Interleukin-2 (IL-2) Regulates the Accessibility of the IL-2-Responsive Enhancer in the IL-2 Receptor α Gene to Transcription Factors

Abstract

Interleukin-2 (IL-2) responsiveness of T lymphocytes is controlled through transcription of the IL-2 receptor (IL-2R) subunit by antigen and by IL-2 itself. IL-2 induces IL-2R transcription via an IL-2-responsive enhancer (IL-2rE), whose activity depends on the cooperative binding of IL-2-induced STAT5 to two sites and of constitutively active Elf-1 to a third one. Here we describe the changes in IL-2rE chromatin that occur in normal T lymphocytes upon activation of IL-2R expression. In cells induced to transiently express IL-2R with concanavalin A (which mimics antigen), none of the IL-2rE sites is occupied despite the presence of Elf-1 and STAT1, which bind to the IL-2rE in vitro. The two STAT binding sites are occupied rapidly upon IL-2 stimulation, concomitantly with STAT5 activation. Occupation of the Elf-1 binding site is delayed, although Elf-1 concentration and binding activity are not modified by IL-2. Digestion of T-cell chromatin with DNase I and micrococcal nuclease shows that IL-2 induces the appearance of nuclease-hypersensitive sites flanking the IL-2rE. Thus IL-2, in addition to activating STAT5, appears to regulate IL-2R transcription by making IL-2R chromatin accessible to transcription factors. Interleukin-2 (IL-2) is the principal growth factor for anti-gen-activated T lymphocytes. It promotes T-cell proliferation by binding to a high-affinity receptor composed of three trans-membrane proteins, the , , and c chains (43). The c chain is shared with the receptors for IL-2,-4,-7,-9, and-15 (22, 33, 50, 54, 55) and is constitutively expressed in mature T cells and their thymic precursors (8, 32, 48). IL-2 receptor (IL-2R) is present on a subpopulation of resting T cells (51, 62). and c chains combine to form an intermediate-affinity IL-2R that can transmit signals (47, 49), but cannot stimulate the proliferation of normal T lymphocytes (7, 38, 59). The chain is undetectable on resting T cells. Its expression is triggered by antigen (53), a stimulus that can be mimicked by lectins such as con-canavalin A (ConA) (31) or by antibodies against the T-cell receptor (TCR) (20). These signals also result in secretion of IL-2, which increases and prolongs IL-2R expression (4, 15, 39), thus acting as a positive feedback regulator of its own high-affinity receptor. IL-2R gene expression is regulated mostly through changes in its rate of transcription (13, 34, 52). In transgenic mice bearing a reporter gene under the control of 2.6 kb of 5 flanking region of the murine IL-2R gene, transgene expression is restricted to lymphoid organs (60). In T cells, the trans-gene can be induced by ConA and IL-2 with kinetics very similar to those of the endogenous gene. The responses of both the human and mouse genes to signals from the TCR depend on cis-acting elements in the promoter-proximal region (1, 5, 10, 28, 61). By transient transfection of IL-2R mutant promoter constructs into a rodent T-cell line, PC60, we identified a 51-nucleotide (nt) enhancer necessary and sufficient for the response of the IL-2R gene to IL-2 1.3 kb upstream of the transcription start site (61). Subsequently the homologue of this IL-2-responsive enhancer (IL-2rE) was found at around 4 kb in the human IL-2R gene (6, 29, 36). The mouse enhancer consists of three separate elements: two binding sites for signal transducers and activators of transcription (STAT) and a consensus motif for Ets family members. In PC60 cells, the STAT motifs (sites I and II) and the Ets binding site (site III) are all required for the response of the enhancer to IL-2. Recently, we have shown that IL-2 stimulation of IL-2rE activity depends on the cooperative binding of two IL-2-induced STAT5 dimers to sites I and II (42). This is concordant with the finding that, in mice lacking STAT5A, IL-2-induced IL-2R expression is severely reduced (46). Site II overlaps with a recognition sequence for GATA proteins, but this motif does not appear to be important for IL-2rE function (42). Elf-1, a member of the Ets family of proteins, contributes to IL-2rE activity by binding to site III (58). Elf-1 binding activity to site III is constitutive in PC60 cells. There is growing evidence that chromatin structure participates in gene regulation, partly by modulating access of transcription factors to DNA (3, 17, 65). Gene activation is often accompanied by perturbation or disruption of the nucleosomes that occupy cis-acting elements. Such an opening of chromatin is generally reflected in a change in its sensitivity to nucleases. Indeed, in mouse T lymphocytes, activation of IL-2R expression results in the appearance of a DNase I-hypersensitive site in, or close to, the IL-2rE (60). Induction of DNase I-hyper-sensitive sites by IL-2 has also been described in the B-cell-specific enhancer of the immunoglobulin J chain gene (30). Here we present the results of an analysis, by in vivo foot-printing and nuclease digestion, of the changes in IL-2rE chro-matin during activation of IL-2R expression in normal mouse T lymphocytes. We find that protein-DNA interactions on all three elements of the IL-2rE depend on stimulation by IL-2, although Elf-1 binding activity is present in resting T lympho-cytes and is not affected by IL-2. Stimulation with ConA, which induces activation of STAT1 but not STAT5, does not result in the occupation of the STAT binding sites in the IL-2rE, even though both site I and site II can bind STAT1 in vitro. Stimulation of ConA-activated cells with IL-2 leads to the appearance of micrococcal nuclease hypersensitive sites flanking the IL-2rE, indicating that IL-2 induces translational positioning of nucleosomes at the borders of the IL-2rE. Our data show that IL-2 regulates the accessibility of the IL-2rE to the tran