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Serine-threonine protein phosphatase 2A (PP2A) is a trimeric holoenzyme that plays an integral role in the regulation of cell growth, differentiation, and apoptosis. The substrate specificity and (sub)cellular localization of the PP2A holoenzymes are highly regulated by interaction with a family of regulatory B subunits (PP2A-Bs). The regulatory subunit PP2A-B/PR55δ (PP2A-Bδ) is involving in the dephosphorylation of PP2A substrates and is crucial for controlling entry into and exit from mitosis. The molecular mechanisms involved in the regulation of expression of PP2A-Bδ gene ( PPP2R2D ) remain largely unknown. To explore genetic variations in the 5′-flanking region of  PPP2R2D  gene as well as their frequent haplotypes in the Han Chinese population and determine whether such variations have an impact on transcriptional activity, DNA samples were collected from 70 healthy Chinese donors and sequenced for identifying genetic variants in the 5′-flanking region of  PPP2R2D . Four genetic variants were identified in the 1836 bp 5′-flanking region of  PPP2R2D . Linkage disequilibrium (LD) patterns and haplotype profiles were constructed for the genetic variants. Using serially truncated human  PPP2R2D  promoter luciferase constructs, we found that a 601 bp (−540 nt to +61 nt) fragment constitutes the core promoter region. The subcloning of individual 5′-flanking fragment revealed the existence of three haplotypes in the distal promoter of  PPP2R2D . The luciferase reporter assay showed that different haplotypes exhibited distinct promoter activities. The EMSA revealed that the −462 G>A variant influences DNA-protein interactions involving the nuclear factor 1 (NF1).  In vitro  reporter gene assay indicated that cotransfection of NF1/B expression plasmid could positively regulate the activity of  PPP2R2D  proximal promoter.  Introduction  of exogenous NF1/B expression plasmid further confirmed that the NF1 involves in the regulation of  PPP2R2D  gene expression. Our findings suggest that functional genetic variants and their haplotypes in the 5′-flanking region of  PPP2R2D  are critical for transcriptional regulation of PP2A-Bδ.

Identification and Functional Analysis of Variant Haplotypes in the 5′-Flanking Region of Protein Phosphatase 2A-Bδ Gene