Legend for Supplemental Figures and Tables

Transcription

1 Legend for Supplemental Figures and Tables Supplemental Fig. 1. Negative regulation of the CYP27B1 promoter in a ligand-dependent manner (A) OK-P cells were transfected with pcdna-trα, pcdna-trβ1 or pcdna3 (mock). After 24 h, cells were harvested and subjected to Western blot analysis using a TRα/β-antibody or β-actin-antibody. (B) ph27b-1.6k was co-transfected with pcmv-β, psg5-rxrα, and pcdna-trβ1 into OK-P cells. The cells were then treated with the indicated concentrations of (B) T 3 or Triac, (C) the TR antagonist 1-85 and/or T 3 (1nM) for 17 h., P <.1 compared with vehicle controls. (D) pnp-2.6k-ex2 was co-transfected with pcmv-β, psg5-rxrα, and pcdna-trβ1 into OK-P cells. The cells were then treated with the indicated concentrations of 1-85 and/or T 3 (1nM) for 17 h. Cell lysates were assayed for β-gal and luciferase activity., P <.1. Supplemental Fig. 2. Regulation of the CYP27B1 promoter by (A) Deletion constructs were generated by PCR amplification or restriction enzyme digestion as described in the Materials and methods section and as illustrated in the left-hand panels. (B) OK-P cells were transfected with ph27b-2 or ph27b-2 del-44-27, pcdna-h (1-449) at increasing concentrations from 2 to 2 ng. (C) The (1-449) fragment was subcloned into the pbapo- CMV expression vector (TaKaRa). Transfection was carried out with 1. μg of pbapro- expression plasmid pre-mixed with Lipofectamine 2, according to the manufacturer s instructions. Twenty-four hours after transfection, cells were treated with 1.8 μg/ml Puromycin dehydrochloride (Sigma). RT-PCR and quantitative PCR analyses were performed as described in the Materials and methods section. (D) ph27b-1.6k was co-transfected with pcdna-trβ1, psg5-rxrα, pcdna h (1-449), and pcmv-β into HKC-8 cells. The cells were then treated with or without T 3 (1nM) for 17 h followed by a luciferase assay. Empty pcdna3 vector was used as a mock control vector to maintain a constant concentration of DNA in all transfections. Each reporter construct was cotransfected with (1-449) or pcdna3 (mock) into OK-P cells. Cell lysates were assayed for β- gal and luciferase activity. Each point represents the average of quadruplicate analysis ± SEM, normalized for β-gal activity. Similar results were obtained from three independent experiments., P <.1 compared with the mock construct. Supplemental Fig. 3. Effects of TRβ1/RXRα on the binding of to the 1α-nTRE (A) The TRα, TRβ1, RXRα and proteins were synthesized separately using the TNT Quick Coupled Transcription/Translation System, pcdna-trα, pcdna-trβ1, psg5-rxrα and pcdna h (1-449). The identities of the in vitro translated proteins were confirmed by Western blotting and by probing with specific antibodies. (B) EMSAs were performed with 32 P-labelled 1α-nTRE as a probe and in vitro synthesized TRβ1, RXRα, and (1-449). (2 μl) was used in lanes 3-6. TRβ1/RXRα (each 2 μl) was used in lane 2, and (each 1, 2 and 3 μl) was used in lanes 4-6. Unprogrammed reticulocyte lysate was added to normalize the amount of lysate in each reaction. The locations of the DNA-protein complex bands are indicated by arrows. Supplemental Fig. 4. Nucleotide sequence of the CYP27B1 promoter region (A) Nucleotide sequence of the human CYP27B1 gene promoter region. Nucleotides are numbered with the transcription start site designated as +1. The underlined ATG under Met indicates the translation start site. Boxes indicate putative binding motifs for various transcription factors and the ntre containing the ER-7 sequence. (B) Nucleotide sequences of the human and mouse CYP27B1 gene promoter regions. Nucleotides are numbered with the transcription start site designated as +1. Boxes indicate putative binding motifs for various transcription factors. The ER-7 in the human CYP27B1 gene promoter is highly homologous to the ER-9 in the mouse CYP27B1gene promoter. The consensus ER-7 and -9 sites in the human and mouse sequences are indicated by arrows. The SRE and TATA box regions have only one or two nucleotide mismatches in the human and mouse CYP27B1 genes. Asterisks indicate consensus nucleotides in the human and mouse CYP27B1 promoter sequences.

2 Supplemental Table 1. Oligonucleotides used in Real-time PCR Supplemental Table 2. Oligonucleotides used in reporter plasmid construction Supplemental Table 3. Oligonucleotides used in mutagenesis Supplemental Table 4. Sequences of the oligonucleotides used for EMSAs

3 Supplymental Figure 1 M.Kozai et al. A TRβ1 TRα Mock TRα TRβ1 5kD β-actin 5kD 37kD B Relative activity (RLU/β-gal (x1 3 )) T 3 Triac Ligand (log M) C 12 1 Relative repression (%) T TR antagonist (1-85)(μM) ph27b-1.6k D Relative luc actyvity (fold increase) T 3 TR antagonist (1-85)(μM) pnp-2.6k-ex2

4 Supplemental Fig. 2 M.Kozai et al. A Relative activity (RLU/β-gal (x1 3 )) k +3 ph27b-1.6k -1.1k ph27b-1.1k -4 ph27b-4-2 ph27b-2-8 ph27b-8-6 ph27b-6 Mock B -2 1α-SRE TATA-box -5-2 ph27b ph27b-2 del Relative activity (RLU/β-gal (x1 3 )) Mock (1-449) TATA-box Mock (1-449) ph27b-2 ph27b-2 del C Relative mrna levels β-actin (1-449) Mock CYP27B1 SCD TRβ RXR T D Relative luciferase activity (fold increase)

5 Supplymental Figure 3 M.Kozai et al. A TRα kDa TRβ kDa 5kDa 5kDa RXRα kDa 75kDa 5kDa 5kDa B Probe TRβ1+RXR 1α-nTRE TRβ1/RXR

6 Suppl. Figure S4 M.Kozai et al. SRE TATA-box A gaaaatcttaggccctccctcccatgagggtcattgcaacatgagacccaa GC-box (Sp1) gggagttgtgaagtcagccccagccccgcctactgttcctgggtgctaatc CRE (CREB) CCAAT-box (NF-Y) cccagcacagaccactcaggaggagggattggctgaggagcttggagaggg ntre (ER7) +1 ggcgtcatcacctcacccaaaggttaaataggggttgagatatgatgctca SRE (SREBP) TATA-box Met GGAGAAGCGCTTTCTTTCGCGAGCACCCTGAACCAGACCATG B CRE (CREB) Human -159 gagacccaagggagttgtgaagtcagccccagccccgcctactgttcctgggtgctaat * * * * * * Mouse -156 CCAAT-box (NF-Y) Human -11 ccccagcacagaccactcaggaggagggattggctgaggagcttggagagggggcgtca * * * * * Mouse gctacacagaccact--tgcaaagggattggctgaagagcttggaga-ggggcgtct ER-7 +1 Human -43 tcacctc-ac-ccaaaggttaaataggggt tgag-atatgatgctcaggaga * * * * * * * * * * * Mouse -49 tcacctccaggaccaagggtatatatgggtccaggcaagtgcagaagatgctctggaaa ER-9 +1

7 Table S1 M.Kozai et al. Table S1. Oligonucleotides used in Real-time PCR Gene Name Accession No. Sense Antisense mouse CYP27B1 NM_19 5'-ATGGTGAAGAATGGCAGAGG-3' 5'-TAGTCGTCGCACAAGGTCAC-3' mouse CYP24A1 NM_9996 5'-TGCCATTCACAACTCGGACCC-3' 5'-TCAAGCCAGCGTTCGGGTCTAA-3' mouse Dio1 NM_786 5'-CCAGGGAGAGTCAAACAGAGCATCC-3' 5'-ACTGGATGCTGAAGAAGGTGGGGAC-3' mouse β-actin NM_7393 5'-CTGACCCTGAAGTACCCCATTGAACA-3' 5'-CTGGGGTGTTGAAGGTCTCAAACATG-3' human CYP27B1 NM_785 5'-TGGCCCAGATCCTAACACATTT-3' 5'-GTCCGGGTCTTGGGTCTAACT-3' human NM_ '-GGAGCCATGGATTGCACTTT-3' 5'-ATGTGGCAGGAGGTGGAGAC-3' human SCD1 NM_563 5'-ACCATTACAGCGCCTCCCTCCAG-3' 5'-TTCCAAGTAGAGGGGCATCGTC-3' human β-actin NM_111 5'-GGCACCACACCTTCTACAATGAGC-3' 5'-AGCCTGGATAGCAACGTACATGGC-3'

8 Table S2 M.Kozai et al. Table S2. Oligonucleotides used in reporter plasmid construction Name Sequence h27b1-1.6k-s h27b1-2-s h27b1-155-s h27b1-114-s h27b1-87-s h27b1-6-s h27b1-44-as h27b1-27-s 5'-GAAGATCTCTCCTCAGCTGGAATGAGG-3' 5'-GAAGATCTTAGGCCCTCCCTCCCATGAGG-3' 5'-GAAGATCTCAAGGGAGTTGTGAAGTCAGC-3' 5'-GAAGATCTGGGTGCTAATCCCCAGCAC-3' 5'-GAAGATCTCAGGAGGAGGGATTGGCTG-3' 5'-GAAGATCTTGGAGAGGGGGCGTCATCACC-3' 5'-TGACGCCCCCTCTCCAAGCTC-3' 5'-TTAAATAGGGGTTGAGATATG-3' h27b1-ex1-as 5'-GAAGATCTGGGTGCTCGCGAAAGAAAGC-3'

9 Table S3 M.Kozai et al. Table S3. Oligonucleotides used in mutagenesis Name GS125-S Sequence 5 -TGCATCACCTGTGGATCCTGCAAGGGCTTC-3 GS125-AS 5 -GAAGCCCTTGCAGGATCCACAGGTGATGCA-3 R429Q-S 5 -GTGACAGACCTGCAGATGATTGGAGCT-3 R429Q-AS 5 -AGCTCCAATCATCTGCAGGTCTGTCAC-3 E457A-S 5 -CCTCTGTTCTTAGCAGTATTTGAGGAC-3 E457A-AS 5 -GTCCTCAAATACTGCTAAGAACAGAGG-3

10 Table S4 M.Kozai et al. Table S4. Sequences of the oligonucleotides used for EMSAs Gene Name Sense Antisense 1α-nTRE 5'-CGTCATCACCTCACCCAAAGGTTAAATAG-3' 5'-CTATTTAACCTTTGGGTGAGGTGATGACG-3' M1 5'-CGTCATCACCTCACCCAAAAATTAAATAG-3' 5'-CTATTTAATTTTTGGGTGAGGTGATGACG-3' M2 5'-CGTCATCACCTCACCCGGAGGTTAAATAG-3' 5'-CTATTTAACCTCCGGGTGAGGTGATGACG-3' M3 5'-CGTCATCACCTCATTCAAAGGTTAAATAG-3' 5'-CTATTTAACCTTTGAATGAGGTGATGACG-3' M4 5'-CGTCATCATTTCACCCAAAGGTTAAATAG-3' 5'-CTATTTAACCTTTGGGTGAAATGATGACG-3' M5 5'-CGTCATTGCCTCACCCAAAGGTTAAATAG-3' 5'-CTATTTAACCTTTGGGTGAGGCAATGACG-3' DR-4 5'-TCGAGCTTCAGGTCACAGGAGGTCAGAGAC-3' 5'-GTCTCTGACCTCCTGTGACCTGAAGCTCGA-3' LDLR-SRE 5'-TTTGAAAATCACCCCACTGCAAACT-3' 5'-AGTTTGCAGTGGGGTGATTTTCAAA-3' FAS-SRE 5'-GCGCGGGCATCACCCCACCGACGGC-3' 5'-GCCGTCGGTGGGGTGATGCCCGCGC-3'