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*Departament de Fisiologia, Universitat de Barcelona, Barcelona, Spain;
Instituto de Salud Carlos III, Majadahonda, Madrid, Spain; and
Department of Immunology, Duke University Medical Center, Durham, NC 27710
CD69 is a type II C-type lectin involved in lymphocyte migration and cytokine secretion. CD69 expression represents one of the earliest available indicators of leukocyte activation and its rapid induction occurs through transcriptional activation. In this study we examined the molecular mechanism underlying mouse CD69 gene transcription in vivo in T and B cells. Analysis of the 45-kb region upstream of the CD69 gene revealed evolutionary conservation at the promoter and at four noncoding sequences (CNS) that were called CNS1, CNS2, CNS3, and CNS4. These regions were found to be hypersensitive sites in DNase I digestion experiments, and chromatin immunoprecipitation assays showed specific epigenetic modifications. CNS2 and CNS4 displayed constitutive and inducible enhancer activity in transient transfection assays in T cells. Using a transgenic approach to test CNS function, we found that the CD69 promoter conferred developmentally regulated expression during positive selection of thymocytes but could not support regulated expression in mature lymphocytes. Inclusion of CNS1 and CNS2 caused suppression of CD69 expression, whereas further addition of CNS3 and CNS4 supported developmental-stage and lineage-specific regulation in T cells but not in B cells. We concluded CNS1–4 are important cis-regulatory elements that interact both positively and negatively with the CD69 promoter and that differentially contribute to CD69 expression in T and B cells.
The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked advertisement in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.
1 This work was supported by Grants SAF2005-04876 and SAF2007-64310 from the Ministerio de Educación, Ciencia y Tecnología. B. Vázquez and T. Laguna were supported by Predoctoral Fellowships from the Spanish Ministry of Education and Science.
2 Address correspondence and reprint requests to Dr. Pilar Lauzurica, Instituto de Salud Carlos III, Centro Nacional de Microbiología, Laboratorio A-20, Carretera Majadahonda a Pozuelo Km 2.2, 28220 Madrid, Spain. E-mail address: lauzurica{at}isciii.es
3 Abbreviations used in this paper: Tg, transgenic; Io, ionomycin; HA, hemagglutinin; CNS, conserved noncoding sequence; ChIP, chromatin immunoprecipitation; CAD, carbamoyl-transferase dihydroorotase; HS, hypersensitive site; DP, double positive; SP, single positive; MFI, mean fluorescence intensity.
4 The online version of this article contains supplemental material.
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