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. 2011 Dec 16;286(50):43429-36.
doi: 10.1074/jbc.M111.299339. Epub 2011 Oct 5.

cAMP-responsive element modulator (CREM)α protein signaling mediates epigenetic remodeling of the human interleukin-2 gene: implications in systemic lupus erythematosus

Christian M Hedrich  1 , Thomas Rauen, George C Tsokos
Affiliations

cAMP-responsive element modulator (CREM)α protein signaling mediates epigenetic remodeling of the human interleukin-2 gene: implications in systemic lupus erythematosus

Christian M Hedrich et al. J Biol Chem. .

Abstract

IL-2 is a key cytokine during proliferation and activation of T lymphocytes and functions as an auto- and paracrine growth factor. Regardless of activating effects on T lymphocytes, the absence of IL-2 has been linked to the development of autoimmune pathology in mice and humans. Systemic lupus erythematosus (SLE) is a multifactorial autoimmune disease and characterized by dysregulation of lymphocyte function, transcription factor and cytokine expression, and antigen presentation. Reduced IL-2 expression is a hallmark of SLE T lymphocytes and results in decreased numbers of regulatory T lymphocytes which play an important role in preventing autoimmunity. Reduced IL-2 expression was linked to overproduction of the transcription regulatory factor cAMP-responsive element modulator (CREM)α in SLE T lymphocytes and subsequent CREMα binding to a CRE site within the IL2 promoter (-180 CRE). In this study, we demonstrate the involvement of CREMα-mediated IL2 silencing in T lymphocytes from SLE patients through a gene-wide histone deacetylase 1-directed deacetylation of histone H3K18 and DNA methyltransferase 3a-directed cytosine phosphate guanosine (CpG)-DNA hypermethylation. For the first time, we provide direct evidence that CREMα mediates silencing of the IL2 gene in SLE T cells though histone deacetylation and CpG-DNA methylation.

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Figures

FIGURE 1.
FIGURE 1.
Alignment of the human and mouse IL2 genes. Pink peaks denote CNS sites, purple peaks are exons with sequence identity of >75% over at least 200 bp. Red squares denote conserved noncoding sequences that were determined regions of interest for further analysis of histone modifications and DNA methylation. The previously reported IL2 −180 CRE site is indicated under the alignment.
FIGURE 2.
FIGURE 2.
CREMα and its effects on IL-2 expression. A, overexpression of CREMα in primary human T lymphocytes (for 5 h) results in reduced IL-2 mRNA expression (n = 4). B, CREMα weakly binds to the IL2 −180 CRE in naïve CD4+ T lymphocytes. CREMα binding is significantly (p = 0.04) increased in response to T cell activation with anti-CD3/CD28 antibodies (for 72 h). Pairs of naïve CD4+ T lymphocytes and activated CD4+ T cells from the same individual are connected by dashed lines. C, CREMα binds to the IL2 −180 CRE of T lymphocytes from healthy control individuals. Corresponding results from age-, gender-, and ethnicity-matched controls are connected to data from SLE T cells by dashed lines. CREMα binding to the IL2 −180 CRE site is significantly (p = 0.03) increased in T lymphocytes from SLE patients. Error bars, S.D.
FIGURE 3.
FIGURE 3.
Histone modifications of the IL2 gene in response to T lymphocyte activation and in SLE T cells. A, activation of naïve CD4+ T lymphocytes with anti-CD3 and anti-CD28 antibodies results in increased histone H3K18 acetylation and decreased histone H3K27 trimethylation. Results are displayed as relative increase in histone H3K18 acetylation and H3K27 trimethylation based on the situation in naïve CD4+ T lymphocytes (indicated by dashed line = 100%). B, T lymphocytes from SLE patients display decreased histone H3K18 acetylation and increased histone H3K27 trimethylation compared with T lymphocytes from healthy controls. Results are displayed as relative histone H3K18 acetylation and H3K27 trimethylation based on the situation in control T lymphocytes (indicated by dashed line = 100%). Error bars, S.D.
FIGURE 4.
FIGURE 4.
HDAC1 recruitment to the IL2 −180 CRE site as detected by ChIP. A, Naïve and activated (anti-CD3/CD28) CD4+ T lymphocytes display no relevant HDAC1 binding. Pairs of naïve CD4+ T lymphocytes and activated CD4+ T cells from the same individual are connected by dashed lines. NS, not significant. B, HDAC1 only weakly binds to the IL2 −180 CRE site in control T lymphocytes. In T lymphocytes from SLE patients, we detected significantly more CREMα recruitment to the −180 CRE site (p = 0.001). Corresponding results from age-, gender-, and ethnicity-matched controls are connected to data from SLE T cells by dashed lines.
FIGURE 5.
FIGURE 5.
CpG-DNA methylation of the IL2 gene as detected by immunoprecipitation of methylated CpG sequences. CNS1–CNS3 are located in the 2 kb spanning proximal promoter, CNS4 is located in the 3′-UTR. Values are normalized to 100% methylated (input) DNA and unmethylated (control) DNA. The IL2 gene T lymphocytes from SLE patients is methylated to a significantly lower degree compared with control T cells (p < 0.001). Error bars, S.D.
FIGURE 6.
FIGURE 6.
Overexpression of DNMT3a in Jurkat T cells suppresses the expression of IL-2 mRNA. Empty vector (EV; 3 μg), or DNMT3a (3 μg) expression plasmids were transfected into Jurkat T cells. Cells were cultured overnight, stimulated with anti-CD3 and anti-CD28 antibodies for 5 h, and harvested. RNA was purified from and IL-2 transcripts were measured and normalized to GAPDH by real-time RT-PCR. The expression levels of the transcripts were significantly decreased in Jurkat T cells transfected with DNMT3a compared with cells transfected with empty control plasmids (p < 0.001). The results represent the mean ± S.D. (error bar) from three independent experiments.
FIGURE 7.
FIGURE 7.
DNMT3a interacts with CREMα and gets recruited to IL2 −180 CRE. A, upper, CREMα and DNMT3a were transfected into HEK293T cells as indicated, and overexpression is shown by immunoblotting of cell lysates. Lower, co-immunoprecipitated (IP) DNMT3a is shown. Both endogenous CREMα (fourth lane 4) and overexpressed CREMα protein (sixth lane) co-immunoprecipitate overexpressed DNMT3a protein. B, SLE T cells that produce significantly less IL-2 compared with controls show increased DNMT3a recruitment to the IL2 −180 CRE site compared with control T lymphocytes. Corresponding results from age-, gender-, and ethnicity-matched controls are connected to data from SLE T cells by dashed lines.
FIGURE 8.
FIGURE 8.
Model for the involvement of CREMα in the regulation of IL2 gene expression under physiologic conditions and in SLE. Under physiologic conditions, CREMα gets expressed in response to T lymphocyte activation. Increased CREMα expression results in binding to the −180 CRE site and competition with the IL2 trans-activator pCREB, an activator of IL-2 expression. This results in an autoregulatory reduction of IL-2 expression. SLE T lymphocytes overexpress CREMα. This results in competition with pCREB and reduced trans-activation of IL2. CREMα binding to the IL2 −180 CRE site results in recruitment of HDAC1 and DNMT3a to the IL2 promoter. This results in epigenetic remodeling and transcriptional repression through histone deacetylation and CpG-DNA methylation.
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