NEW INSIGHTS ON THE TRANSCRIPTIONAL REGULATION OF CD69 1 GENE THROUGH A POTENT ENHANCER LOCATED IN THE CONSERVED 2 NON-CODING SEQUENCE 2 3

22 The CD69 type II C-type lectin is one of the earliest indicators of leukocyte activation 23 acting in lymphocyte migration and cytokine secretion. CD69 expression in 24 hematopoietic lineage undergoes rapid changes depending on the cell-lineage, the 25 activation state or the localization of the cell where it is expressed, suggesting a 26 complex and tightly controlled regulation. Here we provide new insights on the 27 transcriptional regulation of CD69 gene in mammal species. Through in silico studies, 28 we analyzed several regulatory features of the 4 upstream conserved non-coding 29 sequences (CNS 1-4) previously described, confirming a major function of CNS2 in the 30 transcriptional regulation of CD69. In addition, multiple transcription binding sites are 31 identified in the CNS2 region by DNA cross-species conservation analysis. By 32 functional approaches we defined a core region of 226 bp located within CNS2 as the 33 main enhancer element of CD69 transcription in the hematopoietic cells analyzed. By 34 chromatin immunoprecipitation, binding of RUNX1 to the core-CNS2 was shown in a T 35 cell line. In addition, we found an activating but not essential role of RUNX1 in CD69 36 gene transcription by site-directed mutagenesis and RNA silencing, probably through 37 the interaction with this potent enhancer specifically in the hematopoietic lineage. In 38 summary, in this study we contribute with new evidences to the landscape of the 39 transcriptional regulation of the CD69 gene. 40

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