The human PC P2 promoter sequence and its alignment with the rat PC P2 promoter.

<p>Boxes represent the putative transcription factor binding sites for Sp1, FoxA2/HNF3β, USF1/2, and CBF. Identical nucleotides between human and rat sequence are symbolized by an asterisk.</p

Identification of positive regulatory element(s) located between −114 and −39 of the human PC P2 promoter.

<p>(A) Schematic diagram of 15 bp internal deletions of −114/−39 of the human PC P2 promoter. (B) Transient transfections of a series of 15 bp internal deletion constructs into the INS-1 832/13 and non-beta cell HEK293T cell lines were performed to localize the positive regulatory sequence in the human PC P2 promoter. The luciferase activity of each construct was normalized with the β-galactosidase activity. The normalized reporter activity obtained from each construct is shown as a percent relative to those transfected with the wild type −365 hP2 promoter that was arbitrarily set at 100%. *P value <0.05, **P value <0.01. (C) Gel shift and supershift assays of biotin-labeled probe −78 to −54 region of hP2 promoter (−78/−54 CCAAT-probe) using INS-1 832/13 nuclear extract (Lane 1–5) and non-beta cell HEK293T (Lanes 6–10). The nucleotide sequence of wild type and mutant of the hP2 promoter in the −78 to −54 regions are also shown. Lanes 1 and 5 show probes incubated with nuclear extracts from INS-1 832/13 or HEK293T cells; lanes 2 and 6, 10-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 3 and 7, 50-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lane 4 and 9, 50-fold excess amount of mutant unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 5 and 10, nuclear extracts were pre-incubated with anti-NF-Y antibody before the probes were added to the reactions. Arrow represents CCAAT box–NF-Y, complex.</p

RT-PCR analysis of PC mRNA variants in human liver and human pancreatic islets.

<p>(A) Schematic diagram showing alignment of 3 variants of human PC mRNA (GenBank NM_000920.3, NM_022172.2, BC011617.2). (B) Schematic diagram showing the structure of the human PC gene. Two isoforms of human PC mRNA are initiated by two alternative promoters, the proximal (P1) promoter and the distal (P2) promoter. All PC mRNA variants contain the same coding sequences but differ in their 5′-untranslated regions (UTR) produced from different 5′-UTR exons (UE1/UE2, UE3 and UE4) (C) Examination of human PC mRNA in liver and pancreatic islets using RT-PCR. Two sets of primers were used to amplify two different isoforms of human PC mRNA both in human liver and human islets. The 173 bp fragment PCR product of variant 2 and the 200 bp fragment PCR product of variant 1 were amplified by using Primers set no. 1 and primer set no.2, respectively, Lane 1; 1 kb marker, Lane 2; Negative control for primer set no.1, Lane 3; Negative control for primer set no.2, Lane 4; PCR using primer set no.1 and cDNA prepared from human liver, Lane 5; PCR using primer set no.2 and cDNA prepared from human liver, Lane 6; PCR using primer set no.1 and cDNA prepared from human islets, Lane 7; PCR using primer set no.2 and cDNA prepared from human islets.</p

Oligonucleotides used for construction of 5′-trucated hP2 promoter.

*<p>Restriction enzyme recognition sites are underlined.</p

Identification of positive regulatory element(s) located between −365 and −240 of the human PC P2 promoter.

<p>(A) Schematic diagram of 15 bp internal deletions of −114/−39 the human PC P2 promoter. (B) Transient transfections of a series of 25 bp internal deletion constructs into the INS-1 832/13 cell line and non-beta cell HEK293T cell line were performed to identify the positive regulatory sequences in the hP2 promoter. The luciferase activity of each construct was normalized with β-galactosidase activity. The normalized reporter activity obtained from each construct is shown as a percent relative to those transfected with the wild type −365 hP2 promoter, which was arbitrarily set at 100%. *P value <0.05, **P value <0.01. (C) Gel shift and supershift assays of the biotin-labeled probe of the −78 to −54 region of the hP2 promoter (−340/−315 hP2 probe) using an INS-1 832/13 nuclear extract. The nucleotide sequences of the wild type and mutant of the hP2 promoter −78 to −54 regions are also shown. Lane 1 probes incubated with nuclear extracts from INS-1 832/13; lanes 2–3, 10-fold or 50-fold excess wild-type unlabeled oligonucleotides were incubated with nuclear extracts and probes; lane 4, 50-fold excess amount of mutant unlabeled oligonucleotides were incubated with nuclear extracts and probes; lanes 5–7, nuclear extracts were pre-incubated with anti-USF1 or anti-USF2 or both, respectively, before the probes were added to the reactions. Lanes 8–10, nuclear extracts were pre-incubated with anti-Sp1 or anti-Sp3 or both, respectively, before the probes were added to the reactions. Arrow represents DNA-protein complex, SS = supershift band.</p

Identification of HNF4α binding sites in hFBP1 promoter.

<p>(A) Transactivation of 5’-truncated hFBP1 promoter-luciferase reporter construct by HNF4α in HepG2 cells. The 886 nucleotides-hFBP1 promoter-luciferase reporter gene or its 5’-truncated mutants (500, 400, 300, 200 and 100) were transiently co-transfected with empty vector (pcDNA3; grey bar) or plasmid overexpressing HNF4α (pcDNA3-HNF4α; black bar) into HepG2. The luciferase activity of wild type or mutant construct was normalized with β-galactosidase activity and shown as relative luciferase activity. Relative luciferase activity obtained from cells co-transfected with the hFBP1-promoter-luciferase constructs and plasmid encoding HNF4α was presented as “fold change” relative to those transfected with hFBP1-promoter-luciferase construct and empty vector, which was arbitrarily set as 1. (B) Nucleotide sequences of consensus HNF4α binding sites including classical DR1 and H4-SBM and three HNF4α binding sites (HNF4α1 (-566/-554, antisense strand), HNF4α2 (-358/-346; sense strand) and HNF4α3 site (-212/-198; antisense strand)) in hFBP1 promoter also shown (C) Effect of mutations of three HNF4α binding sites on FBP1 promoter activity. Mutations of the HNF4α1, HNF4α2 and HNF4α3 sites were introduced in the 886 FBP1 promoter-reporter construct and co-transfected with empty vector (pcDNA3; grey bars) or vector containing human HNF4α (pcDNA3-HNF4α; black bars) into HepG2 cells. The luciferase activity of each construct was normalized to β-galactosidase activity and expressed as relative luciferase activity. The values obtained from mutated constructs are expressed relative to the corresponding parental or mutant construct which was arbitrarily set as 100%. The values shown are means ± standard deviation (n = 3). *p < 0.01, **p < 0.05, ***p <0.001.</p

Identification of functional C/EBPα binding sites in human FBP1 promoter.

<p>(A) Transactivation of 5’-truncated hFBP1 promoter-luciferase reporter construct by C/EBPα in HepG2 cells. The 886 nucleotides-hFBP1 promoter-luciferase reporter gene or its 5’-truncated constructs (500, 400, 300, 220 and 100 nucleotides) were transiently co-transfected with empty vector (pcDNA3; grey bar) or plasmid overexpressing C/EBPα (pcDNA3-C/EBPα; black bar) into HepG2. The luciferase activity of wild type or mutant construct was normalized with β-galactosidase activity shown as relative luciferase activity. Relative luciferase activity obtained from the cells transfected with the hFBP1-promoter-luciferase constructs and plasmid encoding C/EBPα protein was presented as “fold change” relative to those transfected with hFBP1-promoter-luciferase construct and empty vector, which was arbitrarily set as 1. (B) Nucleotide sequences of two overlapping C/EBPα binding designated C/EBPα1 (-227/-218) and C/EBPα2 (-218/-209) in hFBP1 promoter and its mutagenic sequences in ΔC/EBPα1 and ΔC/EBPα2 or double mutant ΔC/EBPα1 ΔC/EBPα2. Underline indicates nucleotide changes of each mutant. (C) Effect of mutations of two overlapping C/EBPα binding sites on C/EBPα transactivation of FBP1 promoter activity. Single or double mutations of C/EBPα1 and C/EBPα2 sites in 300 hFBP1 promoter-reporter construct and co-transfected with empty vector (pcDNA3; grey bar) or vector containing C/EBPα (pcDNA3-C/EBPα; black bar) into HepG2 cells. The luciferase activity of each construct was normalized to β-galactosidase activity and expressed as relative luciferase activity. Relative luciferase activity obtained from cells transfected with WT or mutated FBP1 promoter-luciferase and plasmid overexpressing C/EBPα was presented as fold change relative to those transfected with the parental or mutated FBP1-luciferase reporter and pcDNA empty vector, which was arbitrarily set as 1. The values shown are means ± standard deviation of three independent experiments (n = 3). The statistical analysis was conducted by ANNOVA test where *p < 0.01, **p < 0.05, ***p < 0.001.</p

Human FBP1 gene promoter and induction of hFBP1 expression in HepG2 by C/EBPα or HNF4α.

<p>(A) Nucleotide sequence of human FBP1 promoter with various putative transcription factor binding sites identified by PROMO [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194252#pone.0194252.ref034" target="_blank">34</a>] and JASPAR databases [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194252#pone.0194252.ref035" target="_blank">35</a>]. The binding sites of previously identified transcription factors including Sp1, USFs, NF-κB [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194252#pone.0194252.ref031" target="_blank">31</a>] and VDRE [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194252#pone.0194252.ref036" target="_blank">36</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0194252#pone.0194252.ref037" target="_blank">37</a>] are also included. (B) Western blot analysis of HepG2 cells transfected with pcDNA3 (empty vector), pcDNA3-C/EBPα or pcDNA3-HNF4α using anti-C/EBPα or anti-HNF4α antibodies. Control loading was assessed by probing the blot with anti-β-actin antibody. (C) qRT-PCR of FBP1 expression of HepG2 cells transfected with empty vector, C/EBPα or HNF4α. Human FBP1 mRNA expression was normalized with that of 18s rRNA, and shown as relative expression. (D) qRT-PCR of FBP1 expression in Huh7 cells transfected with empty vector, C/EBPα or HNF4α. The values obtained from cells transfected with pcDNA3-C/EBPα or pcDNA3-HNF4α were relative to that obtained from cells transfected with empty vector, which was arbitrarily set as 1. The values shown are means ± standard deviation of three independent experiments (n = 3). The statistical analysis was conducted by ANNOVA test where *p < 0.01 and **p < 0.05.</p

Oligonucleotides used for generating wild type and mutant reporter constructs, EMSA and qPCR.

<p>Oligonucleotides used for generating wild type and mutant reporter constructs, EMSA and qPCR.</p