170,870 kb. ZNF44. PSMB1. TBP. 2.09x. 1.14x. 1.24x. F. AIRE-seq PU.1 VDR GABP. A. RNA-seq. ChIP-seq ChIP-seq ChIP-seq. VDR type persistent persistent.
1
Seuter et al.: Suppements: GABPA-VDR crosstalk
SUPPLEMENTARY DATA SUPPLEMENTARY TABLE LEGENDS Table S1: Dicer substrate siRNA oligonucleotides (IDT, Leuven, Belgium). Gene
IDT catalog no.
Oligonucleotide sequences (5’-3’)
GABPA
hs.Ri.GABPA.13.1
GAUUACUCAUGUCAGACCAAGAATT AAUUCUUGGUCUGACAUGAGUAAUCUA
GABPA
hs.Ri.GABPA.13.2
GUAUUGGCAAGUCAAGAACAACAGA UCUGUUGUUCUUGACUUGCCAAUACAU
GABPA
hs.Ri.GABPA.13.3
GUGACUUGAAGACUCUUAUUGGATA UAUCCAAUAAGAGUCUUCAAGUCACAG
negative control
DS NC1
CGUUAAUCGCGUAUAAUACGCGUAT AUACGCGUAUUAUACGCGAUUAACGAC
Table S2: Reverse transcription qPCR primers. Fragment size (bp)
Annealing temperature (°C)
B2M1,2
246
61
GGCTATCCAGCGTACTCCAAA CGGCAGGCATACTCATCTTTTT
CD14
142
60
ACGCCAGAACCTTGTGAGC GCATGGATCTCCACCTCTACTG
FBP12
102
60
AAACACGCCATCATAGTGGAAC TCCAACGGACACAAGGCAATC
GABPA2
133
61
TTGGCAAGTCAAGAACAACAGA GCGCTCTTTGTACTTTGGCT
GAPDH1,2
113
61
CATGAGAAGTATGACAACAGCCTAG TCCTTCCACGATACCAAAGT
HBEGF2
175
60
CAAGGAGGAGCACGGGAAAAG CCCATGACACCTCTCTCCA
HPRT11,3
94
61
TGACACTGGCAAAACAATGCA GGTCCTTTTCACCAGCAAGCT
LRRC254
148
60
CTCCACTCCCGACTATGAGAAC GTTACAGTAGACAGGCTGGGAAG
MPC12
47
64
AGTCTCCAGAGATTATCAGTGGG GCAACAGAGGGCAAATGTCAT
NDUFA22
107
60
GCAGCAAGTCGAGGAGTCG CGTTTCTCAATGAAGTCCCTGA
SLC25A152
212
60
CATCAGGGAAGATAGCCAAGAGC CAAAGGTACAGGGCCTAATTCAT
Gene
1
Primer sequences (5’-3’)
reference gene sequences obtained from PrimerBank (http://pga.mgh.harvard.edu/primerbank) 3 see Vandesompele, Genome Biol 3, R34 (2002) 4 designed with Primer3Plus (http://www.bioinformatics.nl/cgi-bin/primer3plus/primer3plus.cgi) 2
Seuter et al.: Suppements: GABPA-VDR crosstalk
2
Table S3: GABPA cistrome in human monocytes. THP-1 cells were treated for 24 h with 1,25(OH)2D3 (1,25D) or vehicle (EtOH) and chromatin was extracted for GABPA ChIP-seq analysis. The position of the peaks, their binding strength (BS) in the presence and absence of ligand and the resulting FC as well as their overlap with GABPA ChIP-seq from K562 cells are indicated. Based on VDR [29], PU.1 [14], CTCF [42] ChIP-seq the co-localization with the respective transcription factor binding sites are marked as well as the overlap with open chromatin as assessed by FAIRE-seq [30]. Moreover, overlap with a TSS and the distance of the closest 1,25(OH)2D3 target gene (based on RNA-seq data [30]) are indicated. GABPA sites that overlap with VDR alone or with VDR and PU.1 are shaded in green and purple, respectively.
Seuter et al.: Suppements: GABPA-VDR crosstalk
3
SUPPLEMENTARY FIGURE LEGENDS Figure S1: Characterization of the GABPA cistrome in human monocytes. The overlap of the 3,822 GABPA ChIP-seq sites that were detected in THP-1 cells both after 24 h treatment with 1,25(OH)2D3 and vehicle are visualized by Venn diagrams. The absolute number as well as the overlap percentage with open chromatin (A, measured by FAIRE-seq) or PU.1 (B) and CTCF (C) are indicated as well as with persistent (D), transient (E) or “24 h only” (F) VDR peaks. It should be noted that for a group of overlapping peaks the numbers are not exactly the same, since one peak of either transcription factor may overlap with more than one peak of the other.
Figure S2: Example GABPA loci overlapping with VDR sites. THP-1 cells were treated 24 h with vehicle (EtOH) or 1,25(OH)2D3 (1,25D) and ChIP-seq, FAIRE-seq and RNA-seq were performed. The IGV browser [36] was used to visualize the genomic context of GABPA loci overlapping with persistent (A, B), transient (C, D) or “24 h only” (E) VDR binding sites. The peak tracks display data from ChIP-seq for GABPA (blue), VDR (red), PU.1 (purple) and FAIRE-seq (cyan). Gene structures are shown in blue and example peaks are shaded in grey. RNA-seq data indicate the inducibility of the genes after 24 h ligand stimulation 1,25(OH)2D3. Significantly regulated genes (p < 0.05) are highlighted in red.
Figure S3: Pathway analysis based on VDR-PU.1-GABPA co-localization and 1,25(OH)2D3 target genes. GO analysis using GOrilla of 1,25(OH)2D3 target genes in a distance of less than 400 kb from genomic loci, at which VDR and PU.1 either do (left) or do not (right) co-localize with GABPA. GO processes that are significantly (p < 0.0005) associated with the two classes are listed.
Figure S4: Functional impact of GABPA on VDR-regulated transcription. THP-1 cells were transfected with a mixture of three DsiRNA oligonucleotides directed against the
Seuter et al.: Suppements: GABPA-VDR crosstalk
4
GABPA gene or with the negative control DsiRNA (NC1) and after 24 h they were stimulated with vehicle (EtOH) or 100 nM 1,25(OH)2D3 (1,25D) for another 24 h. qPCR was performed in order to determine changes in the expression of GABPA (A) and seven 1,25(OH)2D3 target genes (B) normalized by the three reference genes B2M, GAPDH and HPRT1. The displayed GABPA mRNA expression levels relative to the three housekeeping genes represent the average of four independent experiments and bars indicate standard deviations (A). For the 1,25(OH)2D3 target genes the mean mRNA expression changes after GABPA knockdown are shown (B). Two-tailed Student’s t-tests were performed to determine the significance of the mRNA changes by the GABPA knockdown in reference to control DsiRNA-transfected cells (* p < 0.05).
Fig. S1 A
B
4.7%
47,826
2,385
FAIRE
D
1,437
391
119
87,123
GABPA
3,703
VDRpersistent GABPA
1,828
PU.1
E
23.3%
C
2.1%
1,994
35,593
GABPA
245
23.3%
3,577
VDRtransient GABPA
783
CTCF
F
11.6%
1,864
2.2%
3,039
GABPA
4.2%
8,660
378
23.3%
3,444
VDR”24 h only” GABPA
Fig. S2
A
Chr 19: :
125,470 kb
12,390 kb
12,380 kb
B
Chr 6:
12,400 kb
170,860 kb
170,870 kb
125,570 kb
GABPA ChIP-seq
ETOH
VDR ChIP-seq
[0-3]
ETOH
[0-3]
1,25D [0-10] [0-10]
1,25D ZNF44 2.09x
RNA-seq
PSMB1 1.14x
TBP 1.24x
PU.1 ChIP-seq FAIRE-seq
[0-10]
ETOH
ETOH
[0-10]
1,25D [0-5] [0-5]
1,25D VDR type
C
persistent
Chr 13:
GABPA ChIP-seq
ETOH
VDR ChIP-seq
41,330 kb
ETOH
41,340 kb
41,350 kb
41,360 kb
persistent
41,370 kb 145,550 kb
D
Chr 8:
145,570 kb
145,580 kb
145,590 kb
145,600 kb
[0-3] [0-3]
1,25D [0-5] [0-5]
1,25D MRPS31 1.11x
RNA-seq
SLC25A15 1.26x
TMEM249 SLC52A2 1.01x FBXL6 1.36x 1.19x
PU.1 ChIP-seq FAIRE-seq
[0-10]
ETOH
ETOH
ADCK5 1.02x
[0-10]
1,25D [0-4] [0-4]
1,25D VDR type
transient
E
Chr 6:
99,840 kb
99,830 kb
24 h only
99,850 kb
transient
99,860 kb
transient
99,870 kb
99,880 kb
GABPA ChIP-seq
ETOH
VDR ChIP-seq
[0-3]
ETOH
[0-3]
1,25D [0-5] [0-5]
1,25D COQ3 1.51x
PNISR 0.95x
PU.1 ChIP-seq FAIRE-seq
[0-10]
ETOH
ETOH
[0-10]
1,25D [0-4]
1,25D VDR type
[0-4] 24 h only
24 h only
99,890 kb
Fig. S3 GABPA
no GABPA Tricarboxylic acid cycle
p-value < 0.0005
Citrate metabolic process
p-value = 1
Regulation of interleukin-10 secretion Toll-like receptor 4 signaling pathway Mitochondrial electron transport cytochrome c to oxygen Olfactory bulb development Protein targeting to mitochondrion B cell receptor signaling pathway Positive regulation of mammary gland epithelial cell proliferation Regulation of Notch signaling pathway Brown fat cell differentiation Response to muramyl dipeptide Positive regulation of nitric-oxide synthase biosynthetic process
0.02
0.01
0
DsiRNA -50
1,25D target gene SLC25A15
*
FBP1
*
HBEGF
*
LRRC25
50
MPC1
*
NDUFA2
100
CD14
A
% expression change after KD
* -46% * -42%
0.03
GABPA
NC1
relative expression
Fig. S4
B EtOH
1,25D
* *
0
*
