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1 RESEARCH ARTICLE Open Access
2 Estrogen receptor beta impacts hormone-induced
3 alternative mRNA splicing in breast cancer cells
4Q1 Dougba Noel Dago1,2†
, Claudio Scafoglio3†
, Antonio Rinaldi1
, Domenico Memoli1
, Giorgio Giurato1
, Giovanni Nassa1
5 Maria Ravo1
, Francesca Rizzo1
, Roberta Tarallo1
and Alessandro Weisz1,4*
11 Background: Estrogens play an important role in breast cancer (BC) development and progression; when the two
12 isoforms of the estrogen receptor (ERα and ERβ) are co-expressed each of them mediate specific effects of these
13 hormones in BC cells. ERβ has been suggested to exert an antagonist role toward the oncogenic activities of ERα,
14 and for this reason it is considered an oncosuppressor. As clinical evidence regarding a prognostic role for this
15 receptor subtype in hormone-responsive BC is still limited and conflicting, more knowledge is required on the
16 biological functions of ERβ in cancer cells. We have previously described the ERβ and ERα interactomes from
17 BC cells, identifying specific and distinct patterns of protein interactions for the two receptors. In particular, we
18 identified factors involved in mRNA splicing and maturation as important components of both ERα and ERβ pathways.
19 Guided by these findings, here we performed RNA sequencing to investigate in depth the differences in the early
20 transcriptional events and RNA splicing patterns induced by estradiol in cells expressing ERα alone or ERα and ERβ.
21 Results: Exon skipping was the most abundant splicing event in the post-transcriptional regulation by estradiol. We
22 identified several splicing events induced by ERα alone and by ERα + ERβ, demonstrating for the first time that ERβ
23 significantly affects estrogen-induced splicing in BC cells, as revealed by modification of a subset of ERα-dependent
24 splicing by ERβ, as well as by the presence of splicing isoforms only in ERβ + cells. In particular, we observed that
25 ERβ + BC cell lines exhibited around 2-fold more splicing events than the ERβ- cells. Interestingly, we identified putative
26 direct targets of ERβ-mediated alternative splicing by correlating the genomic locations of ERβ and ERα binding sites
27 with estradiol-induced differential splicing in the corresponding genes.
28 Conclusions: Taken together, these results demonstrate that ERβ significantly affects estrogen-induced early
29 transcription and mRNA splicing in hormone-responsive BC cells, providing novel information on the biological role of
30 ERβ in these tumors.
Keywords: Breast cancer, Estrogen receptor beta, Alternative splicing, Alternative promoters, RNAseq
33 Breast cancer (BC) is the most frequent cancer in women
34 worldwide , and its development and progression are
35 known to rely strongly on the stimulation by female sexual
36 hormones, especially estrogens. Estrogen receptors α (ERα)
37 and β (ERβ) are transcription factors that mediate the
38 actions of estrogens in target cells [2,3]. Ligand binding to
39ERα or ERβ induces receptor dimerization, either as homo-
40dimers (ERα/ERα or ERβ/ERβ) or heterodimers (ERα/ERβ)
41, and promotes its translocation to the nucleus and
42binding of target chromatin sites via Estrogen Response
43Elements (EREs) and other regulatory elements on DNA
44. Although encoded by different genes, ERα and ERβ
45share the same general modular protein structure of the
46nuclear hormone receptor superfamily, and have almost
47100% amino acid sequence homology in their DNA-
48binding domain. They also show 59% amino acid hom-
49ology in their ligand-binding domains [4,5]. The two
50ERs are thus quite similar in sequence and structure,
51but ERβ has considerably different and, in most cases,
52opposite biological effects compared to ERα in BC cells,
* Correspondence: email@example.com
Q2 Laboratory of Molecular Medicine and Genomics, Department of Medicine
and Surgery, University of Salerno, Via S. Allende, 1, Baronissi, SA 84081, Italy
Molecular Pathology and Medical Genomics, “SS. Giovanni di Dio e Ruggi
d’Aragona - Schola Medica Salernitana” Hospital of the University of Salerno,
Full list of author information is available at the end of the article
© 2015 Dago et al.; licensee BioMed Central. This is an Open Access article distributed under the terms of the Creative
Commons Attribution License (http://creativecommons.org/licenses/by/4.0), which permits unrestricted use, distribution, and
reproduction in any medium, provided the original work is properly credited. The Creative Commons Public Domain
Dedication waiver (http://creativecommons.org/publicdomain/zero/1.0/) applies to the data made available in this article,
unless otherwise stated.
Dago et al. BMC Genomics _#####################_
53 both in vivo and in vitro. ERα regulates the transcription
54 of hundreds of genes , enhancing BC cell growth,
55 proliferation and survival in response to estrogens .
56 The specific role of ERβ and its impact in BC are un-
57 clear. This ER subtype is expressed in 70% of human
58 breast tumors in combination with ERα, even if some
59 human breast tumors express only ERβ [8-10]. Several
60 reports have suggested that ERβ has anti-proliferative
61 action in BC cells, by increasing the expression of anti-
62 proliferative genes and/or decreasing the expression of
63 proliferative and anti-apoptotic genes [11-15] and the
64 ERα/ERβ ratio determines the cell-specific response to
65 estrogen. In BC this ratio is higher than in normal tis-
66 sues, due to up-regulation of ERα and down-regulation
67 of ERβ . Loss of ERβ mRNA levels in cancer can
68 occur as a result of promoter methylation . These
69 observations have suggested a positive prognostic value
70 of this receptor subtype [12,18]. However, several studies
71 have reported also a negative prognostic value for ERβ
72 expression [19,20], making the overall contribution of this
73 receptor isoform to BC biology unclear.
74 The exact mechanism of the antagonism between ERβ
75 and ERα is only partially known. As the two receptors
76 share only 30% homology in their transactivation do-
77 main AF1 , it is likely that they show different pat-
78 terns of interaction with coregulatory proteins. Indeed,
79 we have previously reported that the protein interac-
80 tomes of both ERβ and ERα show significant differences
81 of the protein complexes engaged by the two ER sub-
82 types [21-25]. A particularly interesting subset of inter-
83 acting proteins, with only partially overlapping interaction
84 patterns between the two receptors , comprises factors
85 involved in RNA maturation and splicing [Additional
86 file 1: Figure S1] .
87 Alternative splicing is a mechanism by which cells can
88 increase the variability of their proteomes by changing
89 the composition of transcribed genes through differential
90 choice of exons to be included in the final mRNA mol-
91 ecule . Almost 90% of human genes show alternative
92 splicing during development, cell differentiation and
93 disease . Recent studies have shown the existence
94 of cancer-specific splicing events by which transformed
95 cells switch from the adult isoform of the gene to a
96 more embryonic one, contributing to the cancer pheno-
97 type [29-31].
98 Alternative splicing events have been monitored in BC
99 and in numerous tumor types , and ERα itself has
100 been reported to induce alternative splicing of a spe-
101 cific set of genes [33-35]. Here, we investigated the
102 ability of ERβ to regulate mRNA maturation and spli-
103 cing in hormone-responsive BC cells. To this purpose,
104 we performed high-throughput RNA sequencing (RNA-
105 seq) analysis of human MCF-7 cell lines stably trans-
106 fected with ERβ, and compared them with the wild type
107line, expressing only ERα, upon 17β-estradiol (E2)
110High-throughput sequencing in ERβ + and ERβ- human BC
112We have previously established and characterized sub-
113clones of the human BC cell line MCF-7 expressing hu-
114man ERβ fused to a Tandem Affinity Purification (TAP)
115tag, and have shown that the addition of the TAP-tag at
116either the N- or the C-terminus of the protein (indicated
117as Nt-ERβ and Ct-ERβ, respectively) does not alter sig-
118nificantly the receptor function, nor its ability to activate
119transcription or to antagonize ERα-dependent transcrip-
120tion [21,22,36]. In order to study the early events of
121hormone-induced pre-mRNA maturation, we used these
122stable cell lines to perform deep-sequencing analysis of
123estrogen-induced transcriptional events shortly after
124stimulation with 17β-estradiol (2 h), to focus mostly on
125primary transcriptional events [21,36]. For comparison,
126we also performed the experiment in wild-type MCF-7
127cells, which do not express endogenous ERβ.
128Almost 70 million reads/replicate were aligned against
129the reference human genome for ERβ- and ERβ + BC
130cell lines. The number of reads for genes and isoforms
131were normalized to “Fragment Per Kilobases of exon per
132Million of mapped reads” (FPKM). In order to analyze
133genes and isoforms, we set 0.5 FPKM (at least one ana-
134lyzed condition, with/without E2 stimulus) as the expres-
135sion level threshold. In this way, we identified 16,821
136(MCF-7 wt), 16,148 (Ct-ERβ) and 17,135 (Nt-ERβ) genes
137as expressed. The criteria for considering genes and iso-
138forms as significantly regulated by estradiol were: FPKM
139value ≥0.5 in at least one analyzed condition, q-value
140(FDR-adjusted p-value of the test statistics) ≤0.05 and
141|fold-change| (FC) ≥1.3 [Additional file 2: Table S1].
142As shown in Figure Q3 F11A, 895 (MCF-7 wt), 2,899 (Ct-ERβ)
143and 3,043 (Nt-ERβ) genes were detected as significantly
144regulated by E2 in these BC cell lines. Expression of
145ERβ in MCF7 cells significantly affected the estrogen-
146dependent gene expression profile: the regulation of
147around 230 genes (≈25% of E2-regulated genes) was
148lost in both cell lines expressing ERβ, while a large
149number of genes which were not regulated in wt cells
150became significantly regulated in Ct-ERβ (2,396) and
151Nt-ERβ (2,463) clones (Figure 1). The genes regulated
152consistently in both ERβ + lines are reported in Additional
153file 2: Tables S1-D. Interestingly, expression of ERβ in this
154BC cell line had a stronger effect on inhibited genes than
155on the activated ones: regulation of 40% of genes inhibited
156by estradiol in wt cells was lost in both ERβ + cell lines,
157versus 14% for estrogen-activated genes. Gene Ontology
158analysis revealed that among the most enriched functions
159in the group of genes whose regulation by estradiol was
Dago et al. BMC Genomics _#####################_ Page 2 of 13
160 lost in both ERβ + cells there were, as expected, DNA
161 Replication, Recombination and Repair, as well as Cell
162 Cycle and Cell Morphology (data not shown).
163 Estrogen-dependent splicing events in ERβ + and ERβ-
164 human BC cell lines
165 Events of exon skipping, mutually exclusive exons, alterna-
166 tive start, stop splice site and intron retention were anno-
167 tated using the Multivariate Analysis of Transcript Splicing
168 (MATS) software  [Additional file 3: Tables S2]. Exon
169 skipping appeared to be the predominant splice event in all
170 cell lines analyzed. MATS reveled in detail 1,264 (Ct-ERβ),
171 1,402 (Nt-ERβ) and 975 (MCF-7 wt) exon skipping events
172 induced by estradiol, associated with 1,016 (Ct-ERβ), 1,117
173 (Nt-ERβ) and 816 (wt MCF-7) genes (FigureF2 2A). Five
174 hundred seventy-five events were common to all the cell
175 lines analyzed while 115 showed opposite exon inclusion
176 level in ERβ + lines compared to ERβ- wt cells. We also
177 observed high levels of retained intron and mutually ex-
178 clusive exons events, confirming a complex and significant
179 effect of ERs on the regulation of RNA splicing in these
180 cell lines (Figure 2A).
181 To focus on the differences in splicing patterns between
182 ERβ + and ERβ- cell lines, we first looked at the genes
183which were regulated by estradiol in opposite direction in
184ERβ + cells versus wt cells [Additional file 4: Table S3].
185Among 298 regulated genes, 56 also underwent estradiol-
186induced alternative splicing in at least one of the cell lines,
187confirming that pre-mRNA maturation was regulated
188concurrently with transcription in a significant fraction
189of ERβ-regulated genes (Figure 2B). These ERβ-regulated
190genes undergoing alternative splicing included transcrip-
191tional regulators (NCOR2, ZNF189, MLXIP, ANKRD12,
192HSF1), enzymes involved in nucleoside/nucleotide metab-
193olism (GUK1, NME3, NME4), actin remodeling and cellu-
194lar transport processes (TNS3, TRAPPC6A, TMSB15B,
195KIF12), and protein translation (ZNF98, EEF1D, RPL10,
197Estrogen-induced differential splicing has been repor-
198ted also in genes independently on transcriptional regula-
199tion . In order to find the splicing events differentially
200regulated by estradiol in ERβ + compared to wt cells, we
201scanned the whole list of expressed genes for splicing pat-
202terns occurring differentially in ERβ + vs ERβ- cells. In
203addition, we focused on those splicing events whose oc-
204currence significantly altered the ratio between different
205isoforms of the same gene. Therefore, for each isoform we
206calculated the percentage of gene expression associated
207with that particular isoform (FPKM ratio: FPKMisoform/
208FPKMgene %) and selected for further analysis only those
209isoforms in which estradiol induced a change in FPKM
210ratio of at least 10% either in wt MCF-7 or in both ERβ +
211cell lines. Other criteria of inclusion were: occurrence
212of at least one splicing event as detected by MATS;
213at least one isoform of the gene with FPKM value ≥0.5 in
214at least one analyzed condition, q-value ≤0.05 either in wt
215or in both ERβ + cell lines; regulation in both ERβ + lines
216in opposite directions compared to the wt. In this
217way, we identified the quantitatively most relevant
218splicing events differentially regulated by E2 in ERβ +
219versus ERβ- cells, including 35 genes whose isoform
220composition changed significantly after E2 stimulation
221in an ERβ-dependent fashion (Figure F33). Among these, we
222found genes involved in apoptosis (BAD), lipid metabol-
223ism (ACADM, PLSCR1, SLC27A2, STARD4), nutrient
224transport (SLC25A19, SLC35C2), transmembrane receptor
225signaling (IFNGR2, LDLRAD4), Notch signaling (PSEN2,
226POGLUT1, SGK1, SLC35C2), as well as some non-coding
227RNAs (MCM3AP-AS1, SNHG17). An example of a gene
228whose splicing pattern was affected by estradiol in an ERβ-
229dependent fashion is reported in Figure F44A, showing the
230gene SGK1, which encodes a serum and glucocorticoid-
231induced serine/threonine protein kinase involved in ion
232transport affecting many cellular processes such as cell
233growth, proliferation, survival, apoptosis and migration
234[38,39]. The gene was induced by estradiol in both wt and
235ERβ + cells; however, expression of ERβ in the absence of
236hormonal stimulation induced a promoter usage switch
Figure 1 Venn diagram of expressed genes in ERβ + and wt breast
cancer cell lines. The Venn diagram shows the number of genes
regulated by estradiol in the three cell lines, as indicated: wt (parental
MCF-7 cells); Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged
with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably
expressing ERβ tagged with the TAP-tag at the N-terminus). The pie
charts in the lower panels specify the direction of regulation by
estradiol (induction or repression) of the genes whose regulation is
present in wt cells but is lost in both ERβ + cell lines (left panel), or of
the genes whose regulation by estradiol is not present in the wt cells
but appears in both ERβ + lines (right panel).
Dago et al. BMC Genomics _#####################_ Page 3 of 13
Figure 2 (See legend on next page.)
Dago et al. BMC Genomics _#####################_ Page 4 of 13
237 and retention of the first intron causing an alterna-
238 tive translation start site and therefore the expression
239 of an isoform with a different N-terminal sequence
240 (ENST00000367857), compared to the major isoform
241 expressed (ENST00000237305). It has been reported
242 that alternative isoforms at the N-terminus can affect
243 SGK1 localization and protein stability: the ERβ-specific
244 form with intron-retention misses a very crucial sequence
245 involved in targeting to the endoplasmic reticulum as well
246 as in proteasomal degradation .
247 This data suggests that expression of ERβ causes switches
248 in estradiol-induced splicing patterns, potentially affecting
249 expression or function or ER targets.
250 Estrogen-dependent alternative promoter usage in ERβ + vs
251 ERβ- BC cells
252 As the usage of alternative promoters is a major deter-
253 minant of protein diversity, even more than alternative
254 splicing [41,42], we next focused on utilization of mul-
255 tiple promoters. We grouped the primary transcripts of
256 a gene based on the promoter used, and subsequently
257 tested changes in primary transcript abundance by
258 measuring the square root of the Jensen-Shannon diver-
259 gence that occurred within and between the analyzed
260 groups. Finally, we investigated the potential promoter
261 switch regulation for the genes comprising more than
262 one differentially expressed transcript initiating from
263 distinct genomic loci. There were 977 (Ct-ERβ), 402
264 (Nt-ERβ) and 222 (wt MCF-7) distinct promoter-switching
265 genes (FDR ≤ 0.05) in ERβ + and ERβ- BC cell lines, re-
266 spectively [Additional file 5: Tables S4A-C]. Of the 222
267 promoter-switching genes recorded in wt MCF-7 cells,
268 165 did not show promoter switch in both ERβ + cell lines,
269 while 61 new promoter-switching events, not present in
270 wt cells, were detected in both ERβ + lines. These 61 ERβ-
271 specific promoter-switching genes are involved in import-
272 ant cellular functions known to be controlled by E2 in BC
273 cells, such as transcription (FOXJ3, GTF2H, NR2C2AP),
274 DNA metabolism and repair (PRKDC, REV3L, SCAND3),
275 pre-mRNA maturation and splicing (PPM1G, PRPF38B,
276 RNMT, RPRD1A, SMG1), translation (FARSB, RARS,
277 RPS21, UTP20), protein ubiquitination and proteasome
278 pathway (CUL5, KLHL2, PSMB1, USP7), cytoskeleton and
279 cytokinesis (DCTN4, MYL12A, SEPT9, SYDE2), mem-
280 brane metabolism, remodeling and intracellular transport
281 (ATP9A, CAST, MAL2, PSD3, TMEM43, TRAPPC9), cell
282adhesion and polarity (ARHGAP12, CD9, CLDN7,
283EPB41L5, PERP), signal transduction (MAP3K5, NGFRAP1,
284TNFRSF12A, WWC3, ZDHHC5).
285As an example, we focused on the PSD3 gene, predicted
286to be a nucleotide exchange factor for ADP Ribosylation
287Factor (ARF) 6, a member of the RAS family involved in
288vesicular trafficking, remodeling of membrane lipids, and
289signal transduction . As show in Figure 4B, for this
290gene we found 9 distinct primary transcripts (TSS01-
291TSS09) whose usage ratios changed with E2 stimulus
292(right panel) compared to the control untreated cells
293(left panel). In ERβ + cells, estradiol induced a switch
294from promoter TSS08 (ENST00000523619) to the down-
295stream promoter TSS02, resulting in a shorter transcript
296(ENST00000519653), which is predicted to undergo
297nonsense-mediated decay [Additional file 6: Table S5].
298Estrogen-dependent splice ratios in ERβ + vs ERβ- cells
299To obtain a comprehensive view of the estrogen-induced
300differences in the splice ratios between wt, Ct-ERβ and
301Nt-ERβ cells, we employed Cuffdiff v2.1.1 , which
302calculates the changes in splice isoforms abundance, by
303quantifying the square root of the Jensen-Shannon diver-
304gence, considering each primary transcripts able to pro-
305duce multiple isoforms. We determined 217 (Ct-ERβ),
306241 (Nt-ERβ) and 95 (MCF-7 wt) differentially spliced
307genes (DSGs, i.e. genes for which estradiol challenge
308induced at least one splicing event) with a FDR
309value <0.05 [Additional file 5: Tables S4D-F]. Of the
31095 spliced genes detected in wt cells, sixty-nine lacked
311splicing events in both ERβ + cell lines, suggesting inhib-
312ition of ERα-dependent splicing by ERβ. Moreover, 28
313ERβ-specific DSGs, in which no estrogen-induced splicing
314was recorded in wt cells, were found in both ERβ + cell
315lines. These include genes involved in mitosis and cyto-
316kinesis (SEPT9, BICD2, ENSA, PDS5A), cell cycle control
317(CCNJ, RAN), transcription (C11orf30, EIF3M, HIPK1,
318PBX1, ZNF124, ZNF131), protein folding (DNAJB6,
319HSP90B1), ubiquitination and sumoylation (DCUN1D4,
320SENP5, TRIM33), and signal transduction (APBB2, RTKN2).
321Correlation between ER binding and ER-dependent splicing
322In order to identify direct splicing targets of ERα and ERβ,
323we next investigated the presence of ERα and ERβ binding
324sites in genomic locations close to the identified DSGs
325[Additional file 7: Table S6]. To verify the specificity of ER
(See figure on previous page.)
Figure 2 Annotation of splice events in ERα + and ERα + ERβ + BC cell lines. (A) The bar plot shows the number of all alternative splicing events
occurring in the cell lines analyzed. Inclusion and exclusion behavior for each event are shown (FDR ≤ 0.05; c ≤ |0.1|). (B) Genes whose regulation
has opposite direction in the ERβ + lines compared to the wt MCF-7. The heat map on the right side shows the gene expression fold changes
induced by estradiol. The matrix on the left side shows in black those genes for which a splicing event was detected in at least one of the cell
lines. The nomenclature for the cell lines is the following: wt (parental MCF-7 cells); Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with
the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the N-terminus).
Dago et al. BMC Genomics _#####################_ Page 5 of 13
326binding sites, we created a heat-map based on previously
327published ChIP-Seq data [21,45]. The binding densities of
328ERα and ERβ were clustered according to the seqMINER
329platform . In the clustering shown in [Additional file
3308: Figure S2], each line represents a genomic location of a
331binding site with its surrounding ±1.5 kb region. In the left
332panel, ERα binding sites were used as a reference to col-
333lect a ChIP-seq tag densities window in ERβ + and ERβ-
334cell lines, while in the right panel ERβ binding sites were
335used as a reference. The heat map, representing the clus-
336tered density matrix, confirmed that ERβ presence modi-
337fied a significant number of ERα binding sites. In order to
338investigate the correlation between ERα and ERβ DNA
339binding and splicing events, we compared our RNA-Seq
340data with the ChIP-Seq data we had previously obtained
341in these same cell lines . We considered the binding
342sites included within regions spanning 10 kb upstream or
343downstream of all DSGs in each cell line. The Circos plot
344 in Figure F55 shows the DSGs for which estradiol chal-
345lenge induced at least one splicing event in both ERβ + cell
346lines with a nearby binding site for ERα and/or ERβ. The
347outer ring (blue) reports the ERα binding sites, while the
348inner ring (red) shows the ERβ binding sites. Based on
349ERα and ERβ binding, we distinguished three different
350DSGs groups. Group 1 DSGs were associated with both
351ERα and ERβ binding sites (black). Group 2 includes
352DSGs associated with ERα binding sites exclusively (blue)
353and group 3 DSGs associated with ERβ binding sites ex-
354clusively (red). The vast majority of genes exhibited both
355binding sites (Group 1), confirming the competing role of
356ERα and ERβ. Interestingly, among the putative direct
357ERβ splicing targets we found many genes involved in
358transcription: transcription factors (FOXN3, NFIB, TAF6,
359TCF12, ZNF295, ZNF438), histone methyltransferases
Figure 3 Selected isoform switches affected by the expression of
ERβ. The ERβ-dependent differential splicing events that affect most
prominently the balance of different isoforms for each gene were
identified by using the following parameters: (i) isoform ratio
(FPKMisoform/FPKMgene %) changing of at least 10% after estradiol
stimulation, either in the wt cells or in both the ERβ + cells in at least
one of the isoforms of the gene; (ii) at least one isoform of the gene
significantly regulated by estradiol (p-value ≤ 0.05; |FC| ≥ 1.3) either in
the wt cells or in both the ERβ + cells; (iii) isoform ratio changing in
opposite direction in the wt cells compared to the ERβ + cells; (iv) at
least one splicing event identified by MATS analysis. Thirty-five genes
satisfied all the selection requirements; of these, only 2 to 3 isoforms
were selected for presentation, according to the expression levels and
the regulation by estradiol. Left panel: heat map of regulation of the
selected genes by estradiol in: Ct-ERβ (MCF-7 subclone stably expressing
ERβ tagged with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7
subclone stably expressing ERβ tagged with the TAP-tag at the
N-terminus); wt (parental MCF-7 cells). Right panel: heat map with the
change in isoform ratio (FPKMisoform/FPKMgene %) between E2-treated
and non-treated cells in the indicated cell lines; for each gene, at least
two different isoforms are presented, to show estrogen-induced switch
from one isoform to the other.
Dago et al. BMC Genomics _#####################_ Page 6 of 13
Figure 4 Examples of ERβ-specific splicing events. A) Example of alternative splicing in the SGK1 gene. The upper panel shows a schematic
representation of two differentially regulated isoforms of the gene (same as those shown in Figure 3 for the same gene), differing in the transcription
start site, in the inclusion of the first intron (the gene is encoded by the reverse strand) and in the transcription stop site. The lower panels show a
representation of the RNA-Seq reads and junction reads associated with the gene in the different conditions: Ct-ERβ (MCF-7 subclone stably expressing
ERβ tagged with the TAP-tag at the C-terminus) without or with E2 stimulation, and wt (parental MCF-7 line) without or with E2 stimulation. B) Example of
ERβ-specific alternative promoter usage in the gene PSD3. The left panel shows a heat map with the fold change of all the primary transcripts associated
with the gene in: Ct-ERβ (MCF-7 subclone stably expressing ERβ tagged with the TAP-tag at the C-terminus); Nt-ERβ (MCF-7 subclone stably expressing
ERβ tagged with the TAP-tag at the N-terminus); wt (parental MCF-7 cells). The right panels show the logarithm base 10 (log10) FPKM for each of the
different transcript start sites (from TSS01 to TSS09) corresponding with the different gene isoforms in the above described cell lines, without (left) or
with (right) E2 stimulation.
Dago et al. BMC Genomics _#####################_ Page 7 of 13
360 (ASH1L, MLL5, SETD5, SETMAR) and acetyltransferase
361 (YEATS2), other transcriptional regulators (AKNA, BANP,
362 CHD3, MEIS2). Other interesting functions associated with
363 these genes are: apoptosis (BCL2L13, CASP8, C1orf201),
364 autophagy (AMBRA1, ATG12, ATG13, ATG16L2), splicing
365 (HNRNPH3, MBL2), protein ubiquitination (CNOT4,
366 FBXW11, RFWD2, WDR20) cytoskeleton and cytokin-
367 esis (AURKA, EPS8L2, EPB41L2, LARP4, NPHP4, PLEC,
368TLN2), primary cilium biogenesis  (BBIP1, IFT140,
369KIAA0586), intracellular trafficking/membrane traf-
370ficking (ASAP1, KIF13A, MCOLN1, RAB17, TBC1D1,
371VPS29), cell adhesion (ARMC8, CD151, ELMOD3, LLP,
373Taken together, these analyses suggest the strong effect
374of ERs binding on alternative splicing and confirming
375the key role of ERβ in BC cells.
Figure 5 Correlation between ERβ-specific splicing and ER binding. Circos plot of differential spliced genes (DSGs) common to both ERβ + cell
lines which contain at least one ER binding event within a window of 10 kB around the gene. The outer ring shows chromosome ideograms with
the relative genes located in their respective chromosomal locations, with the following color code: the genes that have both ERβ and ERα binding
are in black; the genes which have only ERβ binding are in red; the genes which have only ERα binding are in blue. The two internal rings represent
ERα and ERβ binding events in blue and red, respectively.
Dago et al. BMC Genomics _#####################_ Page 8 of 13
377 In this study we investigated for the first time the effects
378 of ERβ expression on estrogen-dependent pre-mRNA
379 maturation and splicing. We used two different subclones
380 of ERα-positive and estrogen-dependent MCF-7 cells sta-
381 bly expressing ERβ, and we found that expression of ERβ
382 in these cells significantly affected the estradiol-dependent
383 early transcriptional program and splicing pattern. In par-
384 ticular, introduction of ERβ caused loss of regulation of
385 25% of the estrogen-regulated genes. Moreover, a com-
386 parison between the ERβ + and the wt ERβ- cells showed
387 that expression of ERβ caused loss of ERα-induced pro-
388 moter switching in 75% of the genes, and of ERα-induced
389 splicing in 72% of the genes.
390 Besides affecting ERα-dependent transcription and
391 splicing, expression of ERβ caused the appearance of
392 ERβ-specific estrogen-responsive transcription (550 genes)
393 [Additional file 2: Tables S1-D], promoter-switching (61
394 genes) and differential splicing (28 genes) events, counting
395 only the events that were present in both ERβ + lines. The
396 biological functions of the ERβ-specific genes included
397 DNA replication and repair, cell cycle, apoptosis and au-
398 tophagy, DNA transcription, lipid metabolism, membrane
399 metabolism, intracellular trafficking, mRNA maturation
400 and translation, protein ubiquitination and sumoylation,
401 cell signaling, confirming that a wide variety of cellular
402 processes are affected by ERβ.
403 Based on our data, there are at least three different
404 mechanisms by which ERβ can affect ERα-dependent
405 transcription: (1) competition with ERα for binding to
406 target gene promoters, in the form of competitive bind-
407 ing or heterodimerization, which can alter the recruit-
408 ment of coregulators: the comparison between Chip-seq
409 and RNA-seq data showed that the majority of the pri-
410 mary targets identified in this experiment were directly
411 targeted by both ERα and ERβ (Figure S1), as expected
412 also from data in the literature ; (2) gain of new
413 binding sites that are not bound by ERα alone: a subset
414 of binding sites appeared in the ERβ + cells but were not
415 present in wt cells, again consistently with a previous re-
416 port comparing ERα and ERβ genomic binding patterns
417 in MCF-7 cells ; (3) secondary effects: expression of
418 ERβ induced transcription and splicing of transcriptional
419 regulators (most interestingly, the corepressor NCOR2,
420 involved in gene repression by tamoxifen-bound estro-
421 gen receptor and by unliganded NRs such as the retinoic
422 acid receptors) and of splicing factors, which can in turn
423 affect estrogen-induced transcription and pre-mRNA
424 maturation. For instance, ERβ induced promoter switch-
425 ing in the PPM1G gene, encoding a protein phosphatase
426 responsible for dephosphorylation of pre-mRNA splicing
427 factors ; the ERβ-specific alternate protein isoform
428 from this gene (ENST00000350803) has an additional 17
429 amino acids in the N-terminal catalytic domain compared
430to the main isoform (ENST00000544412), which are likely
431to modulate the enzymatic function.
432Even if the sole detection of splicing events does not
433give information on the biological consequences of ERβ
434effects on RNA splicing, it is tempting to speculate on
435the possible implications of ERβ-dependent splicing
436events on BC cells. Changes in splicing patterns can
437affect biological processes by many different mechanisms,
438including gain-of-function or functional switches, altered
439cellular localization, dominant negative effect, changes in
440protein/mRNA stability. For instance, expression of ERβ
441induced a promoter switch in the USP7 gene, resulting in
442a shorter transcript (ENST00000535863) compared to the
443major isoform expressed in wt cells (ENST00000381886).
444This ERβ-specific isoform is missing the N-terminal 84
445amino acids, a region of the protein of critical importance
446for interaction with substrates. This suggests the possibil-
447ity of a switch in substrate affinity induced by ERβ. This is
448particularly interesting as USP7 is a deubiquitinating en-
449zyme, responsible for removing ubiquitin chains from
450both the tumor suppressor p53 and its negative regulator
451Mdm2 , which instead is an ubiquitin ligase inducing
452degradation of p53. As USP7 binds both p53 and Mdm2
453with the same N-terminal domain , the overall effect
454of its enzymatic activity is highly dependent on the relative
455affinity for the two targets. The ERβ-induced switch may
456alter this equilibrium, thus modulating such a relevant
457aspect of cancer biology as p53 stability. In the case of
458IFNγ Receptor 2, ERα induced expression of the full
459length transcript (ENST00000381995), while ERβ favored
460a switch toward truncated forms (ENST00000545369,
461ENST00000405436) lacking the transmembrane domain,
462and therefore predicted to be secreted as soluble forms in
463the extracellular environment, with the potential of acting
464as dominant negative modulators of interferon signaling.
465In another example (the gene PSEN1, involved in intra-
466membrane proteolysis and cleavage of the intracellular
467domain of transmembrane proteins such as amyloid
468precursor protein and Notch and therefore a potential
469therapeutic target in BC ), the ERβ-induced splicing
470switch did not alter the open reading frame but caused
471the expression of a longer mRNA (ENST00000366783)
472compared to the isoform that was favored by ERα
473(ENST00000422240), possibly affecting the rate of transla-
474tion or stability of the mRNA.
475Another way of finding hints to the biological conse-
476quences of ERβ-specific splicing events described here is
477the reconstruction of pathways whose genes are differen-
478tially spliced following ERβ introduction in the cells. For
479instance, we found many genes involved in the Notch sig-
480naling pathway differentially spliced by ERβ: the above-
481mentioned PSEN2 is the catalytic subunit of the γ-secretase
482complex, responsible for intramembrane proteolysis of
483transmembrane receptors including Notch, resulting in
Dago et al. BMC Genomics _#####################_ Page 9 of 13
484 the release of Notch Intracellular Domain (NICD), which
485 migrates to the nucleus and regulates transcription of tar-
486 get genes . Also, NCOR2 is bound to the unliganded
487 CBF-1 transcription factor, a primary effector of Notch
488 signaling which acts as a repressor in unstimulated cells,
489 but is converted to an activator (dismissing the corepres-
490 sor NCOR2) after binding the NICD . Furthermore,
491 POGLUT1 has recently been shown to be an endoplasmic
492 reticulum O-glycosyl-transferase responsible for glycosy-
493 lation of Notch and required for its function , and
494 SLC35C2 is an endoplasmic reticulum transporter respon-
495 sible for accumulation of GDP-fucose, which is used for
496 Notch fucosylation, required for full activation . Fi-
497 nally, SGK1 has recently been shown to be a negative
498 regulator of Notch signaling by inhibiting γ-secretase ac-
499 tivity and promoting Notch degradation , and MAGI1
500 has been shown to recruit Notch ligand Dll1 to cadherin-
501 based adherens junctions, stabilizing it on the cell surface
502 . It is worth noting that the predominant Notch recep-
503 tor expressed in these cell lines was Notch2, which was
504 induced by E2 in wt cells (to a level slightly below the
505 chosen cut-off threshold, but highly statistically signifi-
506 cant: FPKM without E2: 49.826; FPKM with E2: 61.583,
507 FC 1.236, q-value 0.0005), while its basal expression
508 was lowered and its up-regulation smoothened in the
509 ERβ + cells.
510 A possible modulation of the Notch pathway by ERβ is
511 especially interesting as Notch is a known regulator of
512 breast development and maintenance of breast stem
513 cells ; alterations in the Notch pathway have been in-
514 volved in breast carcinogenesis, and in particular the
515 Notch pathway has been implicated in the development
516 of triple negative BC (TNBC), a particularly aggressive
517 form of BC which does not express ERα, progesterone
518 receptor (PR) or HER2, and which has shown resistance
519 to all known therapies . Targeting Notch signaling
520 has been proposed in TNBC. As these cancers are ERα-
521 negative, hormonal treatment is not currently used for
522 these patients; however, ERβ could be expressed in up to
523 50% of TNBCs [10,18], and its expression in TNBC has
524 been associated with better prognosis . Therefore,
525 ERβ may represent a potential new therapeutic target in
526 TNBC. The interrelation between ERβ and Notch in the
527 development and prognosis of triple-negative BC should
528 be investigated further in future research.
530 In conclusion, whole-genome analysis of early transcrip-
531 tion evens and mRNA processing associated with ERβ
532 confirmed a relevant role for this receptor in modulating
533 ERα-dependent transcription and splicing, but also
534 identified novel, ERβ-specific transcription and splicing
535 events, confirming a wide range of actions of ERβ in the
536 biology of BC.
537The data reported here confirm the complexity of
538estrogen action in BC cells and provide a comprehensive
539description of the effects of ERα and by ERβ on early tran-
540scription and splicing in hormone-responsive BC cells.
541More importantly, they provide a starting point to identify
542the events of ERβ-dependent splicing which are most
543significant for cancer biology.
545Human hormone-responsive BC cells
546Stable cell clones expressing either C-TAP-ERβ or N-TAP-
547ERβ (ERβ+) generated as previously described , and
548wild type (wt) MCF7 (ERβ-) cells were used for this
549study. All cell lines were maintained, propagated, hormone-
550starved for 5 days and analyzed for estrogen signaling as
551described earlier [21,45].
552Illumina Genome Sequencing RNA sequencing library
554Total RNA was extracted from hormone-starved cell
555cultures (+Ethanol -E2) or after 2 hours of stimulation
M 17β-estradiol (+E2), as described previously
557. RNA concentration in each sample was deter-
558mined with a NanoDrop-1000 spectrophotometer and
559the quality assessed with the Agilent 2100 Bioanalyzer
560and Agilent RNA 6000nano cartridges (Agilent Technolo-
561gies). Indexed libraries were prepared from 1 μg/ea. of puri-
562fied RNA with TruSeq Stranded total RNA Sample Prep
563Kit (Illumina) according to the manufacturer’s instructions.
564Libraries were sequenced (paired-end, 2×100 cycles) at a
565concentration of 8 pmol/L per lane on HiSeq1500 platform
566(Illumina) with a coverage of >70 million sequence reads/
567sample on average.
568Read alignment and transcript assembly
569TopHat v.2.0.10  was used to align all reads includ-
570ing junction-spanning reads back to the human genome
571(Homo sapiens Ensembl GRCh37, hg19). To identify the
572differentially expressed and spliced genes and isoforms
573between ERβ- and ERβ + cell lines we used Cuffdiff
574v2.1.1 . The parameters to define genes and isoforms
575as differentially expressed were the following: expression
576level threshold of 0.5 FPKM; q-value (FDR-adjusted p-
577value of the test statistic) ≤ 0.05 and |FC| ≥ 1.3. Moreover,
578to detect the ERβ- and ERβ + BC-specific splice events
579such as exon skip, exon inclusion, alternative splice sites
580and intron retention, we performed a direct comparison
581analysis using MATS v3.0.8 . To filter events with at
582least 10% change in exon inclusion level we set the MATS
583cutoff c, representing the extent of splicing change one
584wishes to identify, to 0.1, and FDR ≤ 0.05 to filter the iden-
585tified splice events.
Dago et al. BMC Genomics _#####################_ Page 10 of 13
586 Data access
587 RNA-Seq data have been deposited in the Gene Expression
588 Omnibus genomics data public repository (Q4 http://www.
589 ncbi.nlm.nih.gov/geo/) with Accession Number GSE64590.
590Q5 Additional files
593 Additional file 1: Figure S1. Description: KEGG pathway (Kanehisa et al.
594 Nucleic Acids Res 2014, 42:D199) analysis of ERα and ERβ interactors
595 involved in Spliceosome Pathway. Blue and red boxes highlight ERα and
596 ERβ interacting proteins, respectively.
597 Additional file 2: Tables S1. A: Ct-ERβ gene list. Genes with a FPKM
598 value ≥0.5, q-value ≤0.05 and |FC| ≥ 1.3 in the Ct-ERβ BC cell line. B:
599 Nt-ERβ gene list. Genes with a FPKM value ≥0.5, q-value ≤0.05 and
600 |FC| ≥1.3 in the Nt-ERβ BC cell line. C: MCF-7 wt gene list. Genes with a
601 FPKM value ≥0.5, q-value ≤0.05 and |FC| ≥1.3 in the wt MCF-7 BC cell
602 line. D: Commonly regulated genes in both ERβ + BC cell lines. Genes
603 consistently regulated in both ERβ + BC cells with a q-value ≤0.05 and
604 |FC| ≥1.3. E: Ct-ERβ isoform list. Isoforms with a FPKM value ≥0.5, q-value
605 ≤0.05 and |FC| ≥1.3 in the Ct-ERβ BC cell line. F: Nt-ERβ isoform list.
606 Isoforms with a FPKM value ≥0.5, q-value ≤0.05 and |FC| ≥1.3 in the
607 Nt-ERβ BC cell line. G: MCF-7 wt isoform list. Isoforms with a FPKM
608 value ≥0.5, FDR ≤0.05 and |FC| ≥1.3 in the wt MCF-7 BC cell line.
609Q6 Additional file 3: Tables S2. A: Skipping Exon Ct-ERβ. List of skipping
610 exon events in the Ct-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1). B: Mutually
611 Exclusive Exons Ct-ERβ. List of mutually exclusive exons events in the
612 Ct-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1). C: Retained Intron Ct-ERβ. List of
613 retained intron events in the Ct-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1).
614 D: Alternative 3’ splice site Ct-ERβ. List of Alternative 3’ splice site events in
615 the Ct-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1). E: Alternative 5’ splice site
616 Ct-ERβ. List of Alternative 5’ splice site events in the Ct-ERβ BC cell line
617 (FDR ≤0.05 and |c| ≥0.1). F: Skipping Exon Nt-ERβ. List of skipping exon
618 events in the Nt-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1). G: Mutually Exclusive
619 Exons Nt-ERβ. List of mutually exclusive exons events in the Nt-ERβ BC cell line
620 (FDR ≤0.05 and |c| ≥0.1). H: Retained Intron Nt-ERβ. List of retained intron
621 events in the Nt-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1). I: Alternative 3’ splice
622 site Nt-ERβ. List of Alternative 3’ splice site events in the Nt-ERβ BC cell line
623 (FDR ≤0.05 and |c| ≥0.1). J: Alternative 5’ splice site Nt-ERβ. List of Alternative
624 5’ splice site events in the Nt-ERβ BC cell line (FDR ≤0.05 and |c| ≥0.1). K:
625 Skipping Exon MCF-7 wt. List of skipping exon events in the wt MCF-7 BC cell
626 line (FDR ≤0.05 and |c| ≥0.1). L: Mutually Exclusive Exons MCF-7 wt. List of
627 mutually exclusive exons events in the wt MCF-7 BC cell line (FDR ≤0.05
628 and |c| ≥0.1). M: Retained Intron MCF-7 wt. List of retained intron events in the
629 wt MCF-7 BC cell line (FDR ≤0.05 and |c| ≥0.1). N: Alternative 3’ splice site
630 MCF-7 wt. List of Alternative 3’ splice site events in the wt MCF-7 BC cell line
631 (FDR ≤0.05 and |c| ≥0.1). O: Alternative 5’ splice site MCF-7 wt. List of Alternative
632 5’ splice site events in the wt MCF-7 BC cell line (FDR ≤0.05 and |c| ≥0.1).
633 Additional file 4: Table S3. Discordant Isoforms ERβ+/ERβ-. List of all
634 isoforms with opposite regulation pattern in ERβ+/ERβ-.
635 Additional file 5: Tables S4. A: Differential Promoter Usage Ct-ERβ.
636 Gene list with differential promoter usage (DPU) in the Ct-ERβ BC cell line
637 (FDR ≤0.05). B: Differential Promoter Usage Nt-ERβ. Gene list with DPU in
638 the Nt-ERβ BC cell line (FDR ≤0.05). C: Differential Promoter Usage MCF-7
639 wt. Gene list with DPU in the wt MCF-7 BC cell line (FDR ≤0.05). D: Differential
640 Spliced Gene Ct-ERβ. Gene list differentially spliced (DSGs) in the Ct-ERβ BC
641 cell line (FDR ≤0.05). E: Differential Spliced Gene Nt-ERβ. Gene list differentially
642 spliced (DSGs) in the Nt-ERβ BC cell line (FDR ≤0.05). F: Differential Spliced
643 Gene MCF-7 wt. Gene list differentially spliced (DSGs) in the wt MCF-7 BC cell
644 line (FDR ≤0.05).
645 Additional file 6: Table S5. PSD3 TSSs expression values. Transcript
646 starting sites (TSSs) expression value list of the PSD3 gene, expressed in
647 FPKM with relative FC, FDR and p-value in Ct-ERβ, Nt-ERβ and MCF-7 wt
648 BC cell lines.
649 Additional file 7: Tables S6. A: ERα binding sites in Ct-ERβ. Gene list of
650 DSGs including ERα binding sites in a 10 kb window around the gene in
651 the Ct-ERβ BC cell line. B: ERβ binding sites in Ct-ERβ. Gene list of DSGs
652including ERβ binding sites in a 10 kb window around the gene in the
653Ct-ERβ BC cell line. C: ERα binding sites in Nt-ERβ. Gene list of DSGs including
654ERα binding sites in a 10 kb window around the gene in the Nt-ERβ BC cell
655line. D: ERβ binding sites in Nt-ERβ. Gene list of DSGs including ERβ binding
656sites in a 10 kb window around the gene in the Nt-ERβ BC cell line. E: ERα
657binding sites in MCF-7 wt. Gene list of DSGs including ERα binding sites in a
65810 kb window around the gene in the wt MCF-7 BC cell line.
659Additional file 8: Figure S2. Heat map representing the clustered density
660matrix of ERα and ERβ binding sites. Chromatin immunoprecipitation of ERα
661and ERβ in Ct-ERβ and wt MCF-7 cells show different ER binding profiles
662(highlighted by square brackets). In the clustering, each line represents a
663genomic location of a binding site with its surrounding ±1.5 kb region. This
664matrix was subjected to k-means clustering.
666ER: Estrogen receptor; Nt-ERβ: N-TAP-estrogen receptor beta; Ct-ERβ:
667C-TAP-estrogen receptor beta; E2: 17β-estradiol; BC: Breast cancer;
668FPKM: Fragment per kilobases of exon per million; DSGs: Differentially spliced
669genes; TSSs: Transcripts starting sites; DPU: Differential promoter usage.
671The authors declare that they have no competing interests.
673All authors participated in conception and design of the study and
674manuscript drafting and revision, GN, MR, FR and RT preformed in vitro
675experimental work and sequencing, DND, AR, DM and GG performed the
676statistical and bioinformatic analyses, CS preformed functional investigations
677on alternatively splices genes, CS and AW coordinated and finalized figure
678preparation, manuscript drafting and revision. All authors read and approved
679the final manuscript.
681Work supported by: Italian Association for Cancer Research (Grants IG-13176),
682Italian Ministries of Education, University and Research (Grants 2010LC747T,
683RBFR12W5V5_003 and PON03PE_00146_1) and Health (Young Researcher
684Grants GR-2011-02350476 and GR-2011-02347781), National Research Council
685(Flagship Project Interomics), the University of Salerno (FARB 2014). G.N. is
686supported by a ‘Mario e Valeria Rindi’ fellowship of the Italian Foundation for
687Cancer Research; A.R. is a PhD student of the Research Doctorate in ‘Molecular
688and Translational Oncology and Innovative Medical-Surgical Technologies’,
689University of Catanzaro ‘Magna Graecia’; C.S. is supported by the UCLA Scholars
690in Oncologic Molecular Imaging Fellowship (National Institutes of Health R25
Q2Laboratory of Molecular Medicine and Genomics, Department of Medicine
694and Surgery, University of Salerno, Via S. Allende, 1, Baronissi, SA 84081, Italy.
UFR Sciences Biologiques, Université Peleforo Gon Coulibaly, Korhogo, Ivory
Department of Molecular and Medical Pharmacology, University of
697California, Los Angeles, USA. 4
Molecular Pathology and Medical Genomics,
698“SS. Giovanni di Dio e Ruggi d’Aragona - Schola Medica Salernitana” Hospital
699of the University of Salerno, Salerno, Italy.
700Received: 30 December 2014 Accepted: 17 April 2015
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Dago et al. BMC Genomics _#####################_ Page 13 of 13
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