8Br-cAMP was purchased from Sigma (St. Louis, MO). Silencer Negative Control sirna #1 and

Transcription

1 1 Supplemental information 2 3 Materials and Methods 4 Reagents and animals 5 8Br-cAMP was purchased from Sigma (St. Louis, MO). Silencer Negative Control sirna #1 and 6 Silencer Select Pre-designed sirna for NR5A2 (s5380 and s5381; Ambion, Carlsbad, CA). Immature 7 C57BL/6J mice (21 28 d of age) were purchased from Charles River Laboratory (Wilmington, MA). 8 Fresh-frozen rabbit ovary was purchased from Funakoshi Co. (Tokyo, Japan). Fresh-frozen human ovary 9 was purchased from ILSbio (ILS7190 N03-DS1; Chestertown, MD) RACE 12 Total RNAs were extracted from immature C57BL/6J mice (21 28 d of age) ovary and fresh-frozen 13 rabbit ovary. The 5 RACE-Ready cdnas were synthesized using the GeneRacer Kit (Invitrogen) 14 or SMARTer RACE cdna Amplification Kit (Clontech Laboratories, Inc., Mountain View, CA) 15 according to the manufacturer s instructions. Cap site cdna dt Rat Ovary was purchased from 16 Nippon Gene (Tokyo, Japan). PCR for RACE was performed in a 50 μl reaction mixture comprising 17 KOD -Plus- (Toyobo). The gene-specific primers for RACE are listed in Supplemental Table

2 19 Progesterone production 20 KGN cells were infected with the recombinant adenoviruses. Culture media were collected at 48 h 21 after infection for the measurement of progesterone production by an ELISA kit (Cayman Chemical 22 Co., Ann Arbor, MI) according to the manufacturer s instructions Supplemental figure legends 25 Supplemental FIG. 1. Identification of novel LRH-1 transcripts in rodent and rabbit ovaries. 26 Genomic structure, nucleotide and deduced amino acid sequences of rat (A), mouse (B) and rabbit (C) 27 ovarian LRH-1 are shown. Exons are shown as filled boxes. The novel exon (exon 2o or exon 3o) is 28 shown as an open box. The reported TSS and ovarian TSS are shown as arrows Supplemental FIG. 2. Human ovarian LRH-1 promotes expression of steroidogenesis-related genes 31 in KGN cells. A, Activation of promoter activities of CYP11A1, HSD3B2 and StAR by granulosa 32 cell-derived LRH-1 (gc-lrh-1). KGN cells were transiently transfected with reporter plasmids ( ng) and expression vector (5 ng). KGN cells were treated with or without 8Br-cAMP (1mM) for 12 h 34 before measurements of luciferase activities. Luciferase activities were measured and relative 35 activities are shown. B, Effects of sirna for LRH-1 on endogenous LRH-1 and SF-1 in KGN cells. 36 Synthetic sirnas for LRH-1 (s5380: silrh-1 #1; s5381: silrh-1 #2) or control (sicontrol) were 2

3 37 introduced into KGN cells. C, Effects of sirna on expression of steroidogenesis-related genes in 38 KGN cells. KGN cells were treated with or without 8Br-cAMP (1mM) for 12 h before RNA 39 extraction. The gene-specific primers for RT-PCR are listed in Supplemental Table 1. Lanes C, #1 and 40 #2 represent sicontrol, silrh-1 #1 or #2 transfection. Each value represents the mean ± SE of three 41 independent experiments. *, P < 0.05; and **, P < 0.01 vs. control Supplemental FIG. 3. Identification of the transcription factor binding sites of a GC box (A and B) 44 and a putative SF-1 binding site (C and D) by EMSA. Each end-labeled oligonucleotide was 45 incubated with μg nuclear extracts from KGN cells (A), fresh-frozen human ovary (B), 46 HEK293 cells transfected with pcmv-tag3b-sf-1 (C) or HEK293 cells transfected with 47 pcmv-tag3b-gc-lrh-1 (D). DNA-protein complexes were separated by electrophoresis on a 48 nondenaturing 4% (A) or 6% (B-D) polyacrylamide gel. Unlabeled wild-type (WT) and mutated 49 (Mut) oligonucleotides were used as competitor DNAs. Wild-type and mutated oligonucleotides 50 sequences are shown in the left panel. Bold capital letters indicate mutated sequences. Where 51 indicated, antibodies against Sp1, Sp3 or LRH-1 were used for supershift analysis. Arrows indicate 52 specific DNA-protein complexes. Arrowheads indicate supershifted complexes Supplemental FIG. 4. A, Effect of mutation in the GC box within the promoter region of human 3

4 55 ovarian LRH-1 in KGN cells. The mutant promoter constructs used are drawn schematically. B, 56 Dose-dependent effects of SF-1 on the promoter activity of human ovarian LRH-1 in KGN cells. 57 Reporter plasmids (100 ng) and expression vectors (0, 1, 2.5, 5 ng) were transiently transfected into 58 KGN cells. The total amount of transfected plasmid was adjusted with an empty vector. Luciferase 59 activities were measured and relative activities are shown. Each value represents the mean ± SE of 60 three independent transfection experiments. Letters indicate a significant difference (P < 0.05) Supplemental FIG Progesterone production was augmented with PGC-1α in KGN cells. KGN cells were infected with 64 Adx-GFP or Adx-PGC-1α. Medium was collected at 48 h after infection for measurement of 65 progesterone at the end of culture. Progesterone levels were measured by an ELISA kit. Each value 66 represents the mean ± SE of three independent experiments. N.D., Not detected. 4

5 67 Supplemental Figure 1 68 A new exon (exon 3o) 3o ovarian LRH-1 exon 3 B new exon (exon 3o) 3o ovarian LRH-1 exon 3 C new exon (exon 2o) 2o ovarian LRH-1 exon 2 5

6 69 Supplemental Figure 2 70 A Relative luciferase activity (Fold) * ** CYP11A1 2.3kb HSD3B2 1.25kb ** pcdna3 gc-lrh-1 pcdna3 + camp gc-lrh-1 + camp ** ** ** ** ** StAR 1.3kb B C 1.0 LRH SF-1 Relative mrna level ( LRH-1 / β-actin) sicontrol ** ** silrh-1 #1 silrh-1 #2 Relative mrna level ( SF-1 / β-actin) sicontrol silrh-1 #1 silrh-1 #2 LRH-1 SF-1 CYP11A1 HSD3B2 StAR CYP19A1 β-actin camp(-) camp(+) C #1 #2 C #1 #2 6

7 71 Supplemental Figure 3 72 A GC box WT : gccccgaggaggcggaggca Mut1: gccccttggaggcggaggca Mut2: gccccgattaggcggaggca Mut3: gccccgaggttgcggaggca Mut4: gccccgaggagttggaggca Mut5: gccccgaggaggcttaggca Mut6: gccccgaggatttttaggca probe -72/-53-72/-53 antibody competitor Sp1 Sp3 Sp3 super shift B probe -72/-53 antibody competitor Sp1/Sp3 Sp C probe -157/ /-130 competitor - - D probe -157/-130 antibody competitor - - SF-1 binding sites WT : ttttttaaccctgacctcctcctcgcag Mut1: ttttttaaccctgaaatcctcctcgcag Mut2: ttttttaaaactgacctcctcctcgcag Mut3: ttttttaaaactgaaatcctcctcgcag Mut4: ttttttaaccctgacctaataatcgcag Mut5: ttttttaaaactgaaataataatcgcag SF-1 LRH-1 7

8 73 74 Supplemental Figure 4 A B -67/+68-57/+68 pgl3 Basic Relative luciferase activity (Fold) GC box X a a a a Luc -67/+68 Mut-GC box Luc Luc Luc a a Relative luciferase activity (Fold) b c c b b c SF pgl3 Basic -205/+68 (ng) 8

9 75 Supplemental Figure Progesterone (ng/mg protein) N.D. GFP PGC-1α 9

10 77 Supplemental TABLE 1. Nucleotide sequences of oligonucleotides used in PCR Usage Sense Antisense RACE-PCR Rat 5'-RACE 1 st PCR 5'-GATGCTAGCTGCGAGTCAAGTC-3' 5'-CAGACACTTTATCGCCACACACAGG-3' Rat 5'-RACE 2 nd PCR 5'-CGAGTCAAGTCGACGAAGTGC-3' 5'-GTTCCCCATGCGATCGGACCAGTCC-3' Mouse 5'-RACE 1 st PCR 5'-CGACTGGAGCACGAGGACACTGA-3' 5'-CAGACACTTTATCGCCACACACAGG-3' Mouse 5'-RACE 2 nd PCR 5'-GGACACTGACATGGACTGAAGGAGTA-3' 5'-GTTCCCCATGCGATCGGACCAGTCC-3' Rabbit 5'-RACE 1 st PCR 5'-CTAATACGACTCACTATAGGGC-3' 5'-CATGCGGTCGGCTCTTACAGCTTCC-3' Rabbit 5'-RACE 2 nd PCR 5'-AAGCAGTGGTATCAACGCAGAGT-3' 5'-TCCACACACGGGGCACAGCTCCTCG-3' RT-PCR SF-1 5'-ACCACATCTACCGCCAGGTCCAG-3' 5'-TACTCCTTGGCCTGCATGCTCAG-3' CYP11A1 5'-GAAAGGAAGTGTTCACCACG-3' 5'-TAGTGTCTCCTTGATGCTGG-3' HSD3B2 5'-GTGACAGGAGCAGGAGGG-3' 5'-CTGGGTACCTTTCACATTGACAT-3' StAR 5'-GAGAGTCAGCAGGACAATGG-3' 5'-CTGGTTGATGATGCTCTTGG-3' CYP19A1 5'-GTGGACTTGGTCATGCGCA-3' 5'-TCATCATCACCATGGCGATG-3' 10