Chromatin Remodeling, Measured by a Novel Real-Time Polymerase Chain Reaction Assay, Across the Proximal Promoter Region of the <i>IL-2</i> Gene

Naturally occurring CD4+CD25+ regulatory T cells (Tregs), which play an important role in the maintenance of self-tolerance, proliferate poorly and fail to produce IL-2 following stimulation in vitro with peptide-pulsed or anti-CD3-treated APCs. When TCR proximal and distal signaling events were examined in Tregs, we observed impairments in the amplitude and duration of tyrosine phosphorylation when compared with the response of CD4+CD25− T cells. Defects were also seen in the activity of phospholipase C-γ and in signals downstream of this enzyme including calcium mobilization, NFAT, NF-κB, and Ras-ERK-AP-1 activation. Enhanced stimulation of diacylglycerol-dependent pathways by inhibition of diacylglycerol metabolism could overcome the “anergic state” and support the ability of Tregs to up-regulate CD69, produce IL-2, and proliferate. Our results demonstrate that Tregs maintain their hyporesponsive state by suppressing the induction and propagation of TCR-initiated signals to control the accumulation of second messengers necessary for IL-2 production and proliferation.

Section: Discussionmentioning

confidence: 99%

Defective Activation of Protein Kinase C and Ras-ERK Pathways Limits IL-2 Production and Proliferation by CD4+CD25+ Regulatory T Cells

Hickman

Yang

Thomas

et al. 2006

“…To address the possibility that CREM inhibits IL-2 production by enhancing chromatin condensation, we conducted ChIP and restriction enzyme accessibility experiments (CHART) (24). As shown in Fig.…”

Section: Antisense Crem Treatment Increases Restriction Enzyme Accessmentioning

confidence: 99%

The Cyclic Adenosine 5′-Monophosphate Response Element Modulator Suppresses IL-2 Production in Stimulated T Cells by a Chromatin-Dependent Mechanism

Tenbrock

Juang

Tolnay

et al. 2003

The production of IL-2 is tightly controlled by several transcription factors that bind to the IL-2 promoter. The cAMP response element modulator (CREM) is known to form complexes with CREB and bind to the −180 site of the IL-2 promoter in anergic and in systemic lupus erythematosus T cells. In this study we show that CREM is transcriptionally induced in T cells following stimulation through CD3 and CD28, binds to the IL-2 promoter in vivo, and suppresses IL-2 production. Transfection of an antisense CREM plasmid into T cells blocked the expression and binding of CREM to the IL-2 promoter and the decrease of IL-2 production, which follows the early increase after T cell stimulation with CD3 and CD28. In addition, as assessed by chromatin immunoprecipitation experiments, antisense CREM prevented the binding of protein 300 and cAMP response element binding protein and promoted the acetylation of histones. Antisense CREM also enhanced the accessibility of the IL-2 promoter to endonucleases and prevented the condensation of chromatin in vivo. Our data suggest that upon T cell activation, CREM gradually replaces phosphorylated CREB at the −180 site of the IL-2 promoter. CREM, in turn, binds protein 300 and cAMP response element binding protein, but CREM is unable to activate its histone acetyltransferase activity, which results in condensation of chromatin and down-regulation of IL-2 production.

“…MNase and restriction enzyme accessibility assays were performed as recently described (51). Briefly, Jurkat T cell nuclei were resuspended in either MNase digestion buffer (10 mM Tris, pH 7.5, 15 mM NaCl, 60 mM KCl, 1 mM CaCl 2 ) or restriction enzyme buffer (1ϫ buffer supplied by New England Biolabs (1 l of 2 mg/ml leupeptin, 1 l of 1.8 mg/ml aprotinin, and 1 l of 100 mM PMSF)) and incubated with the appropriate endonuclease.…”

Section: Chromatin Accessibility Using Real-time Pcr (Chart-pcr)mentioning

confidence: 99%

The Human IL-2 Gene Promoter Can Assemble a Positioned Nucleosome That Becomes Remodeled Upon T Cell Activation

Attema

Reeves

Murray

et al. 2002

Self Cite

Controlled production of the cytokine IL-2 plays a key role in the mammalian immune system. Expression from the gene is tightly regulated with no detectable expression in resting T cells and a strong induction following T cell activation. The IL-2 proximal promoter (+1 to −300) contains many well-defined transcriptional activation elements that respond to T cell stimulation. To determine the role of chromatin structure in the regulation of interleukin-2 gene transcription, nucleosome assembly across the IL-2 promoter region was examined using in vitro chromatin reconstitution assays. The IL-2 promoter assembles a nucleosome that is both translationally and rotationally positioned, spanning some of the major functional control elements. The binding of transcription factors to these elements, with the exception of the architectural protein HMGA1, was occluded by the presence of the nucleosome. Analysis of the chromatin architecture of the IL-2 gene in Jurkat T cells provided evidence for the presence of a similarly positioned nucleosome in vivo. The region encompassed by this nucleosome becomes remodeled following activation of Jurkat T cells. These observations suggest that the presence of a positioned nucleosome across the IL-2 proximal promoter may play an important role in maintaining an inactive gene in resting T cells and that remodeling of this nucleosome is important for gene activation.