Determination of cis-acting regulatory elements of the IL-7R alpha gene using flow cytometric reporter assays

Abstract

Interleukin 7 (IL-7) is a cytokine that has an essential role in the development and survival of lymphocytes. IL-7 signaling through its receptor complex IL-7R, has diverse effects in the early stages of development of T-and -lymphocytes. IL-7 also plays a role in the maintance of T-cell homeostasis and survival of memory T-cells. IL-7R is a heterodimer comprised of the common gamma chain (yc) and IL-7R alpha chain (IL-7Ra). As yc is expressed ubiquitously on lymphocytes, IL-7 responsiveness in different developmental stages is controlled by stage specific IL-7Ra gene transcription. Although two different transcription factors, PU.l and GABP were identified as transcriptional regulators of IL-7Ra in earlier studies, the complete transcriptional mechanism still remains to be elucidated. The aim of this study was to determine cis-acting regulatory regions of IL-7Ra. Conserved noncoding sequences in the upstream region of IL-7Ra were identified using bioinformatic tools. Three of the seven identified regions were cloned into a GFP reporter vector and transfected into EL-4 mouse T lymphoma cells. Transcriptional enhancer activities of these regions were assesed using flow cytometry to record GFP levels in live cells. Flow cytometric analyses revealed that one of the constructs showed a higher level of expression compared to background indicating that this CNS contains a potential transcription enhancer element. This study also demonstrated that the constructed GFP reporter vectors and flow cytometric analysis of reporter expression were convenient to identify and characterize putative cis-acting regulatory regions acting on a particular gene of interest.