CpG methylation at GATA elements in the regulatory region of CCR3 positively correlates with CCR3 transcription

DNA methylation may regulate gene expression by restricting the access of transcription factors. We have previously demonstrated that GATA-1 regulates the transcription of the CCR3 gene by dynamically interacting with both positively and negatively acting GATA elements of high affinity binding in the proximal promoter region including exon 1. Exon 1 has three CpG sites, two of which are positioned at the negatively acting GATA elements. We hypothesized that the methylation of these two CpGs sites might preclude GATA-1 binding to the negatively acting GATA elements and, as a result, increase the availability of GATA-1 to the positively acting GATA element, thereby contributing to an increase in GATA-1-mediated transcription of the gene. To this end, we determined the methylation of the three CpG sites by bisulfate pyrosequencing in peripheral blood eosinophils, cord blood (CB)-derived eosinophils, PBMCs, and cell lines that vary in CCR3 mRNA expression. Our results demonstrated that methylation of CpG sites at the negatively acting GATA elements severely reduced GATA-1 binding and augmented transcription activity in vitro. In agreement, methylation of these CpG sites positively correlated with CCR3 mRNA expression in the primary cells and cell lines examined. Interestingly, methylation patterns of these three CpG sites in CB-derived eosinophils mostly resembled those in peripheral blood eosinophils. These results suggest that methylation of CpG sites at the GATA elements in the regulatory regions fine-tunes CCR3 transcription

Eosinophil Development, Regulation of Eosinophil-Specific Genes, and Role of Eosinophils in the Pathogenesis of Asthma

Eosinophils arise from hematopoietic CD34+ stem cells in the bone marrow. They acquire IL-5Rα on their surface at a very early stage during eosinophilopoiesis, and differentiate under the strong influence of interleukin (IL)-5. They then exit to the bloodstream, and enter the lung upon exposure to airway inflammatory signals, including eotaxins. In inflamed tissues, eosinophils act as key mediators of terminal effector functions and innate immunity and in linking to adaptive immune responses. Transcription factors GATA-1, CCAAT/enhancer-binding protein, and PU.1 play instructive roles in eosinophil specification from multipotent stem cells through a network of cooperative and antagonistic interactions. Not surprisingly, the interplay of these transcription factors is instrumental in forming the regulatory circuit of expression of eosinophil-specific genes, encoding eosinophil major basic protein and neurotoxin, CC chemokine receptor 3 eotaxin receptor, and IL-5 receptor alpha. Interestingly, a common feature is that the critical cis-acting elements for these transcription factors are clustered in exon 1 and intron 1 of these genes rather than their promoters. Elucidation of the mechanism of eosinophil development and activation may lead to selective elimination of eosinophils in animals and human subjects. Furthermore, availability of a range of genetically modified mice lacking or overproducing eosinophil-specific genes will facilitate evaluation of the roles of eosinophils in the pathogenesis of asthma. This review summarizes eosinophil biology, focusing on development and regulation of eosinophil-specific genes, with a heavy emphasis on the causative link between eosinophils and pathological development of asthma using genetically modified mice as models of asthma

miR-302 Suppresses the Proliferation, Migration, and Invasion of Breast Cancer Cells by Downregulating ATAD2

Breast cancer is the most common malignant tumor in women. The ATPase family AAA domain-containing protein 2 (ATAD2) contains an ATPase domain and a bromodomain, and is abnormally expressed in various human cancers, including breast cancer. However, the molecular mechanisms underlying the regulation of ATAD2 expression in breast cancer remain unclear. This study aimed to investigate the expression and function of ATAD2 in breast cancer. We found that ATAD2 was highly expressed in human breast cancer tissues and cell lines. ATAD2 depletion via RNA interference inhibited the proliferation, migration, and invasive ability of the SKBR3 and T47D breast cancer cell lines. Furthermore, Western blot analysis and luciferase assay results revealed that ATAD2 is a putative target of miR-302. Transfection with miR-302 mimics markedly reduced cell migration and invasion. These inhibitory effects of miR-302 were restored by ATAD2 overexpression. Moreover, miR-302 overexpression in SKBR3 and T47D cells suppressed tumor growth in the xenograft mouse model. However, ATAD2 overexpression rescued the decreased tumor growth seen after miR-302 overexpression. Our findings indicate that miR-302 plays a prominent role in inhibiting the cancer cell behavior associated with tumor progression by targeting ATAD2, and could thus be a valuable target for breast cancer therapy

Effect of single nucleotide polymorphisms within the interleukin-4 promoter on aspirin intolerance in asthmatics and interleukin-4 promoter activity

Objective Aspirin affects interleukin-4 (IL-4) synthesis; however, the genetic role of IL-4 has not been evaluated in asthmatics with aspirin hypersensitivity. The objective of the study was to examine the influence of single nucleotide polymorphisms (SNPs) in IL-4 gene on aspirin hypersensitivity in asthmatics at the genetic and molecular levels. Methods Aspirin-intolerant (AIA, n = 103) and aspirintolerant asthmatics (n = 270) were genotyped and functional promoter assays were performed. Results Of 15 SNPs tested, seven (-589T>C (rs2243250) in promoter, -33T>C (rs2070874) in the 50-untranslated region, +4047A>G (rs2243266), +4144C>G (rs2243267), +4221C>A (rs2243268), +4367G>A (rs2243270), and +5090A>G (rs2243274) in introns) were significantly associated with AIA risk. The frequency of the rare allele (C) of -589T>C was higher in the AIA group than in the aspirin-tolerant asthmatic group (P(corr) = 0.016), and a gene dose-dependent decline in forced expiratory volume in 1 s was noted after an aspirin challenge (P = 0.0009). Aspirin unregulated IL-4 mRNA production in Jurkat T and K562 leukemia cells. A reporter plasmid assay revealed that aspirin augmented IL-4 promoter transactivation with the -589T>C C and -33T>C C alleles, compared with that bearing the -589T>C T and -33T>C T alleles. Further, electrophoretic mobility shift assay showed the formation of nuclear complexes with -33T>C and -589T>C allele-containing probes; this was augmented by aspirin. The complexes formed with the -33T>C and -589T>C probes were shifted by treatment with anti-CCAAT-enhancer-binding proteins beta and anti-nuclear factor of activated T-cells antibodies, respectively, indicating the inclusion of these transcription factors. Conclusion Aspirin may regulate IL4 expression in an allele-specific manner by altering the availability of transcription factors to the key regulatory elements in the IL4 promoter, leading to aspirin hypersensitivity. Pharmacogenetics and Genomics 20:748-758 (C) 2010 Wolters Kluwer Health vertical bar Lippincott Williams & Wilkins.This study was supported by the BK21, Korea Research Foundation to B.S.K. and J.H.K., partially; and the Korea Health 21 R&D Project, Ministry of Health, Welfare and Family Affairs, Republic of Korea [A010249].Kim TH, 2008, CLIN EXP ALLERGY, V38, P1727, DOI 10.1111/j.1365-2222.2008.03082.xGuo LY, 2008, J IMMUNOL, V181, P3984Hebenstreit D, 2008, J BIOL CHEM, V283, P22490, DOI 10.1074/jbc.M804096200Cormican LJ, 2005, CLIN EXP ALLERGY, V35, P717, DOI 10.1111/j.1365-2222.2005.02261.xCieslik KA, 2005, J BIOL CHEM, V280, P18411, DOI 10.1074/jbc.M410017200Szefler SJ, 2005, J ALLERGY CLIN IMMUN, V115, P233, DOI 10.1016/j.jaci.2004.11.014Adjers K, 2005, INT ARCH ALLERGY IMM, V138, P251, DOI 10.1159/000088726Basehore MJ, 2004, J ALLERGY CLIN IMMUN, V114, P80, DOI 10.1016/j.jaci.2004.05.035Pagani F, 2004, NAT REV GENET, V5, P389, DOI 10.1038/nrg1327Nyholt DR, 2004, AM J HUM GENET, V74, P765Mifflin RC, 2004, MOL PHARMACOL, V65, P470Cohn L, 2004, ANNU REV IMMUNOL, V22, P789, DOI 10.1146/annurev.immunol.22.012703.104716Li-Weber M, 2003, NAT REV IMMUNOL, V3, P534, DOI 10.1038/nri1128Szczeklik A, 2003, J ALLERGY CLIN IMMUN, V111, P913, DOI 10.1067/mai.2003.1487Sousa AR, 2002, NEW ENGL J MED, V347, P1493Nakashima H, 2002, GENES IMMUN, V3, P107, DOI 10.1038/sj/gene/6363820Dahlen SE, 2002, AM J RESP CRIT CARE, V165, P9OLIPHANT A, 2002, BIOTECHNIQUES S, V32, P56*GINA, 2002, GLOB STRAT ASTHM MANPEREZ GM, 2002, J IMMUNOL, V168, P1428Tegeder I, 2001, FASEB J, V15, P2057Stevenson DD, 2001, ANN ALLERG ASTHMA IM, V87, P177Stephens M, 2001, AM J HUM GENET, V68, P978Cianferoni A, 2001, BLOOD, V97, P1742Leff AR, 2001, ANNU REV MED, V52, P1Suzuki I, 2000, CLIN EXP ALLERGY, V30, P1746Berberich-Siebelt F, 2000, EUR J IMMUNOL, V30, P2576Kauffmann F, 1999, CLIN EXP ALLERGY, V29, P17Wiesch DG, 1999, J ALLERGY CLIN IMMUN, V104, P895Varga EM, 1999, EUR RESPIR J, V14, P610Burchard EG, 1999, AM J RESP CRIT CARE, V160, P919Schwenger P, 1999, J CELL PHYSIOL, V179, P109Drazen JM, 1999, NEW ENGL J MED, V340, P197Gerli R, 1998, BLOOD, V92, P2389Noguchi E, 1998, CLIN EXP ALLERGY, V28, P449Dong ZG, 1997, J BIOL CHEM, V272, P9962Marsh DG, 1997, NAT GENET, V15, P389Walley AJ, 1996, J MED GENET, V33, P689Klein SC, 1996, CELL IMMUNOL, V167, P259DAVYDOV IV, 1995, J IMMUNOL, V155, P5273ROSENWASSER LJ, 1995, CLIN EXP ALLERGY, V25, P74*AM THOR SOC, 1995, AM J RESP CRIT CARE, V152, P1107KOPP E, 1994, SCIENCE, V265, P956CRABTREE GR, 1994, ANNU REV BIOCHEM, V63, P1045FLESCHER E, 1991, J IMMUNOL, V146, P2553AKIRA S, 1990, EMBO J, V9, P1897SZCZEKLIK A, 1990, EUR RESPIR J, V3, P588*SENSORMEDICS, 1988, PULM UT OP MAN PREDHEDRICK PW, 1987, GENETICS, V117, P331MORRIS JF, 1976, WESTERN J MED, V125, P110SAMTER M, 1967, J ALLERGY, V40, P281