New PAH gene promoter KLF1 and 3’-region C/EBPalpha motifs influence transcription in vitro
New regulators of PAH gene transcription involve promoter c.-170C variant and 3’-region VNTRs
Journal of Applied Genetics
Supplementary material are intended to be made available through the online edition
Kristel Klaassena, Biljana Stankovica, Nikola Kotura, Maja Djordjevicb, Branka Zukica, Gordana Nikcevica, Milena Ugrina, Vesna Spasovskia, Sanja Srzentica, Sonja Pavlovica,Maja Stojiljkovica
aInstitute of Molecular Genetics and Genetic Engineering, University of Belgrade, Vojvode Stepe 444a, 11010 Belgrade, Serbia; TEL: +(381)11-3976-445; FAX: +(381)11-3975-808
bMother and Child Health Care Institute of Serbia „Dr Vukan Cupic“, School of Medicine, University of Belgrade, Radoja Dakića 6-8,11070 Belgrade, Serbia; TEL: +(381)11-3108-276; FAX: +(381)11-3108-276
Corresponding author: Maja Stojiljkovic, PhD, Senior Research Associate
Institute of Molecular Genetics and Genetic Engineering, University of Belgrade
Vojvode Stepe 444a, 11010 Belgrade, SERBIA
Tel: (+381) 64-2202-373, Fax: (+381) 11-3975-808
E-mail: ;
Oligonucleotides used for PCR and sequencing reactions of selected PAH gene noncoding regions, for reporter construct cloning and for EMSA and supershift analysis are given together with conditions for PCR amplification.
PAHgene promoter region (from c.-40 do c.-551 relative to the transcription initiation site) was amplified using KAPATaq DNA Polymerase (Kapa Biosystems, Wilmington, MA, USA) with specific primers (Table 1). The PCR conditions were as follows: denaturation step at 95 °C for 5 min, 35 cycles of amplification (95 °C for 30 sec, 60 °C for 30 sec and 72 °C for 45 sec) and elongation step for 10 min at 72 °C.
PAH gene VNTR region was amplified with HotStarTaq DNA polymerase (Qiagen, Hilden, Germany) using amplification primers shown in Table 1. The PCR conditions were as follows: denaturation step at 95 °C for 15 min, 50°C for 1 min, 72°C for 2 min, 35 cycles of amplification (92 °C for 25 sec, 50 °C for 25 sec and 72 °C for 40 sec) and elongation step for 7 min at 72 °C.
Table 1. Primers used for PCR and sequencing reactions of selected PAH gene noncoding regions.
Name / Sequence (5´>3´) / Fragment size (bp)PAH prom FW / 5’-CAGCAAGGCAGTGTGCTTAG -3’ / 512
PAH prom RV / 5’-CTCAGGTACAGGCAGGTTTG -3’
VNTR3 FW / 5’-GCTTGAAACTTGAAAGTTGC-3’ / 400 - 550380 - 530
VNTR3 RV / 5’-GGAAACTTAAGAATCCCATC-3’
For expression analysis of PAH promoter and VNTR region, we have used primers shown in Table 2. For PAH promoter, the region from c.-551 to c.-40 was subcloned into pBLCAT5 vector to generate pBLCAT5_-170C construct. The pBLCAT5_-170delC construct was obtained when PAH promoter region c.-173 to -165 (sequence ACAACGCCCA) harboring the binding site was removed from the pBLCAT5_-170C fragment in two consecutive PCR reactions.
Table 2. Primers used for reporter construct cloning.
Name / Sequence (5´>3´)PAH prom FWHindIII upstream / 5’-CCCAAGCTTGCAGCAAGGCAGTGTGCTTAG -3’
PAH prom RV upstream / 5'- CCTGACGCAGGAGGCCAGGGCAGCCTG -3'
PAH prom FW downstream / 5’-CTGCGTCAGGCGAGGGGCGTTACTGTGC-3’
PAH prom RVXbaI downstream / 5’-GCTCTAGAGCCTCAGGTACAGGCAGGTTTG -3’
HindVNTR F / 5’-CCCAAGCTTGGCTTGAAACTTGAAAGTTGC-3’
XbaVNTR R / 5’-GCTCTAGAGCGGAAACTTAAGAATCCCATC-3’
Note:HindIII and XbaI restriction sites in oligonucleotides are underlined.
For EMSA and supershift analysis of PAH promoter and VNTR region, we have used oligonucleotides shown in Table 3.All the oligonucleotides used are listed; with the oligonucleotides marked a1, a2, b2, b5, c1 and c3 representing the individual repeats which comprise the VNTR alleles detected in Serbian PKU patients. The oligonucleotides marked EBPa1, EBPa2 and EBPb2 correspond to the junctions of the individual repeats. As for the marks coding (or cod), they refer to the DNA strand to be 5' end-labeled with [γ-32P]ATP and subsequently annealed with noncoding (or noncod) strand to generate double-stranded EMSA probes.
Table 3. Oligonucleotides used for EMSA and supershift analysis
Name / Sequence (5´>3´)-170C_cod / 5’-GTCAGGACAACGCCCACGAG-3’
-170C_noncod / 5’-CTCGTGGGCGTTGTCCTGAC-3’
a1 coding / 5’-CACATATATGTATATGCATATGTACGTATG-3’
a1 noncoding / 5’-CATACGTACATATGCATATACATATATGTG-3’
a2 coding / 5’-CACATATATGTATATGCATACGTACGTATG-3’
a2 noncoding / 5’-CATACGTACGTATGCATATACATATATGTG-3’
b2 coding / 5’-CACATATATGTATGTGCATATGTACATAGG-3’
b2 noncoding / 5’-CCTATGTACATATGCACATACATATATGTG-3’
b5 coding / 5’-CACATATATGTATGTGCATATGTACGTATG-3’
b5 noncoding / 5’-CATACGTACATATGCACATACATATATGTG-3’
c1 coding / 5’-CACATATATGTATGTGCATATGTATGTATA-3’
c1 noncoding / 5’-TATACATACATATGCACATACATATATGTG-3’
c3 noncoding / 5’-CACATATATGTATGTGCATATGTATGTATG-3’
c3 noncoding / 5’-CATACATACATATGCACATACATATATGTG-3’
EBPa1 coding / 5’-TGCATATGTACGTATGCACATATATGTAT-3’
EBPa1 noncoding / 5’-ATACATATATGTGCATACGTACATATGCA-3’
EBPa2 coding / 5’-TGCATACGTACGTATGCACATATATGTAT-3’
EBPa2 noncoding / 5’-ATACATATATGTGCATACGTACGTATGCA-3’
EBPb2 coding / 5’-TGCATATGTACATAGGCACATATATGTAT-3’
EBPb2 noncoding / 5’-ATACATATATGTGCCTATGTACATATGCA-3’
VNTR3
coding / 5’- CACATATATGTATATGCATACGTACGTATGCACATATATGTATGTGCATATGTACATAGGCACATATATGTATGTGCATATGTATGTATA-3’
VNTR3 noncoding / 5’-TATACATACATATGCACATACATATATGTGCCTATGTACATATGCACATACATATATGTGCATACGTACGTATGCATATACATATATGTG-3’
Furthermore, a detailed scheme representing fragments studied in this manuscript (in the context of other 5'and 3' noncoding regions and identified TF-binding sites published by other authors), with fragments corresponding to probes used in EMSA assays is shown in Figure S1.
The results of the self-competition assays are shown in Figure S2.
Figure Captions
Fig. S1 A scheme representing PAH locus focusing on 5' and 3' noncoding regions. Blue boxes 1-13 represent PAH gene exons. The numbering of the promoter region is relative to the transcription initiation site, while the numbering of the 3’ region refers to the PAH genomic sequence. Known transcription factor binding sites in the proximal promoter (Konecki et al. 1992, Wang et al. 1992, Wang et al. 1994) as well as the new KLF1 motif are boxed. Probes used in EMSA assays are shown in different colors (-170C probe in PAH promoter is shown in red, while VNTR3 probe in 3' region is shown with reference to individual repeats comprising this allele: a2 repeat is shown in green, b2 in blue and c1 in red); EBPa2 and EBPb2 probes which are located at the junctions of a2 with b2 and b2 with c1, respectively, are underlined. The 3'noncoding PAH gene region which was amplified and analyzed ranged from 380 to 530 bp, according to VNTR alleles’ size present in Serbian PKU patients.
Fig. S2 (a) Competition EMSA assay of the PAH gene promoter region. A 20bp long PAH promoter region probe comprising KLF1 motif was incubated with whole nuclear extract from HepG2 cell line, and also with an unlabeled self-competitor. The supershift assay with KLF1 specific antibody is also present on the same gel. Specific complex between PAH promoter region of interest and HepG2 nuclear extract, SC. (b) Competition EMSA assay of PAH gene VNTR3 region. A 90bp long PAH gene VNTR3 region probe was incubated with whole nuclear extract from HepG2 cell line and also with an unlabeled self-competitor. Specific complex between PAH VNTR3 and HepG2 nuclear extract, SC.