Supplementary Information
ASXL2 promotes proliferation of breast cancer cells by
linking ERα to histone methylation
Ui-Hyun Park, Moo-Rim Kang, Eun-Joo Kim, Young-Soo Kwon, Wonhee Hur, Seung Kew
Yoon, Byung-Joo Song, Jae Ho Park, Jin-Taek Hwang, and Soo-Jong Um*
*To whom correspondence should be addressed. E-mail: umsj@sejong.ac.kr
1
MATERIALS AND METHODS
Cell line and cell culture
MCF7 and HEK293 cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM)
(WelGENE Inc.) containing 5% fetal bovine serum (FBS) and an antibiotic–antimycotic mixture
(Invitrogen) in a 5% CO2 atmosphere 37°C. For transcription assays, MCF7 cells were
maintained in DMEM phenol red-free media with charcoal-treated FBS.
Plasmids and cDNA constructions
All cDNA was constructed according to standard methods and verified by sequencing. The
multicopy yeast expression plasmids used in the two-hybrid assays were described elsewhere.1
Deletion or point mutants of the desired genes were created by PCR amplification and
subcloned into the pBTM116 or pASV3 vector. Flag (2X)-tagged hASXL2, mASXL2 (or PHDdeleted mutant ASXL2ΔPHD), LSD1, and UTX wild-type (wt) and mutant (mt: H1146A) were
placed on pcDNA3 vector (Invitrogen). For GST-fused proteins, pGEX4T-1 (GE Healthcare)
were used. For His-tagged ERα and LSD1 cDNA, an E. coli expression system was used
(Novagen). Details of plasmid constructions are available upon request.
Yeast-two hybrid (Y2H) assays
Yeast reporter strain L40 was used to Y2H assays. As prey, human full-length ASXL2 and its
deletion derivatives were introduced in pASV3 yeast expression vector containing VP16-AD
domain. As bait, ERα was introduced into pBTM116 yeast expression vector coding LexADBD domain. Cotransformation of both bait and prey were performed into L40. After culture of
L40 in the absence and presence of cognate ligands (1 μM of E2), L40 cells were extracted
using lysis buffer (250 mM Tris-Cl, pH 8.0, 1 mM EDTA, and 1 mM PMSF). The level of
interaction was determined by quantitative β-galactosidase (β-gal) assays.
Glutathione S-transferase (GST) pull-down assay
ERα and LSD1 proteins were induced in E. coli (BL21) using pET15b His-tag vector (Novagen)
and purified with Ni-NTA affinity column (GE Healthcare) by standard methods. Either GST or
GST-fused ASXL2 (amino acids 916–1083, 1-703, or 319-703) was expressed in E. coli and
purified on glutathione-Sepharose beads (GE Healthcare) using standard methods. An
approximately equal amount of GST or GST-fusion was mixed with His-tagged protein in the
2
absence or presence of 17β-estradiol (E2, Sigma-Aldrich) and 4-hydroxytamoxifen (OHT,
Sigma-Aldrich) depending on experimental conditions. Bound proteins were detected by
Western blotting (WB) analysis using indicated antibodies.
Glycerol gradient ultracentrifugation
Dialysis buffers (10 mM Tris-Cl pH 7.9, 150 mM NaCl, 1.5 mM MgCl2, 0.2 mM EDTA, 1 mM
PMSF, and protease inhibitor) containing 50, 40, 30 and 20% glycerol were prepared and
layered from bottom to top in 50 ml of ultracentrifuge tube (Beckman). Total 4 mg of nuclear
extracts were loaded on the top of the glycerol gradient. Tube was centrifuged using Ti75 rotor
18000rpm for 18 h at 4°C by ultracentrifuge (Beckman). Fractions (0.5 ml) were obtained and
visualized by resolving SDS-PAGE and WB using indicated antibodies.
Western blotting (WB) and immunoprecipitation (IP) assays
WB and IP were performed as reported previously.2 For WB analysis, cells were lysed in TENmodified buffer (50 mM Tris-Cl, pH 7.5, 150 mM NaCl, 0.1% Nonidet P-40, 5 mM EDTA, and
1 mM PMSF) supplemented with protease inhibitors (Roche Molecular Biochemicals). Proteins
were separated by electrophoresis on SDS-polyacrylamide gels, transferred to PVDF membrane
(Millipore), and incubated with primary antibodies as indicated. The blots were then incubated
with HRP-conjugated mouse or rabbit IgG secondary antibody (Invitrogen). Protein bands were
detected using the WEST-ZOL plus Western Blot Detection System (Intron Biotechnology). For
IP assays, HEK293 cells were transfected with the indicated plasmid vectors, washed with icecold PBS, and lysed in TEN-modified buffer supplemented with a protease inhibitor cocktail.
The lysates were pre-cleared for 90 min with protein A/G PLUS agarose beads (Santa Cruz
Biotechnology), and incubated with beads and a 1:200 dilution of the indicated antibodies for 2
h at 4°C. After washing beads once with TEN and twice with PBS, the immune complexes were
released from the beads by boiling and analyzed by WB using the indicated antibodies. The
endogenous interaction between ERα and ASXL2 in MCF7 cells was determined by IP with
anti-ASXL2 antibody followed by WB using anti-LSD1, anti-UTX or anti-ERα antibody.
Antibodies
Antibodies used in the study were as follows: anti-Flag, anti-β-actin (Sigma-Aldrich), anti-ERα
(H-184), anti-HSP70, anti-RIF1, anti-BAP1, anti-GST (Santa Cruz Biotechnology), anti-LSD1,
anti-UTX, anti-MLL2, anti-H3K9me3, anti-H3K9me2, anti-H3K27me3, anti-H3K27me2
3
(Abcam), anti-H3K4me3, antiH3K4me2 (Millipore), anti-Myc (abm), anti-ER (GeneTax) and
anti-ASXL2 (mouse monoclonal antibody raised against amino acids 1213–1396, Bethyl).
Luciferase reporter gene assay
Either MCF7 or HEK293 cells were seeded in a 12-well culture plate, and transiently
transfected with ERα expression vector, ERE-tk-luciferase reporter, and SV40-drivengalactosidase expression vector as an internal control using Lipofectamine Plus reagent
(Invitrogen). After 4 h transfection, cells were washed, fed with DMEM phenol red-free
medium containing 5% charcoal-striped FBS, and incubated for an additional 16 h. Depending
on the experimental conditions, 10 nM E2 or various concentrations of OHT was treated. Cells
were then washed with cold PBS, resuspended in luciferase lysis buffer (Promega), and
subjected to three freeze-thaw cycles. Luciferase (Luc) activity was measured using an
analytical luminescence luminometer (Promega) according to the manufacturer’s instructions. βGalactosidase (β-gal) activity was determined using a microplate reader at 405 nm. Luciferase
activity was normalized to β-gal activity.
RNA interference (RNAi)
Strand sequences of the custom siRNA duplex against ASXL2 (Invitrogen) were shown in
Supplementary Table S6b. Transfection of the siRNA was performed with Lipofectamine 2000
in Opti-MEM I reduced-serum medium (Invitrogen) according to the manufacturer’s
instructions. For the depletion of ASXL2, LSD1, UTX and MLL2 using small hairpin RNA (sh
RNA), the synthetic oligonucleotides were shown in Supplementary Table S6c. Each duplex
was formed and digested with HindIII and BamHI and ligated into the digested pSilencer 2.1U6 hygro (Ambion). pSilencer hygro luciferase was used as a control (shLuc). The MCF7derived knockdown stable cell lines were generated by selecting resistant colonies against
hygromycin (A.G. Scientific) at 0.1 mg/ml. The knockdown efficiency was monitored by WB
analysis using anti-AXL2 antibody.
Chromatin immunoprecipitation (ChIP) assays
ChIP assay was performed as described previously.16 Either mock or ASXL2-depleted MCF7
cells were adapted in DMEM phenol red-free medium containing 5% charcoal-striped FBS for 2
days, and treated with 10 nM E2 or 100 μM OHT for 30 min. Cross-linked, sheared, chromatin
complexes were recovered by IP with indicated antibodies. Cross-linking was then reversed
4
according to Upstate’s protocol. The DNA pellets were recovered and analyzed by qPCR using
a primer pair that encompasses the EREs of the indicated gene promoters (Supplementary Table
S6d). Ratios of fold enrichment from each antibody were calculated from Ct values normalized
against Ct of IgG. Percentages of input were calculated and displayed.
Microarray analysis
Total RNA was extracted from either control (shLuc) or stably ASXL2-depleted MCF7 cells.
RNA samples with an RNA integrity number (RIN) greater than 9 were used for microarray
experiment using Agilent Whole Human Genome Microarray Kit, 4x44K. Equal amounts of
total RNA were amplified, labeled, hybridized, washed, and scanned. The LOWESS (locally
weighted linear regression curve fit) and normalization methods were applied to the ratio of the
signal intensities generated in the microarrays. Results were filtered and the cut off was set 2
fold difference. Clustering analysis and Heat map generation were performed with Java Tree
View 1.1.6 (Sun Microsystems). Genes exhibiting significant differences in expression level
were
classified
into
Gene
(http://www.geneontology.org),
Ontology
KEGG
(GO)-based
functional
(http://www.genome.jp/kegg/)
and
categories
DAVID
Bioinformatics Resources (http://david.abcc.ncifcrf.gov/).
Gene set enrichment analysis (GSEA)
GSEA was performed with Java GSEA software v2.0.13 (http://www.broadinstitute.org/gsea).
Normalized gene expression profiles were ranked with signal to noise metric and enrichment
scores (ES) were calculated with random gene set permutation 1000. Gene sets were created
with genes identified as common target of ERα and ASXL2 (ER.ASXL2.CHIPSEQ gene set)
within range of -50kb and +50kb from TSS. Genes changed 2 fold of expression by E2
stimulation were obtained from our results and GSE2225, which were created with gene sets of
ER.2FOLD.ARRAY and GSE2225E2UPDN. Created gene sets were added to gene set file
msigdb.v4.0.symbols.gmt for GSEA. Significance were considered in case of less than 0.05 of
nominal p-value (Nom p-value) and 0.25 of false discovery rate (FDR).
MTT assays
The proliferation of ASXL2-depleted MCF7 cells was monitored by 3-(4,5-dimethylthiazol-2yl)-2,5-diphenyltetrazolium bromide (MTT) (Sigma-Aldrich) assay. Either control (mock and
shLuc) or ASXL2-depleted cells were plated at 500 cells/well in 96-well tissue culture plate,
5
treated with DMSO or 10 nM E2, and incubated for 7 days under hygromycin selection (0.1
mg/ml). Cells were switched to fresh medium every 2 days. Then, 50 μl of MTT solution (2
mg/ml in PBS) was added to the culture medium and the reaction mixture was incubated at
37C in a 5% CO2 atmosphere for 4 h. The optical density was measured spectrophotometrically
at 550 nm. Each experiment was performed in triplicate and repeated a minimum of three times.
Colony formation assays
Either control (mock and shLuc) or ASXL2-depleted MCF7 cells were seeded at 2,000
cells/well in a 6-well plate, treated with DMSO or 10 nM E2 for 12 days under 0.1 mg/ml
hygromycin selection. Cells were switched to fresh medium every 2 days, fixed with 3.7%
paraformaldehyde for 10 min, stained with 0.05% of crystal violet (CV) for 20 min, washed
with dilute water, and photographed. After photograph, CV stain were solved in 99% methanol
for 5 min and measured in OD. 540.
Confocal Microscopy
MCF7 cells were treated with 10 nM of E2 and incubated for 24h. Cells were washed with PBS,
fixed with 4% paraformaldehyde in PBS for 10 min and permeabilized with 0.1% Triton X-100
in PBS. Cells were blocked for 60 min in PBS containing 2% bovine serum albumin and then
incubated for 2h with rabbit anti-ERα (1:100) and mouse anti-ASXL2 (1:25, mouse monoclonal
antibody). Cells were then incubated with Texas Red-conjugated anti-rabbit antibody (1:200:
Santa Cruz Biotechnology) and FITC-conjugated anti-rabbit antibody (1:200; Santa Santa Cruz
Biotechnology) for 1h in dark, stained with DAPI (1μg/ml) for 5min, mounted with VectaShield,
and observed by confocal microscope (Leica)
Cell invasion assay
Cells were suspended and plated in 24-well BME coated chamber (Trevigan, Gaithersburg, MD)
and allowed to migrate for 24 h. Cells migrated though BME chambers were fixed with 4%
paraformaldehyde for 10 min and stained with 0.05% CV for 30 min at RT. Invasive cells were
monitored by microscopy and reported as percent control.
Cell migration assays
MCF7 cells were seeded in 6-well plates and grown to 90–95% confluency. Subsequently, two
6
scratches were placed in the middle of the well with a sterile 200µl pipette tip. After washing
once growth media, cells were incubated for 48 h and photographed by microscopy (Leica).
Pictures were manually processed using ImageJ3 and reported as percent of control.
Survival analysis
Published microarray results for breast tumor tissues (GSE4922 datasets) were downloaded
from Gene Expression Omnibus (GEO, www.ncbi.nlm.nih.gov/geo). Kaplan-Meier survival
analysis was performed with survival data from downloaded data sets using R v2.14.1, Survival
package, and BioConductor software (www.bioconductor.org). Log-ranked p-values were
calculated with the survdiff function. All plots for survival rates were generated using R. Other
experimental procedures are listed in the Supplemental Information.
Exome sequencing
MCF7 cells were prepared according to an Agilent SureSelect Target Enrichment Kit
preparation guide. The libraries were sequenced with Illumina HiSeq 2000/2500 sequencer.
Exome-sequencing data of T47D cells was prepared using GSE48215 dataset. Sequencing data
were
analyzed
using
BWA
bwa.sourceforge.net/bwa.shtml),
(Genome
Analysis
Toolkit,
(Burrows-Wheeler
Picard
Alignment
Tool,
(http://broadinstitute.github.io/picard/),
https://www.broadinstitute.org/gatk/),
(http://snpeff.sourceforge.net/SnpEff.html)
7
and
http://bioGATK
SnpEff
Supplementary Figure 1 (Related to Figure 1). E2-dependent interaction of ERα with ASXL2.
(a) Schematic diagram of ASXL2 and its deletion derivatives. Three conserved ASXN, ASXM,
8
and plant homeodomain domains (PHDs) among the ASXL family are represented by closed
boxes. The previously identified NR-binding motif is indicated by a gray box.
(b) E2-dependent interaction between ERα and ASXL2 in yeast. Yeast two-hybrid and β-gal
assays were performed using LexA DBD-fused ERα and VP16 AD-ASXL2 (and its truncations)
in the absence and presence of E2 (1 μM). Fold change in β-gal activity is represented by the
average of three independent experiments (mean ± s.d.). Abbreviations: e, VP16 AD empty
vector; L2, VP16 AD-fused hASXL2 full-length; I–VI, shown in Supplementary Fig S1A.
(c) Schematic diagram of regions responsible for the ERα-ASXL2 interaction.
(d) Subcellular colocalization of ERα and ASXL2 in MCF7 cells. ERα and ASXL2 were
visualized by staining with rabbit anti-ERα polyclonal antibody and ASXL2 mouse monoclonal
antibody. Each bar represents 10 μm.
(e) Expression of ASXL1, ASXL3, and ERα in four breast cancer cell lines
(f) Interaction with ERα and ASXL1 or ASXL3. HEK293 cells were transiently transfected with
expression vectors for Flag-ASXL1 and Flag-ASXL3 C-terminal fragment (amino acids 1457–
2248). IP using anti-Flag antibody was followed by WB using anti-ERα antibody.
(g) Interaction with ERβ. MCF7 cell lysates were immunoprecipitated with anti-ASXL2
antibody in the presence of 10 nM E2. Subsequently, WB was followed using anti-ERα and
anti-ERβ antibodies.
(h) Interaction in vitro. GST pull-down assays were employed using a purified GST-ASXL2
fragment (amino acids 916–1083) and in vitro-translated ERβ in the presence of 5 μM E2.
(i) OHT sensitivity in yeast. Yeast two-hybrid assays were performed using LexA DBD-fused
ERα and VP16 AD-ASXL2 in the presence of E2 (1 μM), OHT (5 μM), and E2+OHT (1 μM +
5 μM). Fold change in β-gal activity is represented by the average of three independent
experiments (mean ± s.d.).
(j) OHT sensitivity in vitro. GST pull-down assays were performed using GST-fusion ASXL2
fragment and in vitro-translated ERα in the absence and presence of E2 and OHT.
(k) Effect of ASXL2 depletion on the expression of E2/ERα target genes in T47D cells. Under
two different ASXL2 depletion conditions using specific sh-RNA for mouse and human (mh),
and for human only (h), RT-qPCR was performed using primer sets specific for TFF1 and
GREB1c. Error bars represent the mean ± s.d. (n = 3, *p < 0.05).
9
Supplementary Figure 2 (Related to Figure 2). ChIP-sequencing analysis.
(a) Venn diagram of co-bound peaks of ERα and ASXL2 upon E2 stimulation.
10
(b) Venn diagram of genes identified as co-bound peaks of GEO1ER and ERα (left) and ASXL2
(right) upon E2 stimulation.
(c) Genome distribution of GEO1ER-bound peaks upon E2 stimulation.
(d) Cumulative level of ASXL2, H3K4me3, H3K9Ac, H3K9me3, H3K27me3, GEO1ER tag
counts per base pair are shown around ERα peak positions.
(e) Validation of ChIP-seq data. Immunoprecipitated DNAs were subjected to quantitative PCR
using primer sets specific to the putative ERE-containing region of indicated genes.
Supplementary Figure 3 (Related to Figure 3). Validation of ASXL2 complex.
(a) Schematic representation of the ASXL2 complex.
(b) Endogenous interaction between ASXL2 and LSD1 (or UTX). MCF7 cell lysates were
prepared and immunoprecipitated with pre-immune serum (IgG) or an anti-ASXL2 antibody in
the presence of 10 nM E2. Subsequent WB was followed using anti-LSD1 and anti-UTC
antibodies.
(c) Direct interaction between ASXL2 and LSD1. GST pull-down assays were employed using
purified His-LSD1 and GST-ASXL2 fragments (aa 319–703 and 1–703). Bound protein was
visualized by WB using an anti-LSD1 antibody.
11
Supplementary Figure 4 (Related to Figure 4). Positive role of ASXL2 complex in ERα
activation.
(a) Effect of ASXL2 knockdown on LSD1-enhanced ERα activation. Luciferase assays were
performed according to the conditions indicated.
(b) Effect of pargyline on ERα activation. MCF7 cells were treated with 10 nM E2 and/or 0.25
mM (+) and 1 mM (++) of pargyline, an inhibitor of LSD1.
(c) Effect of UTX on ERα activation. HEK293 cells were transfected according to the
conditions indicated.
(d) Knockdowns of LSD1, UTX, and MLL2. Depletion was monitored by RT-qPCR. Error bars
represent the mean ± s.d ( n = 3, *p < 0.01).
(e, f) Knockdown effect of LSD1, UTX, and MLL2 on the expression of CTSD (e) and GREB1
(f). RT-qPCR was performed using primer sets specific for CTSD and GREB1. Error bars
represent the mean ± s.d. (n = 3, *p < 0.01).
12
Supplementary Figure 5 (Related to Figure 5). ASXL2-regulated histone H3 methylation at
the ERα target promoter.
(a) ASXL2-dependent recruitment of LSD1 and UTX to the TFF1 promoter. T47D cells
transiently transfected with either shLuc or shASXL2 were used for ChIP assays.
(b) Effect of ASXL2 knockdown on the recruitment of LSD1 and UTX. GREB1c was used
instead of TFF1 in MCF7 cells.
(c) Effect of ASXL2 knockdown on the occupancies of di- and trimethylated (me2 and me3,
respectively) histone H3K9 and H3K27 in GREB1c promoter.
13
Supplementary Figure 6 (Related to Figure 6). Critical role of ASXL2 PHD finger in ERα
activation and regulation of H3K5 methylation.
(a) Expression of Flag-tagged ASXL2 wild-type (Wt) and mutant (ΔPHD).
(b) Preferential binding of the ASXL2 PHD finger to H3K4me2. Purified GST-ASXL2 PHD
(aa 1213–1435) was incubated with 0.5 mg biotinylated histone tail modified in H3K4me1, me2,
me3, and an unmodified H3 tail. Immunoprecipitated beads were washed, eluted by boiling, and
visualized using an anti-GST antibody.
(c) Effect of LSD1 knockdown on the occupancies of ASXL2, H3K4me2, and H3K9me2 at
TFF1 promoter.
(d) Effect of MLL2 knockdown on the occupancy of H3K4me3 at TFF1 promoter. Error bars
represent the mean ± s.d. (n = 3, *p < 0.01 and **p < 0.05).
14
Supplementary Figure 7 (Related to Figure 7). Effect of ASXL2 depletion on cell fate.
(a) Effects of ASXL2 depletion on cell migration. MCF7 mock, shLuci, and shASXL2 stable
cells (90~95% confluence) were scratched using 200 μl tip, and incubated for 48 h. Captured
images were analyzed by ImageJ. Error bars represent the mean ± s.d. (n = 3, * p<0.05
compared to mock).
(b) Effects of ASXL2 knockdown on invasion. Indicated cells were incubated for 24 h, stained
for 30 min by CV staining and stained cells were analyzed.
(c) Quantification of colony formation assays. Crystal violet was solved in 99% methanol for 5
min and measured in OD at 540nm. Error bars represent the mean ± s.d. *p<0.05.
15
Supplementary Figure 8 (Related to Figure 8). Correlation of LSD1 and UTX with ERα in
breast cancer.
(a) Positive correlation of ERα expression with LSD1 and UTX. Expression patterns of LSD1
and UTX mRNA were adopted from a published GEO data set (GSE2990) of patients with
breast cancer.
(b) Kaplan-Meier survival analysis of patients with ERα-positive breast cancer using the
GSE4922 dataset.
(c) GO analysis of co-regulated genes.
(d–e) Validation of microarray-ChIP seq combined data. Effect of ASXL2 knockdown on the
E2-dependent expression of NCOA3 (d), CA4 (f), and RSP6KB1 (h). Expression was measured
by RT-qPCR. Effect of ASXL2 knockdown on the LSD1 and UTX binding to the NCOA3
promoter (e). Binding of LSD1 and UTX to the CA4 (g) and RSP6KB1 (i) promoters. Promoter
binding was analyzed by ChIP-qPCR.
16
Supplementary Table S6. Primer and strand sequences used for assays.
Table S6a. Primer sequences used for RT-qPCR
Gene
Forward primer (5’ to 3’)
Reverse primer (5’ to 3’)
TFF1
CCATGGAGAACAAGGTGATCTG
GTGACACCAGGAAAACCACAATT
CTSD
CCAGAACATCTTCTCCTTCTACCTGA
GTAATACTTGGAGTCTGTGCCACC
ASXL2
CAGCACCAGCAGCCATTTCAG
GGAAAACGAGCCCTGGGAGAG
LSD1
GGAAATGACTATGATTTAATGGCTCA
GC
GTATGTTCTCCMGCAAAGAAGAGT
C
ERα
GATCCACCTGATGGCCAA
GCTCCATGCCTTTGTTACTCA
NCOA3
ATGAGACCCCGGACAAACAC
TGGGCGACCATTTGAGCAT
GREB1c
GCAAAGATTCCCCGAAGTGC
AAGGTGACTGAAGCTGGTCC
CA4
CACTGGTGCTACGAGGTTCA
TTTGCCTTGGTGGTGACGAT
BMP7
GAGTGTGCCTTCCCTCTGAAC
GAATTCTCGGAGGAGCTAGTGG
RPS6KB1
TACAGAGACCTGAAGCCGGA
ACTCCACCAATCCACAGCAC
GAPDH
CTGCACCACCAACTGCTTAGC
GGGCCATCCACAGTCTTCTGG
MLL2
TTCAGCGTTGAGGCAGAGAG
CTGCTGGTGGTAACGGAACT
UTX
AGCATTTGTGAAGTGGAGGTT
AGACTTGCATCAGGTCCTCC
Table S6b. Strand sequences of siRNA
Gene
Sense strand (5’ to 3’)
Antisense strand (5’ to 3’)
ASXL2
GAGCUUUAGGAGGACCCAUUCUGUA
Control
GAGGAUUGGAGCCCACUUAUUCGUA
17
UACAGAAUGGGUCCUCCUAAAGCU
C
UACGAAUAAGUGGGCUCCAAUCCU
C
Table S6c. Strand sequences used for shRNA expression
Gene
Sense strand (5’ to 3’)
Antisense strand (5’ to 3’)
ASXL2
(human/
mouse)
ASXL2
(humanspecific)
GATCCAACCCCAGGAACTTCTTTA
TTCAAGAGATAAAGAAGTTCCTGG
GGTTTTTTTTGGAAA
GATCCAGGGGGATGGGAGCGCTCA
TTCAAGAGATGAGCGCTCCCATCC
CCCTTTTTTTGGAAA
GATCCGTTACTCGAGAAATATGATT
CTCAAGAGAAATCATATTTCTCGAG
TAATTTTTTGGAA A
GATCCGCTATGAAGACAACGACTAT
GCGAACATAGTCGTTGTCTTCATAG
C TTTTGGAAA
GATCCGCTGGCCAAGATCAAGCAA
ATTCAAGAGATTTGCTTGATCTTGG
CCAGTTTTTTGGAAA
GATCCGCACAGTTCAACTATACATG
CTCAAGAGAGTGTCAAGTTGATAT
GTACTTTTTTGGAAA
AGCTTTTCCAAAAAAAACCCCAGGA
ACTTCTTTATCTCTTGAATAAAGAA
GTTCCTGGGGTTG
Luciferase
MLL2
LSD1
UTX
18
AGCTTTTCCAAAAAAAGGGGGATGG
GAGCGCTCATCTCTTGAATGAGCGC
TCCCATCCCCCTG
AGCTTTTCCAAAAAATTACTCGAGA
AATATGATTTCTCTTGAGAATCATATT
TCTCGAGTAACG
AGCTTTTCCAAAAGCTATGAAGACA
ACGACTATGTTCGCATAGTCGTTGTC
TTCATAGC CG
AGCTTTTCCAAAAAACTGGCCAAGA
TCAAGCAAATCTCTTGAATTTGCTTG
ATCTTGGCCAG CG
AGCTTTTCCAAAAAAGTACATATCA
ACTTGACACTCTCTTGAGCATGTATA
GTTGAACTGTGCG
Table S6D. Primer sequences used for ChIP-qPCR analysis
Forward primer (5’ to 3’)
Reverse primer (5’ to 3’)
Distal
AGTCCAGGGGACCAACTG
GGAGGTTAAGTCAGGAGAAT
ERE
GGCCATCTCTCACTATGAATC
GGCAGGCTCTGTTTGCTTAAA
Coding
ATCCCTGACTCGGGGTCG
TGGCACAAAACAGGTGCTCA
GREB1c
GTGGCAACTGGGTCATTCTGA
CGACCCACAGAAATGAAAAGG
APPBP2
AGTCAGCAGAGATGACCAGA
TTGAGTCCCCATCCTGCCT
CA4
TTAGACGAGCAGGGACAGC
GGGAGAATCAGAGCCTGGA
BMP7
GAGCC ATTTCCTCACCTG
ATAGTGGTCAGGGTGGTCTT
CYP24A1
CTGGTTGGGCTTCATCAGA
GTGGCCAGTAGAAAAGCCC
BCAS1
TGCCTGTTTCTCAGGCTCA
GGCAAGAGGCAACATGACT
RPS6KB1
CTGGCTTATTCTGGGCAACT
GACCTGCCAAGGGCTCCT
PFDN4
ATGTGCAGTGATGCTAATGAAT
TGCCTGTGGCCAGTAGAAAA
BRIP1
GAAGGGATTAGTTGAAATGTCA
GTAATGTGAATGTGGGCATG
Locus
TFF1
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