR (PRbs), which mostly correspond to independent sites but both adjacent to sites bound by PAX2. Analysis of long-range interactions by Hi-C showed enrichment of regions co-bound by PR and PAX2 inside TADs that contain dif- ferentially progestin-regulated . These regions, which we call “progestin control regions” (PgCRs), exhibit an open chromatin state prior to the exposure to the hormone. Our observations suggest that endometrial response to progestins in differentiated endometrial tumor cells results in part from binding of PR together with partner transcription factors to PgCRs, compartmentalizing hormone-independent open chromatin. Keywords: steroid receptors, gene regulation, endometrial cancer, ChIPseq, Hi-C, ATACseq, , , PAX2

1 Introduction 8 gies and functions. During pregnancy, Pg pre-

pares the uterine epithelium to receive the em- 9 2 Progesterone (Pg) is a key regulator in bryo and initiates the process of differentiation 10 3 the female reproductive tract, including uter- of stromal cells towards their decidual phe- 11 4 ine and mammary gland development (Lydon notype. In the mammary gland and in coor- 12 5 et al., 1995). Endometrial and breast tissues dination with prolactin, Pg stimulates epithe- 13 6 exhibit significantly different responses to hor- lial proliferation and differentiation of alveolar 14 7 mones, resulting in very distinctive morpholo- Preprint submitted to BioRxiv December 30, 2020 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

lobes in the mammary gland (Mulac-Jericevic 5815 often associated with shorter progression-free

and Conneely, 2004). Unlike Pg, estradiol 5916 survival and overal survival rates (Leslie

17 (E2) is the main proliferative signal in the uter- et al., 1997; Miyamoto et al., 2004; Sak- 60

18 ine epithelium and exerts its function through aguchi et al., 2004; Jongen et al., 2009; 61

19 activating estrogen receptor (ER) alpha and Kreizman-Shefer et al., 2014). The absence 62

20 beta (ERalpha and β, respectively) (Ishiwata of PR may be attributed to 63

21 et al., 1997; Kayisli et al., 2004). hypermethylation of CpG islands within the 64

22 The physiological role of Pg is mediated by promoter or first exon regions of the PR gene 65

23 the interaction and consequent activation of or to the presence of associated deacetylated 66

24 isoforms A (PRA) and B (PRB) of the pro- histones. These modifications were reported 67

25 gesterone receptor (PR), which are transcribed for endometrial cancer cell lines as well as 68

26 from alternate promoters of the gene (Hov- tumor samples and may be exclusive to PRB 69

27 land et al., 1998). While PRA is more abun- (Sasaki et al., 2001; Xiong et al., 2005; Ren 70

28 dant in stromal endometrial cells, PRB is the et al., 2007). Treatment of such cells with 71

29 most representative isoform in ephitelial cells DNA methyltranferase or histone deacetylase 72

30 of endometrium. Steroid hormones exert their inhibitors can restore both PRB expression 73

31 transcriptional effects through binding of the and its regulation of target genes such as 74

32 steroid receptors (SR) to specific DNA se- FOXO1, p21 (CDKN1A), p27 (CDKN1B), 75

33 quences in the promoters or enhancers of tar- and cyclin D1 (CCND1) (Xiong et al., 2005; 76

34 get genes known as “hormone response ele- Yang et al., 2014). Down-regulation of 77

35 ments” (HRE). Estradiol exposure triggers ER PR by post-transcriptional mechanisms and 78

36 binding to estrogen response elements (ERE) through pos-translational modifications of 79

37 regulating target genes such as PGR. Previous PR may contribute to progesterone resistance 80

38 work showed E2-dependent upregulation of in endometrial cancer but have not been 81

39 PR in many different target cells, species and extensively explored in the context of en- 82

40 pathological conditions (Graham et al., 1995; dometrial cancer. It is known that oncogenic 83

41 Kraus and Katzenellenbogen, 1993). Expo- activation of KRAS, PI3K or AKT and/or 84

42 sure to progestins triggers binding of PR to loss of functional tumor suppressors such as 85

43 PRE. Once bound to their HREs the hormone PTEN are common genetic alterations (Hecht 86

44 receptors interact with other transcription fac- and Mutter, 2006), toghether with ARID1A 87

45 tors, co-regulators (Beato et al., 1995), such as (Liang et al., 2012), all of them observed 88

46 the p160 family of co-activators of steroid re- in endometrial cancer. Although there are 89

47 ceptors SRC-1-3, and chromatin remodelling numerous reports of hormonally regulated 90

48 enzymes. This evidence favors tissue specific enhancers and super-enhancers in mam- 91

49 roles of PR isoforms and their co-regulators mary cancer cells (see in dbsuperenahncer, 92

50 orientated towards differential transactivation http://bioinfo.au.tsinghua.edu.cn/dbsuper/) 93

51 of target genes. (Khan and Zhang, 2016; Hnisz et al., 2015), 94

52 High levels of PRA and PRB have been de- there is a void of information about their 95

53 scribed in endometrial hyperplasia (Miyamoto presence in endometrial cells. 96

54 et al., 2004) while low and high-grade en- To better understand the response to pro- 97

55 dometrial cancers reveal reduced or absent gestin in endometrial cancer cells, we have 98

56 expression of one or both isoforms in epithelia studied the genomic binding of ER and PR, 99

57 or stroma (Shao, 2013). This PR decrease is the global gene expression changes and the 100 2 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

Figure 1. R5020 inhibits E2-induced Ishikawa cell proliferation through an active PR that is capable of transactivating an exogenous MMTV promoter sequence and an endogenous enhancer sequence lo- cated 50kb upstream of EGFR gene. (A) Proliferation of Ishikawa cells either pretreated with E2 10nM for 12h (preE2) or not (no preE2) and later treated with vehicle (OH), E2 10nM (E2), R5020 10nM (R5020), E2 combined with R5020 (E2+R5020) and FBS (10%FBS), expressed as mean number of cells ± SE of three independent experiments. (***) p<0.001. (B) Immunofluorescence of PR in untreated (T0; top left), 60min R5020-treated (R5020; bottom left), 12h E2-pretreated (top right) and 12h E2-pretreated 60min R5020-treated (bottom right) Ishikawa cells. Scale bar is equivalent to 30µm. Mean nuclear signal of PR for every cell in all images was determined and shown to the right of the images as arbitrary units (log a.u.). Horizontal dashed lines in boxplots indicate background signal for secondary antibody. (***) p<0.001. (C and D) Gene set enrichment analysis (GSEA) results using R5020- and E2-treated Ishikawa expression profiles as discrete phenotypes for classification of normal endometrium (proliferative and secretory) samples. Enrichment profile (green) shows correlation of normal samples at the top or bottom of a ranked gene list (phenotypes). Normalized enrichment scores (NES) and nominal p values (nom.p) are shown in the graphs. (E) Ishikawa cells transfected with an MMTV-Luciferase reporter gene and treated with vehicle (OH) and R5020 10nM (R5020) for 18h. Diagram at the top depicts MMTV LTR promoter features, including several hormone response elements (HRE) and a nuclear factor 1 (NF1) binding site within nucleosome B (dark grey circle and magnification). Numbers in the diagram indicate position relative to transcription start site (TSS). Results are expressed as relative units (r.u.) of Luciferase activity. (F) Representation of EGFR TSS and the enhancer sequence located 50kb up- stream used to evaluate PR recruitment. Black arrows indicate position of qPCR primers employed on samples treated or not (0) with R5020 for 5, 30 and 60min. Unspecific immunoprecipitation of chromatin was performed in parallel with normal rabbit IgG (IgG). Results are expressed as %input DNA and bars represent mean fold change in PR enrichment relative to time 0 (untreated cells) ± SE of two independent experiments. (*) p<0.05.

3 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

state of chromatin by ATACseq as well as 142101 R5020 on proliferation were corroborated by

the genomic interactions by HiC in Ishikawa 102 BrdU incorporation and phase anal- 143

103 cells exposed to progestin or estrogen, and ysis 18h after hormone exposure (Supplemen- 144

104 also in cells exposed to progestin after a pe- tary Fig. S1C and S1D). E2 increased the 145

105 riod of estradiol pretreatment. Inside TADs number of BrdU positive cells and percent- 146

106 with progestin regulated genes, we identified age of cells in S phase compared to untreated 147

107 regions that we named “progestin control re- control cells and to cell exposed to the ve- 148

108 gions” (PgCRs) that correlate with the open hicle (OH), and these increments were inhib- 149

109 chromatin compartment independently of hor- ited by R5020. Treatment with the histone 150

110 monal stimuli and include binding sites for the deacetylase inhibitor Trichostatin A 250nM 151

111 partner PAX2. (TSA 250nM) was used as negative control for 152

BrdU incorporation and cell cycle progression 153

154 112 Results (Supplementary Fig. S1C). Ishikawa cells contain isoforms A and B of 155

113 Ishikawa endometrial epithelial cells respond PR (PRA and PRB), both of which increased 156

114 to R5020 through activation of PR, whose lev- their steady state levels by treating cells with 157

115 els increase upon exposure to E2 E2 10nM for 12h (Supplementary Fig. S1E 158

116 Endometrial epithelial cells respond to ovar- and S1F). Pretreating cells with E2 for 12h 159

117 ian steroid hormones -progesterone (Pg) and (preE2) had little effect on the proliferative re- 160

118 estradiol (E2)-, E2 being the main prolifer- sponse to R5020 (Figure 1A), while E2 pre- 161

119 ative stimulus and Pg its antagonist. Af- treatment for 48h significantly increased the 162

120 ter treating Ishikawa cells with E2 10nM for proliferative effect of E2 exposure, compared 163

121 48h we observed an increment in number of to non-pretreated cells (FC 1.47±0.08 v. no 164

122 cells compared to vehicle (OH) (FC 1.78±0.08 preE2). The percentage of cells exhibiting nu- 165

123 v. OH) that was suppressed by addition of clear localization of PR increased upon E2 pre- 166

124 R5020 10nM (FC 1.15±0.08 v. OH) (Fig- treatment prior to R5020 exposure (T0). Upon 167

125 ure 1A). Treatment with R5020 10nM alone exposure to R5020 for 60min the percentage 168

126 did not induce proliferation on Ishikawa cells of cells exhibiting nuclear PR was not affected 169

127 (FC 0.77±0.08 v. OH) (Figure 1A). E2- by E2 pretreatment, though the intensity of the 170

128 induced cell proliferation was also abrogated fluorescence signal increased in E2-pretreated 171

129 by pre-incubation with estrogen receptor (ER) cells (Figure 1B). Ishikawa cells express con- 172 −6 130 antagonist ICI182780 1µM (ICI 10 M) (FC siderably higher levels of ERalpha than of ERβ 173

131 1.05±0.05 v. OH) (Supplementary Fig. S1A), (Supplementary Fig. S1G), suggesting that 174

132 but not pre-incubation with PR antagonist the proliferative effect of E2 was mediated 175 −6 133 RU486 1µM (RU486 10 M) (FC 1.42±0.07 by ERalpha. R5020 increased nuclear ERal- 176

134 v. OH) (Supplementary Fig. S1B), proving pha, suggesting a functional PR-ER crosstalk 177

135 that ER but not PR was directly involved in in response to hormonal stimuli (Supplemen- 178

136 the proliferative response to E2. Suppression tary Fig. S1H). Such interactions have already 179

137 of E2-induced cell proliferation by R5020 was been proven in breast cancer T47D cells (Bal- 180

138 inhibited by pre-incubation with RU486 (FC lare´ et al., 2003) and in UIII rat endometrial 181

139 1.50±0.06 v. OH), indicating that R5020 effect stromal cells (Vallejo et al., 2005), though in 182

140 was mediated by PR in Ishikawa cells (Sup- the latter PR remains strictly cytoplasmatic. 183

141 plementary Fig. S1B). The effects of E2 and Treatment with hormones during 12h pro- 184 4 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

A D Number of PRbs TGFA R5020 (min) no preE2 preE2 FPR 2 _ 2 _ 2 _ 0 331 346 476 PR T0 PR T0 PR T0 5 275 - 499 T0 0 _ 0 _ 0 _ 30 1446 2745 3913 2 _ 2 _ 2 _ PR 60 1491 5701 3128 PR R60 R60 PR R60 PR R60 0 _ 0 _ 0 _ 2 _ 2 _ 2 _ PRbs prePRbs Number of ERbs prePR T0 prePR T0 prePR T0 31 T0 0 _ 0 _ 0 _ E2 (min) ER 2 _ 2 _ ERbs 1188 2 _ 0 25 198 prePR R60 prePR R60 prePR R60 prePR R60 5 178 3765 0 _ 0 _ 0 _ 924 403 2 _ 2 _ 2 _ 60 1591 FPR T0 FPR T0 FPR T0 360 1973 T0 0 _ 0 _ 0 _ 2 _ 2 _ 2 _ FPR FPR R60 R60 FPR R60 FPR R60 B 0 _ 0 _ 0 _ prePRbs 2 _ 2 _ 2 _

2933 ER T0 ER T0 ERbs T0 ER T0 0 _ 0 _ 0 _ 2 _ 2 _ 2 _ 311 ER ER E60 E60 ER E60 ER E60 901 290 2020 FPRbs 0 _ 0 _ 0 _ 42 _ 61 _ 116 _

GSE132713-3Prolif. GSE132713-3 GSE132713-3 619 endom. 23 1 _ 1 _ 1 _ 42 _ 29 _ 126 _ PR

GSE132713-4Secr. GSE132713-4 GSE132713-4 C unique common ERbs endom. 1 _ 1 _ 1 _

prePRbs_s

FPRbs_s 1.00 0.75 0.50 0.25 0.00 Fraction of peaks

Figure 2. Estradiol induces R5020-dependent PR binding to specific regions in chromatin. (A) Upper table shows total number of PRbs obtained by ChIPseq for untreated (0min) and R5020-treated (5, 30 and 60min) endometrial Ishikawa cells under three different conditions: non-pretreated with E2 (PR), pretreated with E2 for 12h (prePR) and exogenous expression of PR (FPR). Lower table shows number of ERbs using anti-ERalpha antibody on untreated (0min) and E2-treated (5, 60 and 360min) Ishikawa cells. Venn Diagram shows shared binding sites among PRbs (red), prePRbs (blue) and ERbs (green) at 60min. (B) Venn Diagram shows intersection between ERbs (green), FPRbs (dark grey) and prePRbs (blue) at 60min. (C) Fraction of peaks in FPR and prePR after substraction of shared PRbs (FPR s and prePR s, respectively) that are not shared with each other (unique), that are common to each other (common) and that are common with ER (ERbs). (D) Normalized coverage of PR and ERalpha binding in untreated (T0) and 60min hormone-treated (R60 and E60) Ishikawa cells and PR binding in proliferative (GSE1327133) and secretory (GSE1327134) endometrium. Black arrow indicates peak of interest. R60: 60min R5020 10nM; E60: 60min E2 10nM. The three regions displayed include TGFA, PGR and CCND2 genes (indicated at the top). An estrogen response element (ERE) and a half ERE are indicated below the peaks.

duced transcriptomic changes consistent with 191185 12h treatments with R5020 10nM regulated

186 the physiological stages of normal cycling en- a gene expression profile similar to a mid- 192

187 dometrial tissue (Chi et al., 2020). RNAseq re- secretory phase (Figure 1D). In line with these 193

188 sults from Ishikawa cells exposed to E2 10nM findings, among the top overrepresented bio- 194

189 for 12h showed a significant resemblance to logical processes for E2-treated Ishikawa cells 195

190 proliferative endometrium (Figure 1C), while showed angiogenesis and positive regulation 196

5 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

La_Greca_Fig3

n=1342

n=1072

n=633

n=3162

PRbs genes R5020

224 1443 1662 D

ERbs genes n=198 McLean et al. Nat Biotechnol, 2010 E2 n=112 199 n=24 1495 751 n=329 n=918

Figure 3. A fraction of E2-induced PRbs localize on ERbs and contain half ERE motifs. (A) Classification of steroid receptor binding relative to genomic features expressed as percentage (%) of peaks after 60min of hormone treatment inside each feature. Legend at the top right corner indicates the color key for ERbs (green dots) and three conditions of PR binding: non-pretreated with E2 (PRbs, red dots), pretreated with E2 for 12h (prePRbs, blue dots) and exogenous expression of PR (FPRbs, grey dots). The table below shows percentages represented in the plot. (B) To the left: Representation of GREAT tool association rules adapted with modifi- cations. To the right: Venn diagrams show intersection between PRbs-associated genes and R5020-regulated genes (top), and ERbs-associated genes and E2-regulated genes (bottom). (C) Peak signals in PRbs, FPRbs and prePRbs from 60min R5020-treated Ishikawa cells were plotted as heatmaps. Regions were defined inside a window of 10kb centered in peak summit (±5kb) and intensity of the signal correspond to number of reads in each region. Heatmap is subdivided into 4 mutually exclusive groups depending on shared/partly shared/non- shared binding sites: a (n= 1342), sites shared by all three conditions of PR binding; b (n=1072), sites uniquely found in FPR and prePR; c (n=633), sites found only in FPR; and d (n=3162), sites found only in prePR. De novo motif discovery (MEME) was performed on all groups and results are indicated as sequence logos to the right of the map, including the name of the most related known motif. PRE: progesterone response element. (D) Peak signals in PRbs, FPRbs and prePRbs as in (C), and ERbs from 60min E2-treated Ishikawa cells. Heatmap was subdivided into 5 mutually exclusive groups: e (n=198), sites shared by all three conditions of PR binding and ER binding; f (n=112), sites shared by FPRbs, prePRbs and ERbs; g (n=24), sites shared by FPRbs and ERbs; h (n=329), sites shared by prePRbs and ERbs; and i (n=918), sites uniquely found in ERbs. Motif dis- covery was performed as in A for all groups and results are shown to the right of the map, including the most related known motif. ERE: estrogen response element; 1/2 ERE: half ERE.

6 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

of smooth muscle cell proliferation and for 239197 tif), nor new motif different from PRE, but re-

198 R5020-treated cells processes like tar- vealed a higher abundance of PREs in com- 240

199 geting to Golgi and SRP-dependent cotrans- mon and unique 60min datasets, yielding 1.72 241

200 lational protein targeting to membrane were fold and 1.78 relative unique sites in 30min re- 242

201 found (Supplemenatry Fig. S2A). In addi- spectively. Thus PR could bind as monomer 243

202 tion, the majority of regulated genes (81% of isoforms at 30min and as dimer isoforms at 244

203 R5020 and 63% of E2) were not shared by 60min, providing more probability of active 245

204 both hormones (Supplementary Fig. S2B). PR at 60 than at 30min of R5020 treatment. 246

205 Genes like PGR (progesterone receptor) and qPCR performed on six regions in the vecin- 247

206 cell-cycle regulator CCND2 (cyclin d2) were ity of hormone regulated genes and occupied 248

207 upregulated by E2 but not by R5020, while by PR at 30 and 60min of R5020 exposure 249

208 TGFA (transforming growth factor alfa) was validated ChIPseq results (Supplementary Fig. 250

209 upregulated by both hormones (Supplemen- S4B). These regions were selected according 251

210 tary Fig. 2B and C). to differentially expressed genes from RNAseq 252

data and top-ranked by peak signal. These re- 253

211 Binding of PR and ERalpha to the Ishikawa sults indicate that hormone-dependent PR oc- 254

212 endometrial cancer genome cupancy increased 5-fold by 30min and stabi- 255

213 To explore the genome-wide distribution of lized between 30 and 60min of treatment, in 256

214 PR and ERalpha binding (PRbs and ERbs re- accordance with qPCR results (Supplementary 257

215 spectively) in Ishikawa cells, ChIPseq was per- Fig. S4C). 258

216 formed in different conditions (Figure 2A and Next, we explored the recruitment of ERal- 259

217 Supplementary Fig. S4). First, we analyzed pha to chromatin of Ishikawa cells exposed to 260

218 untreated cells (T0) and cells exposed for 5, E2 (10nM) for 5, 60 and 360min. Poor ERal- 261

219 30, and 60min to 10 nM R5020 using a specific pha binding was detected at T0 (25 sites), of 262

220 antibody to PR that detects both isoforms PRA which 90% remained occupied throughout all 263

221 and PRB. Results showed robust PR bind- times of treatment with E2. Exposure to E2 re- 264

222 ing after 30 min of R5020 treatment (R5020 sulted in the detection of 178 ERalpha binding 265

223 30min) with 1,446 sites, of which 322 sites sites (ERbs) at 5min, 1,591 at 60min and 1,973 266

224 (22%) were present in untreated cells (PRbs at 360min (Figure 2A and Supplementary Fig. 267

225 at time zero, T0=331). After 60min of treat- S4D). The majority (85%) of ERbs found at 268

226 ment with R5020 (R5020 60min), the major- 60min was also identified at 360min (Supple- 269

227 ity of sites identified at 30min were still ev- mentary Fig. S4D). ERalpha binding at 0, 60 270

228 ident (78%), with 336 sites gained and 307 and 360min of E2 treatment was confirmed by 271

229 sites lost (Figure 2A and Supplementary Fig. qPCR on four of the sites identified (Supple- 272

230 S4A). The representation of PREs in 22% of mentary Fig. S4E). ChIPseq results point to 273

231 the PR binding sites that were lost between a clear and sustained E2-dependent enhance- 274

232 30 and 60 min of R5020 treatment was ana- ment of ERalpha binding (Supplementary Fig. 275

233 lyzed taking into account common, and unique S4F). 276

234 30min or 60min PRbs. De novo motif dis- De novo motif discovery confirmed that PR 277

235 covery, analysis of information content and binding occurred mostly through PREs ex- 278

236 quantification occurrences of PRE motifs in hibiting the complete palindromic response 279

237 such regions did not show differences in the elements (Supplementary Fig. S4G), while 280

238 information content (the strength of PRE mo- ER binding sites were enriched in half- 281 7 295 294 293 292 291 290 289 288 287 286 285 284 283 bioRxiv preprint was notcertifiedbypeerreview)istheauthor/funder,whohasgrantedbioRxivalicensetodisplaypreprintinperpetuity.Itmade ie ihol afo h hrceitcpalin- characteristic mostly the drome. of binds half ERalpha only with cells sites Ishikawa in identity, line but cell of ERbs). regardless through PREs 490 binds complete IV: PR that group revealed PRbs; Classification lines 896 cell T47D II: sites and (group and Ishikawa ERbs) both 1101 in III: present group PRbs, 595 (group cells I; respec- Ishikawa S4H, for specific and sites tively): clases S4G two into Fig. ERbs (Supplementary and clus- PRbs enabled both 2016) of al., tering et in (Nacht findings cells previous T47D with Comparison Fig. S4H). (Supplementary motifs ERE palindromic 52-adE-euae ee rmRAe eut xrse s-o(-au) rosidct oiinof position indicate Arrows (p<0.05)for -log(p-value). ER. predicted as and TFBS expressed motif) analysed 20 binding and results were (PR-like top RNAseq ESR1 cells GR the from PAX2, Ishikawa SOX9, shows genes in 9), Heatmap 10nM E2-regulated (TFBS) and E2 and tool. Factor and 5 R5020- Transcription 10nM enrichment 2, R5020 UCSC functional with (1, Predicted web-based motifs treatments DAVID (C) family Relevant 12h using PAX by ERbs. (RARr). regulated the and receptor genes of prePRbs acid on between members retinoic binding the (A) and NR3C1-4, of in (ESRr) members to as NR3C1-4, related Comparison correspond to estrogen (B) plot correspond SOX9. plot the and the on 9) hormone- on and pointed pointed and 5 at motifs displayed 2, Relevant binding key (1, color plot. ERalpha family the the PAX through of and indicated corner are PR right analyses lower enrichment with the for p-values associated Combined are PRbs. and sites prePRbs cells. binding Ishikawa in PAX2 genes Putative regulated 4. Figure doi: https://doi.org/10.1101/739466 C A

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282 MIF1 MEIS1BHOXA9 30 2(ipe l,21) oepoeteef- the explore To 2016). with al., treated et cells (Diep result Ishikawa E2 similar in reported A and was Kraus 1995; 1993). al., Katzenellenbogen, et upregulate (Graham levels expression E2 endometrium, higher functional and the the promotes of cycle proliferation the of phase follicular mid-to- late the during estradiol circulating changes. Rising endometrial for princi- drivers the hormonal as pal serve cycle progesterone) menstrual the and during (estrogen steroids ovarian o Rbnigt h noera genome endometrial the to binding PR for landscape the defines environment Estrogenic IRF2 HEN1

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E2 R5020 PGR gene 305 309 308 307 306 304 303 302 301 300 299 298 297 296 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

310 fect of E2 on PR binding to DNA we per- hallmark phosphorylation of serine 294 in 353

311 formed PR ChIPseq analyses on Ishikawa cells PR (Supplementary Fig. S5G), indicating 354

312 exposed to E2 10nM for 12h (preE2) before that FPR cells were capable of responding 355

313 treatment with R5020 for 30 and 60min. Pre- to hormone. Upon hormone exposure, FPR 356

314 treatment with E2 significantly increased the cells exhibited rapid binding of PR to the 357

315 number of R5020-dependent PRbs (prePRbs), EGFR enhancer sequence (Supplementary 358

316 which included most of PRbs already iden- Fig. S5H). ChIPseq experiments after R5020 359

317 tified in non-pretreated Ishikawa cells (Fig- exposure showed twice the number of PRbs 360

318 ure 2A, Table and Venn Diagram). Quantita- in FPR cells compared to parental Ishikawa 361

319 tive real-time PCR validations performed on 6 cells. The majority of PRbs identified in 362

320 sites occupied by PR confirmed positioning of Ishikawa cells (>90%) were also detected in 363

321 the receptor in both non-preE2 (non E2 pre- FPR cells (Supplementary Fig. S5I), meaning 364

322 treatment) and preE2 conditions (Supplemen- that PR overexpression reflected mostly on an 365

323 tary Fig. S5A). It also showed that E2 pre- increase in number of binding sites. 366

324 treatment augments both number of PRbs and Upon hormone induction, sites engaged by 367

325 occupancy of the receptor (signal). Contrary PR in Ishikawa cells were also occupied in 368

326 to PRbs in non-pretreated cells, the number of FPR and pretreated cells, denoting a strong 369

327 PRbs doubled between 30 and 60min of R5020 similarity between them (Supplementary Fig. 370

328 in preE2 cells, reaching 5,701 sites (Figure 2A S5I). Although a small number of binding sites 371

329 and Supplementary Fig. S5B, S5C and S5D). was shared between ERalpha and PR in all 372

330 Sequencing experiments performed on three conditions, PR binding in pretreated cells 373

331 T47D cells exposed to 10nM R5020 revealed exhibited a higher degree of similarity to ER- 374

332 over 25,000 PRbs (Ballare´ et al., 2013; Nacht alpha binding than FPRbs (Figure 2B). More- 375

333 et al., 2016), likely reflecting the high content over, subtracting PRbs from FPRbs (FPR s) 376

334 of PR in these cells. However, a large propor- and prePRbs (prePR s) heightens this differ- 377

335 tion of these PRbs was considered functionally ence, with a much larger fraction of bind- 378

336 irrelevant as indicated by the lack of nucle- ing sites shared with ERalpha in the case 379

337 osome remodelling in response to hormone of prePRbs (Figure 2C). Among these sites, 380

338 treatment (Ballare´ et al., 2013). More recent one located close to the promoter of TGFA 381

339 experiments in T47D exposed to subnanomo- gene, identified as an ERbs, showed signifi- 382

340 lar R5020 revealed that around 2,000 PRbs cant PR binding only in preE2 Ishikawa cells, 383

341 are sufficient to evoke a functional response but not in FPR (Figure 2D, left panel). ERE- 384

342 (Zaurin et al, 2020, personal communication). containing ERbs, such as the ones found in the 385

343 Hence, the number of PRbs found in Ishikawa transcription termination site of PGR gene and 386

344 cells probably reflects the low concentration immediately upstream of CCND2 promoter, 387

345 of PR, which is compatible with a functional were occupied by R5020-bound PR in preE2 388

346 response to progestins. To test this possibility Ishikawa cells (Figure 2D, middle and right 389

347 we increased the levels of PR in Ishikawa panels). These three genes were upregulated 390

348 cells by expressing a recombinant FLAG-PR by E2 treatment in RNAseq experiments per- 391

349 vector (Supplementary Fig. S5E). These cells, formed on Ishikawa cells, while only TGFA 392

350 FPR Ishikawa (FPR), expressed levels of PR was also upregulated by R5020 (Supplemen- 393

351 comparable to T47D cells (Supplementary tary Fig. S2C). 394

352 Fig. S5F) and showed no impairment in The distribution of PRbs and ERbs in non- 395 9 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

396 pretreated Ishikwa cells, in FPR cells and cells similar to the motif observed in sites uniquely 439

397 pretreated with E2 (prePRbs) relative to TSS found in Ishikawa ERbs (from Supplementary 440

398 of regulated genes was consistent with previ- Fig. S4H, group III). Sites shared by ERal- 441

399 ous reports in oher cell lines (Ballare´ et al., pha and PR in all three conditions resulted in 442

400 2013; Need et al., 2015), in that they were an unclear combination of PRE and ERE mo- 443

401 enriched in intronic and distal intergenic re- tifs (Figure 3D, group e-g). Degenerated mo- 444

402 gions (Figure 3A). Nearly 50% of binding sites tif logo in group g showed no association to 445

403 localized to distal regions (>50Kb) and ap- any known motif, probably due to a corrupt 446

404 proximately 30% to introns other than the first analysis performed on insufficient data, and 447

405 intron, indicating that regulation of gene ex- the partially degenerated motif logo in group 448

406 pression by the steroid receptors PR and ER- e showed limited association to both PRE and 449

407 alpha is not mediated through proximal pro- ERE (PRE/ERE). 450

408 moters but mostly by distal enhancer/silencer Taken together, this evidence suggests 451

409 sequences. We corroborated these results that, provided there is an estrogenic back- 452

410 employing another strategy based on binding ground, activated PR could regulate estrogen- 453

411 site-gene association using the GREAT web dependent Ishikawa-specific transcriptome by 454

412 tool (see Methods for further details (McLean binding sites already or formerly bound by 455

413 et al., 2010)). First, we defined a set of genes ERalpha. 456

414 associated to binding sites with a basal plus

415 extension rule (extended up to 100kb away) PAX2 binds chromatin in close proximity to 457

416 and then we intersected this group of genes ERalpha and PR binding sites in Ishikawa 458

417 with R5020- or E2-regulated genes. Of the cells 459

418 1,886 genes regulated by R5020, only 224 Evidence described so far partially explains 460

419 (12%) were potentially associated to PRbs, cell type specific hormone-dependent gene 461

420 while only 199 of the 950 genes regulated by regulation, though it is not sufficient to under- 462

421 E2 (21%) proved to be associated to ERbs stand the mechanisms underlying differential 463

422 (Figure 3B). binding of hormone receptors to chromatin. 464

423 As expected, from the sequences contained Initially, we addressed this by contrasting the 465

424 in 10kb windows centered in peak summits of sequences of ERbs and PRbs from groups I- 466

425 PRbs, FPRbs and prePRbs, the PRE emerged IV, i.e. hormone regulated Ishikawa specific, 467

426 as the most representative binding motif (Fig- (from Supplementary Fig. S4G and S4H) with 468

427 ure 3C), including sites uniquely found in FPR an array of 1,395 known TF binding motifs 469

428 (group c: 633) or preE2 (group d: 3,162) cells. (see Methods). Results revealed an enrich- 470 −4 429 While comparison between ERalpha and PR ment (p-value<1e ) of multiple members of 471

430 ChIPseq results showed few similarities re- the PAX family -including variants 2, 5, 6 472

431 garding identity of binding sites, with a set of and 9- in groups I and III, i.e. In Ishikawa 473

432 216 shared by both hormone receptors, pre- specific PRbs and ERbs (Supplementary Fig. 474

433 treatment with E2 added nearly twice as many S6A and S6B, respectively), suggesting that 475

434 binding sites to the pool shared with ERalpha members of the Pax family may be involved 476

435 (from Figure 2A, Venn Diagram). The most in PR and ERalpha action in Ishikawa cells. 477

436 representative motif discovered in these sites - Unbiased comparison (all sites) of enrichment 478

437 only shared by ERalpha and prePR- was a half in TF binding motifs between Ishikawa and 479

438 ERE (Figure 3D, group h: 329) that was highly T47D PRbs showed similar results for PRbs, 480 10 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

La_Greca_Fig5

A non-pretreated E2-pretreated B T0 R5020 T0 R5020 Number of PAX2 chr14 54,300 kb binding sites PAX2 T0 43

R60 201 PAX2 PR E60 208

PR

merge prePR

FPR

DAPI E60

C D PAX2 binding sites: R5020 60min PAX2 binding sites: E2 60min 0.4 Shuffled PAX2 0.4 Shuffled PAX2 binding sites binding sites 0.3 0.3

0.2 0.2

0.1 0.1

-5kbshuffled 5kb -5kbshuffled 5kb PAX2 PAX2 Reads per million Reads per million

-5kb PAX2 peaks 5kb -5kb PAX2 peaks 5kb

Figure 5. PAX2 co-localizes with PR and ERalpha in nuclei of Ishikawa cells and it is positioned primarily in the vicinity of receptors binding sites. (A) Immunofluorescent detection of PR (green) and PAX2 (red) in untreated (T0) and 60min R5020-treated (R5020) Ishikawa cells which were pretreated or not with E2 for 12h (non-pretreated, E2-pretreated). Images were merged for co-localization analysis (merge). Scale bar is shown in the panels and is equivalent to 30µm. (B) PAX2 binding profile and peak calling output (thicks below peaks) inside a region of 70kb of 12. Number of PAX2 binding sites for untreated Ishikawa cells and treated with R5020 for 60min or E2 for 60min is shown to the right of the profiles. Tracks for PRbs, prePRbs, FPRbs and ERbs are displayed below the profiles for the same region. (C) Binding profiles of ER (green), PR (red), FPR (black) and prePR (blue) on PAX2 binding sites of 60min R5020-treated Ishikawa cells. PAX2 binding after 60min E2 treatment was included (purple). Inset shows signal profiles centered on shuffled R5020-dependent PAX2 binding sites. (D) Binding profiles as in (C) on PAX2 binding sites of 60min E2-treated Ishikawa cells. PAX2 binding after 60min R5020 treatment was included (orange). As in (C), inset shows signal profiles centered on shuffled E2-dependent PAX2 binding sites.

11 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

481 although the enrichment was less significant results in PAX2 localization were obtained af- 524

482 (Supplementary Fig. S6C). Moreover, while ter treating Ishikawa cells with E2 for 60min 525

483 enrichment of PAX motifs was also observed (Supplementary Fig. S6G). The increase in 526

484 around ERbs in Ishikawa cells (Supplemen- nuclear PAX2 signal is not due to changes 527

485 tary Fig. S6D), this was not the case with in protein levels, which were not affected by 528

486 T47D cells, in which examples like the well- treatment with either R5020 or E2 (Supple- 529

487 known breast-related pioneer transcription fac- mentary Fig. S6H). In accordance to motif 530

488 tor FOXA1, were found instead (Supplemen- analysis results, PAX2 was not detected in nu- 531

489 tary Fig. S6E). clei of T47D cells after hormonal treatments 532

490 Enrichment of NR3C1-4 (mineralocorti- (Supplementary Fig. S6I). 533

491 coid, glucocorticoid, progesterone and an- To extend these findings, we performed 534

492 drogen receptors) and ESR1 motifs included PAX2 ChIPseq experiments on untreated cells 535

493 into the 1,395 known motifs corroborated de and in cells exposed for 60min to either R5020 536

494 novo discovery performed with MEME in both or E2. The results confirmed PAX2 binding to 537

495 Ishikawa and T47D cells. Stronger enrichment chromatin following hormonal treatment (Fig- 538

496 of PAX motifs was observed in prePRbs com- ure 5B). Even though identified PAXbs were 539

497 pared to PRbs (Figure 4A), indicating that PR few (T0: 43, R60: 201 and E60: 208), most 540

498 binding to regions potentially bound by PAX of PAX2 binding occurred after R5020 and 541

499 is favored after E2 pretreatment. Coherently, E2 treatments. Moreover, PAX2 binding was 542

500 while equivalent fold enrichment values were not stochastically distributed in the genome 543

501 detected when comparing prePRbs to ERbs of Ishikawa cells but rather partially associ- 544

502 (Figure 4B), comparison between prePRbs and ated to ERbs and PRbs. This association was 545

503 FPRbs showed that increased PR levels alone stronger for PR binding in cells pre-teated with 546

504 were not sufficient for a greater association E2 than in non-pretreated cells or in cells over- 547

505 to PAX binding motifs (Supplementary Fig. expressing recombinant PR (Figure 5C). Simi- 548

506 S6F). Consistently, RNAseq experiments on lar results were observed for ERbs in response 549

507 Ishikawa cells treated either with R5020 10nM to E2 (Figure 5D), indicating that PAX2, and 550

508 or E2 10nM for 12h showed putative PAX2 possibly other members of the PAX family 551

509 binding sites among the top 20 significantly may co-operate with PR and ERalpha for bind- 552

510 enriched TFs (DAVID web-based tool (Huang ing to chromatin in Ishikawa cells but not in 553

511 et al., 2009)) on differentially regulated genes T47D cells, in which neither enrichment for 554

512 (Figure 4C). ER was also predicted to bind PAX binding motif nor nuclear localization of 555

513 on E2-responsive genes, while glucocorticoid PAX2 was detected. 556

514 receptor (GR) motif (PR-like motif) was de-

515 tected on R5020-responsive genes. Under estrogenic conditions, PR and PAX2 557 558 516 PAX association to PR and ERalpha action conform endometrial regulatory domains in 559 517 was also evaluated by immunofluorescence open chromatin compartments

518 against PAX2. Nuclear localization of PAX2 Nuclear architecture is a major determinant 560

519 was observed predominantly after 60min of of hormonal gene regulatory patterns (Le Dily 561

520 R5020 in pretreated and non-pretreated PR+ et al., 2014). Therefore, we used in nucleo Hi- 562

521 cells (Figure 5A), indicating that hormonal C technology to study the folding of chromatin 563

522 treatment promotes co-localization of PAX2 across the genome of Ishikawa cells by gen- 564

523 and PR in nuclei of Ishikawa cells. Similar erating genome-wide contact datasets of cells 565 12 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

La_Greca_Fig6

ATACseq A B PgCRs Shuffled Regions PgCRs Defini�on 0.30 T0 PGR chr11 ALPP chr2 R5020 PAX2 <=25kb 0.25 preE2 T0 PR preE2 R5020 0.20

FPR 0.25 input 0.15 PR prePR

0.20 Reads per million 0.10

0.15 0.05 -1.5 5'end 3'end 1.5 -1.5 5'end 3'end 1.5 0.10 Hi-C

Reads per million GENCODEv29 R5020 E2 0.05 Nucleo In 1.0 -1.5 5'end 3'end 1.5Kb 0.8 zoom on 0.6 PgCRs 0.4 T0_atac R5020_atac preT0_atac 0.2 preR5020_atac Reads per million -1.5 Promoters 1.5 -1.5 Promoters 1.5-1.5 Promoters 1.5 Compart. Chromatin PR C 5 R5020 E2 FPR PGR chr11

prePR 0 PAX2 2 2

log2 FC vT0 1.5 1.5 p=0.054 ER −5 p=4.4e-3 PgCRs ALPP chr2 1 1 Genes 0.5 0.5 close far close far

−50 0 50 −50 0 50 Distance to PgCRs (bp x 1e06) D E

F

13 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

Figure 6. Convergence of PR and PAX2 binding in TADs with regulated genes defines potential en- dometrial regulatory domains. Convergence of PR and PAX2 binding in TADs with regulated genes defines potential endometrial regulatory domains. (A) Upper panel shows the contact matrices at a resolution of 20kb obtained by In Nucleo Hi-C in PGR and ALPP loci. Middle panel shows the spatial segregation of chromatin as open or closed compartments inside TADs (green bars: A compartment; white bars: B compartment - see methods section). The bottom panels show ChIPseq signal distribution of PR, FPR, prePR, PAX2 and ERalpha as well as the location of PgCRs and genes over the region. The dashed rectangle restricts the TAD of interest and the vertical arrow marks the TSS of PGR and ALPP. Definition of PgCR: Coverage profiles of PR (red), FPR (black) and prePR (blue) binding on Progesterone Control Regions (PgCRs) delimited by the start and end labels, and flanked upstream and downstream by 1.5Kb regions. Input sample (grey) was included in the plot. Rules for qualifying as a control region are depicted on top of the profile plot. Magnified images over Control Regions are shown to the right (zoom on PgCRs). (B) ATACseq peaks from cells untreated (T0), treated with R5020 for 60min, 12h E2-pretreated (preE2 T0) and E2-pretreated followed by 60min treatment with R5020. Signal was plotted over Control Regions, shuffled Control Regions (Shuffled Regions), promoters of all anno- tated genes from GENCODE database (GENCODEv29) and promoters of genes regulated by 12h treatments with R5020 or E2. (C) Plot shows fold change values of genes regulated by R5020 and E2 (v. untreated cells) relative to Control Regions. Genes located upstream of PgCRs are represented with negative distance values. Dashed horizontal lines mark fold change cut-off points (|log2FC|=0.8) and vertical lines are placed at position -1 and 1Mb. Insets depict comparison of fold change values (absolute values) between genes located beneath (close) and over (far) a 1Mb distance from PgCRs. Statistical significance for this comparison was determined with Welch Two Sample t-test and is represented by a p value on the plot. (D) Top panel: Hi-C contact map at 5kb resolution of Chromosome 2 (70,200,000-71,200,000) obtained in Ishikawa cells and showing the organi- zation around TGFA gene locus. Middle panel: Virtual 4C profile at 5kb resolution (expressed as normalized counts per thousands within the region depicted above) using the TGFA promoter as bait and showing the con- tacts engaged between TGFA promoter and the PgCR detected in this region (highlighted in green). Arrow on top panel highlights the position of the loop in the map. Bottom panel shows the positions of genes in the region depicted. (E) Distributions of observed versus expected interactions established between promoters (red - left), between PgCRs (blue - middle) and between Promoters and PgCRs (purple - right) located within a same TAD in Ishikawa cells treated as indicated below. (F) Representation of a chromatin loop involving a PgCR and the promoter of a regulated gene. Initially, the gene is transcriptionally inactive even though the loop is already formed. After hormone induction (E2 pretreatment followed by R5020), PR, PAX2 and in some cases ERalpha occupy open chromatin compartments in contact with promoters resulting in transcriptional activation.

566 untreated (T0) or pretreated with E2 for 12h, periments were more closely related to each 580

567 and exposed to R5020 or E2 for 60min. A other than any of them to a T47D sample (Sup- 581

568 comparison of contact matrices at 20 kb res- plementary Fig. S7C and S7D). However, A/B 582

569 olution of untreated Ishikawa cells to T47D profile distribution in Ishikawa cells was in- 583

570 cells confirmed the high degree of conserva- dependent from hormonal treatments (Supple- 584

571 tion on the borders of topologically associating mentary Fig. S7B and S7E), meaning that 585

572 domains (TADs) (Supplementary Fig. S7A). chromatin was in a primed state that condi- 586

573 TADs are grouped into two chromatin com- tioned hormone-dependent regulation of gene 587

574 partments A and B, which represent the active expression. Detailed analysis revealed that 588

575 open chromatin (A) and the closed inactive 7% of A domains in Ishikawa cells were B in 589

576 chromatin (B) respectively. Analysis of such T47D cells, and 12% of B domains in Ishikawa 590

577 compartments showed a cell type-specific pat- cells were A in T47D cells (Supplementary 591

578 terning (Supplementary Fig. S7B), in which Fig. S7F). A total of 861 genes encompassed 592

579 Ishikawa samples from two independent ex- in the A compartment in Ishikawa cells be- 593

14 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

594 long in the B compartment in T47D cells, and FPR cells did not show similar results (Figure 637

595 1,438 genes in B compartaments in Ishikawa 6A: PgCRs Definition). 638

596 cells belong in A in T47D cells (12%), sug- Considering that TAD borders may act as 639

597 gesting that distribution of A and B compart- regulatory barriers, we removed from further 640

598 ments could in part explain cell type specific analysis any region that, in spite of satisfy- 641

599 gene expression profiles. ing the rules for being a PgCR, was local- 642

600 To evaluate whether chromatin states are ized across a barrier as well. In agreement 643

601 related to gene expression through differen- with this restriction, the sizes of PgCR -with 644

602 tial binding of hormone receptors to DNA, an average of 25kb- were smaller than TADs 645

603 we intersected PR and ERalpha ChIPseq re- -with an average of 1000kb- (Supplementary 646

604 sults with the A/B compartment coordinates. Fig. S7I). In addition, the majority of the 121 647

605 Both transcription factors, PR and ERalpha, identified PgCRs (coordinates in hg38 can be 648

606 bound A compartments more frequently than found as Supplementary Data) were not lo- 649

607 B, meaning that open genomic regions in cated near the TAD borders, but in the TAD 650

608 Ishikawa showed preferential binding of the center (Supplementary Fig. S7J), where most 651

609 hormone receptors (Supplementary Fig. S7G). non-housekeeping genes are found (Le Dily 652

610 Neither pre-treatment with E2 nor expression et al., 2019). Moreover, PgCRs seem to be 653

611 of recombinant PR modified the preferential located in A compartments in the vecinity of 654

612 binding of the PR to the A compartments. hormone-regulated genes like PGR and ALPP 655

613 As mentioned above, PAX2 binding occurs (Figure 6A). Expression of these genes was 656

614 mostly in close proximity to PR and ERal- analyzed by qPCR of total RNA samples of 657

615 pha binding sites. In fact, distances between Ishikawa cells exposed to hormone for 12h, 658

616 PAXbs and PRbs were remarkably shorter in which showed that ALPP is induced by both 659

617 E2 pretreated cells than in any other condi- hormones and PGR is only induced by E2 660

618 tion (Supplementary Fig. S7H). This raised (Supplementary Fig. S7K). 661

619 the question of whether recruitment of PR to- As was mentioned before, the Hi-C matri- 662

620 gether with PAX2 to open chromatin com- ces were used to determine the spatial segre- 663

621 partments facilitates regulation of gene expres- gation of chromatin in both open and closed 664

622 sion. To study this notion, we defined puta- chromatin compartments (A/B), and the A:B 665

623 tive endometrial regulatory domains that we ratio was independent of hormone treatment. 666

624 named “Progestin Control Regions” (PgCR) Consistent with these results, ATACseq signal 667

625 with the capacity to potentially regulate nearby on PgCRs remained unchanged upon hormone 668

626 genes. The restrictions for being a regulatory exposure, but it decreased after shuffling the 669

627 domain, which consisted in containing at least coordinates for PgCRs, indicating that chro- 670

628 two PRbs separated by a maximun distance of matin was readily and non-randomly accessi- 671

629 25kb and a PAXbs (represented in Figure 6A: ble to TFs in these locations (Figure 6B, top 672

630 PgCRs Definition), were met mostly under E2 panels). Although ATACseq peaks were also 673

631 pretreated conditions. This outcome was due detected on promoters of hormone-regulated 674

632 to the strong association between prePRbs and genes, the signal did not differ after hormone 675

633 PAXbs, though it may have been aided by the exposure (Figure 6B, bottom panels), imply- 676

634 increased PR protein levels. However, the sole ing that treatments were not responsible for 677

635 increment in PR protein levels was not enough opening the chromatin in these regions. In 678

636 to force an association to PAXbs, given that addition, both R5020- and E2-regulated genes 679 15 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

680 with highest FC values (v. T0) were concen- amined the genes carrying the most frequent 722

681 trated under 1Mb (“close”) away from PgCRs mutations in a cohort of 403 cases diagnosed 723

682 (Figure 6C), though the comparison between with endometrial adenocarcinoma (data avail- 724

683 FC values of “close” and “far” (over 1Mb) reg- able in The Cancer Genome Atlas, TCGA, 725

684 ulated genes was significant only in the case of Project TCGA-UCEC). The top 1000 most fre- 726 −3 685 R5020 (p=4.4e ; Figure 6C, inset). quent somatic mutations in these cases were 727

686 Further analysis on Hi-C contact matri- distributed among 837 genes, 33 of which 728

687 ces revealed that PgCRs preferentially interact belonged to PgCR-containing TADs (Figure 729

688 with promoters of hormone-regulated genes 7A), comprising 6% of the 517 protein cod- 730

689 (Figure 6D). Although PgCR-promoter inter- ing genes that may be regulated by direct in- 731

690 actions were non-random and mostly intra- teractions with a corresponding PgCR (PgCR- 732

691 TAD, we found no difference in contact en- genes). In fact, pathway analysis of these 733

692 richment between treated and untreated cells 517 genes revealed a clear bias towards reg- 734

693 (Figure 6E). These results are consistent ulation of immunological processes and tran- 735

694 with ATACseq profiles and imply that chro- scriptional alterations in cancer (Figure 7B), 736

695 matin would be pre-assembled into regulatory suggesting that PgCR-genes may participate in 737

696 loops -involving PgCRs and promoters- which key steps of tumor onset and progression. 738

697 are transcriptionally inactive until hormone- We also studied the expression profile of 739

698 dependent binding of steroid receptors and genes involved in enriched pathways (40 740

699 PAX2 triggers PolII activation (Figure 6F). genes) using 423 Endometrioid adenocarci- 741

700 These results suggest that specific binding noma RNAseq samples previously classified 742

701 of PR, PAX2 and ERalpha to chromatin oc- into stages (Stage I: 300, Stage II: 34, Stage 743

702 curs in compartments that are present in a per- III: 76 and Stage IV: 13) according to the 744

703 missive (open) or restrictive (closed) status de- FIGO system (International Federation of Gy- 745

704 pending on the cell line, and are not modified necology and Obstetrics). Considering the in- 746

705 by short term hormone exposure (Figure 6F). herently heterogeneous nature of tissue sam- 747

706 However, it is not yet clear the role of PAX2 in ples, we intentionally set a permissive fold 748

707 PR binding to PgCRs. Summing up, PR and change cut-off value when comparing Stage I 749

708 ER bind mostly to non-common sites that ex- to Stage IV to detect probable subtle differ- 750

709 hibit the corresponding consensus sequences, ences. The analysis showed that 22 of the 40 751

710 and are adjacent to PAX2 binding. Therefore, genes tended to decrease their expression lev- 752

711 the endometrial specific hormone response re- els with stage progression (Stage IV v. Stage I: 753

712 sults in part from specific chromatin compart- log2FC<-0.5, downregulated genes) including 754

713 ments, unique receptor binding sites and se- the frequently mutated genes CXCL5, PLK2, 755

714 lective TFs binding partners to regulate gene AFF1 and MEIS1, 7 genes had no clear ten- 756

715 expression. dency and 11 genes increased their expression 757

levels like chemokines CXCL9/10/11 (Stage 758 716 Genes contained in TADs with PgCRs are as- IV vs. Stage I: log2FC>0.5, upregulated 759 717 sociated to endometrial tumor progression genes) (Figure 7C). Among the genes included 760

718 To explore the possibility that alterations in in the pathway “Transcriptional misregulation 761

719 the expression profile of genes under the in- in cancer” (14 genes), there were 7 downregu- 762

720 fluence of PgCRs were related to disease pro- lated genes during tumor progression -CXCL8, 763

721 gression such as endometrial cancer, we ex- IGFBP3, MDM2, TGFBR2, AFF1, MEIS1 764 16 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

A B Most frequent somatic mutations in KEGG Pathways Endometrial cancer samples from TCGA − interaction Somatic PgCR MAPK signaling pathway mutations genes Viral protein interaction with 33 cytokine and cytokine receptor Count 804 484 Transcriptional 5 misregulation in cancer 10 15

Chemokine signaling pathway 20

p.adjust MEIS1 RSBN1L NEXN PEX2 PRAMEF17 TNF signaling pathway MKLN1 GRIA2 ARL4C CXCL5 SRPK1 0.01 ZFP36L1 BAZ2B DTNA RBMS3 TMEM14C 0.02 NUP153 MAP4K4 MAK SOX9 USP33 IL−17 signaling pathway AKAP6 NOL4 CCNI BCLAF1 BMP4 0.03 RYR3 SLCO3A1 AFF1 PLK2 FoxO signaling pathway 0.04 FKBP5 NFIA ROBO1 AKAP7 0.04 0.06 0.08 0.10 GeneRatio

C Expression profile of genes from KEGG pathways in endometrioid adenocarcinoma TCGA samples

CXCL5 * CXCL2 CXCL8 IL18R1 MECOM TGFA IGFBP3 MAP3K1 CXCL3 MAPK10 MDM2 VAV3 IL1R1 TGFBR2 TEK CXCL1 PLK2 CCNG2* JAG1 AFF1 *MEIS1 * SS18

MYC IL1R2 n.c. MAP4K4 * SKP2 ERBB4 CDKN1A PDGFC

MAPK14 HMGA2 ETV4 DUSP3 MAPK13

Transcr. misreg. in cancer CXCL11 CXCL10 ETV7 EFNA5 CXCL9 GZMB

Stage I (n=300) Stage III (n=76) Stage II (n=34) Stage IV (n=13) −4−2 0 2 4

D Principal Component Analysis E Pearson correlation analysis Stage I Stage II ALPP −0.73*** 0.23***−0.07 0.09 0.8 (n=300) (n=34) NPAS3 −0.7***−0.06 0.29***−0.04 ALPG −0.66*** 0.27***−0.04 0.07 PGR Stage III Stage IV 16 PGR −0.66***−0.25*** 0.27***−0.11* TMEM232 −0.65***−0.17*** 0.07−0.1* (n=76) (n=13) 12 LY75 −0.61*** 0.06−0.03 0.41*** RFX3 −0.6***−0.06−0.03 0.07 0.6 20 8 GRIA2 −0.58***−0.24*** 0.16** 0.15** ERBB4 −0.51*** 0.07 0 0.3*** CXCL5 −0.47*** 0.06−0.53***−0.22*** 4 CXCL1 −0.38*** 0.06−0.53*** 0 ITGB8 −0.37*** 0.04−0.31*** 0.32*** 0 ACADL −0.37***−0.16*** 0.3*** 0.04 0.4 ESR1 SOWAHB −0.31*** 0.27***−0.23*** 0.08 15 PRSS56 −0.29*** 0.15**−0.01−0.53*** ABCG2 −0.29***−0.72*** 0.13**−0.06 −20 12 CRISPLD1 −0.28***−0.15**−0.12*−0.33*** PTPRC −0.26***−0.37***−0.43*** 0.36*** 9 RASEF −0.26*** 0.32***−0.14** 0.14** 0.2 PC2, 9.48% variation MAPK10 −0.22***−0.56***−0.01−0.04 6 ZNF608 −0.2***−0.14**−0.17***−0.09 −40 CXCL10 −0.14** 0.02−0.43*** 0.38*** 3 −20−10 0 10 20 30 CXCL11 −0.14**−0.05−0.43*** 0.35*** TGFA −0.13** 0.19***−0.53***−0.27*** PC1, 12.43% variation RBMS3 −0.1*−0.72***−0.03−0.02 0.0 LYZ −0.1*−0.19***−0.35*** 0.37*** Correlation DMRTA1 −0.06−0.06 0.26*** 0.13** F RYR3 −0.02−0.67*** 0.09 0.07 ZFHX4 FKBP5 MSRB3 NEXN with progression SPARCL1 0−0.8***−0.03 0.06 12 14 PI16 0.01−0.56*** 0.09 0.01 12.5 stage IV −0.2 12 12 CXCL9 0.06−0.07−0.32*** 0.38*** 8 EPHA3 0.07−0.55***−0.15**−0.12* 10 10 MTFR2 0.07 0.56***−0.15** 0.38*** 7.5 4 GZMB 0.09 0.06−0.43*** 0.09 8 8 Positive DTNA 0.12*−0.6***−0.1*−0.07 VAX2 0.13** 0.42***−0.1*−0.09 6 6 −0.4 0 2.5 stage I BMP4 0.13**−0.17***−0.12*−0.69*** DDR2 0.15**−0.78***−0.2***−0.02 ALPP ALPG CXCL5 NPAS3 for variation 15% genes responsible Top/bottom ZIC2 0.16*** 0.19***−0.37*** 0.09 15 15 stage I MAP2 0.16**−0.11*−0.28***−0.05 HMGA2 0.19*** 0.25***−0.41***−0.27*** 15 Negative 10 10 10 NEXN 0.27***−0.75***−0.14** 0.11* −0.6 10 MSRB3 0.27***−0.77***−0.17***−0.2*** CPS1 0.3***−0.33***−0.12* 0.2*** 5 5 Log2 normalized expression 5 5 ETV4 0.37*** 0.35***−0.32***−0.35*** FKBP5 0.41***−0.33***−0.31*** 0.12* 0 0 0 stage IV ZFHX4 0.43***−0.39***−0.1*−0.01 stage 0.23***−0.04−0.09 0.04 −0.8 Stage I Stage II Stage III Stage IV PC1 PC2 PC3 PC4

17 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

Figure 7. Altered expression of genes contained in TADs with PgCRs correlates with drivers of endome- trial tumor progression. (A) Venn diagram of genes carrying the top 1000 most frequent mutations in a cohort of 403 cases of endometrial adenocarcinomas from TCGA (n=837) and protein coding genes contained in TADs with PgCRs (n=517). Names of genes located in the intersection of the two groups (n=33) are detailed below the diagram. (B) Enriched KEGG pathways for all protein coding genes included in TADs containing PgCRs. Number of genes in each category and adjusted p-value (Benjamini-Hochberg) are indicated in the plot. (C) Heatmap of genes from enriched pathways using normalized counts from 423 endometrioid adenocarcinoma samples (TCGA) classified according to the FIGO system (Stage I to IV). Top panels show genes that decrease expression with stage and bottom panels genes that increase expression levels with stage. Genes in the middle panels do not show a clear expression pattern (n.c.: not clear). Each cell in the heatmap represents the mean expression value of three samples (bin). Genes frequently mutated in endometrial adenocarcinomas are marked with an asterisk (*) and genes belonging to pathway transcriptional misregulation in cancer are indicated by a dendrogram. (D) Bi-plot of PCA results depicting scores of components 1 and 2 (PC1 and PC2). Dots repre- sent the samples included in the analysis (n=423) and color identifies the tumor stage. Density marginal plots represent distribution of scores for each stage. (E) Correlation of variables (genes) and stage to principal com- ponents (PC1 to PC4). Pearson correlation scores are shown inside the cells and represented in a color scale (red as positively correlated and blue as negatively correlated). The 29 genes displayed in the matrix are included among the 15% of genes that give rise to the variation in principal components (PC1 to PC4). Significant results are indicated in the cells: **** p<0.0001, *** p<0.001, ** p<0.01, * p<0.05. (F) Distribution of normalized counts (log2) for genes positively (top row) and negatively (bottom row) correlated with endometrial cancer stage progression. Dashed line indicates position of the median in Stage I.

765 and SS18-, 3 genes with ambiguous behavior depending on the presence of these molecules 788

766 among samples -, ILR2 and CDKN1A- to tune a specific response. Finally, we iden- 789

767 and 4 upregulated genes in Stage IV tumors tified the genes that contributed the most to 790

768 -HMGA2, ETV4, ETV7 and GZMB-. inter-stage variation (top/bottom 15%), con- 791

firming that PGR (r=-0.66) was indeed nega- 792 769 To determine if the PgCR-genes could be tively correlated with progression (Figure 7E), 793 770 drivers of progression in endometrial adeno- as well as ALPP (r=-0.73), NPAS3 (r=-0.7), 794 771 carcinoma, we performed Principal Compo- ALPG/ALPPL2 (r=-0.66) and CXCL5 (r=- 795 772 nent Analysis (PCA) on the 423 Endometri- 0.47) among others, while ZFHX4 (r=0.43), 796 773 oid adenocarcinoma RNAseq samples using FKBP5 (r=0.41), MSRB3 (r=0.27) and NEXN 797 774 the 517 PgCR-genes as variables. Assessment (r=0.27) were positively correlated with the 798 775 of PCA results revealed that inter-stage varia- stage. Correlation results for these genes were 799 776 tion was mostly explained within the first two consistent with the distribution of their nor- 800 777 components (PC1: 12.43% and PC2: 9.48%) malized expression values across stages (Fig- 801 778 and notably, this variation was accompanied ure 7F). 802 779 by a considerable change in PGR mRNA lev-

780 els (Figure 7D), which could partly account for Discussion 803 781 differences between stages. Although ESR1 is

782 not directly influenced by PgCRs, we detected There seems to be consensus that the way 804

783 that its mRNA levels were also reduced with in which combinations of TFs assemble their 805

784 stage progression. The signature of genes reg- binding sites contributes to the folding of 806

785 ulated in conjunction with loss of hormonal the genome in cell type specific patterns that 807

786 regulation could assign novel markers in order orchestrate the physiological coordination of 808

787 to differentiate the evolution of malignancies gene expression programs required for the 809 18 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

810 proper development and function of complex 2016), in endometrial cells PR binding has lit- 853

811 organisms (Lambert et al., 2018; Stadhouders tle influence on ERalpha binding. In Ishikawa 854

812 et al., 2019). There is evidence that the same cells, binding of ER and PR occurs mainly at 855

813 TF can regulate different gene sets in different ERE and PRE sequences, respectively, in re- 856

814 cell types (Gertz et al., 2012), but the mech- gions that are also enriched in PAX response 857

815 anisms through which hormone receptors reg- elements. Ishikawa cells are rich in PAX TF 858

816 ulate endometrial specific gene networks had and PAX ChIPseq shows a similar overlapping 859

817 not been previously deciphered. Here, we de- with ERbs and PRbs. 860

818 scribe ERalpha and PR binding to the genome When we analyzed chromatin topology of 861

819 of endometrial cancer cells and analyze their Ishikawa cells using Hi-C we found that PRbs 862

820 specific chromatin context. In this genomic and ERbs are enriched in Topologically Asso- 863

821 study we used Ishikawa cells, given that they ciating Domains (TADs) containing hormone 864

822 are a good model of Type I epithelial endome- regulated genes. These TADs were predomi- 865

823 trial cancer [37] containing ERalpha and PR. nantly part of the open (A) chromosome com- 866

824 It was reported that in Pgr Knockout partment, even in cells not exposed to hor- 867

825 (PRKO) mice the absence of PR results in un- mone. This was confirmed by ATACseq re- 868

826 opposed estrogen-induced endometrial hyper- sults showing that the sites where the hormone 869

827 plasia (Lydon et al., 1995). As for the two receptors will bind were already more accessi- 870

828 isoforms of PR, the PRB isoform is consid- ble for enzyme cleavage, suggesting that hor- 871

829 ered a strong transcriptional activator while mone independent mechanisms were respon- 872

830 PRA can function as a transcriptional inhibitor sible for the generation and maintenance of 873

831 of PRB activity(Mulac-Jericevic et al., 2000). the hormone responsive TADs. In that re- 874

832 Selective ablation of PRA in mice results in spect, it is interesting that we found an enrich- 875

833 a PRB dependent gain of function, with en- ment of PAXbs near PRbs in these TADs con- 876

834 hanced estradiol-induced endometrial prolifer- taining progesterone regulated genes, suggest- 877

835 ation (Conneely et al., 2003). Ishikawa cells ing that PAX2 could generate the open chro- 878

836 express more PRB than PRA, coherent with matin conformation that enables PR binding 879

837 PRB dominance in glandular epithelial cells and facilitates the interacting loops detected 880

838 (Mote et al., 1999). To explore the mecha- in Hi-C experiments. Loss of PAX2 expres- 881

839 nism underlying the endometrial specific re- sion has been implicated in the development 882

840 sponse to ovarian steroids hormones, we stud- of endometrial intraepithelial neoplasia (EIN) 883

841 ied the genomic binding of ERalpha and PR by (Sanderson et al., 2017) and PAX2 is poten- 884

842 ChIPseq in hormone untreated Ishikawa cells tially useful in the diagnostic of difficult EIN 885

843 and in cells exposed to hormone for differ- cases (e.g. where there is no “normal” tis- 886

844 ent time periods. We discovered that the ma- sue available to act as an internal control when 887

845 jority (67%) of PRbs after estradiol pretreat- assessing nuclear morphology) (Quick et al., 888

846 ment were new sites not present in untreated 2012). Our results connect PR response ele- 889

847 cells and different as well from ERbs occupied ments with PAX2 and 3D chromatin confor- 890

848 after estradiol treatment. Just 639 PR bind- mation, which is consistent with the preser- 891

849 ing sites (11% of all PRbs) were the same for vation of progestin regulation in differenti- 892

850 both PR and ERalpha. This indicates that con- ated cancer cells expressing hormone recep- 893

851 trary to what was described in breast cancer tors and may be lost in undifferentiated tumor 894

852 cells (Mohammed et al., 2015; Singhal et al., cells, which do not express hormone recep- 895 19 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

896 tors. We hypothesize that PR-PAX-PR bind- cell type specific expression of different gene 939

897 ing sites containing regulatory domains that regulatory networks in part by generating and 940

898 we name PgCRs could reflect PR shadow en- maintaining different genome topologies. 941

899 hancers (Cannavo` et al., 2016) in endometrial Droog et al. highlights that “the diver- 942

900 cells. gence between endometrial tumors that arise 943

901 The redundancy of PRbs associated to en- in different hormonal conditions and shows 944

902 dometrial specific gene expression may rein- that ERalpha enhancer use in human can- 945

903 force a genetic mechanism to ensure progestin cer differs in the presence of nonphysiolog- 946

904 regulation in tissue under hormonal influence, ical endocrine stimuli” (Droog et al., 2017). 947

905 in periods in which there is low or no circu- They reported that ERalpha-binding sites in 948

906 lating hormone. Notably, the only described tamoxifen-associated endometrial tumors are 949

907 super-enhancer in endometrial carcinomas is different from those in the tumors from 950

908 the Myc super-enhancer and is not hormonally nonusers. It has yet to be explored whether 951

909 regulated (Zhang et al., 2016). We postulate the response to progesterone and sinthetic 952

910 the existence of a novel subset of 121 strate- progestins, used in treatments of hormone- 953

911 gical endometrial regulatory domains in this dependent endometrial cancers, is affected by 954

912 hormonally responsive endometrial cancer cell the changes resulting from the use of tamox- 955

913 line. Among them the TGFA gene presents one ifen. 956

914 of PgCR-promoter interaction that could ex- On the other hand, estrogen receptor a 957

915 plain hormone regulation previously reported (ER) and (GR) are ex- 958

916 in this cells (Hata et al., 1993). This con- pressed in the uterus and have differential ef- 959

917 cept could be exploited to guide treatments ori- fects on growth (Vahrenkamp et al., 2018). Ex- 960

918 ented to recover progestin regulation over es- pression of both receptors was associated with 961

919 trogen proliferative effects in endometrial ma- poor outcome in endometrial cancer and the 962

920 lignancy. simultaneous induction of ER and GR leads 963

921 Previous results in T47D mammary can- to molecular interplay between the receptors 964

922 cer cells have shown Hormone Control Re- (Vahrenkamp et al., 2018). In our conditions, 965

923 gions, which include ERbs and PRbs acting in R5020 induces genes with GR/PR putative 966

924 conjunction with FOXA1 and C/EBPa (Nacht binding sites, enabling regulation that could 967

925 et al., 2019) interact with promoters of hor- result in a similar ER-GR pathological out- 968

926 mone regulated genes in hormone responsive come. 969

927 TADs and organize the high level folding of Regarding genes under PgCRs regulation, 970

928 the genome (Le Dily et al., 2019). Although ETV4 was one of the most frequent genes en- 971

929 the analysis of interaction between PgCR and compassed in a PgCR giving rise to the vari- 972

930 different ERalpha enriched binding regions in ation of PCA applied to endometrial adeno- 973

931 endometrial cells remains to be performed, carcinoma tumors. This gene was recently 974

932 our present study proposes that PR binding reported as playing a major role in control- 975

933 sites originated under estrogenic conditions ling the activity of ER and the growth of 976

934 and acting in conjunction with PAX2, fulfil endometrial cancer cells (Rodriguez et al., 977

935 a similar function in differentiated hormone- 2020). Like ETV4, other genes such as MEIS1, 978

936 responsive endometrial cancer cells. Thus ZFHX4, FKPB5, TGFBR2 are under regu- 979

937 combinations of the same hormone receptors lation of PR specific endometrial enhancers 980

938 and different transcription factors account for present in PgCRs and could be responsible 981 20 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

982 for the advance in malignancy of endome- Transfection with flag-tagged PR (FPR 1020

983 trial cancer through a progressive repression Ishikawa cells) 1021

984 of the immune response together with an in- Plasmid p3xFLAG-CMV-14 carrying the 1022

985 creased EMT-based metastatic/invasive poten- complete sequence for progesterone receptor 1023

986 tial (Bhanvadia et al., 2018; Alfaro et al., 2017; gene (HindIII924 - 938EcoRI) was introduced 1024

987 Bai et al., 2019; Cancer Genome Atlas Re- in Ishikawa cells using Lipofectamine 2000 1025

988 search Network et al., 2017; Monsivais et al., (Thermo Fisher Scientific) following manufac- 1026

989 2019; Dufait et al., 2019; Ma et al., 2018; turer recommendations. After 24h of transfec- 1027

990 Deshmukh et al., 2018; Harwood et al., 2018). tion, cells were exposed to 0.6mg/ml G418 for 1028

991 In sum, our results suggest that loss of PR and selection. Then on, every two passages, FRP 1029

992 ER signaling in endometrial cells may lead to cells were exposed to a reduced concentration 1030

993 the aberrant expression of the genes located in of G418 (0.4mg/ml), except during hormonal 1031

994 TADs with PgCRs (PgCR-genes), which could treatments. 1032 995 contribute to tumor progression. Proliferation assay 1033 4 Ishikawa cells were seeded at 5x10 1034

996 Materials and Methods cells/plate density in 35mm dish plates. Af- 1035 ter 48h in 5% DCC-FCS, the medium was re- 1036

placed for 1% DCC-FCS for 18h. Treatments 1037 997 Cell culture and hormonal treatments were performed for 48h and cells were then 1038

collected using trypsin (0.25%). Antagonists 1039

998 Endometrial adenocarcinoma Ishikawa cells for ER and PR, ICI182780 and RU486 1µM 1040

999 and FPR Ishikawa cells were cultured in respectively, were added for 60min and re- 1041

1000 phenol red DMEM/F12 medium (GIBCO, moved before hormonal treatments. The num- 1042

1001 Thermo Fisher Scientific) supplemented with ber of live cells was determined using try- 1043

1002 10% FCS (GreinerBioOne) and gentamycin pan blue (0.1%) in Neubauer chamber, repeat- 1044 ◦ 1003 (Thermo Fisher Scientific) at 37 C and 5% ing the procedure sixteen times for each sam- 1045

1004 carbon dioxide to maintain cell line stock. ple and performing three independent experi- 1046

1005 Before each experiment, cells were plated ments. 1047 1006 in phenol red-free DMEM/F12 medium sup-

1007 plemented with 5% dextran-coated charcoal- BrdU incorporation assay and cell cycle anal- 1048

1008 treated (DCC)-FCS and gentamycin for 48h. ysis 1049

1009 Then, the medium was replaced by serum-free Ishikawa cells were seeded and prepared for 1050

1010 DMEM/F12 and kept in it for 18h (overnight). hormonal treatments as described for Prolifer- 1051

1011 Treatments were performed with R5020 and ation assay. Treatments were carried out for 1052

1012 E2 to a final concentration of 10nM and 15h, the last two hours of which includes incu- 1053

1013 ethanol (vehicle) for the times indicated for bation with BrdU. Cells were treated with cell 1054

1014 each experiment. When indicated, pretreat- cycle inhibitor TSA A 250nM as negative con- 1055

1015 ment with E2 consisted of a single administra- trol of BrdU incorporation. After collecting 1056

1016 tion of E2 to a final concentration of 10nM 12h cells in trypsin and washing them with PBS, 1057

1017 before hormonal treatments. T47D cells were ethanol 70% was added to fix and permeabilize 1058

1018 cultured in RPMI 1640 medium as previously them. DNA denaturation was achieved with 1059

1019 described (Nacht et al., 2016). 0.5% BSA and 2M HCl after which cells were 1060 21 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

1061 incubated in 1:2000 solution of anti-BrdU (BD 488 and Alexa 555 (Thermo Fisher Scientific, 1102

1062 Pharmingen) for 1h at RT. FITC secondary Thermo Fisher Scientific) diluted 1:1000 in 1103

1063 antibody (Dako) was incubated for 1h in ob- 10% BSA 0.1% Tween-PBS for 1h at room 1104

1064 scurity at RT followed by propidium iodide temperature using DAPI to reveal nuclei. Cov- 1105

1065 for 5min. BrdU incorporation and cell cycle erslips were mounted on slides with Mowiol 1106

1066 phases were evaluated by flow cytometry (BD mounting medium (Sigma Aldrich) and ana- 1107

1067 FACS Canto II) in three replicates. lyzed in TIRF Olympus DSU IX83 (Olympus 1108

Life Sciences Solutions). Quantification of nu- 1109

1068 Western blot clear fluorescence was done with ImageJ soft- 1110

1069 Cell extracts were collected at the times ware after generating a binary mask in dapi 1111

1070 indicated by the experiment with 1% SDS, images. 1112

1071 25mM Tris-HCl pH 7.8, 1mM EDTA, 1mM 1113 1072 EGTA and protease and phosphatase in- qRTPCR

1073 hibitors. Total protein extracts were loaded in After 12h of treatment with R5020 and 1114

1074 8% SDS-PAGE and incubated with the follow- E2, cell extracts were collected in dena- 1115

1075 ing antibodies: PR (H190, Santa Cruz Bio.), turing solution (4M Guanidine thiocyanate, 1116

1076 ERalpha (HC-20, Santa Cruz Bio.) and alpha- 25mM Sodium citrate pH 7, 0.1M 2- 1117

1077 tubulin (Sigma Aldrich). Quantification of gel Mercaptoethanol, 0.5% Sarkosyl) and to- 1118

1078 images was performed with ImageJ software tal RNA was prepared following phenol- 1119

1079 and expressed as abundance in relative units to chloroform protocol (Chomczynski and Sac- 1120

1080 alpha-tubulin. chi, 1987). Integrity-checked RNA was used 1121

to synthesize cDNA with oligodT (Biodynam- 1122

1081 Immunofluorescence ics) and MMLV reverse transcriptase (Thermo 1123

1082 Cells were seeded onto coverslips in six- Fisher Scientific). Quantification of candidate 1124 3 1083 well plates in a density of 10 cells/150µl using gene products was assessed by real-time PCR. 1125

1084 the protocol described in Cell culture and hor- Expression values were corrected by GAPDH 1126

1085 monal treatments and either pretreated or not and expressed as mRNA levels over time zero 1127

1086 with E2 10nM during the last 12h of serum- (T0). Primer sequences are available on re- 1128

1087 free culture. After hormonal treatments cells quest. 1129

1088 were washed with ice cold PBS followed by 1130 1089 fixation and permeabilization by incubation in Luciferase reporter assay ◦ 1090 70% ethanol for 12h at -20 C. After rinsing Ishikawa cells were seeded and prepared for 1131

1091 three times for 5min in 0.1% Tween-PBS, the hormonal treatments as described for Prolif- 1132

1092 coverslips were incubated for 2h with 10% eration assay without addition of gentamycin. 1133

1093 BSA in 0.1% Tween-PBS to reduce nonspe- Cells were co-transfected with MMTV LTR- 1134

1094 cific staining. To detect PR (H-190 Santa Firefly Luciferase (pAGMMTVLu, gift from 1135

1095 Cruz Bio.), phosphoserine 294 PR (S294 Cell Laboratory of Patricia Elizalde) and CMV- 1136

1096 Signaling), ERalpha (HC-20 Santa Cruz Bio.) Renilla luciferase (pRL-CMV, Promega) plas- 1137

1097 and PAX2 (Biolegends) cells were incubated mids using lipofectamine plus 2000 (Thermo 1138

1098 with corresponding antibodies diluted in 10% Fisher Scientific). After 5h, media were re- 1139 ◦ 1099 BSA 0.1%Tween-PBS at 4 C overnight. Af- newed with the addition of antibiotics and 12h 1140

1100 ter several washes in Tween-PBS, coverslips later cells were treated with vehicle (ethanol) 1141

1101 were exposed to secondary antibodies Alexa and R5020 for 20h. Firefly and Renilla 1142 22 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

1143 activities (arbitrary units) were determined using Ishikawa RNAseq results, labelling 1184

1144 with Dual-Luciferase Reporter assay system samples as “R5020” and “E2” for cate- 1185

1145 (Promega) and expressed as Firefly units - gorical classification (phenotypes). Gene 1186

1146 tive to internal control Renilla for each sample sets were constructed from proliferative 1187 4 1147 (Firefly x10 /Renilla). (SRR9298724, SRR9298725, SRR9298726 1188

and SRR9298727) and mid-secretory 1189

1148 RNAseq (SRR9298728, SRR9298729, SRR9298730, 1190

1149 Total RNA was collected from untreated SRR9298731 and SRR9298732) normal en- 1191

1150 (T0) and 12h R5020- and E2-treated Ishikawa dometrial RNAseq samples (Chi et al., 2020). 1192

1151 cells using RNeasy Plus Mini Kit (QIAGEN) Differential expression analysis to extract 1193

1152 and subjected to high-throughput sequencing genes representative of each stage was per- 1194

1153 in Illumina HiSeq 2000 and 2500. Poly-A- formed with DESeq2 package (|log2FC|>2.5, 1195

1154 enriched RNA was used to prepare libraries p<0.05) (Love et al., 2014). 1196

1155 with TruSeq RNA Sample Preparation kit v2

1156 y v4 (ref. RS-122-2001/2, Illumina) accord- Endometrial cancer samples (TCGA) 1197 1157 ing to instructions from manufacturer followed Raw count data from endometrial cancer 1198 1158 by single-end (run1) and paired-end (run2) RNAseq samples (n=575) were downloaded 1199 1159 sequencing. Good quality 50bp reads were from The Cancer Genome Atlas (TCGA), 1200 1160 aligned to the reference (hg19, project TCGA-UCEC. Endometrioid adeno- 1201 1161 UCSC) using Tophat software (Trapnell et al., carcinoma samples (n=423) were selected us- 1202 1162 2009) keeping those that mapped uniquely ing associated clinical data and only protein 1203 1163 to the reference with up to two mismatches coding genes above arbitrary threshold (mean 1204 1164 and transcript assembly, abundance quantifica- > 100 counts) were kept for further analyses. 1205 1165 tion and differential expression analyses were Raw counts were normalized in DESeq2 pack- 1206 1166 performed with the Cufflinks tool (Trapnell age and later used for heatmaps (pheatmap R 1207 1167 et al., 2010). Genes under 200bp in length or package (Kolde and Kolde, 2015)) and Princi- 1208 1168 with FPKM values below 0,1 were excluded pal Component Analysis in PCAtools package 1209 1169 from downstream analyses. Genes were clas- (Blighe et al., 2019). 1210 1170 sified into induced, repressed or non-regulated

1171 depending on log2FC value relative to un- Chromatin immunoprecipitation (ChIP) 1211 1172 treated cells (T0). Threshold value was ar-

1173 bitrarily set at log2FC = ± 0.8 and q<0.05 ChIP experiments were performed as de- 1212

1174 (FDR). Enriched terms and TFBS were deter- scribed in (Strutt and Paro, 1999) and (Vicent 1213

1175 mined through RDAVIDWebservice (Fresno et al., 2011). Antibodies used for immunopre- 1214

1176 and Fernandez,´ 2013) and DAVID web-based cipitation were PR (H190, Santa Cruz Bio.), 1215

1177 tool (Huang et al., 2009) under standard pa- ERalpha (HC-20X and H184X, Santa Cruz 1216

1178 rameter settings for each tool. Bio.), PAX2 (PRB-276P, BioLegend) and nor- 1217

mal rabbit IgG (sc-2027, Santa Cruz Bio.). En- 1218 1179 Gene Set Enrichment Analysis (GSEA) richment to DNA was expressed as percentage 1219

1180 GSEA tool was implemented follow- of input (non-immunoprecipitated chromatin) 1220

1181 ing instructions from developers under relative to untreated Ishikawa cells (T0) using 1221

1182 default parameters (Subramanian et al., the comparative Ct method. Ct values were ac- 1222

1183 2005). The expression dataset was created quired with BioRad CFX Manager software. 1223 23 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

1224 ChIPseq overlap of at least one bp was considered 1266

1225 After minor modifications to the ChIP pro- positive. De novo motif discovery (MEME 1267

1226 tocol described in (Vicent et al., 2011), pu- software) performed on sequences contained 1268

1227 rified ChIP-DNA was submitted to deep se- in 10kb windows centered in peak summits. 1269

1228 quencing using Illumina HiSeq-2000. Li- Graphs, correlation tests, non-linear regression 1270

1229 braries were prepared by the Genomics unit and statistical analyses in general were per- 1271

1230 of the CRG Core Facility (Centre for Ge- formed for common peaks between ChIPseq 1272

1231 nomic Regulation, Barcelona, Spain) with samples using R (R Development Core Team). 1273

1232 NEBNext ChIPseq Library Prep Reagent Set Heatmaps were plotted using the summit of the 1274

1233 (ref. E6200S, Illumina) and 50bp sequenc- peaks as a reference central position. Refer- 1275

1234 ing reads were trimmed to remove Illumina ence positions were taken from common and 1276

1235 adapters and low-quality ends using Trimmo- exclusive peaks within experiments and were 1277

1236 matic (Bolger et al., 2014) version 0.33 in sorted by height of the peak. Genome aligned 1278

1237 single-end mode. Good quality reads were reads occurring between −5000 and +5000 1279

1238 aligned to the reference human genome (hg19, bp from reference sites were mapped using 1280

1239 UCSC) with BWA (Li and Durbin, 2009) count occurences program (Kremsky et al., 1281

1240 v0.7.12 (BWA MEM algorithm with default 2015) and the number of reads per bins of 1282

1241 parameters) keeping alignments that mapped 200bp was used for the color intensity of 1283

1242 uniquely to the genome sequence (Samtools heatmap cells with R. For Motif discovery, 1284

1243 version 1.2, (Li and Durbin, 2009)). Over- genomic regions of top 500 peaks ranked 1285

1244 lapping reads were clustered and significant by their height were extracted from each set 1286

1245 signal enrichments (peaks) were identified by and regions that overlap with repeats, low 1287

1246 MACS2 v2.1.0 (Zhang et al., 2008) using in- complexity regions or transposable elements 1288

1247 put as background signal. FDR value during (extracted from the UCSC genome browser, 1289

1248 initial peak calling steps was set to 0.05 (q), hg19 human assembly), were removed from 1290

1249 though downstream analyses included only the analysis. Motif discovery was performed 1291 −5 1250 those with q<10 . Replication of binding using MEME program suite executed with 1292

1251 sites was evaluated among treatments (time of the following parameters: -maxsize 250000 - 1293

1252 exposure to hormone) and conditions (no pre- revcomp -dna -nmotifs 3 -mod oops (Bailey 1294

1253 treated, pretreated and FPR) using scatter plots et al., 2015). Motif enrichments were eval- 1295

1254 and venn diagrams. Selected sites were val- uated with the procedure and statistics de- 1296

1255 idated by qPCR. When necessary peak files scribed in (Agirre et al., 2015). Addition- 1297

1256 were converted to hg38 coordinates using the ally, the analysis utilized a 5mers collection 1298

1257 batch conversion tool from UCSC. ChIPseq of 1,395 human position frequency matrices 1299

1258 coverage data of proliferative and secretory modelling transcription factors binding sites 1300

1259 normal endometrium were downloaded from (Weirauch et al., 2014), which were scanned 1301 −4 1260 GEO (GSE132713, (Chi et al., 2020)). (p-value<1e ) and their enrichment evaluated 1302 in regions of 200bp centered in the summits of 1303

1261 Heatmaps, Scatterplots and Motif analysis whole peaks sets. To uncover motif profiles, 1304

1262 Overlap of ChIPseq peak regions defined by discovered and library motifs were whole- 1305 −4 1263 upstream peak calling procedures (MACS2) genome scanned (p-value<1e ). Their occur- 1306

1264 were determined using intersectBed program rences around the sets of summits were ob- 1307

1265 from the bedTools suite (Quinlan, 2014). An tained with count occurrences (±2000bp, bin 1308 24 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

1309 size=200bp) and the profiles showing the pro- 2009; Rao et al., 2014). Adherent cells were 1350

1310 portion of regions per bin having at least one directly cross-linked on the plates with 1% 1351

1311 match were plotted using R. formaldehyde for 10min at room temperature. 1352

After addition of glycine (125mM final) to 1353 1312 Binding site-gene association stop the reaction, cells were washed with PBS 1354 1313 Genomic coordinates of PR and ERalpha and recovered by scrapping. Cross-linked cells 1355 1314 binding sites (hg38) were fed to GREAT web were incubated 30min on ice in 3C lysis Buffer 1356 1315 tool (McLean et al., 2010) to identify potential (10mM Tris-HCl pH=8, 10mM NaCl, 0.2% 1357 1316 cis-regulatory interactions. Association was NP40, 1X anti-protease cocktail), centrifuged 1358 1317 determined in a “basal plus extension” process 5min at 3,000 rpm and resuspended in 190µl 1359 1318 using a proximal regulatory domain of 5kb up- of NEBuffer2 1X (New England Biolabs - 1360 1319 stream and 1kb downstream from each TSS NEB). 10µl of 10% SDS were added and cells 1361 1320 (GRCh38, UCSC hg38) and an extension of ◦ were incubated for 10min at 65 C. After addi- 1362 1321 100kb in both directions. The group of genes tion of Triton X-100 and 15min incubation at 1363 1322 associated with PRbs or ERbs were respec- ◦ 37 C, nuclei were centrifuged 5min at 3,000 1364 1323 tively intersected to R5020 and E2 RNAseq re- rpm and resuspended in 300µl of NEBuffer2 1365 1324 sults, employing simple python scripting. 1X. Digestion was performed overnight using 1366

400U MboI restriction enzyme (NEB). To fill- 1367 1325 ATACseq in the generated ends with biotinylated-dATP, 1368 1326 ATACseq was performed as previously nuclei were pelleted and resuspended in fresh 1369 1327 described (Buenrostro et al., 2013). Briefly, repair buffer 1x (1.5µl of 10mM dCTP; 1.5µl 1370 1328 50,000 cells were lyzed with 50µl cold lysis of 10mM dGTP; 1.5µl of 10mM dTTP; 37.5µl 1371 1329 buffer (Tris-Cl pH 7.4 10mM; NaCl 10mM; of 0.4mM Biotin-dATP; 50U of DNA Poly- 1372 1330 MgCl2 3mM; NP-40 0.1% v/v) and cen- ◦ merase I Large (Klenow) fragment in 300µl 1373 1331 trifuged at 500xg for 10min at 4 C. Nuclei NEBuffer2 1X). After 45min incubation at 1374 1332 were resuspended in TD Buffer with 1.5µl ◦ 37 C, nuclei were centrifuged 5min at 3,000 1375 1333 Tn5 Transposase (Nextera, Illumina) and ◦ ◦ rpm and ligation was performed 4h at 16 C us- 1376 1334 incubated 15 minutes at 37 C. DNA was ing 10,000 cohesive end units of T4 DNA lig- 1377 1335 isolated using Qiagen MinElute column and ase (NEB) in 1.2ml of ligation buffer (120µl 1378 1336 submitted to 10 cycles of PCR amplifica- of 10X T4 DNA Ligase Buffer; 100µl of 10% 1379 1337 tion using NEBNext High-Fidelity 2X PCR Triton X-100; 12µl of 10mg/mL BSA; 963µl 1380 1338 Master Mix (Univ. primer: AATGATACG- of H2O). After reversion of the cross-link, 1381 1339 GCGACCACCGAGATCTACACTCGTCG- DNA was purified by phenol extraction and 1382 1340 GCAGCGTCAGATGTG ; Indexed primers: EtOH precipitation. Purified DNA was soni- 1383 1341 CAAGCAGAAGACGGCATACGA- cated to obtain fragments of an average size 1384 1342 GATNNNNNNNNGTCTCGTGGGCTCG- of 300-400bp using a Bioruptor Pico (Diagen- 1385 1343 GAGATGT). Library were size selected ode; 8 cycles; 20s on and 60s off). 3µg of 1386 1344 using AMPure XP beads and sequenced on a sonicated DNA was used for library prepara- 1387 1345 NextSeq 500 instrument (2x75nt). tion. Briefly, biotinylated DNA was pulled 1388

1346 Hi-C down using 20µL of Dynabeads Myone T1 1389

1347 High-throughput chromosome conforma- streptavidine beads in Binding Buffer (5mM 1390

1348 tion capture assays were performed as pre- Tris-HCl pH7.5; 0.5mM EDTA; 1M NaCl). 1391

1349 viously described (Lieberman-Aiden et al., End-repair and A-tailing were performed on 1392 25 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

1393 beads using NEBnext library preparation end- Virtual 4C 1432

1394 repair and A-tailing modules (NEB). Illumina Hi-C matrices were normalized for sequenc- 1433 1395 adaptors were ligated and libraries were am- ing depth and genomic biases using OneD (Vi- 1434 1396 plified by 8 cycles of PCR. Resulting Hi-C li- dal et al., 2018) and further smoothed using 1435 1397 braries were first controlled for quality by low a focal average. Virtual 4C plots were gener- 1436 1398 sequencing depth on a NextSeq500 prior to ated from the matrices locally normalized and 1437 1399 higher sequencing depth on HiSeq2000. Hi-C expressed as normalized counts per thousands 1438 1400 data were processed using an in-house pipeline within the region. 1439 1401 based on TADbit (Serra et al., 2017). Reads

1402 were mapped according to a fragment-based Intra-TAD interactions between specific loci 1440 1403 strategy: each side of the sequenced read was Each bin of a TAD was labeled as part of a 1441 1404 mapped in full length to the reference genome PgCR or TSS (or “others” if they did not be- 1442 1405 Human Dec. 2013 (GRCh38/hg38). In the long to the previous types). We collected the 1443 1406 case reads were not mapped when intra-read observed contacts between the different types 1444 1407 ligation sites were found, they were split. Indi- of bins and computed the expected contacts 1445 1408 vidual split read fragments were then mapped frequencies based on the genomic distance that 1446 1409 independently. We used the TADbit filtering separate each pair. In the figure, results are ex- 1447 1410 module to remove non-informative contacts pressed as Log2 of the ratio of observed con- 1448 1411 and to create contact matrices as previously tacts between the different types of pairs above 1449 1412 described (Serra et al., 2017) PCR duplicates the intra-TAD background. 1450 1413 were removed and the Hi-C filters applied

1414 corresponded to potential non-digested frag- Data Availability 1451 1415 ments (extra-dandling ends), non-ligated frag-

1416 ments (dandling-ends), self-circles and ran- All raw and processed sequencing data gen- 1452 1417 dom breaks. erated in this study have been submitted to 1453

the NCBI Gene Expression Omnibus (GEO; 1454

https://www.ncbi.nlm.nih.gov/geo/) under ac- 1455

1418 CNV cession number GSE139398. 1456

Acknowledgments 1457 1419 The copy number variation (CNV) analy-

1420 sis was estimated comparing the coverage ob- We are grateful to members of the Beato and 1458

1421 tained in the Hi-C datasets with the expected Saragueta¨ laboratories for help and sugges- 1459

1422 coverage for a diploid genome based on the tions. 1460 1423 density of restriction sites and genomic biases

1424 (Vidal et al., 2018). Indeed, the linear corre- Author Contributions 1461 1425 lation between number of Hi-C contacts and

1426 number of restriction sites is lost in case of ALG, FLD, MB and PS design experiments. 1462

1427 altered copy number allowing the estimation ALG, FLD, RJ, GV and ITR performed cell 1463

1428 of a relative number of copy as compared to culture and experiments. ALG, NB, RJ, GM, 1464

1429 diploid in each dataset. Such CF, JQO, EV and FLD performed bioinfor- 1465

1430 estimations are consistent with other analyses matic analyses. RJ, JLV, EV and FLD ana- 1466

1431 and with karyotyping (Le Dily et al., 2014). lyzed Hi-C results. GV, EF, GPV, MB, ALG 1467 26 bioRxiv preprint doi: https://doi.org/10.1101/739466; this version posted December 30, 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.

1468 and PS discussed experiments and manuscript. Bai R, Cui Z, Ma Y, Wu Y, Wang N, Huang L, Yao

1469 MB and PS provided fundings for this paper. Q, Sun J. 2019. The nf-κb-modulated mir-19a-3p enhances malignancy of human ovarian cancer cells 1470 ALG, MB and PS wrote the manuscript. through inhibition of igfbp-3 expression. Mol Car- cinog 58, 2254–2265. doi:10.1002/mc.23113. 1471 Funding Bailey TL, Johnson J, Grant CE, Noble WS. 2015. The meme suite. Nucleic Acids Res 43, W39–49. doi:10. 1472 This work was supported by the Na- 1093/nar/gkv416. 1473 tional Scientific and Technical Research Coun- Ballare´ C, Castellano G, Gaveglia L, Althammer S,

1474 cil (CONICET), Grant/Award Number: PIP Gonzalez-Vallinas´ J, Eyras E, Le Dily F, Zaurin R, Soronellas D, Vicent GP, et al.. 2013. Nucleosome- 1475 2015-682; Scientific and Technical Research driven transcription factor binding and gene regula- 1476 Fund (FONCyT), Grant/Award Number: PICT tion. Mol Cell 49, 67–79. doi:10.1016/j.molcel. 1477 2015-3426. Doctoral Fellowship from CON- 2012.10.019. 1478 ICET, Argentina, awarded to ALG. PS is PI Ballare´ C, Uhrig M, Bechtold T, Sancho E, Di Domenico M, Migliaccio A, Auricchio F, 1479 from CONICET. Research in the Beato’s labo- Beato M. 2003. Two domains of the progesterone 1480 ratory receives funding from the European Re- receptor interact with the estrogen receptor and are 1481 search Council under the European Union’s required for progesterone activation of the c-src/erk 1482 Seventh Framework Programme (FP7/2007- pathway in mammalian cells. Mol Cell Biol 23, 1483 2013)/ERC Synergy grant agreement 609989 1994–2008. Beato M, Herrlich P, Schutz¨ G. 1995. Steroid hormone 1484 (4DGenome). The content of this manuscript receptors: many actors in search of a plot. Cell 83, 1485 reflects only the author’s views and the Union 851–7. 1486 is not liable for any use that may be made of Bhanvadia RR, VanOpstall C, Brechka H, Barashi NS, 1487 the information contained therein. We also Gillard M, McAuley EM, Vasquez JM, Paner G,

1488 acknowledge support of the Spanish Ministry Chan WC, Andrade J, et al.. 2018. Meis1 and expression and prostate cancer progression: A role 1489 of Economy and Competitiveness, “Centro for binding partners in metastatic disease. 1490 de Excelencia Severo Ochoa 2013-2017” and Clin Cancer Res 24, 3668–3680. doi:10.1158/ 1491 Plan Nacional (SAF2016-75006-P), as well as 1078-0432.CCR-17-3673. 1492 support of the CERCA Programme / General- Blighe K, Lewis M, Lun A, Blighe MK. 2019. Package

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