Table 1.
Primers used for cloning and sequencing of PPP2R2D gene promoter and NFI/B gene.
Figure 1.
The four identified variants in the 5′-flanking region of PPP2R2D.
The 5′-flanking fragment containing 1836 bp (−1775 nt to +61 nt) around the transcription start site (TSS) was amplified by PCR and resequenced with an ABI 3730 automatic sequencer. The genomic DNA samples collected from 70 healthy donors in southern Han Chinese population. The direct DNA resequencing revealed 4 genetic variations located in this 5′-flanking region of PPP2R2D. A. were the sequences for −1684 AA and −1684 AG genotypes; B. were the sequences for −1610 AA and −1610 AT genotypes; C. were the sequences for −1196 CC and −1196 CT genotypes; D. were the sequences for −462 GG and −462 GA genotypes. The arrows indicated the nucleotide variants.
Table 2.
The characteristics of the 4 variations identified in the 5′-flanking region of PPP2R2D in 70 southern Han Chinese subjects.
Table 3.
Comparison for MAF of variants in the 5′-flanking region of PPP2R2D between southern Han Chinese and HapMap populations.
Figure 2.
The LD structure of genetic variations in the 5′-flanking region of PPP2R2D.
Pairwise linkage disequilibrium (LD) values between variants were calculated using Haploview in current Han Chinese population. The relative locations of variants are indicated along the top. The value within each diamond represents the pairwise correlation between variant sites (measured as r2) defined by the upper left and the upper right sides of the diamond. The diamond without a number corresponds to D′ = 1. Red diamonds indicate statistically significant allelic association between the pairs of variations, as measured by D′; Blue diamonds indicate pairwise D′ values of 1, but without statistical significance.
Table 4.
Haplotypes formed by genetic variants in the distal promoter of PPP2R2D.
Figure 3.
Scheme of the 5′-flanking region of PPP2R2D and identification of the active core promoter.
A. Human PPP2R2D 5′-flanking region identified in the NCBI database. The box with shadow represents exon 1. The four variants identified in current study, −1684 A>G (rs11146169), −1610 A>T (rs77523588), −1196 C>T (rs9419202), and −462 G>A (rs7074421), are located in the 5′-flanking region (−1775 nt to +61 nt) of PPP2R2D. The PPP2R2D genomic reference sequence with GeneBank ID no. 55844 was used with the first nucleotide of the RNA transcript as +1 nt. Other numbers represent primer positions for cloning reporter constructs. B. Fragments F1, F2, and F3 were amplified by PCR to make reporter constructs, and their positions and lengths are shown in parentheses. Each fragment with wild-type alleles was cloned into the pGL3-Basic vector. C. Relative luciferase activity of series truncated constructs of 5′-flanking region of PPP2R2D in human L02 cells. Each construct or empty vector was transfected into L02 cells with pRL-TK plasmid as an internal intrinsic control of transfection efficiency. After 24 hours, the luciferase activities from each testing construct and also from the internal control plasmid were measured using Dual-Luciferase Reporter Assay System. RLU indicates relative light units. The relative luciferase activity was normalized to Renilla activity and was relative to pGL3-Basic control (Ctrl), which was set as 1.0 RLU. Compared with the basal activity of Ctrl, the promoter activity of all constructs was increased at statistically significant levels (*P<0.05). Compared with the activity of the F3 construct, all constructs showed significant lower activity (#P<0.05). The results represent the mean ± SD of three independent experiments.
Figure 4.
Functional analysis for −462 G>A in PPP2R2D proximal promoter.
Transcriptional activity of F3 fragment bearing PPP2R2D proximal promoter with variant allelotype of −462 G or −462 A was measured by Dual Luciferase Assays. Results are expressed as fold increases in relative light units (RLU) over the empty pGL3-Basic vector (Ctrl). Compared with the basal activity of Ctrl, the promoter activity of the pGL3b-2R2Dp-462G or −462A was higher (*P<0.05). Construct with the −462 G allele had higher activity compared with the −462 A allele (#P<0.05). The results represent the mean ± SD of three independent experiments.
Figure 5.
Transcriptional activity of the PPP2R2D promoter with variant haplotypes.
A. Human PPP2R2D 5′-flanking region identified in the NCBI database. These four variants, −1684 A>G (rs11146169), −1610 A>T (rs77523588), −1196 C>T (rs9419202), and −462 G>A (rs7074421), are located in the distal promoter of PPP2R2D (−1775 nt to +61 nt). B. F1 fragment harboring different haplotypes (F1H1, F1H2, and F1H3) were amplified by PCR to make reporter constructs, and their various haplotypes are shown above the line. Each fragment was cloned into the pGL3-Basic vector and transfected into L02 cells. C. Transcriptional activities of PPP2R2D promoter with various haplotypes (F1Hs) were measured by Dual Luciferase Assays. Results are expressed as fold increases in relative light units (RLU) over the empty pGL3-Basic vector (Ctrl). Compared with the basal activity of Ctrl, the promoter activity of all the F1Hs was higher (*P<0.05). Compared with the F1H1 activity, F1H2 and F1H3 showed significant lower activity (#P<0.05). The results represent the mean ± SD of three independent experiments.
Figure 6.
EMSA of PPP2R2D core proximal promoter region containing the −462 G>A variation.
A. 5′-end biotin-labeled double-stranded oligonucleotides probes including −462 G (lane 1–3) or −462 A (lane 4–6) were incubated with 10 µg of nuclear protein extracted from human L02 cells. The DNA-protein complex (indicated by arrow) showed difference between the −462 G and −462 A allele. For competition EMSA, a 200-fold molar excess of unlabeled/cold −462 G probe (lane 2) or −462 A probe (lane 5) was included in the binding reactions as specific competitors. A 200-fold molar excess of unlabeled consensus NF1 oligonucleotides (lane 3 and 6) were used as competitors to determine the critical binding site. Labeled consensus NF1 probe was included as a positive control (lane 7). B. For cross-competition and supershift assays, a 200-fold molar excess of unlabeled/cold −462 G probe (lane 3), −462 A probe (lane 4) or consensus NF1 oligonucleotides (lane 5) or anti-NF1 antibody (lane 7) was preincubated 30 minutes with nuclear protein. The arrow indicates the DNA-protein complex. The asterisk indicates the supershifted complexes. The sequences of all probes used in the experiment were shown in methods.
Figure 7.
Effects of NF1 on the transcriptional activity and gene expression of PPP2R2D.
A. Expression of the exogenous NF1/B in L02 cells induced dose-dependent increase in the luciferase reporter activity within 250 ng to 1000 ng of the pEGFP-NF1/B plasmid (*P<0.05). B. Transfection of pEGFP-NF1/B or its corresponding empty vector (pEGFP-N1) into L02 cells and cells were harvested 24 hours after transfection. PPP2R2D transcripts were measured and normalized to ACTB by real-time RT-PCR. The relative ratio of PPP2R2D mRNA in L02 cells transfected with empty vector was set as 1.0 for a control. The relative expression levels of the endogenous PPP2R2D were increased in a dose-dependent manner after introduction of 250 ng to 1000 ng pEGFP-NF1/B plasmid (*P<0.05). C. Overexpression of the exogenous NF1/B in L02 cells induced dose-dependent enhanced expression of the endogenous PP2A-Bδ (*P<0.05). The results represent the mean ± SD of three independent experiments.