Regulation of P2X(7) gene transcription

Abstract

The pro-apoptotic P2X(7) receptor regulates growth of epithelial cells. The objectives of the study were to understand P2X(7) gene transcription; to identify the active promoter and the transcription initiation site (TpIS); and to begin understanding regulation of P2X(7) gene transcription. Experiments in vitro utilized normal and cancerous cultured human uterine cervical epithelial cells, and HEK293 cells overexpressing P2X(7)-luciferase reporters. Experiments in vivo used surgical specimen of normal and cancerous uterine cervix. Assays involved DNA, RNA, and protein techniques. (a) The P2X(7) TpIS was localized to adenine (+1) at nt 1683 of the human P2X(7) gene [GenBank Y12851]), with a TTAAA sequence at nt -32/-28 and an active promoter region within nt -158/+32. (b) P2X(7) transcription was found to be regulated by two enhancers located at nt + 222/+232 and +401/+573 regions downstream of the active P2X(7) promoter. (c) The putative enhancer regions formed four DNA-protein complexes. (d) P2X(7) transcription was found to be controlled by hypermethylated cytosines at cytosine-phosphodiester-guanosines (CpG) that cluster or co-localize with the enhancers' sites. (e) We identified nine CpGs as inhibitory cis elements, and three CpG sites that are hypermethylated in cultured cervical epithelial cells and in cervix epithelia in vivo. (f) In cancer cervical cells, the degree of hypermethylation of the CpG sites was greater than in the normal cervical cells. Expression of the P2X(7) receptor is controlled by hypermethylated CpGs that flank transcription enhancers located within a 547-nt region downstream of the promoter.

Fig. 1

Sequence of the 5′ region of the human P2X₇, containing the active promoter (white symbols on black background, nt −158/+32); a 547-nt CpG-rich region (underlined, nt +26/+573) downstream of the promoter; Exon 1 (underlined and italics, nt +92/+216); and the proximal part of intron-1 (distal to Exon 1, underlined, beginning at nt +217). Nucleotides were numbered relative to the Transcription Initiation Start Site (TpIS; +1; nt 1683 according to GenBank Y12851). TpISs- and TATA-like sequences within the active promoter bases/regions are bolded and doubly underlined. CpG dinucleotides are bolded and doubly underlined. Vertical thick empty arrows point to MaeII-sensitive CpG sites (nt +193/+194, +211/+212, and +330/+331). The vertical thick-filled arrow points to a BstUI-sensitive CpG–CpG site (nt +461/+462 and +463/+464). For DNA methylation experiments the 547-nt CpG-rich region was subdivided into segment 1 (nt +26/+247), segment 2 (nt +223/+399), and segment 3 (nt +352/+573)

Fig. 2

a–b. Elucidation of the P2X₇ Active Promoter Region. a cDNA fragments corresponding to regions within a 1.7-kb DNA segment of the 5′ region of the human P2X₇ gene were inserted into luciferase vector; the P2X₇-luciferase reporters were transfected into HEK293 and P2X₇ promoter activity was determined in terms of changes in luciferase activity (Fluc/Rluc). Data (means ± SD, three to five experiments in triplicates) were normalized (=1) to Fluc/Rluc recorded in cells transfected with empty vector. *p < 0.01 compared to the rest. b Confirmation of P2X₇ TpIS. Two potential TpISs and their related TATA-like regions were mutated and effects on P2X₇ transcription were determined as in a (means±SD, three to five experiments in triplicates). *p < 0.01 compared to −158/+32. c P2X₇ transcription is modulated by effectors downstream of the active promoter. P2X₇ −158/+32 or −158/+573 luciferase reporters were transfected into HEK293 cells and P2X₇ promoter activity was determined in terms of changes in luciferase activity (Fluc/Rluc, upper panel) or in terms of changes in Fluc/GAPDH mRNA (lower panel). Shown are means (±SD) of one to three experiments in triplicates. Data of Fluc/GAPDH mRNA were normalized (=0) to those recorded in cells transfected with empty vector. *p < 0.01

Fig. 3

Effects of treatments with Aza-dC (1 μM) on steady-state levels of P2X₇ mRNA (a), P2X₇ receptor protein levels (b), and BzATP-induced apoptosis (in arbitrary units [A.U.]) (c). Means ± SD of three to six experiments in triplicates. Insert in b shows immunofluorescence data (×20). Data in a and b were normalized (=1) to levels in hEVEC cells at t = 0. The time-related increases in P2X₇ mRNA (a) and P2X₇ receptor protein levels (b) for both hEVEC and HeLa cells were significant (p < 0.01). Protein levels of P2X₇ remained higher (p < 0.01) in hEVEC than in HeLa cells throughout the length of the experiment. In c, treatments with Aza-dC were followed by 100 μM BzATP (added for 8 additional hours). The degree of apoptosis (in arbitrary units [A.U.]) was normalized to levels determined in non-treated cells. In c, *p < 0.05; **p < 0.01

Fig. 4

Effects of hypermethylation and de-methylation on changes in transcription in HEK293 cells transfected with the luciferase −158/+32 or −158/+573 reporters (means±SD of two experiments in triplicates). Hypermethylation assays were done by incubating the test plasmids prior to transfections with the CpG-Methylase M.SssI and changes in transcription were determined in terms of changes in luciferase activity (Fluc/Rluc). De-methylation assays were done by treating transfected cells with Aza-dC (1 μM for 48 h). Changes in transcription were determined in terms of changes in Fluc/GAPDH mRNA levels. Data were normalized (=1) to levels in control cells. *p < 0.01

Fig. 5

Elucidation of transcription regulatory cis elements within a CpG-rich 547-nt region downstream of the P2X₇ promoter. a cDNA fragments were constructed containing the P2X₇ active promoter (nt −158/+32) attached with one of the shown segments of the 547-nt region downstream of the promoter (Fig. 1). b Effects of mutations in the CpG sites within the 547-nt region downstream of the promoter on P2X₇ transcription. WT wild-type. Mutations are described in the section of “Methods” section. For both a and b cDNA fragments composed of the P2X₇ active promoter attached with one of the shown segments were inserted into a luciferase vector and transfected into HEK293 cells. Promoter activity was determined in terms of changes in luciferase activity (Fluc/Rluc, means ± SD, of two experiments in triplicates). In a *p < 0.01 compared to −158/+32; **p < 0.05–0.01 compared to −158/+232. In b *p < 0.01 compared to the wild-type sequence in each case

Fig. 6

a Effects of treatment with Aza-dC in HeLa cells (1 μM, 48 h) on methylation of cytosines in CpG sites +193/+194 (and/or +211/+212), +330/+331, and +461/+462 (and/or +463/+464), within segments 1, 2, and 3 respectively of a 547-nt region downstream of the P2X₇ active promoter (Fig. 1). Methylation of cytosines in CpG sites was determined in terms of cleavage at CpG sites using the genomic DNA bisulfite conversion method followed by gene-specific PCR and restriction enzyme cutting. Left-pointing arrows show bands corresponding to uncleaved (broken lines) and cleaved fractions (continuous lines) at the CpG sites. M markers. Controls were aliquots of human placental genomic DNA (H.G.-DNA) treated in vitro with the CpG-methylase SssI. b Aliquots of human placental genomic DNA were mixed with different molar concentrations of SssI and the degree of cleavage at CpG sites +193/+194 (and/or +211/+212) within segment 1 was determined as in a. Data were normalized to the effect (100%) obtained in a reaction mixture containing 1 μg DNA. c–h Methylation status of cytosines in CpG sites in cultured human epithelial uterine cervical cells (c–e) and in human uterine cervix tissues in vivo (f–h). Experiments were done as in a, and data on the degree of the cleaved fractions (in terms of the ratio of densitometry of the cleaved, versus the uncleaved plus cleaved bands [%]) are summarized in Table 1. i Degree of cleavage at CpG sites in tissues of human cervix. Data were compiled from ten sets of paired cervical specimens, including in each case normal and squamous cell carcinoma tissues. Available for analysis were nine cases for segments 1 and 3, and eight cases for segment 2. Experiments and data evaluation were done as above. Lines connect paired tissues (Normal [N] and Cancer [Ca]) from the same patient. Data are explained in the section of “Results” section

Fig. 7

Elucidation of DNA–protein binding within the 547-nt region downstream of the P2X₇ promoter. cDNA fragments were constructed containing the indicated P2X₇ gene segments (Table 5). Electrophoretic mobility shift assays (EMSA) were used to detect DNA–protein complexes. Right-pointing arrows indicate shifted bands. The experiment was repeated twice

Fig. 8

Schema of the CpG-rich 547-nt DNA region (+26/+573) downstream of the active promoter of the P2X₇ gene. Filled ellipses denote CpG sites that were found experimentally to inhibit P2X₇ transcription. Upwards pointing arrows denote CpG sites that were found experimentally to be hypermethylated in cultured cervical cells and in cervix epithelial tissues in vivo. Hatched squares are cis regions that were found experimentally to possess transcription enhancer activity. Horizontal bi-directional arrows show putative sites within the cis-enhancer regions that were found to form DNA–protein complexes