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

Abstract

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.

Highly expressed genes within hippocampal sector CA1: implications for the physiology of memory

“…Of note, Hui-Feng Chen et al 21 estimated overall that Protein phosphatase 2A (PP2A) accounts for up to 1% of cellular protein, and that certain functional variants in haplotypes and nuclear factor 1 may regulate the transcription of the regulatory subunit gene PPP2R2D (protein phosphatase 2, regulatory subunit B, delta).…”

Role of a Novel Functional Variant in the PPP2R1A Promoter on the Regulation of PP2A-Aalpha and the Risk of Hepatocellular Carcinoma

“…After incubation with transient transfection treatment for 24 h, the cells were harvested and assayed for firefly and Renilla luciferase activities with a Dual-Luciferase Reporter Assay System (Promega), as in our previous study [16]. All transfections were performed in triplicate and repeated at least thrice in independent experiments.…”

“…The study subjects consisted of 251 HCC case patients and 252 cancer-free controls recruited from The First Affiliated Hospital, Sun Yat-sen University, between January 2008 and October 2011. All subjects in the study were unrelated ethnic Han Chinese confirmed by ID cards and “Household Register books”, which serve as identification in China as described in our previous study [16]. The individuals were living in Guangzhou and its surrounding regions (Guangdong Province, China) in southern China, where HCC is prevalent and HBV infection has been shown to be the main attributable risk factor [17], [18].…”

“…We used a short questionnaire to obtain demographic and risk factor information and frequency matched the controls to the cases by age (±5 years) and sex. After physical examination, 5 ml of blood was drawn, labeled, and delivered to the lab for subsequent genomic DNA extraction, as in our previous study [16].…”

Molecular and clinical analyses with neuropsychological assessment of a case of del(10)(q26.2qter) without intellectual disability: Genomic and transcriptomic combined approach and review of the literature

“…Indeed, PTPRE (encoded a member of the protein tyrosine phosphatase family) [Kabir et al, 2013], GLRX3 (member of the glutaredoxin oxidoreductase family) [Ohayon et al, 2010], and PPP2R2D (delta Serine-threonine protein phosphatase 2A) [Chen et al, 2012] had been reported in somatic cancer pathologies. MTG1codes for a conserved protein required for mitochondrial translation but remains still poorly understood [Barrientos et al, 2003].…”