Supplementary Figure Legends
Supplementary Figure S1 (a) T47D cells transfected with control or SUPT6H
siRNA or treated with ICI 182780 were analyzed by qPCR for TTP and DSP gene
expression. (b-d) MCF7 cells transfected with control or single siRNAs for SUPT6H
were analyzed by qPCR (b-c) and Western blot (d). ESR1 mRNA levels (c) as well
as ERα protein levels (d) were not affected with SUPT6H knockdown. HSC70 is
shown as a loading control.
For RNA experiment, MCF7 cells transfected with
control or SUPT6H siRNAs, were grown for 8 h and then switched to hormone
deprived medium for 14 h. Cells were then stimulated with 10 nmol/L 17β-estradiol
(E2) for 2 h and expression levels were analyzed. Gene expression levels were
normalized to 18S ribosomal mRNA, graphed relative to the control sample and
expressed as “Relative mRNA Expression”; mean values + s.d., n = 3. For protein
samples, MCF7 cells transfected with control or SUPT6H siRNA pool or single
SUPT6H siRNAs were grown for 24 h and then harvested. (e) SUPT6H recruitment
on the 3’ ends of estrogen target genes, GREB1 and PGR. MCF7 cells were
transfected and cultured as in Figure 1(a), except that cells were stimulated with 10
nmol/L 17β-estradiol (E2) for 2 h. ChIP samples were normalized to input samples
and expressed as “% Input”; mean values + s.d., n = 3. The dotted line indicates the
background binding as measured by the average signal of non-specific IgG binding
across all samples and sites.
Supplementary Figure S2 MNase samples were also analyzed with a Bioanalyzer
and the nucleosome profile was plotted.
Supplementary Figure S3 SUPT6H knockdown affects RNF40 and H2Bub1 levels
in ERα negative breast cancer MCF10A (mammary epithelial) cells. RNF40 mRNA
levels were modestly affected with SUPT6H knockdown. MCF10A cells transfected
with control or SUPT6H siRNAs were grown for 48h and then analyzed by Western
blot and qPCR. Gene expression levels were normalized to 18S ribosomal mRNA,
graphed relative to the control sample and expressed as “Relative mRNA
Expression”; mean values + s.d., n = 3.
Supplementary Figure S4 Immunohistochemical analysis of human breast tissue
sections.
(a) Staining intensities of SUPT6H and H2Bub1 in individual tissue
sections with hormone receptor status. (b) Overall intensity gradient of SUPT6H and
H2Bub1 in tissue sections classified on the basis of receptor status. (c) Intensity
gradient and (d) 2D plot of SUPT6H and H2Bub1 and in tissue sections divided into
various grades. (e) Expression levels of ACTA2 and CK19 in T47D cells upon ICI
182780 treatment. T47D cells were transfected with control or treated with ICI
182780, grown for 24 h before switching to hormone-deprived medium and grown for
another 24 h. Cells were then stimulated with 10 nmol/L 17β-estradiol (E2) for 48 h
and the expression levels of ACTA2 and CK19 were analyzed by qPCR. Gene
expression levels were normalized to 18S ribosomal mRNA, graphed relative to the
control sample and expressed as “Relative mRNA Expression”; mean values + s.d.,
n = 3.
Supplementary Figure S5 H3 ChIP was performed on MCF7 cell extracts from
Figure 1c. (a) H3 levels on TSS of various estrogen target genes, CXCL12, GREB1,
PGR and TFF1. (b) H3K27me3 levels shown in Figure 6a were normalized to total
H3 levels. ChIP samples were normalized to input samples and expressed as “%
Input”; mean values + s.d., n = 3. The dotted line indicates the background binding
as measured by the average signal of non-specific IgG binding across all samples
and
sites.
Supplementary Table S1.
List of ChIP Primers used in this Study
Gene
Forward Primer (5´to 3´)
Reverse Primer (5´to 3´)
Source
GREB1 ERE
CCTGGGAATGGAGATTTTGATA
GAGCTGCGAGTCCCTAACAG
(15)
PGR ERE
GGCCAGCAGTCCTGCAACAGTC
CCCAAGCTTGTCCGCAGCCTT
(15)
CXCL12 TSS
GCAGTGCGCTCCGGCCTTT
CCTCACTGCAGACCGGGCCA
(15)
GREB1 TSS
GCCAAATGGAAGAAGGACAG
ACCACCTACCTCCAGTCACC
(15)
PGR TSS
GTGCGTGTGGGTGGCATTCTC
GCGGGAGCACTAGCCGCC
This study
TFF1 TSS
CCTGGATTAAGGTCAGGTTGGA
TCTTGGCTGAGGGATCTGAGA
(15)
PDK4 BV
GCGTCGAGGCTCCAGGGCT
GCCCAAGCTGGGTCCTAGGGTT
(18)
PPARG BV
AGCCGCTCCGGGGGAACTT
ACAGGGCCTGGCCAGCTACAA
(18)
RASD1 BV
CGGCCACCCTCACCTTCTCCT
GATCTGCTGCCTGAGCCGCTG
(18)
Supplementary Table S2.
List of qPCR Primers used in this Study
Gene
Forward Primer (5´to 3´)
Reverse Primer (5´ to 3´)
Source
ACTA2
ACCTTTGGCTTGGCTTGTCA
GGAAGCTTTAGGGTCGCTGG
This study
CK19
GAATCGCAGCTTCTGAGACCA
CTGGCGATAGCTGTAGGAAGTC
This study
CXCL12
TGCCAGAGCCAACGTCAAGCATC CGGGTCAATGCACACTTGTCTGTTGT
(15)
DSP
AAAGCCCAGCAGATCCACT
TGGCGGCGTTTAGCATCATA
This study
GREB1
GTGGTAGCCGAGTGGACAAT
ATTTGTTTCCAGCCCTCCTT
(15)
HNRNPK
ATCCGCCCCTGAACGCCCAT
ACATACCGCTCGGGGCCACT
(18)
PGR
TCCACCCCGGTCGCTGTAGG
TAGAGCGGGCGGCTGGAAGT
(15)
PPARG
ACCTCCGGGCCCTGGCAAAA
TGCTCTGCTCCTGCAGGGGG
(18)
RNF40
AGTACAAGGCGCGGTTGA
GAAGCAGAAAACGTGGAAGC
(15)
RUNX2
GCGGTGCAAACTTTCTCCAG
GCAGCCTTAAATGACTCTGTTGG
This study
SUPT6H
GAAAACGCACCTCTTTTGATG
CGTCCTCGTCATCTGACATTT
This study
TTP
ACCTCTCCTGAGGGGGAATC
GCAACGGCTTTGGCTACTTG
This study
18S rRNA
AACTGAGGCCATGATTAA
GGAACTACGACGGTATCTGA
This study